This Issue is Dedicated to
Professor Franco F. Vincieri
on the Occasion of his 70th Birthday
Volume 3. Issue 12. Pages 1941-2166. 2008
ISSN 1934-578X (printed); ISSN 1555-9475 (online)
www.naturalproduct.us
NPC
Natural Product Communications
EDITOR-IN-CHIEF
DR. PAWAN K AGRAWAL
Natural Product Inc.
7963, Anderson Park Lane,
Westerville, Ohio 43081, USA
agrawal@naturalproduct.us
EDITORS
PROFESSOR GERALD BLUNDEN
The School of Pharmacy & Biomedical Sciences,
University of Portsmouth,
Portsmouth, PO1 2DT U.K.
axuf64@dsl.pipex.com
PROFESSOR ALESSANDRA BRACA
Dipartimento di Chimica Bioorganicae Biofarmacia,
Universita di Pisa,
via Bonanno 33, 56126 Pisa, Italy
braca@farm.unipi.it
PROFESSOR DEAN GUO
State Key Laboratory of Natural and Biomimetic Drugs,
School of Pharmaceutical Sciences,
Peking University,
Beijing 100083, China
gda5958@163.com
PROFESSOR J. ALBERTO MARCO
Departamento de Quimica Organica,
Universidade de Valencia,
E-46100 Burjassot, Valencia, Spain
alberto.marco@uv.es
PROFESSOR YOSHIHIRO MIMAKI
School of Pharmacy,
Tokyo University of Pharmacy and Life Sciences,
Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan
mimakiy@ps.toyaku.ac.jp
PROFESSOR STEPHEN G. PYNE
Department of Chemistry
University of Wollongong
Wollongong, New South Wales, 2522, Australia
spyne@uow.edu.au
PROFESSOR MANFRED G. REINECKE
Department of Chemistry,
Texas Christian University,
Forts Worth, TX 76129, USA
m.reinecke@tcu.edu
PROFESSOR WILLIAM N. SETZER
Department of Chemistry
The University of Alabama in Huntsville
Huntsville, AL 35809, USA
wsetzer@chemistry.uah.edu
PROFESSOR YASUHIRO TEZUKA
Institute of Natural Medicine
Institute of Natural Medicine, University of Toyama,
2630-Sugitani, Toyama 930-0194, Japan
tezuka@inm.u-toyama.ac.jp
ADVISORY BOARD
Prof. Viqar Uddin Ahmad
Karachi, Pakistan
Prof. Øyvind M. Andersen
Bergen, Norway
Prof. Giovanni Appendino
Novara, Italy
Prof. Yoshinori Asakawa
Tokushima, Japan
Prof. Maurizio Bruno
Palermo, Italy
Prof. Carlos Cerda-Garcia-Rojas
Mexico city, Mexico
Prof. Josep Coll
Barcelona, Spain
Prof. Geoffrey Cordell
Chicago, IL, USA
Prof. Samuel Danishefsky
New York, NY, USA
Dr. Biswanath Das
Hyderabad, India
Prof. A.A. Leslie Gunatilaka
Tucson, AZ, USA
Prof. Stephen Hanessian
Montreal, Canada
Prof. Michael Heinrich
London, UK
Prof. Kurt Hostettmann
Lausanne, Switzerland
Prof. Martin A. Iglesias Arteaga
Mexico, D. F, Mexico
Prof. Jerzy Jaroszewski
Copenhagen, Denmark
Prof. Teodoro Kaufman
Rosario, Argentina
Prof. Norbert De Kimpe
Gent, Belgium
Prof. Hartmut Laatsch
Gottingen, Germany
Prof. Marie Lacaille-Dubois
Dijon, France
Prof. Shoei-Sheng Lee
Taipei, Taiwan
Prof. Francisco Macias
Cadiz, Spain
Prof. Anita Marsaioli
Campinas, Brazil
Prof. Imre Mathe
Szeged, Hungary
Prof. Joseph Michael
Johannesburg, South Africa
Prof. Ermino Murano
Trieste, Italy
Prof. Virinder Parmar
Delhi, India
Prof. Luc Pieters
Antwerp, Belgium
Prof. Om Prakash
Manhattan, KS, USA
Prof. Peter Proksch
Düsseldorf, Germany
Prof. William Reynolds
Toronto, Canada
Prof. Raffaele Riccio
Salerno, Italy
Prof. Ricardo Riguera
Santiago de Compostela, Spain
Prof. Satyajit Sarker
Coleraine, UK
Prof. Monique Simmonds
Richmond, UK
Prof. Valentin Stonik
Vladivostok, Russia
Prof. Hermann Stuppner
Innsbruck, Austria
Prof. Apichart Suksamrarn
Bangkock, Thailand
Prof. Hiromitsu Takayama
Chiba, Japan
Prof. Karin Valant-Vetschera
Vienna, Austria
Prof. Peter G. Waterman
Lismore, Australia
Prof. Paul Wender
Stanford, USA
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Natural Product Communications Vol. 3 (12) 2008
Published online (www.naturalproduct.us)
Editorial
In Honor of the 70th Birthday of Professor Franco Francesco Vincieri
It is our great privilege and pleasure to introduce this issue, which is dedicated to Professor Franco Francesco
Vincieri, Faculty of Pharmacy, Department of Pharmaceutical Sciences, of the University of Florence, on the
occasion of his 70th birthday.
His research has been based on numerous aspects of modern pharmacognosy, offering valuable contributions to
pharmacognosy (pharmaceutical biology) and analytical phytochemistry, and more recently pharmaceutical
technology applied to herbal drug preparations. He is author of more than 180 publications in international
scientific journals, several books and chapters of books and a great number of contributions in congress
proceedings.
Prof. Vincieri’s efforts to promote quality and modernization, including the technological aspects of herbal
medicinal products, have been acknowledged by the Italian Government through the financing of projects related to
optimization of the technological and biopharmaceutical aspects of Herbal Medicinal Products and Botanical
Health Products to improve their quality and safety of use, as well as awards from national and international
scientific societies including ESCOP (European Scientific Cooperative on Phytotherapy) and APV
(Abeitsgemeinschaft für Pharmazeuitsche Verfahrenstechnik E.V.).
This issue, on occasion of his 70th Birthday on October 12th 2008, represents a tribute for his outstanding scientific
contributions and an opportunity to express our congratulations and warm wishes from us, as well as his colleagues
and friends. Our personal thanks go also to all the authors, mostly of them Franco’s friends, and reviewers who
have contributed to the success of this special issue.
Anna Rita Bilia
Pawan K. Agrawal
Editor in chief
Natural Product Communications Vol. 3 (12) 2008
Published online (www.naturalproduct.us)
Preface
Franco Francesco Vincieri: A succesful example of youthful enthusiasm
for research and education
This issue of Natural Product Communications recognizes Professor Franco Francesco Vincieri on the occasion of
his 70th birthday celebrated on October 12, 2008.
I have invited some dear colleagues to present him in writing. First of all, the Director of the Department of
Pharmaceutical Sciences, Prof. Massimo Bambagiotti Alberti, and the Dean of the Faculty of Pharmacy,
Prof. Sergio Pinzauti, who have also been good friends since the beginning of his academic career. Prof. Vincieri
also served for many years in the Italian Society of Phytochemistry (SIF) and the European Scientific Committee
(ESCOP) and thus I asked the SIF president, Prof. Cosimo Pizza, and the Chair of ESCOP, Dr. Barbara Steinhoff,
to comment on his role in these organizations.
Prof. Anna Rita Bilia
University of Florence, Department of Pharmaceutical Sciences
Via Ugo Schiff 6, 50019 Sesto Fiorentino (FI), Italy
I met Franco F. Vincieri for the first time in 1964 when I entered the Institute of Pharmaceutical and Toxicological
Chemistry, University of Florence, nowadays called the Department of Pharmaceutical Sciences. Being young and
avid researchers, we soon became interested in modern analytical chemistry. As a matter of fact, in the following
few years we introduced Gas Chromatography into our Institute with the first instrument acquired by the University
of Florence. The analysis of terpenoids and related volatile compounds was the starting point for our ever
increasing interest in separation science. Gas Chromatography-Mass Spectrometry (GC-MS) was the outcome of
the longing to improve our scientific tooling. Again, the acquired instrument was the first GC-MS in the University
of Florence. Our research capabilities were boosted accordingly and a new generation of mass spectrometrists was
trained and formed in our lab.
After 1990 we entered quite different careers, but as we have remained in the same Department, we have proceeded
with our collaboration and solid friendship. During that period Franco became increasingly interested in quality
aspects of herbal medicinal products and was first involved in the Italian Pharmacopoeia as a member of the
Permanent Commission (since 1993) where, since January 2004, he has been President of the Working Group
“Herbal Drugs”. He is an expert member of “Consiglio Superiore di Sanità” for the three-year period 2006-2009.
Prof. Vincieri was the Italian Delegate, first at HMPWG (Herbal Medicinal Products Working Group) and then at
HMPWP (Herbal Medicinal Products Working Party) from 1997 to 2004 at the EMEA.
He was assessor of the “Certification procedure for Chemical quality and microbiological purity evaluation” of
EDQM (European Directorate for the Quality of Medicines) from 1998 to 2004 and assessor of the “Certification
procedure for Herbal Drugs and Herbal Drug Preparations evaluation” of EDQM since 2004. Beginning this year
(2008), he is a member of the European Pharmacopoeia Commission of the Traditional Chinese Medicine working
Party.
Prof. Massimo Bambagiotti
Head of Department
University of Florence, Department of Pharmaceutical Sciences
Via Ugo Schiff 6, 50019 Sesto Fiorentino (FI), Italy
Natural Product Communications Vol. 3 (12) 2008
Published online (www.naturalproduct.us)
The first time I met Franco F. Vincieri was in June 1967 when I entered as a CNR fellow in the Institute of
Pharmaceutical and Toxicological Chemistry. During that time he was involved in the elementary analysis of
various products synthetised in the Institute. A strong friendship was quickly created between us and we discovered
sharing, apart from our great enthusiasm for synthesis and analysis of new drugs, a passion for photography which
led to experimentation of numerous photographical processes of developing and printing. It was a “chemical” way
to cultivate through images, during our free time, our aesthetics, which I recall as distinguishing.
In the Seventies he passed from being professor in charge of the "Laboratory of Extractive and Synthetic Drug
Preparation" to being an associate, then supernumerary professor of "Phytopharmacy". Now he is full professor of
Pharmaceutical Technology, Socio-economics and Legislation. Over the last 45 years his teaching skills have been
very much appreciated by generations of students. He is quite active in the University of Florence, taking on
various institutional roles such as Vice Dean of the Faculty of Pharmacy since 2001, Director of the graduate school
of “Herbal Technology” from 2000 to 2006, Director of the Professional Specialization Course “Expert Technician
in Officinal Herbs” (collaboration between the University of Florence and the Region of Tuscany) from 2005 to
2006, Director of the post-graduate course “Herbal Medicinal Products of Traditional Chinese Medicine” since
2005 and Director of the post-graduate school of “Hospital Pharmacy ” from 1994 to 2007.
His research has been based on numerous aspects and he is author or coauthor of more than 180 publications in
international scientific journals, several books and chapters of books, and a great number of contributions in
congress proceedings.
Prof. Sergio Pinzauti
Editor, Journal of Pharmaceutical and Biomedical Analysis
Dean, Faculty of Pharmacy
University of Florence, Department of Pharmaceutical Sciences
Via Ugo Schiff 6, 50019 Sesto Fiorentino (FI), Italy
It is an honour and a pleasure for me, as Chairman of the Italian Society of Phytochemistry (SIF) and also as a
colleague and friend, to send wishes to Prof. Franco Vincieri, also on behalf of the Society, on the occasion of his
70th birthday.
Prof. Vincieri, at the beginning of 80’s founded the Italian Society of Phytochemistry together with other
distinguished scientists in the field and he was a member of the board for many years. The Italian Society of
Phytochemistry has the aim of promoting knowledge of the plant kingdom by phytochemical, pharmacological,
toxicological, food, industrial and ecological point of view and the cooperation with other national and international
organizations pursuing the same objectives.
Prof. Franco Vincieri has always participated in all the SIF activities, contributing with his considerable scientific
profile and his ability as organizer to various national and international events, paying particular attention to the
initiatives devoted to young scientists. In the period 1998-2000 he was chairman of our Society, promoting a
number of successful scientific activities and workshops, for example on Ginkgo biloba, on Analytical Methods in
Phytochemistry and on Cell Cultures, which attracted a large number of new members to SIF.
To Franco, a very outstanding scientist, sincere thanks for all he has done and will continue to do in the future in
the field of phytochemistry.
Prof. Cosimo Pizza
Chairman of Italian Society of Phytochemistry
University of Salerno, Department of Pharmaceutical Sciences
via Ponte Don Melillo 84084 Fisciano (SA), Italy
Natural Product Communications Vol. 3 (12) 2008
Published online (www.naturalproduct.us)
For many years, Professor Franco F. Vincieri has been very active in ESCOP, the European Scientific Cooperative
on Phytotherapy. Starting already in 1991 at an early stage of this scientific organisation, he represented the Società
Italiana di Fitochimica as host of the 2nd ESCOP Symposium which took place in Milan in March 1992 and
chaired the meeting of the ESCOP Council.
The colleagues from the Scientific Committee remember well the fruitful ESCOP meetings consisting of detailed
work and long discussions on draft monographs which are intended to provide harmonised assessment criteria for
efficacy and safety of herbal medicinal products in Europe, but also beautiful visits to botanical gardens and other
places of interest. They gratefully accepted his kind invitation to the Florence meeting in May 1996 which took
place in the Scuolà di Sanità Militare, the concert of "I Mandolinisti Fiorentini" in the evening being one of the
special highlights of this weekend.
When Franco Vincieri handed over his ESCOP seat to Professor Anna Rita Bilia in 1997, ESCOP offered him to
continue his activities as a member of the Board of Supervising Editors which he kindly accepted. Since 2004 he is
also member of the ESCOP Research Committee. In this position he reviews all the monographs produced by the
Scientific Committee and comments on each of them before publication. Due to his long-term experience in
pharmacognosy and related scientific areas his suggestions for modifications are highly appreciated by the
Committee.
The members of the ESCOP Scientific Committee would like to congratulate Franco Vincieri on his 70th birthday
and express their sincere thanks for all his contributions to the work of ESCOP. They are looking forward to further
enjoyable collaboration in the future and would like to thank him for being such a good colleague.
Dr. Barbara Steinhoff
Bundesverband der Arzneimittel-Hersteller e.V.
Postfach 20 12 55
53142 Bonn, Germany
Happy birthday from all of us, colleagues and friends!
Natural Product Communications
2008
Volume 3, Number 12
Contents
Page
1968-2008: 40 Years of Franco F. Vincieri’s Natural Products Research
Anna Rita Bilia
1941
Effects of Terpenoids from Salvia willeana in Delayed-type Hypersensitivity, Human
Lymphocyte Proliferation and Cytokine Production
Anna Vonaparti, Anastasia Karioti, María C. Recio, Salvador Máñez, José L. Ríos, Eleani Skaltsa and
Rosa M. Giner
1953
Characterization of By-products of Saffron (Crocus sativus L.) Production
Pamela Vignolini, Daniela Heimler, Patrizia Pinelli, Francesca Ieri, Arturo Sciullo and Annalisa Romani
1959
Antitrypanosomal and Antileishmanial Activities of Organic and Aqueous Extracts of
Artemisia annua
Anna Rita Bilia, Marcel Kaiser, Franco Francesco Vincieri and Deniz Tasdemir
1963
Secondary Metabolites from the Roots of Salvia palaestina Bentham
Antonio Vassallo, Ammar Bader, Alessandra Braca, Angela Bisio, Luca Rastrelli, Francesco De Simone
and Nunziatina De Tommasi
1967
Cancer Chemopreventive Potential of Humulones and Isohumulones (Hops α- and Iso-α-acids):
Induction of NAD(P)H:Quinone Reductase as a Novel Mechanism
Gregor Bohr, Karin Klimo, Josef Zapp, Hans Becker and Clarissa Gerhäuser
1971
A Polar Cannabinoid from Cannabis sativa var. Carma
Giovanni Appendino, Anna Giana, Simon Gibbons, Massimo Maffei, Giorgio Gnavi,
Gianpaolo Grassi and Olov Sterner
1977
HPLC-DAD-MS Fingerprint of Andrographis paniculata (Burn. f.) Nees (Acanthaceae)
Sabrina Arpini, Nicola Fuzzati, Andrea Giori, Emanuela Martino, Giacomo Mombelli, Luca Pagni
and Giuseppe Ramaschi
1981
Diterpenoid Alkaloids and Phenol Glycosides from Aconitum naviculare (Brühl) Stapf.
Stefano Dall’Acqua, Bharat B. Shrestha, Mohan Bikram Gewali, Pramod Kumar Jha , Maria Carrara
and Gabbriella Innocenti
1985
Inhibition of PGHS-1 and PGHS-2 by Triterpenoid Acids from Chaenomelis fructus
Eveline Reininger and Rudolf Bauer
1991
Preparative Isolation of Antimycobacterial Shoreic Acid from Cabralea canjerana by High Speed
Countercurrent Chromatography
Gilda G. Leitão, Lisandra F. Abreu, Fernanda N. Costa, Thiago B. Brum, Daniela Fernandes Ramos,
Pedro Eduardo A. Silva, Maria Cristina S. Lourenço and Suzana G. Leitão
1995
Antiplasmodial Effects of a few Selected Natural Flavonoids and their Modulation of
Artemisinin Activity
Anna Rita Bilia, Anna Rosa Sannella, Franco Francesco Vincieri, Luigi Messori, Angela Casini,
Chiara Gabbiani , Carlo Severini and Giancarlo Majori
1999
Comparative Analysis of Antimalarial Principles in Artemisia annua L. Herbal Drugs from
East Africa
Silvia Lapenna, Maria Camilla Bergonzi, Franco Francesco Vincieri and Anna Rita Bilia
2003
Continued Overleaf
Natural Product Communications Vol. 3 (12) 2008
Published online (www.naturalproduct.us)
In vitro Apoptotic Bioactivity of Flavonoids from Astragalus verrucosus Moris
Joseph A. Buhagiar, Alessandra Bertoli, Marie Therese Camilleri-Podesta and Luisa Pistelli
2007
Qualitative Profile and Quantitative Determination of Flavonoids from Crocus sativus L. Petals
by LC-MS/MS
Paola Montoro, Carlo I. G. Tuberoso, Mariateresa Maldini, Paolo Cabras and Cosimo Pizza
2013
HPLC/DAD/ESI-MS Analysis of Non-volatile Constituents of Three Brazilian Chemotypes of
Lippia alba (Mill.) N. E. Brown
Patrícia Timóteo, Anastasia Karioti, Suzana G. Leitão, Franco Francesco Vincieri and Anna Rita Bilia
2017
Optimization and Validation of an HPLC–Method for Quality Control of Pueraria lobata Root
Lidiya Bebrevska, Mart Theunis, Arnold Vlietinck, Luc Pieters and Sandra Apers
2021
Pharmacokinetics of Luteolin and Metabolites in Rats
Sasiporn Sarawek, Hartmut Derendorf and Veronika Butterweck
2029
Complete Characterization of Extracts of Onopordum illyricum L. (Asteraceae) by
HPLC/PDA/ESIMS and NMR
Luisella Verotta, Laura Belvisi, Vittorio Bertacche and Maria Cecilia Loi
2037
Phenolic Profiles of Four Processed Tropical Green Leafy Vegetables Commonly Used as Food
Sule Ola Salawu, Marzia Innocenti, Catia Giaccherini, Afolabi Akintunde Akindahunsi and
Nadia Mulinacci
2043
(Bio)Sensor Approach in the Evaluation of Polyphenols in Vegetal Matrices
M. Camilla Bergonzi, Maria Minunni and Anna Rita Bilia
2049
In vitro Radical Scavenging and Anti-Yeast Activity of Extracts from Leaves of Aloe Species
Growing in Congo
Annalisa Romani, Pamela Vignolini, Laura Isolani, Sara Tombelli, Daniela Heimler,
Benedetta Turchetti and Pietro Buzzini
2061
Antioxidant Principles and Volatile Constituents from the North-western Iberian mint
“erva-peixeira”, Mentha cervina
Matteo Politi, César L Rodrigues, Maria S Gião, Manuela E Pintado and Paula ML Castro
2065
Chemical Composition of Thymus serrulatus Hochst. ex Benth. Essential Oils from Ethiopia:
a Statistical Approach
Bruno Tirillini, Roberto Maria Pellegrino, Mario Chessa and Giorgio Pintore
2069
GC MS Analysis of the Volatile Constituents of Essential Oil and Aromatic Waters of Artemisia
annua L. at Different Developmental Stages
Anna Rita Bilia, Guido Flamini, Fabrizio Morgenni, Benedetta Isacchi and Franco FrancescoVincieri
2075
Do Non-Aromatic Labiatae Produce Essential Oil? The Case Study of Prasium majus L.
Claudia Giuliani, Roberto Maria Pellegrino, Bruno Tirillini and Laura Maleci Bini
2079
Olive-oil Phenolics and Health: Potential Biological Properties
Francesco Visioli, Francesca Ieri, Nadia Mulinacci, Franco F. Vincieri and Annalisa Romani
2085
Traceability of Secondary Metabolites in Eucalyptus and Fagus Wood derived Pulp and Fiber
Aline Lamien-Meda, Karin Zitterl-Eglseer, Heidrun Fuchs and Chlodwig Franz
2089
Potential Anticancer Activity Against Human Epithelial Cancer Cells of Peumus boldus Leaf Extract
Juan Garbarino, Nicolas Troncoso, Giuseppina Frasca, Venera Cardile and Alessandra Russo
2095
Antihyperalgesic Effect of Eschscholzia californica in Rat Models of Neuropathic Pain
Elisa Vivoli, Anna Maidecchi, Anna Rita Bilia, Nicoletta Galeotti, Monica Norcini and Carla Ghelardini
2099
Natural Product Communications Vol. 3 (12) 2008
Published online (www.naturalproduct.us)
Problems in Evaluating Herbal Medicinal Products
Jozef Corthout
2103
Impurities in Herbal Substances, Herbal Preparations and Herbal Medicinal Products, IV.
Heavy (toxic) Metals
SFSTP Commission, Didier Guédon, Michèle Brum, Jean-Marc Seigneuret, Danièle Bizet,
Serge Bizot, Edmond Bourny, Pierre-Albert Compagnon, Hélène Kergosien, Luis Georges Quintelas,
Jerôme Respaud, Olivier Saperas, Khalil Taoubi and Pascale Urizzi
2107
Review/Account
A Fresh Insight into the Interaction of Natural Products with Pregnane X Receptor
Salvador Máñez
2123
Natural Products as Gastroprotective and Antiulcer Agents: Recent Developments
Rosa Tundis, Monica R Loizzo, Marco Bonesi, Federica Menichini, FilomenaConforti,
Giancarlo Statti and Francesco Menichini
2129
Phytochemistry and Pharmacology of Boronia pinnata Sm.
MassimoCurini, Salvatore Genovese, Luigi Menghini, Maria Carla Marcotullio and Francesco Epifano
2145
Therapeutic Potential of Kalanchoe Species: Flavonoids and other Secondary Metabolites
Sônia S. Costa, Michelle F. Muzitano, Luiza M. M. Camargo and Marcela A. S. Coutinho
2151
Manuscripts in Press
2165
Natural Product Communications Vol. 3 (12) 2008
Published online (www.naturalproduct.us)
LIST OF AUTHORS
Abreu, LF ................. 1995
Acqua, SD ................ 1985
Akindahunsi, AA...... 2043
Apers, S .................... 2021
Appendino, G ........... 1977
Arpini, S ................... 1981
Bader, A ................... 1967
Bauer, R.................... 1991
Bebrevska, L............. 2021
Becker, H.................. 1971
Belvisi, L .................. 2037
Bergonzi, MC .. 2003,2049
Bertacche, V ............. 2037
Bertoli, A .................. 2007
Bilia, AR. 1941,1963,1999
2003,2017,2049,2075,2099
Bini, LM ................... 2079
Bisio, A..................... 1967
Bizet, D..................... 2107
Bizot, S ..................... 2107
Bohr, G ..................... 1971
Bonesi, M ................. 2129
Bourny, E ................. 2107
Braca, A.................... 1967
Brum, M ................... 2107
Brum, TB.................. 1995
Buhagiar, JA............. 2007
Butterweck, V .......... 2029
Buzzini, P ................. 2061
Cabras, P................... 2013
Camargo, LMM........ 2151
Cardile, V ................. 2095
Carrara, M ................ 1985
Casini, A................... 1999
Castro, PML ............. 2065
Chessa, M ................. 2069
Commission, SFSTP 2107
Compagnon, P .......... 2107
Conforti, F ................ 2129
Corthout, J ................ 2103
Costa, FN.................. 1995
Costa, SS .................. 2151
Coutinho, MAS
2151
Curini, M .................. 2145
Derendorf, H .............2029
Epifano, F..................2145
Flamini, G .................2075
Franz, C .....................2089
Frasca, G ...................2095
Fuchs, H ....................2089
Fuzzati, N ..................1981
Gabbiani , C...............1999
Galeotti, N ................2099
Garbarino, J ...............2095
Genovese, S...............2145
Gerhäuser, C..............1971
Gewali, MB ...............1985
Ghelardini, C .............2099
Giaccherini, C ...........2043
Giana, A ....................1977
Gião, MS ...................2065
Gibbons, S .................1977
Giner, RM .................1953
Giori, A......................1981
Giuliani, C .................2079
Gnavi, G ....................1977
Grassi, G....................1977
Guédon, D .................2107
Heimler, D.................1959
Heimler, D.................2061
Ieri, F ............... 1959,2085
Innocenti, G...............1985
Innocenti, M ..............2043
Isacchi, B...................2075
Isolani, L ...................2061
Leitão, SG1995,2003,2017
Loi, MC .....................2037
Loizzo, MR ...............2129
Lourenço, MCS.........1995
Maffei, M ..................1977
Maidecchi, A.............2099
Majori, G ...................1999
Maldini, M ................2013
Máñez, S....................1953
Máñez, S....................2123
Marcotullio, MC .......2145
Martino, E .................1981
Menghini, L...............2145
Menichini, F ..............2129
Messori, L .................1999
Minunni, M ...............2049
Mombelli, G ..............1981
Montoro, P ................2013
Morgenni, F...............2075
Mulinacci, N.....2043,2085
Muzitano, MF............2151
Norcini, M ................2099
Pagni, L .....................1981
Pellegrino, RM .2069,2079
Pieters, L ...................2021
Pinelli, P ....................1959
Pintado, ME ..............2065
Pintore, G ..................2069
Pistelli, L ...................2007
Pizza, C .....................2013
Podesta, MTC............2007
Politi, M.....................2065
Quintelas, LG ............2107
Jha, PK ......................1985
Kaiser, M...................1963
Karioti, A......... 1953,2017
Kergosien, H .............2107
Klimo, K....................1971
Lamien-Meda, A .......2089
Lapenna, S.................2003
Ramaschi, G ..............1981
Ramos, DF ................1995
Rastrelli, L.................1967
Recio, MC .................1953
Reininger, E ..............1991
Respaud, J .................2107
Ríos, JL .....................1953
Rodrigues, CL ...........2065
Romani, A1959,2061,2085
Russo, A ....................2095
Salawu, SO................2043
Sannella, AR .............1999
Saperas, O .................2107
Sarawek, S.................2029
Sciullo, A ..................1959
Seigneuret, J ..............2107
Severini , C ................1999
Shrestha, BB .............1985
Silva, PE....................1995
Simone, FD ...............1967
Skaltsa, E...................1953
Statti, G .....................2129
Sterner, O ..................1977
Taoubi, K ..................2107
Tasdemir, D...............1963
Theunis, M ................2021
Timóteo, P.................2017
Tirillini, B.........2069,2079
Tombelli, S................2061
Tommasi, ND............1967
Troncoso, N...............2095
Tuberoso, CIG...........2013
Tundis, R...................2129
Turchetti, B ...............2061
Urizzi, P ....................2107
Vassallo, A ................1967
Verotta, L ..................2037
Vignolini, P ......1959,2061
Vincieri, FF .....1963,1999,
2003 ,2017,2075,2085
Visioli, F....................2085
Vivoli, E ....................2099
Vlietinck, A...............2021
Vonaparti, AV...........1953
Zapp, J.......................1971
Zitterl-Eglseer, K ......2089
NPC
Natural Product Communications
1968-2008: 40 Years of Franco F. Vincieri’s Natural
Products Research
2008
Vol. 3
No. 12
1941 - 1952
Anna Rita Bilia
Department of Pharmaceutical Sciences, University of Florence, via Ugo Schiff,
8-50019. Sesto Fiorentino, Florence, Italy
ar.bilia@unifi.it
Received: November 6th, 2008; Accepted: November 14th, 2008
This paper presents an overview of Prof. Vincieri’s accomplishments in his career as a researcher in the field of
pharmacognosy (pharmaceutical biology), analytical phytochemistry and pharmaceutical technology applied to herbal drug
preparations at the Department of Pharmaceutical Sciences of the University of Florence. This article is a recognition of his
valuable contributions to these research fields, especially for his outstanding and innovative interdisciplinary studies on the
quality control of herbal drugs, herbal drug preparations, herbal medicinal products, botanical food supplements, and some
“special foods” such as grapes, wines, olives and olive oil.
Keywords: Franco Francesco Vincieri, Department of Pharmaceutical Sciences, University of Florence, pharmacognosy
(pharmaceutical biology), analytical phytochemistry and pharmaceutical technology, herbal drug preparations.
This issue of Natural Product Communications is
dedicated to the 70th birthday of Franco Francesco
Vincieri, Full Professor at the University of Florence,
Department of Pharmaceutical Sciences. It has been
my special honor to prepare this paper concerning
some of his outstanding achievements in the field of
pharmacognosy (pharmaceutical biology), analytical
phytochemistry and pharmaceutical technology
applied to herbal drug preparations, especially for his
important and innovative interdisciplinary studies on
the quality control of herbal drugs, herbal drug
preparations, herbal medicinal products, botanical
food supplements and some “special foods” such as
grapes, wines, olives and olive oil. I am grateful for
the comments and contributions of all colleagues,
former and present students, and friends who
participated in this special issue for their comments
and contributions, and in particular for the
construction and revision of this article. My special
thanks to Anastasia Karioti for her patient
collaboration in reading and commenting on some
parts of the paper.
Scientifically Prof. Vincieri is a very polyhedral and
curious scientist, “a sort of active vulcan”, ready to
start a new experience, even if it is an expedition to
the African desert (Figure 1). Most of his success has
come from his own intense efforts and his extreme
Figure 1: Franco duing an expedition through Sahara from Algeria to
Giordania. The Sheik, Jeleil al-Deisah, with one of his wives, Hadra, and
his childrem is recognising some herbal drugs collected by Franco (in the
middle) and his assistent Giacalone (on the right).
versatility and passion for research, which led to his
many accomplishments, awards and memberships
which highlight his extraordinary abilities, not only
as a scientist, but also as a person.
For these reasons, in 1997, when as a post-doc I
moved from the University of Pisa for a permanent
position as Researcher at the Department of
Pharmaceutical Sciences in Florence, he has had a
deep personal and professional impact on my life, a
sentiment that is shared by all his former and present
students, some of whom are now either post-docs or
colleagues in this Department.
1942 Natural Product Communications Vol. 3 (12) 2008
Bilia
Figure 3: Sedum telephium L. ssp. maximum Schinz & Thell.
Figure 2: ESCOP meeting at Hailsham (UK) in June 1995. Franco
talking to the late Prof. Hein Zeylstra (on the left of the picture) and Prof.
Finn Sandberg.
Among his publications, including several chapters
and books (most of them used as text books for
undergraduate, postgraduate, master’s and PhD
students), I have selected papers concerning several
projects which represent the scope and breadth of his
work. In addition, these publications are considered
milestones for the development of many scientific
activities, not only of his research group but also of
the Department of Pharmaceutical Sciences, which
attracted many young researchers from all parts of
the world, and led to finance for his work from
private and public pharmaceutical companies. His
firm belief that both basic and applied research are
equally important is reflected in his efforts in 1981
when he was instrumental in organizing the Italian
Society of Phytochemistry (Società Italiana di
Fitochimica, SIF), an association of both university
and company researchers. He was for many years the
Italian delegate of the EMEA (the European
Medicines Agency) and ESCOP (the European
Scientific Cooperative on Phytotherapy) Scientific
Committee working to assess criteria for efficacy and
safety of herbal medicinal products in Europe.
At the beginning of his academic career (in the late
Sixties-early Seventies) he worked with his colleague
and friend, Prof. Massimo Bambagiotti Alberti (the
current Director of the Department), on the analysis
of terpenoids and related volatile compounds which
marked the starting point for their ever-increasing
interest in separation science. After a few years, Prof.
Silvia Coran joined them, and later on Prof. Gloriano
Moneti and Prof. Valerio Giannellini. In a short
timethe group became well known at the University
of Florence for their modern analytical chemistry,
thanks to the introduction of the first GC- MS at the
University of Florence. There are several papers [1-7]
related to volatiles from Pinus species using diverse
spectroscopic approaches for the structural
elucidation of the components In the same period his
friendship and collaboration [8,9] with Prof. Sergio
Pinzauti, today the Dean of the Faculty of Pharmacy,
also began.
Prof. Vincieri’s instrumental ability attracted research
groups of the University of Florence interested in
diverse disciplines, and in particular Prof. Maria
Teresa Vincenzini, one of the most outstanding
scientists in Florence, interested in the effects of
some natural constituents on the biochemistry of
enzymatic systems (dehydrogenases, lyases),
especially on germination of several medicinal plants.
Their collaboration was confirmed by many papers
published during the Sevetnties [10-16].
The studies of Sedum telephium L. ssp. maximum
Schinz & Thell. and Oenanthe aquatica (L.) Poiret
marked the beginning of analysis of natural
constituents from the plant kingdom for the
Department, in addition to the analysis of
pharmaceutical products.
S. telephium (Crassulaceae) (Figure 3) is largely
diffused in traditional medicine, especially in
Tuscany, as a remedy for the local treatment of
wounds and inflammatory diseases of the skin [17].
The term “telephium” is probably related to its
vulnerary properties, introduced by Plinius, who first
reported this plant as the herbal drug used to cure the
leg wounds of King Telephium. The leaves (without
the external cuticle, Figure 4) are reported in “The
medicatis herbarum facultatibus” by Fulgenzo
Vitman (1770), a monk of Vallombrosa (Florence),
with this description: "Ulcera detergit...(clean
ulcers)...et ad cicatricem perducit ... ( and support
Franco F. Vincieri’s natural product research
Natural Product Communications Vol. 3 (12) 2008 1943
two new ones, kaempferol-3-O-β-neohesperidoside7-O-α-rhamnoside
and
quercetin-3-O-βneohesperidoside-7-O-α-rhamnoside, which could
contribute to the anti-inflammatory activity of Sedum
[21].
Figure 4: Preparation of Sedum leave before its application.
cicatrization) ..tumorum suppurationem promovet ...
(favour the suppurative phlogistic process) .. et
dolores mitigat (and sooth pain)”.
At the beginning of the Eighties, the properties of
either the fresh or deep-frozen leaves, with the peel
of the inferior part removed, were rediscovered and
confirmed by a clinician, Dr Sergio Balatri, first at
the Emergency Unit of the Torte Galli Hospital and
later at San Giovanni di Dio Hospital, Florence in the
treatment of various local inflammatory conditions,
including whitlow, abscesses, complicated wounds,
burns, poor cicatrisation, cysts, ulcerous phlebitis,
and horniness (Figure 5) [18].
Figure 5: Application of the fresh peeled leaves of Sedum telephium to a
withlow resulting in rapid healing, shown in "before" and "after" pictures.
Dr Balatri put forward to Prof. Vincieri the need for
chemical analysis of the plant, the testing of its
extracts to recognise the compounds responsible for
the activity, and the preparation of modern
formulations for use in the clinic. Studies started with
the degree thesis investigations of Nadia Mulinacci
[19], currently associate professor in the Vincieri’s
team, and collaboration with Prof. Hildeberg Wagner
(University of Munich, Germany). This led to the
isolation and identification of polysaccharides from
the leaf tissue which had anti-inflammatory potential,
including an anticomplementary effect in vitro,
induction of TNF-alpha-production, increasing
phagocytosis in vitro and in vivo [20]. Two years
later the flavonoids were also identified, including
Negative-ion
fast-atom
bombardment
mass
spectrometry was employed in the identification of
flavonol glycosides directly in the juice [22]. Other
methodologies were proposed for the qualitative and
quantitative determination of flavonol glycosides, in
particular a study in collaboration with Prof.
Hermann Stuppner and coworkers (University of
Innsbruck, Austria), which used MEKC analysis
which was compared with HPLC-MS using
electrospray ionization (ESI) interface [23]. In
another collaborative study with Prof. Francesco
Bonina (University of Catania, Italy), in-vitro and invivo studies suggested that, both the total lyophilized
juice and, in particular, the lyophilized flavonoidic
fraction, but not the lyophilized polysaccharidic
fraction of the leaves, have photoprotective effects
against UVB-induced skin damage [24]. Further
studies with Prof. Renato Pirisino, Prof. Laura
Raimondi and Prof. Maria Grazia Banchelli of the
Department of Preclinical and Clinical Pharmacology
of the University of Florence revealed that total
Sedum juice strongly inhibited cell adhesion to
laminin and fibronectin (EC50 1.03±0.12 mg mL-1).
This anti-adhesive feature was concentrated mainly
in the two polysaccharide fractions (EC50 values
between 0.09 and 0.44 mg mL-1). The flavonol
fractions did not seem to contribute to this effect
[25].
According to the phytochemical, biological and
pharmacological findings, some simple preparations
were developed using either the fresh or lyophilized
juice, and fractions. However, in contrast to the fact
that extracts can represent the best way to select
constituents of plants to be used for medicinal
purposes, in the case of Sedum it was not possible to
obtain the active fraction or constituents and prepare
a valid formulation from them. The leaves still
represent the best form for application, as a natural
plaster. All efforts to update this simple formulation
have been fruitless.
Another interesting medicinal plant investigated by
Prof. Vincieri is O. aquatica (Apiaceae, Figure 6).
Fruits, their alcoholic extract and essential oil are
widely reported as a valuable remedy for dyspepsia,
for the treatment of chronic pectoral affections as an
1944 Natural Product Communications Vol. 3 (12) 2008
Figure 6: Oenanthe aquatica (L.) Poiret.
expectorant, for intermittent fevers, as a diuretic and
for obstinate ulcers [26]. The infusion of the fruits was
also reported by the “Farmacopea italiana del Regno”
until the 4th Edition (1920). However, it has been
known for a long time that the fruits can cause vertigo,
dizziness, inebriation, dull pains in the head and other
narcotic effects, as reported in “A Further Account of
the Poisonous Effects of Oenanthe aquatica Succo
Viroso Crocante of Lobel, or Hemlock Dropwort”
[27].
Problems related to this herbal drug and preparations
were diverse, all related to its safe use. Initial studies
by Prof. Vincieri provided definitive information on
the composition of the essential oil and the light
petroleum (40-60°) extract of the fruit by a
combination of different techniques: GLC, UV and
IR spectroscopy and MS [28]. A group of C15
hydrocarbon and oxygenated polyacetylenes,
including three new compounds, were also isolated
and structurally identified on the basis of
spectroscopic evidence. Due to their extreme
sensitivity to air, heat and light, removal of the
solvents and spectroscopic manipulation of the
sample was performed by a home-made device to
directly measure NMR spectra, similar to the modern
concept of the hyphenated systems. IR spectra were
performed in the crystalline state at liquid nitrogen
temperature to observe the out-of-plane vibration
band of the cis-double bond which, in the liquid
phase, forms one broad band with the -CH2-rocking
vibration [29,30]. With the aim of finding a rapid
method of characterization of polyacetylenes,
second-derivative UV spectra of polyacetylenes were
studied with Prof. Mario Pio Marzocchi and Prof.
Giulietta Smulevich, colleagues in the Department of
Chemistry of the University of Florence. This method
was useful for the complete structural identification
Bilia
of their chromophores, including the stereoisomerism
of the double bonds [31]. The second-derivative
technique was also applied to a series of related
naturally-occurring polyenynes, whose chromophoric
fragments ranged from three to six conjugated
groups. These results account for the structural
characteristics, including steroisomerism, of a given
sequence of triple and double bonds providing a
complete fingerprint of the polyenyne chromophore
[32]. In another paper, the application of the MS-MS
technique for the rapid monitoring of some
γ-butyrolactone ring and related lignans of
O. aquatica fruits infusion was reported [33]. Finally,
the GC-MS technique led to the identification of the
constituents of the fruit tincture.Ten polyacetylenes,
three lignans (derivatives of matairesinol), numerous
monoterpenes, including phellandrene and cryptone,
dillapiole and small amounts of sterols were
identified [34]. Due to the low stability of the
constituents of the tincture, especially polyacetylenes,
a novel system of stabilization of the active principles
of tinctures by means of cyclodextrins (α, β, γ-CyD)
was also proposed. Microinclusion of the components
was found to be incomplete with all three
cyclodextrins, however, β-CyD was the most
efficient and the stabilization of the most unstable
microincluded active principles was verified by
means of artificial ageing studies. These studies were
possible thanks to an expert pharmaceutical
technologist, Prof. Giovanni Mazzi, who joined the
Vincieri group during that period. Further studies
through artificial membranes also provided evidence
for an increase in the permeability of the constituents.
These studies on O. aquatica represent a good
example of an interdisciplinary approach, including
the enhanced biopharmaceutical properties of the
formulated phytocomplex, representing a true
milestone in this field of research [35,36].
During the 1980s-1990s, Vincieri expanded his
chemistry program to cover many other herbal
products and in particular vegetal matrices of interest
in the biological and/or alimentary fields aiming at
the development of specific methods of extraction,
fractionation, isolation and characterization of
potentially interesting secondary metabolites for the
pharmaceutical, alimentary and cosmetic fields. Two
typical Tuscan species with agro-alimentary interest,
Vitis vinifera L. (leaves, fruits, wines) and Olea
europaea L. (leaves, olives, oil, olive residues and
waste waters), have been included in his
investigations. Other plants belonging to the
Mediterranean maquis, such as Myrtus communis L.,
Franco F. Vincieri’s natural product research
Natural Product Communications Vol. 3 (12) 2008 1945
Figure 7: Sangiovese, a typical grape variety of Chanti DOCG wine.
Pistacia lentiscus L., Phillyrea latifolia L., Ligustrum
vulgare L., Ligustrum sinensis L., Fraxinum ornus
L., Arbutus unedo L. were also studied [37-39].
Studies of polyphenols in wines and grapes were
developed by Dr Alessandro Baldi and Prof. Annalisa
Romani, former PhD students, and Romani later as a
post-doc, researcher and associate professor. The key
studies are represented by the development of an
analytical method for anthocyanins of Vitis vinifera
L. (Vitaceae) [40,41].The pool of anthocyanins
contained in the berry skins of different cultivars of
V. vinifera was taken as a research model to
investigate the possible application of HPLC/MS to
anthocyanins. The interface chosen was the API
(atmospheric pressure ionization) ion spray interface
coupled with a quadrupole mass spectrometer, which
allows ambient pressure ionization and the use of any
aqueous eluent.The use of this technique made it
possible to obtain the mass spectra of all the
anthocyanin compounds present in the extracts under
investigation, even those occurring in traces or some
coeluted ones. Studies were first carried out on
certificated clones belonged to two varieties
commonly used for the production of the Chianti
DOCG red wine: Sangiovese (clone SS-F9-A5-48,
[40] and Colorino (clone Nipozzano 6) [41].
The developed HPLC method led to the identification
of the 3-glucosides, the 3-acetylglucosides, and the 3p-coumaroylglucosides of delphinidin, cyanidin,
petunidin, peonidin, and malvidin, already known in
the literature. Two 3-caffeoylglucoside derivatives
were identified too, and for the first time, some 3,5diglucosides. The investigated cultivars showed the
same anthocyanin profile, but dramatic quantitative
differences, i.e. the cultivar Sangiovese showed a
lower amount of the acylated compounds. This
analytical application was, therefore, useful as a
supporting technique for the structural investigation
Figure 8: Structure of the characteristic phenols of O. europea.
of the polyphenolic compounds of different cultivars
used in the production of red wines [40,41].
Numerous studies have been published on the
analysis of olives, olive oil, waste waters (OMWW)
and solid olive residue (SOR), experimental or
commercial ones, from cultivars of different origins,
stoned or whole fruits, and overall, there are more
than 50 publications by the group up to now.
However, in this case, I have selected some key
studies carried out mainly by Prof. Nadia Mulinacci
and Annalisa Romani, and more recently, by Prof.
Patrizia Pinelli, as a graduate then PhD student, and
as a postdoc. Olive oil is obtained from the olive
(Olea europaea L., Oleaceae), a traditional tree crop
of the Mediterranean Basin. On a European scale, 3
million tons of olives are processed for olive oil per
year (with an oil yield of about 60,000 tons) [42].
After the epidemiological evidence of a lower
incidence of CHD in the Mediterranean area [43] and
certain types of cancers [44], there was an increasing
popularity of the Mediterranean diet, in which olive
oil is the major oil component, and its consumption is
expanding to non producer countries such as the
United States, Canada, and Japan. Olives and olive
oil contain phenolic compounds, which not only
influence the sensory properties, but are also
important markers for type, biodiversity and quality
determination of this product. These polyphenols
have been shown to exert potent biological activities,
including principally, but not limited to, antioxidant
and free radical scavenging actions [42,45]. Some
of the most representative phenolic compounds
are hydroxytyrosol (3,4-dihydroxyphenylethanol),
1946 Natural Product Communications Vol. 3 (12) 2008
Bilia
Antioxidant and anti-inflammatory properties were
proven. A study on experimental and commercial
OMWW from four Mediterranean countries (Italy,
Spain, France, and Portugal) [52] was also carried
out. The results demonstrated that Italian commercial
OMWWs were the richest in total polyphenolic
compounds with amounts between 150 and 400
mg/100 mL of waste waters. These raw, as yet
unused, matrices were found to be an interesting and
alternative source of biologically active polyphenols.
Figure 9: Frantoio, a typical variety of Tuscan olive oil
tyrosol, oleuropein, verbascoside and luteolin and its
derivatives (Figure 8). Studies were first focused on
the extraction of the minor polar compounds from
olive fruit [46] and from extra virgin olive oils
[47,48]. A solid-liquid extraction (LSE) procedure
(Extrelut cartridge, diatomaceous earth), followed by
HPLC-DAD-MS analysis led to the characterization
of the polyphenolic content of different Tuscan olive
cultivars (Frantoio, reported in Figure 9, Rossellino,
Ciliegino, Cuoricino, and Grossolana), including
phenolic acids, verbascoside, oleuropein derivatives,
flavons and flavonol glycosides [46].
Verbascoside was proposed as a chemotaxonomic
marker of different cultivars. Numerous comparative
studies [47,48] were also carried out on extra-virgin
oils from different parts of Italy and obtained from
several harvest years (1999-2002) from both stoned
and whole fruits. A higher antioxidant capacity of the
oils from stoned olives was found. At the end of the
‘90s, a project supported by the European
Community entitled "Natural antioxidants from olive
oil processing waste waters" (FAIR PL 973039) was
commenced with the aim of evaluating the
polyphenolic contents in different samples of olive
mill waste waters (OMWWs), and the possibility of
recovering them. It is well-known that OMWWs
contain powerful pollutants [49] which are acidic (pH
5-5.5) and malodorous, containing potassium and
phosphorus salts and organic substances, such as fats,
proteins, sugars, organic acids, but also polyphenols.
For this purpose a preliminary qualitative screening
of the polyphenols was performed working on waste
waters obtained from an experimental mill in
Tuscany [50], noting antioxidant and other biological
activities of extracts obtained from this matrix [51].
Extracts presented both phenolic polymers and
low and medium molecular weight phenols such
as elenolic acid, hydroxytyrosol, and tyrosol.
A further work was aimed at investigating the
phenolic content of another by-product, the solid
olive residue (SOR). The aim of this investigation
was the selection of the best extraction procedure to
increase the yields of phenylpropanoidic derivatives
in the obtained extracts [53]. Soxhlet extraction with
ethanol was identified as the first step of purification
and was followed by either a liquid/liquid extraction
with ethyl acetate or fractionation using an ionexchange resin. The total concentration of the
phenolic compounds ranged between 1.1 and 6.23
mg/g of fresh SOR. Phenolic distribution among the
different chemical classes was due to several factors:
type of cultivar, degree of ripening, different milling
processes, and pedoclimatic factors. The greatest
differences among the samples were observed for
verbascoside, which ranged between 0.15 and 4.15
mg g-1, and for its less abundant analogues [53].
When in 1997 I joined the group of Prof. Vincieri,
my research studies were mostly directed toward
pharmaceutical and technological aspects of herbal
drugs, herbal drug preparations and herbal medicinal
products. The first person I met in the Department
was Dr. Sandra Gallori, an expert technician with a
deep artistic sense. She was able to transform a
simple poster into a work of art and the Vincieri
group has been known for a long time in the
scientific community also for these artistic posters,
obtaining several awards. In many occasions she
has prepared leaflets for congresses. An example of
her work is a special picture representing the
Vincieri group as an anthill (Figure 10).
In 1998 Prof. Maria Camilla Bergonzi joined the
group in order to start her PhD and during the
following years, first as a post-doc and since 2005 as
an aggregate professor, she has strongly supported
my research. Authentication, quality control and
stability testing have largely been performed using
not only conventional [54-72] but also non
conventional methods such as 1D- and 2D-NMR,
NIR, and biosensors [73-77].
Franco F. Vincieri’s natural product research
Natural Product Communications Vol. 3 (12) 2008 1947
S taff:
Sandra
Nadia
Annalisa
Anna Rita
Patrizia
Catia
Vania
Marzia
Giovanni
Camilla
Carlotta
Prof.
Figure 10: A portrait of Vincieri research team.
At the same time my scientific interest was driven by
the improvement of bioavailability and technological
features of extracts and HDPs, HMPs by formulation
with liposomes, micelles, supramolecular complexes
with cyclodextrins and their characterization by
conventional methods (HPLC, DSC, UV, light
scattering, dissolution tests) and innovative ones,
such as NMR (ROESY and DOSY) [78-84].
Our publications represented the first studies on the
stability and compatibility with excipients of
important dried commercial extracts, such as St.
John’s wort (SJW), and results were dramatic when
the studies were carried out using ICH guidelines
[56]. Other studies reported on the quality and/or
stability of decoctions, teas and infusions, aromatic
waters, tinctures and mother tinctures, commercial
instant teas of common herbal drugs and obtained
important information about their preparation,
content and shelf-lives.
Due to my long experience as a phytochemist and
having at my disposal the excellent NMR facilities of
Florence (CERM), one of the best equipped NMR
groups in the world, I started a project using
multidimensional NMR methods aimed at the
analysis of complex spectra, such as those of plant
extracts for their authentication, quality control and
stability testing.
Studies were performed by the direct NMR analysis
of complex plant mixtures, without purification or
fractionation steps. Many matrices were used, such as
SJW, ginkgo and ginseng extracts, an innovative
supercritical carbon dioxide (CO2) commercial
extract of arnica, and samples of kava-kava herbal
drugs and extracts [73-75].
Conventional methods used in herbal drug analysis
(HPLC, HPTLC, GC, EC) can give a fingerprint of
the markers or active constituents (and their
percentage), but no information about the other
metabolites of the extract, which can represent up to
95% of the total. This is true, especially in the
unconventional extracts such as the innovative
supercritical CO2 extracts. The extract or the finely
powdered herbal drug were directly dissolved in
hexadeuterated dimethylsulfoxide and analyzed after
filtration by NMR spectroscopy.
Spectral assignments of the constituents were carried
out according to the data (chemical shifts and
1948 Natural Product Communications Vol. 3 (12) 2008
Bilia
coupling constants) found in the literature and by
means of 1D- and 2D-NMR spectra, which were
found to be a valid alternative method to obtain a
fingerprint for the assurance of content and stability
and, as a consequence, safety and efficacy of extracts
and herbal drugs. These studies represent a combination of fingerprint and semiquantitative analyses
and although quantitation was only a minor aspect of
these studies, the suitability of qHNMR to address
questions of extract stability, as exemplified by the
unstable Hypericum phloroglucinol derivatives, such
as hyperforin, was clearly pointed out [56,73].
It was demonstrated that NMR experiments can
provide a real and complete fingerprint of the extract,
as required especially for innovative ones, to have a
global vision and a “separation” of all constituents.
All the molecules, as well as possible unknown or
unexpected compounds can be detected. In addition,
they can be used in the authentication of herbal drug
material and to compare extracts manufactured with
different processes and batch-to-batch analysis in the
industry. NMR experiments can be considered a very
simple, widely applicable and rapid analytical
instrument, readily performed without pre-treatment,
with tremendous versatility, not depending on the
nature of the extract, inexpensive, perhaps with a
lower precision than other methods but sufficient for
pharmaceutical applications.
Another important line of research was dedicated to
the application of biosensors. This important
application was possible thanks to collaboration with
a colleague and dear friend, Prof. Maria Minunni, and
her chief, Prof. Marco Mascini, of the Department of
Chemistry of the University of Florence. Our studies
proved the applicability of sensors and biosensors for
analysis in the search for new active constituents
from plants, for the quality-control of HDs, HDPs
and HMPs related not only to active or marker
constituents but also to other substances, such as
heavy metals and pesticides. Different biosensors
(based on electrochemical transduction or on optical
detection) were employed to evaluate the content of
alkaloids of different extracts of Chelidonium majus
L. (Papavaraceae), fractions obtained during
“biosensor
assay-guided”
fractionation,
pure
constituents and on extracts submitted for stability
testing. In addition, disposable sensors were used to
detect heavy metals in samples of St. John’s wort. A
good correlation between the results obtained with
the electrochemical devices and those from A.A.S.
was observed [76,77].
Figure 11: Formation of liposome, a typical pharmaceutical carrier, in
aqueous solutions.
An important part of my research concerns the
improvement of bioavailability and technological
features of extracts, HDPs and HMPs by innovative
methods (liposomes, ethosomes, supramolecular
complexes, micelles) for which my thanks go also to
Prof. Maria Camilla Bergonzi and many graduating
and PhD students, among them Benedetta Isacchi,
whom I consider to be my right arm. These
preparations were analysed both by conventional
methods (HPLC, DSC, UV, light scattering,
dissolution tests) [78,80,81] and innovative ones,
such as NMR (ROESY and DOSY) [79,82-84].
These studies were carried out on several
supramolecular complexes between preparations of
St. John’s wort, kava-kava and cyclodextrins,
micelles and liposomes (Figure 11).
I am really grateful to Prof. Gareth Morris of the
University of Manchester (UK) for having applied
and developed my initial idea of using diffusionordered spectroscopy (DOSY) methods for the
analysis of micellar dispersions (octanoyl-6-Oascorbic acid, SDS) and included molecules, such as
artemisinin,
curcumin,
phloroglucinols,
and
anthocyanins. The investigations showed that DOSY
experiments can yield both qualitative and
quantitative information on the solubilization of
nonpolar species by surfactants and the
supramolecular complexes.
Finally, I should like to report some studies [85-89]
with Artemisia annua L.. (Asteraceae, Figure 12) and
artemisinin, a promising and potent antimalarial drug.
These studies were carried out from the beginning in
the framework of collaboration with a dear friend and
colleague Prof. Luigi Messori of the Department of
Analytical Chemistry of the University of Florence.
Franco F. Vincieri’s natural product research
Natural Product Communications Vol. 3 (12) 2008 1949
Artemisia annua L.
Figure 12: Artemisia annua L.
The interaction with hemin was first evaluated by
UV/Vis spectrophotometry and HPLC/DAD/MS and
the suitability of these simple methods for selection
of new antimalarial compounds having similar
properties was assessed. Furthermore the flavonoids
isolated from A. annua were found to increase the
rate of the reaction [85,86]. NMR studies of the
supramolecular complex formed in the reaction were
also carried out in collaboration with Prof. Paola
Turano of the CERM [87]. Interesting results were
also obtained using in the reaction hemoglobin
instead of hemin [88]. Recent findings led to the
discovery that green tea is also active against
Plasmodium, and more importantly, its characteristic
constituents, catechins, have a synergistic effect if
administered with artemisinin [89]. Other studies on
artemisinin and its extracts are also reported in this
special issue.
This project still represents one of the most visible
for the group, involving many other scientists and
dear friends including Dr Carlo Severini and Dr Anna
Rosa Sannella of the Istituto Superiore di Sanità,
Rome, Italy, and Prof. Deniz Tasdemir of the School
of Pharmacy, University of London (UK), and also
attracting funds from the Sigma Tau (Pomezia,
Rome, Italy), Ente Cassa di Risparmio di Firenze
(Florence, Italy) and Toscana Life Sciences
Foundation (Siena, Italy). Recently, a collaborative
study with Prof. Carla Ghelardini and Prof. Nicoletta
Galeotti of the Department of Preclinical and Clinical
Pharmacology of the University of Florence has
begun, with the aim of investigating antineuropathic
activity of extracts, fractions and pure compounds
from herbal drugs (a paper concerning these studies
is reported in this issue). Finally, last but not
the least, I would like to remember Prof. Vincieri’s
Figure 13: From the left side of the picture: Prof. K. Głowniak; Prof. T.
Efferth, Prof. M. Hamburger, Prof. B.J. Baker, Prof. V. Butterweck, Prof.
I. Merfort, Prof. L. Skaltsounis, Prof. R. Verpoorte, Prof. A. Nahrstedt,
Prof. Prof. D. Guo, Prof. L. Pieters, Prof. A.R. Bilia and in the centre
Prof. F.F. Vincieri on occasion of the celebration of his birthady during
the 2008 Shangai International Conference on TCM and Natural
Medicine.
collaboration with Prof. Anacleto Minghetti and Dr.
Nicoletta (Nicky) Crespi Perellino, wonderful people
and outstanding scientists who started their sincere
friendship with Franco from the first time they met.
They are also very active people and their expertise
lies in the field of cell cultures [90] in particular.
The Vincieri research team is nowadays quite large,
including three associate professors, three aggregate
professors, four post-doctoral positions and about
twenty students (including PhD, master’s and
graduate students) having two principal lines of
research. The first is related to the development of
specific methods of extraction, fractionation,
isolation and characterization of potentially
interesting secondary metabolites for pharmaceutical,
alimentary and cosmetic fields. Other studies are
directed towards the optimization of stability and
technological and biopharmaceutical characteristics
of herbal products, their extracts and commercial
preparations.
I would like to conclude with a sentence to describe
Prof Vincieri’s profile: “an inspration to scientists,
young and old, in all fields of research”.
Figure 13 is a photo of Prof. Vincieri in Shangai for
the International Conference on Traditional Chinese
Medicine and Natural Medicine taken on his
birthday, 12th October 2008. At that time he had a
nice surprise and I hope this issue will be another
even a greater one!
Happy birthday from all of us
1950 Natural Product Communications Vol. 3 (12) 2008
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from stoned olives. Journal of the Science of Food and Agriculture, 85, 662-670.
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olive mill waste waters. Journal of Agricultural and Food Chemistry, 47, 3397-3401.
Mulinacci N, Romani A, Galardi C, Pinelli P, Giaccherini C, Vincieri FF. (2001) Polyphenolic content in olive oil waste waters and
related olive samples. Journal of Agricultural and Food Chemistry, 49, 3509-3514.
Mulinacci N, Innocenti M, la Marca G, Mercalli E, Giaccherini C, Romani A, Erica S, Vincieri FF. (2005) Solid olive residue: an
insight on their polyphenolic composition. Journal of Agricultural and Food Chemistry, 53, 8963-8969.
Bilia AR, Fumarola M, Gallori S, Mazzi G, Vincieri FF. (2000) Identification by HPLC-DAD and HPLC-MS analyses and
quantification of constituents of fennel teas and decoctions. Journal of Agricultural and Food Chemistry, 48, 4734-4738.
Bilia AR, Salvini D, Mazzi G, Vincieri FF. (2001) Characterization of calendula, milk-thistle and passionflower tinctures by
HPLC-DAD and HPLC-MS. Chromatographia, 53, 210-215.
Bilia AR, Bergonzi MC, Morgenni F, Mazzi G, Vincieri FF. (2001) Evaluation of stability of St. John’s wort commercial extract
and some preparations. International Journal of Pharmaceutics, 213, 199-208.
Bergonzi MC, Bilia AR, Gallori S, Guerrini D, Vincieri FF. (2001) Variability in the content of the constituents of Hypericum
perforatum and some commercial extracts. Drug Development and Industrial Pharmacy, 27, 491-97.
Gallori S, Flamini G, Bilia AR, Morelli I, Landini A, Vincieri FF. (2001) Chemical composition of some traditional herbal drug
preparations: essential oil and aromatic water of Costmary (Balsamita suaveolens Pers.). Journal of Agricultural and Food
Chemistry, 49, 5907-5910.
Bilia AR, Flamini G, Taglioli V, Morelli I, Vincieri FF. (2002) GC-MS analysis of essential oil of some commercial Fennel teas.
Food Chemistry, 76, 307-310.
1952 Natural Product Communications Vol. 3 (12) 2008
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Bilia
Bilia AR, Bergonzi MC, Mazzi G, Vincieri FF. (2002) HPLC-DAD and HPLC-MS analysis and stability of the constituents of
Artichoke and St. John’s wort tinctures. Drug Development and Industrial Pharmacy, 28, 609-619
Bilia AR, Bergonzi MC, Mazzi G, Vincieri FF. (2002) Stability of the constituents of Calendula, Milk-thistle and Passionflower
tinctures by HPLC-DAD and HPLC-MS analyses. Journal of Pharmaceutical and Biomedical Analysis, 30, 613-617.
Sturm S, Taglioli V, Bilia AR, Vincieri FF, Stuppner H. (2003) Analysis of alkaloids in Ipecacuanhae radix and preparations by
capillary zone electrophoresis. Journal of Separation Science, 26, 1175-1179.
Gallori S, Bilia AR, Bergonzi MC, Barbosa WLR, Vincieri.FF. (2004) Polyphenolic constituents of fruit juice of Euterpe oleracea
Mart. (Açai palm). Chromatographia, 59, 739-743.
Gallori G, Giaccherini C, Bilia AR, Mulinacci N, Vincieri FF. (2004) Identification of anthocyanins in Amorphophallus titanum
(Becc.) Becc.. Chromatographia, 59, 647-651.
Bilia AR, Melillo de Malgalhaes P, Bergonzi MC, Vincieri FF. (2006) Simultaneous analysis of artemisinin and flavonoids of
several extracts of Artemisia annua L. obtained from a commercial sample and a selected cultivar. Phytomedicine, 13, 487-493.
Karioti, A, Chatzopoulou A, Bilia AR, Liakopoulos G, Stavrianakou S, Skaltsa S. (2006) Novel secoiridoid glucosides in Olea
europaea leaves suffering from boron deficiency. Bioscience, Biotechnology and Biochemistry, 70, 1898-1903.
Bilia AR, Gabriele C, Bergonzi MC, Melillo de Malgalhaes P, Vincieri FF. (2006) Variation in artemisinin and flavonoids content
in different extracts of Artemisia annua L. Natural Product Communications, 1, 1111-1115.
Bilia AR, Eterno F, Bergonzi MC, Mazzi G, Vincieri FF. (2007) Evaluation of the content and stability of the constituents of
mother tinctures and tinctures: the case of Crataegus oxyacantha L. and Hieracium pilosella L.. Journal of Pharmaceutical and
Biomedical Analysis, 44, 70-78.
Isacchi B, Bergonzi MC, Carnevali F, van der Esch SA, Vincieri FF, Bilia AR. (2007) Analysis and stability of the constituents of
St. John's wort oils prepared with different methods. Journal of Pharmaceutical and Biomedical Analysis, 45, 756-761.
Baraldi R, Isacchi B, Predieri S, Marconi G, Vincieri FF, Bilia AR. (2008) Distribution of artemisinin and bioactive flavonoids
from Artemisia annua L. during plant growth. Biochemical Systematics and Ecology, 36, 340-348.
Bilia AR, Giomi M, Innocenti M, Gallori S, Vincieri FF. (2008) HPLC-DAD-ESI-MS analysis of the constituents of aqueous
preparations of verbena and lemon verbena and evaluation of the antioxidant activity. Journal of Pharmaceutical and Biomedical
Analysis, 46, 463-470.
Birilli R, Ferretti R, Gallinella B, Bilia AR, Vincieri FF, La Torre F. (2008) Enantioseparation of kavain on Chiralpak IA under
normal-phase, polar organic and reversed-phase conditions. Journal of Separation Science, 31, 2206-2210.
Bilia AR, Bergonzi MC, Mazzi G, Vincieri FF. (2001) Analysis of plant complex matrices by use of nuclear magnetic resonance
spectroscopy: St. John’s wort extract. Journal of Agricultural and Food Chemistry, 49, 2115-2124.
Bilia AR, Bergonzi MC, Lazari D, Vincieri FF. (2002) Characterisation of commercial kava-kava herbal drug and herbal drug
preparations by means of nuclear magnetic resonance spectroscopy. Journal of Agricultural and Food Chemistry, 50, 5016-5025.
Bilia AR, Bergonzi MC, Mazzi G, Vincieri FF. (2002) Analysis of plant complex matrices by use of nuclear magnetic resonance
spectroscopy: supercritical carbon dioxide arnica extract. Journal of Pharmaceutical and Biomedical Analysis, 30, 321-330.
Palchetti I, Mascini M, Minunni M, Bilia AR, Vincieri FF. (2003) Disposable electrochemical sensor for rapid determination of
heavy metals in herbal drugs. Journal of Pharmaceutical and Biomedical Analysis, 32, 251-256.
Minunni M, Tombelli S, Marrazza G, Palchetti I, Mascini M, Bilia AR, Bergonzi MC, Vincieri FF. (2005) An optical DNA-based
biosensor for the analysis of bioactive constituents with application in drug and herbal drug screening. Talanta, 65, 578-585.
Taglioli V, Bilia AR, Ghiara C, Mazzi G, Mercati V, Vincieri FF. (2001) Evaluation of the dissolution behaviours of some
commercial herbal drugs and their preparations. Pharmazie, 56, 868-870.
Bilia AR, Bergonzi MC, Morris GA, Lo Nostro P, Vincieri FF. (2002) A diffusion-ordered NMR spectroscopy study of the
solubilization of artemisinin by octanoyl-6-O-ascorbic acid. Journal of Pharmaceutical Sciences, 91, 2265-2270.
Bergonzi MC, Bilia AR, Casiraghi A, Cilurzo F, Minghetti P, Montanari L, Vincieri FF. (2005) Evaluation of skin permeability of
sesquiterpenes of an innovative supercritical carbon dioxide arnica extract by HPLC/DAD/MS. Die Pharmazie, 60, 36-38.
Bilia AR, Bergonzi MC, Mazzi G, Vincieri FF. (2006) Development and stability of semisolid preparations based on a supercritical
CO2 arnica extract. Journal of Pharmaceutical and Biomedical Analysis, 41, 449-54.
Bergonzi MC, Bilia AR, Di Bari L, Mazzi G, Vincieri FF. (2007) Studies on the interactions between some flavonols and
cyclodextrins. Bioorganic & Medicinal Chemistry Letters, 17, 5744-5748.
Sinico C, Caddeo C, Valenti D, Fadda AM, Bilia AR, Vincieri FF. (2008) Liposomes as carriers for verbascoside: stability and
skin permeation studies. Journal of Liposome Research, 18, 83-90
Bilia AR, Scalise L, Bergonzi MC, Vincieri FF. (2008) Effect of surfactants and solutes (glucose and NaCl) on solubility of
kavain. American Association of Pharmaceutical Scientists-(AAPS Pharmscitech), 9, 444-448.
Bilia AR, Lazari D, Messori L, Taglioli V, Temperini C, Vincieri FF. (2002) Simple and rapid physico-chemical methods to
examine action of antiimalarial drugs: its application to Artemisia annua L. constituents. Life Sciences, 70, 769-778.
Messori L, Piccioli F, Eitler B, Bergonzi MC, Bilia AR, Vincieri FF. (2003) Spectrophotometric and ESI-MS/HPLC studies reveal
a common mechanism for the reaction of various artemisinin analogues with hemin. Bioorganic and Medicinal Chemistry letters,
13, 4055-4057.
Messori L, Piccioli F, Temperini C, Bilia AR, Vincieri FF, Allegrozzi M, Turano P. (2004) The reaction of artemisinin with hemin:
a further insight into the mechanism. Inorganica Chimica Acta, 357, 4602-4606.
Messori L, Gabbiani C, Casini A, Siragusa M, Vincieri FF, Bilia AR. (2006) The reaction of artemisinins with hemoglobin: A
unified picture. Bioorganic & Medicinal Chemistry, 14, 2972-2977.
Sannella AR, Messori L., Casini A, Vincieri FF, Bilia AR, Majori G, Severini C. (2007) Antimalarial properties of green tea.
Biochemical and Biophysical Research Communications, 353, 177-181.
Mulinacci N, la Marca G, Innocenti M, Vincieri FF, Crespi Perellino N, Minghetti A. (2005) Cell cultures of Ajuga reptans L. to
bioconvert emodin and aloe-emodin: an HPLC/ESI/MS investigation. Enzyme and Microbial Technology, 36, 399-408.
NPC
Natural Product Communications
Effects of Terpenoids from Salvia willeana in Delayed-type
Hypersensitivity, Human Lymphocyte Proliferation and
Cytokine Production
2008
Vol. 3
No. 12
1953 - 1958
Anna Vonapartib, Anastasia Kariotib, María C. Recioa, Salvador Máñeza, José L. Ríosa,
Eleani Skaltsab and Rosa M. Ginera,*
a
Departament de Farmacologia, Facultat de Farmàcia, Universitat de València,
Av. Vicent Andrés Estellés s/n, 46100 Burjassot, Spain
b
Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy,
University of Athens, Panepistimiopolis, Zografou 157 71, Athens, Greece
Rosa.M.Giner@uv.es
Received: June 24th, 2008; Accepted: October 16th, 2008
The effect of the lipophilic extract of S. willeana and three terpenoids isolated therefrom, camphor, lupeol and oleanolic acid,
on oxazolone-induced hypersensitivity was evaluated. The extract reduced the ear edema by 46% at 24 h after challenge. All
three terpenoids inhibited the edema and suppressed cytokines release at different rates. Lupeol inhibited the swelling by over
50% and reduced the production of IL-1β by 62%. Camphor caused inhibition of the efferent phase (45% inhibition at 72 h)
and the levels of IL-1β, IL-4 and TNF-α (around 80% inhibition). Oleanolic acid diminished moderately the reaction and the
levels of IL-4 and TNF-α. We also demonstrated that the three terpenoids inhibited human T-lymphocytes proliferation in a
concentration-dependent manner and induced their apoptosis. Thus, these terpenoids could be considered anti-inflammatory
constituents of S. willeana.
Keywords: Salvia willeana, oleanolic acid, lupeol, camphor, oxazolone, lymphocytes, cytokines.
The genus Salvia comprises about 900 species, many
of which are used in traditional medicine to treat
various disorders [1,2]. S. willeana (Holmboe) Hedge
(Lamiaceae), an aromatic herb endemic to Cyprus,
and locally common in the Troodos range [3],
possesses pharmacological properties. Its extracts or
infusions are used as tonics and anti-diarrhoeal
agents. The plant can also be used to halt milk
production in nursing mothers and has a strong
antiseptic action [4,5]. This species has been
characterized by the presence of the triterpenoids,
ursolic and oleanolic acids and urs-12-ene-3α,11αdiol, the diterpenoids, carnosic acid and isorosmanol
and the flavonoid salvigenin [6]. These classes of
compounds have demonstrated anti-inflammatory
activity in different experimental models of
inflammation.
In the present study we report the activity of the
lipophilic extract of the aerial parts of S. willeana
and the three isolated compounds, camphor, lupeol
and oleanolic acid, on a delayed-type hypersensitivity
reaction induced by oxazolone, specifically on edema
formation and their effect on different proinflammatory mediators involved in this reaction, as
well as their influence on both T lymphocyte
proliferation and the cell cycle.
The lipophilic extract, which was topically applied at
a dose of 1 mg/ear after oxazolone challenge, reduced
the ear edema by 46% at 24 h, but exhibited only a
weak effect during the later stages of the process
(22% and 20% at 48 h and 72 h, respectively). When
topically applied at a dose of 0.5 mg/ear, the isolated
compounds clearly and significantly reduced the
hypersensitivity reaction in mouse ears (Figure 1).
Lupeol was the most active compound, inhibiting
the swelling by 58% 24 h after challenge and
maintaining the effect after 72 h (50% and 52% at
48 and 72 h, respectively). Camphor caused a slight
1954 Natural Product Communications Vol. 3 (12) 2008
Vonaparti et al.
Control
1000
175
Lupeol
250
800
**
Oleanolic acid
150
**
125
**
**
100
Dexamethasone
**
75
50
**
25
**
**
TNF-α and IL-4
(pg/mL)
200
300
200
600
150
100
**
50
**
400
**
** **
10
20
30
40
50
60
70
Time (h)
Figure 1: Effect of the extract, the isolated compounds and
dexamethasone on the delayed-type hypersensitivity ear swelling induced
by oxazolone, measured 24, 48 and 72 h after challenge. Increase in ear
thickness is expressed as mean ± SEM. ** P < 0.01 after Dunnett’s test as
compared with control group.
inhibition of the elicitation phase (28% at 24 h), but
its effect intensified at 72 h (45% inhibition), whereas
oleanolic acid produced a moderate reduction of the
reaction (37%, 33%, and 30% at 24 h, 48 h, and 72 h,
respectively) (Figure 1).
A critical event during the development of cutaneous
immune responses, including those provoked by
exposure to a contact allergen such as oxazolone, is
the mobilization of epidermal Langerhans cells (LC).
These cells act as sentinels of the immune system in
the skin, responding to a variety of local injuries with
migration and the delivery of potentially foreign
signals leading to the draining of the lymph nodes.
IL-1β and TNF-α are known to play pivotal roles in
the stimulation of LC migration. Thus, with regard to
the effect of the compounds on the protein level of
such cytokines, IL-1β, TNF-α, and IL-4 production
were measured in ear homogenates 72 h after
challenge with oxazolone with the aid of an ELISA
analysis. The effectiveness of the compounds varied
depending on the cytokine, with the inhibitory
activity on the liberation of all three cytokines being
especially marked for camphor (around 80%
inhibition with respect to those in the acetone-treated
group). In contrast, the other compounds exhibited
different behaviors. Oleanolic acid, for example
reduced the levels of IL-4 and TNF-α by 42% and
46%, respectively, but did not modify the
concentration of IL-1β. Lupeol notably reduced the
production of IL-1β by 62%, moderately reduced that
of IL-4 by 36%, but had no significant effect on
TNF-α levels. In the dexamethasone-treated group, a
decrease in the production of cytokines to basal
levels was observed (Figure 2).
0
Bl
an
0
200
IL-1β
k
Co
nt
ro
l
Ca
m
ph
or
Lu
pe
O
le
ol
an
ol
i
c
D
ac
ex
id
am
et
ha
so
ne
0
0
**
IL-4
IL-1β (pg/mL)
ΔEar swelling (μm ± SEM)
TNF-α
Extract
Camphor
225
Figure 2: Effect of isolated compounds and dexamethasone on IL-1β,
TNF-α and IL-4 production in ear homogenates. Data represent mean ±
SEM of at least three independent experiments. ** P < 0.01 after
Dunnett’s test, as compared with control group.
The study of cell viability and toxicity, assessed by
examining the mitochondrial reduction of MTT after
24 h, showed that at 100 μM, none of the tested
compounds was toxic to either human lymphocytes
or murine RAW 264.7 macrophages (data not
shown). In LPS-stimulated macrophages, lupeol at a
final concentration of 100 μM reduced the nitrite
production in the culture medium by 55% while
camphor only exhibited a slight inhibition (25%).
These results indicate an effect on the production of
nitric oxide, which plays a relevant role in contact
dermatitis and contributes to the swelling and
infiltration of effector cells.
All three tested compounds, camphor, lupeol, and
oleanolic acid, inhibited T-cell proliferation in a
concentration dependent manner 72 h after PHA
stimulation, with IC50 values of 3.7, 1.6, and 3.3 μM,
respectively. We then set out to determine the phases
in which the cell cycle was modified. After
incubation, either with or without the compounds, the
cell cycle was analyzed with propidium iodide
reagent and subsequent flow cytometry analysis. The
analysis of the cell cycle at different times indicated
that resting T-cells stayed mainly in the G0/G1 phase,
whereas PHA-stimulated cells went from the G0/G1
phase to the S phase and then on to the G2/M phase.
When PHA-stimulated cells were treated with the
isolated compounds, the passage to the S and M
phases was reduced, with the maximum effect
appearing 72 h after stimulation. These compounds
actually induced the apoptosis of human
lymphocytes, with the percentage of cells in the subG0 phase higher at 24 and 72 h with respect to the
control group (Figure 3).
Effects of terpenoids of Salvia willeana on hypersensitivity
Natural Product Communications Vol. 3 (12) 2008 1955
A)
edema, and lupeol was active against 12deoxyphorbol-13-phenylacetate- and bryostatin-1induced edemas. These findings indicate that the
inhibition of protein kinase C plays a role in their
anti-inflammatory
mechanism
[7].
Oral
administration of lupeol inhibited CD4+ and CD8+ T
cells as well as cytokines IL-2, IL-4, and IFN-γ in a
DTH reaction induced by ovalbumin in mice [8].
Oleanolic acid exhibited moderate activity on the
DTH reaction induced by dinitrofluorobenzene,
suppressing the edema by 32% 96 h after challenge
[9]. This compound also enhanced the total white
blood cell count [10] and increased the total antibody
production in the same way as the monoterpenes
carvone, limonene, and perillic acid, indicating its
potentiating effect on the immune system [11].
However, due to their irritant properties, camphor
and limonene were unable to induce an immunostimulatory response in the popliteal lymph node
assay in rats [12]. A case report of an allergic contact
dermatitis from rectified camphor oil as a component
of a topical medicine has been previously published
[13]. Still, it has been reported that T cell activation,
proliferation, and cytokine gene transcription are all
regulated by several transcription factors in which
NF-κB shows a significant participation. In this
context, in a search for inhibitory natural products
from medicinal plants against NF-AT transcription
factor, it was found that oleanolic acid showed an
IC50 > 50 μM [14].
100
Percentage ± SEM
Sub G0
Go
75
S
M
50
25
c
A
PH
A
+
O
PH
A
+
le
an
ol
ic
Lu
pe
ol
ph
or
C
am
PH
A
PH
A
+
B
la
nk
0
B)
Percentage ± SEM
90
80
Sub G0
70
Go
60
S
50
M
40
30
20
10
A
c
O
le
an
ol
ic
Lu
pe
ol
+
PH
A
PH
A
PH
+
A
C
+
am
ph
or
A
PH
B
la
nk
0
C)
100
Percentage ± SEM
Sub G0
Go
75
S
M
50
25
A
c
O
le
an
ol
ic
Lu
pe
ol
+
PH
A
PH
A
C
+
A
PH
+
am
ph
or
A
PH
B
la
nk
0
Figure 3: Effect of isolated compounds on the cell cycle phases at 12 h
(A), 24 h (B) and 72 h (C) after PHA-stimulation. Time is expressed as
hours after addition of 8 μM isolated compounds subsequent to PHA
stimulation. Values represent the percentage of cells in each phase of the
cell cycle ± SEM.
A number of triterpenoids, including oleanolic acid
and lupeol, exhibit marked anti-inflammatory activity
and have been found to modulate the immune system.
We found both triterpenoids to be effective when
applied topically against various protein kinsase C
activators. For example, both inhibited 12deoxyphorbol-13-tetradecanoate-induced
edema,
while oleanolic acid reduced merezein-induced
Considering the variety of bioactive triterpenoids
from nearly 100 Salvia species reported in the
literature [15], it seems clear that this kind of
compound is fairly representative within the genus.
So, taking into account the results obtained for the
terpenoids assayed in this study, we must assume that
the pharmacological properties attributed to Salvia
willeana correlate with the occurrence of these
constituents, which support its use in traditional
medicine and which could be extended to other
species of Salvia with the same constituents.
In conclusion, this study demonstrates that S.
willeana possesses anti-inflammatory activity and
that lupeol, oleanolic acid, and camphor could be
considered as a part of its active constituents. They
are most likely responsible for the plant’s potential
therapeutic benefits since they attenuate the
inflammatory reaction induced by oxazolone and
exert a proliferation-suppressive action, in part
through a reduction in the release of inflammatory
cytokines.
1956 Natural Product Communications Vol. 3 (12) 2008
Experimental
General experimental procedures: 1H, 13C and 2D
NMR spectra were recorded in CDCl3 on Bruker
DRX 400 and Bruker AC 200 (50.3 MHz for 13C
NMR) instruments at 295° K. Chemical shifts are
given in ppm (δ) and were referenced to the solvent
signals at δ 7.24 and 77.0 for 1H and 13C NMR,
respectively. IR spectra were obtained on a PerkinElmer PARAGON 500 FT-IR spectrophotometer.
The [α]D values were obtained in CDCl3 at 20ºC on a
Perkin - Elmer 341 polarimeter. Vacuum liquid
chromatography: silica gel 60H (Merck); Column
chromatography (CC): silica gel 60 (SDS, 40-63
μm), gradient elution with the solvent mixtures
indicated in each case; Sephadex LH-20 (Pharmacia)
with MeOH; cyclohexane-CH2Cl2-MeOH. TLC:
Merck silica gel 60 F254. Detection: UV-light, spray
reagent (vanillin-H2SO4; anisaldehyde-H2SO4).
Plant material: Aerial parts of Salvia willeana
(Holmboe) Hedge were collected on Troodos
Mountain (Cyprus), in April 2004. A voucher
specimen has been deposited in the Agricultural
Research Institute Herbarium of Nicosia [nº ARI
3213].
Extraction and isolation: The air-dried powdered
aerial parts of S. willeana (0.43 kg) were successively
extracted at room temperature with cyclohexane,
dichloromethane, MeOH and MeOH/H2O (5/1) (2 L
of each solvent, twice for 48 h). The combined dried
cyclohexane and dichloromethane extracts (45.0 g)
were subjected to vacuum liquid chromatography
(VLC) over silica gel (8 x 6.0 cm) with
cyclohexane/CH2Cl2 (90:10-10:90); CH2Cl2-MeOH
mixtures (99:1-80:20) of increasing polarity to yield
eighteen fractions (A-R) of 500 mL. Fraction F (0.36
g, eluted with cyclohexane/CH2Cl2 50:50), was
identified as camphor (1). Fraction H (0.80 g, eluted
with cyclohexane/CH2Cl2 30:70) was further
subjected to repeated CC over silica gel using
cyclohexane-EtOAc mixtures of increasing polarity
(99:1-96:4)
and
Sephadex
LH-20
(cyclohexane/CH2Cl2/MeOH 7:4:0.2) and afforded
lupeol (2; 30.5 mg). Fraction N (3.0 g, eluted with
CH2Cl2-MeOH 97:3) was further purified on
Sephadex LH 20 (CH2Cl2-MeOH mixtures of
increasing polarity) yielding oleanolic acid (3; 31.0
mg). These compounds were identified by comparing
their chromatographic and spectroscopic data with
those of pure standards.
Vonaparti et al.
Oxazolone-induced delayed-type hypersensitivity
[16]: Female CD-1 mice (Harlan Interfauna Iberica,
Barcelona, Spain), weighing 25–30 g, were randomly
distributed into groups of 6 animals and fed with a
standard diet and water ad libitum. Housing
conditions and the protocol were approved by the
Ethical Committee of the Faculty of Pharmacy in
accordance with the guidelines established by the
European Union on Animal Care (CEE Council
86/609). At the beginning of the experiment, on day
1, mice were sensitized by means of topical
application to the shaved abdomen of 150 μL of a 3%
(w/v) solution of oxazolone in acetone. On day 2, ear
thicknesses were measured to obtain data for ears
with no inflammation. On day 6, challenge was
performed by applying 20 μL of 1% (w/v) oxazolone
in acetone to both the inner and outer surfaces of both
ears, after which the extract (1 mg/ear) and test
compounds (compounds 1, 2 and 3 at 0.5 mg/ear and
dexamethasone 0.025 mg/ear), dissolved in either
acetone or EtOH/H2O (7:3) were applied topically
(20 μL) to the ears either 1, 24 or 48 h after
challenge. Ear thickness of both the treated and
control groups was measured with a micrometer
(Mitutoyo Series 293) and the edema was calculated
as the difference in thickness before treatment and
24, 48 and 72 h after challenge. The control group
was treated only with oxazolone. Inhibition was
expressed as a percentage of control. The mice were
killed by means of cervical dislocation at 72 h and
ear punches from each animal were used for
determination of cytokine production.
Determination of cytokine production in ear
homogenates [16]: Ear samples were homogenized
with a Polytron (Kinematica AG, Lucerne,
Switzerland) in a buffer solution (10 mM HEPES
pH = 7.6, 10 mM KCl, 1.5 mM MgCl2, 0.8%
Triton X-100, 1 mM dithiotreitol, 2 mM
phenylmethanesulfonyl fluoride, protease inhibitors
cocktail, Roche), sonicated (3 x 10 s) and centrifuged
at 14,000 rpm at 4ºC for 15 min. Supernatants were
analyzed for protein content with Bradford reagent
and frozen at –80ºC. IL-1β, IL-4 and TNF-α were
quantified in homogenates in triplicate by a specific
enzyme immunoassay kit used according to the
manufacturer’s instructions (eBiosciences). After
overnight incubation at 4ºC with the capture
antibody, microplate wells were washed 3 times with
washing buffer. Afterwards, the same operation was
repeated after 1h incubation with diluent. Standards
and test solutions (100 μL) were uploaded and
incubated for 2 h at room temperature. Thereafter,
Effects of terpenoids of Salvia willeana on hypersensitivity
Natural Product Communications Vol. 3 (12) 2008 1957
two further steps consisting of the addition of biotin
conjugate anti-mouse polyclonal antibody (100 μL, 1
h incubation) and avidin-peroxidase (100 μL, 30 min
incubation) preceded the termination of the reaction
with 1M H2SO4. Each incubation was followed by
aspiration and repeated washing with the buffer.
Final absorbance was read at 450 nm. Once a
standard curve was obtained with different cytokine
concentrations ranging from 7.81-1000 pg/mL,
experimental values were calculated by means of
interpolation. Inhibition percentage was defined as
the difference between the mean value of control and
test value absorbance, divided by control value, and
multiplied by 100.
obtained from the human blood of healthy volunteers.
Cells were isolated by the Ficoll-Paque gradient
density method (GE Healthcare). T lymphocytes
were isolated by depletion of adherent cells on plastic
dishes (95% purity) and resuspended in RPMI 1640
medium supplemented with 10% fetal bovine serum,
100 U/mL penicillin and 100 μg/mL streptomycin
(Invitrogen Gibco, Langley, OK, USA).
Determination of cell viability [17]: The cytotoxicity
of the test compounds was performed by the 3-[4, 5dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium
bromide (MTT) assay. Murine RAW 264.7
macrophages and human lymphocytes were exposed
to test compounds at concentrations of 100, 50 and
25 μM in a microplate for various lengths of time and
then 100 μL per well of a 0.5 mg/mL solution of
MTT was added and incubated at 37°C until blue
deposits were visible. The blue metabolite was
dissolved in dimethyl sulfoxide (DMSO).
Absorbance was measured at 570 nm using a
Labsystems Multiskan EX plate reader (Midland,
Canada). Results were expressed in absolute
absorbance readings, with a decrease indicating a
reduction in cell viability.
A volume of 200 μL of T-lymphocyte suspension (1
x 106 cells/mL) was applied to each well of a 96-well
plate with 25 μg/mL phytohemagglutinin (PHA)
alone or with test compounds at different
concentrations (5–10 μM) and dexamethasone (5
μM) as a positive control. The plates were incubated
in a 5% CO2/air humidified atmosphere at 37°C for 3
days, after which T-cell proliferation was determined
with a modified colorimetric MTT assay. The
formazan product formed was dissolved in DMSO by
shaking it. The absorbance was measured at 570 nm
using a Labsystems Multiskan EX plate reader
(Midland, Canada). Results were expressed in
absolute absorbance readings; a decrease indicated a
reduction in cell viability. Controls consisted either
of lymphocytes with PHA (100% activity), with
medium (0% activity) or samples with nonstimulated lymphocytes.
Nitrite production in intact RAW 264.7
macrophages [18]: Murine RAW 264.7 macrophages
were cultured in Dulbecco's Modified Eagle Medium
(DMEM) containing 2 mM L-glutamine, 100 U/mL
penicillin, 100 μg/mL streptomycin and 10% fetal
bovine serum (all from Invitrogen). Cells were
removed from the tissue culture flask with a cell
scraper and resuspended to a final concentration of
1×106 cells/mL. Nitrite production was assessed as
the index of nitric oxide generation in the induction
phase. Thus, RAW 264.7 macrophages (1×106
cells/mL) were co-incubated in a 96-well culture
plate (200 μL) with 1 μg/mL of lipopolysaccharide
(Sigma-Aldrich) at 37° C for 24 h in the presence of
test compounds (100, 50, 25 and 12.5 μM) or vehicle
(phosphate-buffered saline). Nitrite concentration in
culture supernatant was determined by using Griess
reagent (Sigma-Aldrich).
T-lymphocytes (1 x 106 cells/mL) were co-incubated
in a 6-well plate with or without 25 μg/mL PHA after
treatment with the compounds. Different experiments
concerning the cell cycle were performed to see the
effect on cell cycle after PHA stimulation and the
effect of isolated compounds on PHA-stimulated
cells at different times. Isolated compounds (6-8 μM)
were added to the cells, after which the plates were
incubated in a 5% CO2-air-humidified atmosphere at
37°C for 12, 48 and 72 h. In these experiments we
see at what point the cell cycle is blocked and we
view the subG0 peak as the index of the apoptosis
event, and then the G1, G2 and S peaks. The cells
were harvested by means of centrifugation, washed in
phosphate-buffered saline (pH 7.2) and then fixed in
70% ethanol for 30 min at −20° C. After washing the
cells once with phosphate-buffered saline, the DNA
was stained with propidium iodide (4 μg/mL)
containing 100 μg/mL of ribonuclease A. Flow
cytometry analysis was conducted with an EPICS
XCL (Beckman, Fullerton, CA, USA); 1 x 104 cells
for each test sample were counted.
T-lymphocyte isolation, proliferation and cell cycle
analysis [19,20]: Peripheral lymphocytes were
Statistics: Data were expressed as mean ± S.E.M.
Statistical analysis involved carrying out a one-way
1958 Natural Product Communications Vol. 3 (12) 2008
analysis of variance (ANOVA) followed by Dunnett's
t-test for multiple comparisons. In comparisons
against the control group, values of P less than 0.05
were considered significant. Inhibition percentages
(%I) were calculated from the differences between
drug-treated groups and control animals treated only
with the inflammatory agent.
Vonaparti et al.
Acknowledgments – This work was supported by
the Spanish Ministry of Education and Science
(FEDER) (SAF 2006-06726). The authors wish to
thank Dr T. Vrahimi-Hadjilouca and Dr D.
Droushiotis (Agricultural Research Institute, Ministry
of Agriculture, Natural Resources & Environment,
Nicosia, Cyprus) for providing us the plant material.
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Olmos A, Giner RM, Recio MC, Ríos JL, Cerdá-Nicolás JM, Máñez S. (2007) Effects of plant alkylphenols on cytokine
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NPC
Natural Product Communications
Characterization of By-products of Saffron
(Crocus sativus L.) Production
2008
Vol. 3
No. 12
1959 - 1962
Pamela Vignolinia, Daniela Heimlerb, Patrizia Pinellia, Francesca Ieria, Arturo Sciulloc and
Annalisa Romani*
a
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Firenze, via U. Schiff 6,
50019 Sesto Fiorentino, Italy
b
Dipartimento di Scienza del Suolo e Nutrizione della Pianta, Università degli Studi di Firenze,
P.le delle Cascine 18, 50144 Firenze, Italy
c
ARPAT, Agenzia Regionale per la Protezione Ambientale della Toscana, via Ponte alle Mosse 211,
50144 Firenze, Italy
annalisa.romani@unifi.it
Received: July 28th, 2008; Accepted: October 31st, 2008
The stigma, stamens and sepals of Crocus sativus L,. from two different geographical origins, were analyzed for their crocin
and flavonol contents. Identification of crocins, safranal, picrocrocin, and flavonols was carried out by HPLC/DAD and
HPLC/MS analysis. Both stigma samples, grown under natural conditions, exhibited high crocin contents (between 342 and
231 mg/g), while the stamens and sepals were rich in flavonols (between 6 and 10 mg/g). The stamens contain mainly
kaempferol- 3-O-sophoroside, whereas the sepals contain mainly quercetin and methyl-quercetin glycosides. These data
may be useful in order to find a possible exploitation of the by-products of saffron production, in which large quantities of
C. sativus flowers are available.
Keywords: Crocins, flavonols, HPLC/DAD/MS, sepals, stamens, stigma.
The dried, red stigmas of Crocus sativus L. are a very
expensive spice known as saffron, which is used as a
food flavoring and coloring agent and as a traditional
herbal medicine [1a]. Crocus is cultivated in India,
Iran, Spain, Greece and Italy. The production process
involves a large amount of manual work and cannot
be completely mechanized. In Italy, from a 1000 m2
area, about 120,000-150,000 flowers can be obtained
(4000-5000 kg), which give rise to 5-7 kg of fresh
stigma, i.e. 1.0-1.3 kg of dried product.
Many papers deal with methods for the
separation and determination of the biologically
active [1b-1f] and aroma components [2a-2c]. The
quality control of commercial saffron is checked
using spectrophotometric [3a,3b], TLC [3c], GC [3d],
HPLC [3e], and CE [3f] methods.
The purpose of this paper is the analysis of stigmas
from C. sativus cultivated in Italy (Perugia and
Fiesole) in order to characterize this commercial
saffron from a quality point of view. In these areas,
cultivation is effected under natural conditions and
without the use of any chemical product in the drying
and conservation phases.
However, the most important part deals with the
characterization
of
the
biologically
active
components of the stamens and sepals in order to find
a possible use for this material, which forms the
major part of C. sativus flowers. The exploitation of
stamens and sepals, notwithstanding their availability
as by-products in the production of saffron, has not
been taken into account, with the exception of one
paper dealing with the isolation of flavonoids from
crocus petals to study their tyrosinase inhibition
action [4a]. Notwithstanding the lack of information
on the polyphenol content of these tissues, petal
extracts were used to control rat blood pressure [4b]
and to test their antitussive effect in guinea pigs [1b].
The major biologically active components of saffron
are crocin analogues, which are all glycosides of
1960 Natural Product Communications Vol. 3 (12) 2008
O-R2
CH3
CH3
O
Table 1: Quantitative data for dried stigma. Average value ± SD of three
samples. Data are expressed as mg/g fresh sample.
O
O-R1
CH3
COMPOUNDS
(Rt)
CH3
COMPOUND
R1
R2
MW
Crocin-5 ;
C50H74O29
Crocin-4 ;
C44H64O24
Crocin-3 ;
C38H54O19
Crocin-2 ;
C33H44O14
Crocin-2’ ;
C33H44O14
Crocin-1 ;
C26H34O9
Crocetin ;
C20H24O4
Three β-glucosyl
β-D-gentiobiosyl
1138
β-D-gentiobiosyl
β-D-gentiobiosyl
976
β-D-gentiobiosyl
β-D-glucosyl
814
β-D-gentiobiosyl
H
652
β-D-glucosyl
β-D-glucosyl
652
β-D-glucosyl
β-D-glucosyl
490
H
H
328
CHO
CHO
HO
Gluc.-O
Safranal C10H14O
MW= 150
Picrocrocin C16H26O7
MW= 330
OH
HO
O
R
OH
OH
Vignolini et al.
COMPOUND
R
MW
Quercetin ; C15H10O7
ΟΗ
302
Kaempferol ; C15H10O6
Η
286
O
Figure 1: Chemical structures of saffron components
trans-crocetin, a carotenoid derivative, and which are
responsible for the color. Safranal (2,6,6-trimethyl1,3-cyclohexadien-1-carboxaldehyde),
which
is
responsible for the characteristic aroma of saffron, is
formed during storage by dehydration of picocrocin,
which is responsible for its bitter taste. Flavonoids
are found in stigma, sepals, and stamens (Figure 1).
As regards stigma, the composition of the extract was
similar to that found by other authors regarding
crocins, picocrocins, and safranal. Three kaempferol
derivatives (two triglycosides and one diglycoside)
were identified, according to previous findings
[1f,5a]. In the case of stamens, a lesser number of
crocins was found and quercetin, as well as
kaempferol derivatives were detected. Also, methylquercetin derivatives in quite large amounts were
recorded. There were no differences, from a
qualitative point of view, between the two sampling
zones; in fact only a quantitative variation was found
in the samples from the different geographic regions
[5a].
Table 1 reports the quantitative data for the dried
stigma. It should be noted that the two samples differ
are present in largest amount in the two samples.
These compounds, together with cis-crocin 4, were
trans crocin-5 (10.30)
crocin derivative (11.14)
crocin derivative (11.46)
crocin derivative (11.87)
trans crocin-4 (12.84)
crocin derivative (13.88)
trans crocin-3 (14.39)
crocin derivative (14.99)
crocin derivative (15.90)
trans crocin-2' (16.17)
crocin derivative (17.37)
cis crocin-4 (17.79)
trans crocin-2 (19.33)
crocin derivative (20.40)
crocin derivative (21.11)
cis crocin-1 (22.02)
crocin derivative (22.81)
crocin derivative (23.17)
TOTAL
Picrocrocin (6.34)
Safranal (24.87)
K-3-sophoriside -7- glucoside (3.78)
K -3,7,4'-triglucoside (5.90)
K-3-sophoroside (8.49)
TOTAL
Stigma
(FI)
Stigma
(PG)
2.4±0.09
2.1±0.08
0.3±0.01
0.3±0.009
238.9±2.86
1.3±0.06
65.6±1.84
0.2±0.01
0.6±0.03
2.1±0.07
0.3±0.01
9.5±0.33
16.9±0.51
0.2±0.009
0.3±0.01
1.0±0.05
0.2±0.01
traces
342.02
111.1±2.33
2.2±0.09
4.7±0.2
1.2±0.05
6.2±0.22
2.0±0.07
0.8±0.04
0.3±0.01
0.1±0.007
148.5±2.66
0.5±0.02
46.2±1.38
0.2±0.009
0.5±0.02
1.5±0.06
0.3±0.009
14.1±0.49
14.8±0.50
traces
traces
0.8±0.04
traces
0.5±0.02
231.1
68.9±1.79
2.6±0.09
3.3±0.14
0.9±0.04
5.4±0.17
12.1
9.64
mainly in trans-crocin 4, trans-crocin 3 and
picrocrocin contents, i.e. the three compounds which
also the main compounds found by Caballero-Ortega
et al. [5b] in a study of 11 saffron samples from
different origins. The crocins content of the two
samples is quite high giving evidence for the very
good quality of the two samples. Among flavonols,
kaempferol-3-O-sophoroside
was
the
main
compound reported for a Spanish sample analyzed by
Carmona et al. [5a].
Table 2 reports the crocin contents of sepals and
stamens. The amount of crocins is low, while that of
flavonols (Table 3) ranged from 10.1 to 6.1 mg/g.
Stamens and sepals differ mainly in their kaempferol3-O-sophoroside content, which is the most abundant
flavonol in the sepals.
The flavonols composition of the two tissues is
different: in sepals, kaempferol derivatives ranged
between 91 -93 %, whereas in stamens, quercetin and
methyl-quercetin derivatives ranged between 5271%. From all these data the possible exploitation of
alternative tissues like stamens and sepals as
phytochemical resources can be pointed out. For each
kg of stigma, about 1000 kg of flowers are processed;
therefore, sepals and stamens are important
by-products of saffron production and their use could
increase the economic value of C. sativus flowers.
Characterization of byproducts of saffron
Natural Product Communications Vol. 3 (12) 2008 1961
Table 2: Crocins content of sepals and stamens. Average value ± SD
of three samples.
COMPOUNDS
(Rt)
trans crocin-4
(12.84)
crocin der.
(13.88)
trans crocin-3
(14.39)
crocin der.
(14.99)
crocin der.
(15.99)
trans crocin-2'
(16.17)
cis crocin-4
(17.79)
trans crocin-2
(19.33)
cis crocin-1
(22.02)
crocin der.
(22.81)
crocin der.
(23.17)
cis crocin-2
(24.82)
Sepals
(FI)
Sepals
(PG)
Stamens
(FI)
Stamens
(PG)
3.1±0.17
traces
112.2±5.65
4.0±0.19
1.7±0.09
traces
33.4±1.74
traces
traces
traces
0.8±0.04
traces
1.3±0.07
traces
traces
traces
3.3±0.18
traces
22.0±1.14
0.1±0.006
20.7±1.07
1.3±0.08
7.0±0.38
traces
0.3±0.02
traces
0.1±0.008
traces
0.3±0.02
TOTAL
4.2
0.6±0.03
traces
196.3
5.4
Experimental
Sample preparation: Sepals, stamens and dried
stigma samples were obtained from plants harvested
in 2005 from Fiesole (FI, Italy) and Perugia (PG,
Italy). Sepals and stamens (500 mg) were suspended
in 50 mL of 70% ethanol, adjusted to pH 2.0 with
formic acid, and left overnight. After extraction, the
samples were filtered to eliminate plant residues, and
the filtrate evaporated to dryness under vacuum at
room temperature. The residue was redissolved in
EtOH/H2O (70:30) and adjusted to pH 2.0 with
formic acid to a final volume of 3 mL.
Saffron stigmas (50 mg) were extracted with 10 mL
of 70% ethanol, adjusted to pH 2.0 with formic acid,
left overnight and then filtered to eliminate plant
residues. The extracts were analysed by
HPLC/DAD/MS for the determination of saffron
components.
Authentic standards of crocin were purchased from
Fluka (St. Louis, USA), safranal from Sigma-Aldrich
(St. Louis, USA), and p-hydroxybenzoic acid,
kaempferol 3-O-glucoside, rutin and curcumin from
Extrasynthèse S.A. (Lyon, France). All solvents were
of HPLC grade purity (BDH Laboratory Supplies,
United Kingdom).
HPLC/DAD analysis:. Analysis for flavonols and
crocins was carried out using a HP 1100L liquid
chromatograph equipped with a DAD detector and
managed by a HP 9000 workstation (Agilent
Technologies, Palo Alto, CA, USA). Flavonols and
crocins were separated by using a 150 × 3.9 mm i.d. 4
μm Nova-Pak C18 column (Waters) operating at
27°C. UV/Vis spectra were recorded in the 190-600
nm range and the chromatograms were acquired at
250, 308, 350 and 440 nm. The mobile phase was a
Table 3: Flavonols content of sepals and stamens. Average value ± SD of three samples. Data are expressed as μg/g fresh sample.
COMPOUNDS (Rt)
Kaempferol derivative (3.71)
Kaempferol-3-sophoroside-7-glucoside (3.78)
Kaempferol derivative (5.81)
Kaempferol diglucoside (5.89)
Kaempferol derivative (6.49)
Kaempferol diglucoside (7.30)
Quercetin diglucoside (7.30)
Methyl quercetin diglucoside (7.82)
Quercetin derivative (8.15)
Methyl quercetin di glucoside (8.42)
Kaempferol-3-sophoroside (8.49)
Kaempferol glucosyl rhamnoside (9.29)
Methyl quercetin derivative (9.34)
Quercetin derivative (9.44)
Quercetin diglucoside (9.58)
Kaempferol sinapoyl glucoside (10.59)
Kaempferol derivative (10.86)
Kaempferol glucoside (10.98)
Methyl quercetin glucoside (11.13)
Quercetin derivatives (11.55-12.21)
Kaempferol derivative (12.99)
Quercetin p-cumaroyl glucoside (13.76)
Quercetin derivative (14.09)
Kaempferol p-cumaroyl glucoside (15.42)
Methyl quercetin p-cumaroyl glucoside (15.61)
Kaempferol (18.43)
TOTAL
Sepals (FI)
76±4.10
Sepals (PG)
97±4.85
15±1.03
113±5.6
34±1.83
480±22.08
82±4.16
738±32.16
84±4.21
6415±192.45
41±2.13
8304±215.9
66±3.20
24±1.27
306±14.38
60±3.18
309±14.25
421±19.78
399±18.75
Stamen(FI)
Stamen(PG)
511±22.84
24±1.18
923±41.53
77±4.15
416±21.16
1037±37.32
628±28.88
27±1.15
209±10.03
1702±64.7
755±33.75
1227±47.81
2091±61.74
39±2.14
249±11.73
377±17.72
691±31.09
239±11.47
1188±46.32
303±14.54
140±5.81
39±2.25
21±1.15
93±4.65
52±2.75
26±1.19
17±0.078
199±9.75
4±0.22
35±2.05
26±1.21
20±1.16
7998
176±8.62
66±3.43
14±0.74
10138
6059
237±110.61
26±1.20
52±2.65
40±2.12
8±0.44
7873
1962 Natural Product Communications Vol. 3 (12) 2008
one-step linear solvent gradient system, starting from
90% H2O (adjusted to pH 3.2 with HCOOH) up to
100% CH3CN during a 60-min period; flow rate
0.8 mL min-1.
HPLC/MS analysis: HPLC/MS analysis was
performed using a HP 1100L liquid chromatograph
linked to a HP 1100 MSD mass spectrometer with an
API/electrospray interface (Agilent Technologies,
Palo Alto, CA, USA). The mass spectrometer
operating conditions were: gas temperature, 350°C;
nitrogen flow rate, 10.5 L/min, nebulizer pressure,
40 psi; quadrupole temperature, 30°C; and capillary
voltage, 3500 V. The mass spectrometer was
operated in positive mode at 120 eV.
Identification and quantification of individual
polyphenols: Quantification of individual compounds
was directly performed by HPLC/DAD using a
five-point regression curve (r2 ≥ 0.998) in the range
Vignolini et al.
0-30 μg on the basis of authentic standards. In
particular, crocin derivatives were determined at 440
nm using curcumin as reference compound; safranal
was determined at 308 nm using safranal as reference
compound and picrocrocin was determined at 250 nm
using p-hydroxybenzoic acid as reference compound.
Flavonols, like kaempferol and quercetin derivatives,
were determined at 350 nm using kaempferol-3-Oglucoside and rutin, respectively, as reference
compounds. In all cases, actual concentrations of the
derivatives were calculated after applying corrections
for differences in molecular weight.
Acknowledgments - The authors wish to express
their sincere gratitude to the Cassa di Risparmio di
Firenze that contributed to the acquisition of a part of
the instrumentation used for this work. We express
sincere thanks to Mr Piscolla, Azienda Agricola
Poggio al Sole (Fiesole, FI) for the supply of saffron
samples from Fiesole.
References
[1]
[2]
[3]
[4]
[5]
(a) Xi L, Qian Z. (2006) Pharmacological poperties of crocetin and crocin (digentiobiosyl) ester of crocetin from. saffron. Natural
Product Communications, 1, 65-75; (b) Alonso GL, Salinas M., Garijo J, Sanchez-Fernandez MA. (2001) Composition of crocins
and picocrocin from Spanish saffron (Crocus sativus L.), Journal of Food Quality, 24, 219-233; (c) Li N, Lin G, Kwan YW, Min
ZD. (1999) Simultaneous quantification of five major biologically active ingredients of saffron by high-performance liquid
chromatography. Journal of Chromatography A, 849, 349-355; (d) Pfander H, Rychener M. (1982) Separation of crocetin glycosyl
esters by high-performance liquid chromatography. Journal of Chromatography A, 234, 443-447; (e) Tarantilis PA, Polissiou M,
Manfait M. (1994) Separation of picrocrocin, cis-trans-crocins and safranal of saffron using high-performance liquid
chromatography with photodiode-array detection. Journal of Chromatography A, 664, 55-61; (f) Tarantilis PA, Tsoupras G,
Polissiou M. (1995) Determination of saffron (Crocus sativus L.) components in crude plant extract using high-performance liquid
chromatography-UV-visible photodiode-array detection-mass spectrometry. Journal of Chromatography A, 699, 107-118
(a) Lozano P, Delgado D, Gomez D, Rubio M, Iborra JL. (2000) A non-destructive method to determine the safranal content of
saffron (Crocus sativus L.) by supercritical carbon dioxide extraction combined with high performance liquid chromatography and
gas chromatography. Journal of Biochemical and Biophysical Methods, 43, 367-378; (b) Loskutov AV, Beninger CW, Hosfield
GL, Sink KC. (2000) Development of an improved procedure for extraction and quantitation of safranal in stigmas of Crocus
sativus L. using high performance liquid chromatography. Food Chemistry, 69, 87-95; (c) Straubinger M, Bau B, Eckstein S, Fink
M, Winterhalter P. (1998) Identification of novel glycosidic aroma precursors in saffron (Crocus sativus L.). Journal of
Agricultural and Food Chemistry, 46, 3238-3243.
(a) Carmona M, Carrion ME, Zalacain A, Alonso GL. (2004) Detection of adulterated saffron through UV-Vis spectral analysis.
Journal of Food Science & Technology, 41, 451-455; (b) Zalacain A, Ordoudi SA, Blazquez I, Diaz-Plaza EM, Carmona M,
Tsimidou MZ, Alonso GL. (2005) Screening method for the detection of artificial colours in saffron using derivative UV-Vis
spectrometry after precipitation of crocetin. Food Additives and Contaminants, 22, 607-615; (c) Corti P, Mazzei E, Ferri S,
Granchi GG, Dreassi E. (1996) High performance thin layer chromatographic quantitative analysis of picrocrocin and crocetin,
active principles of saffron (Crocus sativus L.): a new method. Phytochemical Analysis, 7, 201-203; (d) Alonso GL, Salinas MR,
Garijo J. (1998) Method to determine the authenticity of aroma of saffron (Crocus sativus L.). Journal of Food Protection, 61,
1525-1528; (e) Lozano P, Castellar MR, Simancas MJ, Iborra JL. (1999) Quantitative high-performance liquid chromatographic
method to analyse commercial saffron (Crocus sativus L.) products. Journal of Chromatography A, 830, 477-483; (f) Zougagh M,
Simonet BM, Rios A, Valcarcel M. (2005) Use of non-aqueous capillary electrophoresis for the quality control of commercial
saffron samples. Journal of Chromatography A, 1085, 293-298.
(a) Kubo I, Kinst-Hori I. (1999) Flavonols from saffron flower: tyrosinase inhibitory activity and inhibition mechanism. Journal of
Agricultural and Food Chemistry, 47, 4121-4125; (b) Fatehi M, Rashidabady T, Fatehi-Hassanabad Z. (2003) Effects of Crocus
sativus petals extract on rat blood pressure and on responses induced by electrical field stimulation in the rat isolated vas deferens
and guinea pig ileum. Journal of Ethnopharmacology, 84, 199-203; (c) Hosseinzadeh H, Ghenaati J. (2006) Evaluation of the
antitussive effect of stigma and petals of saffron (Crocus sativus) and its components safranal and crocin in guinea pig. Fitoterapia,
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(a) Carmona M, Sanchez AM, Ferreres F, Zalacain A, Tomas-Barberan F, Alonso GL. (2007) Identification of the flavonoids
fraction in saffron spice by LC/DAD/MS/MS: comparative study of samples from different geographic origins. Food Chemistry,
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NPC
Natural Product Communications
Antitrypanosomal and Antileishmanial Activities of Organic
and Aqueous Extracts of Artemisia annua
2008
Vol. 3
No. 12
1963 - 1966
Anna Rita Biliaa, Marcel Kaiserb, Franco Francesco Vincieria and Deniz Tasdemirc,*
a
Department of Pharmaceutical Sciences, University of Florence, 50019 Sesto Fiorentino, Florence, Italy
b
Department of Medical Parasitology, Swiss Tropical Institute, 4002 Basel, Switzerland
c
Centre for Pharmacognosy and Phytotherapy, School of Pharmacy, University of London,
London WC1N 1AX, UK
deniz.tasdemir@pharmacy.ac.uk
Received: July 22nd, 2008; Accepted: October 17th, 2008
Artemisia annua is an herbal drug with profound antimalarial activity, which can be ascribed to the sesquiterpene lactone artemisinin.
Artemisinin also shows efficacy against other parasitic protozoan species, such as Trypanosoma and Leishmania, however
trypanocidal and leishmanicidal effects of A. annua extracts have not been reported so far. In the current study, we evaluated the in
vitro growth inhibitory activity of a number of organic and aqueous A. annua extracts, including tinctures, infusions and decoctions
against three parasitic protozoa, T. brucei rhodesiense, T. cruzi and L. donovani. Artemisinin content of these extracts was determined
by HPLC/DAD/MS. Artemisinin was also evaluated for its antiparasitic activity for comparison. Among the tested extracts, the
acetone- and the n-hexane-solubles of A. annua were the most potent against T. b. rhodesiense with IC50 values of 0.30 and 0.455
μg/mL, respectively, whereas the other extracts were ten- to fifty-fold less potent. Neither of the extracts nor artemisinin had
trypanocidal activity against T. cruzi (IC50 > 30 μg/mL). Only the organic extracts of A. annua arrested the growth of L. donovani
with modest IC50 values (5.1 to 9.0 μg/mL) comparable to that of artemisinin (IC50 8.8 μg/mL). This study highlights significant
variations in the artemisinin content of A. annua extracts and underlines the potential of A. annua extracts and artemisinin in the
treatment of trypanosomal and leishmanial infections.
Keywords: Artemisia annua, organic and aqueous extracts, artemisinin, Trypanosoma, Leishmania.
Artemisia annua L. (Asteraceae) is an annual herb
that has been used against fever in traditional Chinese
medicine for over 2,000 years [1a]. The plant is
nowadays listed in the Pharmacopoeia of the
Republic of China for the treatment of fever and
malaria. The recommended daily dose is specified as
4.5 to 9 g of dried herb prepared as a tea (infusion)
with boiling water [1b]. After the discovery of
artemisinin (1) as the antimalarial principle of the
plant, all clinical studies have focused on this
compound. Currently artemisinin-based combination
therapy (ACT) is the most effective treatment of
drug-resistant malaria and recommended by the
WHO. A. annua is the sole source of artemisinin,
which occurs in the plant in very low concentrations,
resulting in high costs of therapy. Thus, there is an
increasing interest in the use of A. annua teas in
malaria endemic areas where large scale
pharmaceutical production is not possible.
Accordingly, the consumption of A. annua tea or
decoctions by patients in clinical studies has been
shown to reduce parasitaemia significantly [1c].
Trypanosomiasis and leishmaniasis are insect-borne
parasitic diseases that represent a significant and
neglected public health problem worldwide.
Trypanosoma brucei rhodesiense is the causative
agent of African trypanosomiasis (sleeping sickness)
in South and East Africa, infecting 50,000 people
every year, primarily in the poorest rural populations
of sub-Saharan Africa where the tsetse fly vector is
common [1d]. The incidence of the disease may
approach 500,000 cases per year. The American
trypanosomiasis, also known as Chagas’ disease
caused by T. cruzi is a major endemic disease in
South and Central America. It is estimated that 18
million people are infected with the disease and
50,000 individuals die of it annually [2].
Leishmaniasis is a tropical and subtropical disease
affecting nearly 88 countries, with an estimated
global prevalence of 12 million cases annually. It is
caused by protozoal parasites of the genus
Leishmania and occurs in three different clinical
forms (cutaneous, muco-cutaneous and visceral),
where visceral leishmaniasis (Kala azar), due to
1964 Natural Product Communications Vol. 3 (12) 2008
L. donovani and L. infantum, is the most severe form.
Leishmaniasis presents as an opportunistic infection
in association with immunodepression, in particular
AIDS [3a]. Both trypanosomiasis and visceral
leishmaniasis are invariably fatal if untreated. There
is no prophylactic chemotherapy and little or no
prospect of a vaccine. Moreover, current treatments
of these diseases are far from being ideal because of
the high costs, long treatment courses, high clinical
failure and serious toxic, even fatal side effects. Thus,
it is obvious that new drugs and therapy regimes are
needed for the treatment of these diseases.
Besides its profound antimalarial effect, artemisinin
has been shown to possess growth inhibitory effects
against pathogenic Trypanosoma and Leishmania
species [3b,3c]. However, the effects of different
A. annua extracts have not been studied. In a
previous study on a selected high-yield Brazilian
cultivar of A. annua, we determined the artemisinin
content of a number of solvent extracts, such as
aqueous EtOH (tinctures AA-T40, AA-T60),
infusions (AA-I1, AA-I2, AA-I3) and decoctions
(AA-D1, AA-D2) by HPLC/DAD/MS and compared
these with the artemisinin concentration in the
n-hexane extract (AA-Hexane) [3d]. In the
continuation of our studies on this A. annua cultivar,
we have now prepared additional solvent extracts,
namely toluene (AA-Toluene), dichloromethane
(AA-DCM), acetone (AA-Acetone) and 100%
ethanol (AA-EtOH). Their artemisinin content was
determined by HPLC/DAD/MS, as previously
reported [3d,3e]. All available extracts, as well as
artemisinin were tested for in vitro activity against T.
brucei rhodesiense, T. cruzi and L. donovani.
Table 1 displays the quantitative analysis results on
artemisinin content of all twelve extracts
investigated. Percentages are given on the dried
extracts obtained by evaporation of the organic
solvent or freeze-dried aqueous solutions, and do not
reflect the content of artemisinin in the dried herbal
drug, which is about 0.52%. Very different
artemisinin percentages were detected in the
investigated extracts, with AA-DCM being the
richest with 3.68% artemisinin. This extract was
followed by EtOH, n-hexane and acetone extracts
(2.64%, 2.28% and 1.82, respectively), whereas the
toluene extract was the poorest (0.57%). The
tinctures prepared with 40% v/v (AA-T40) and 60%
v/v (AA-T60) EtOH had also lower artemisinin
yields (0.75% and 1.08%), similar to those of A.
annua infusions or decoctions, which contained
roughly 0.7-0.8% artemisinin.
Bilia et al.
Table 1: Artemisinin content, trypanocidal and leishmanicidal activities
of different solvent extracts of A. annua and artemisinin (IC50 values are
in μg/mL and mean values from at least two replicates, i.e. the variation is
a maximum of 20%).
Extract/
Compound
AA-Toluene
AA-Acetone
AA-Hexane
AA-DCM
AA-EtOH
AA-T40
AA-T60
AA-I1
AA-I2
AA-I3
AA-D1
AA-D2
Artemisinin
Reference
Extraction
yield (g)
3.01
2.47
2.28
0.68
1.04
1.80a
1.90a
2.24a
2.10a
2.78a
2.36a
2.31a
Artemisinin
(%±SEM)
0.57±0.07
1.82±0.11
2.28±0.98
3.68±1.01
2.64±0.93
0.75±0.16a
1.08±0.20a
0.72±0.11a
0.68±0.23a
0.80±0.14 a
0.68±0.13a
0.81±0.15 a
T. b.
rhodesiense
3.31
0.30
0.455
3.82
14.57
13.31
23.48
21.15
22.15
22.58
22.96
24.40
24.40
0.004a
T.
cruzi
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
0.22b
L.
donovani
9.0
5.10
5.64
8.50
>30
>30
>30
>30
>30
>30
>30
>30
8.80
0.10c
a
: These results were obtained in our previous study [3d]
Reference compounds: amelarsoprol, bbenznidazole, cmiltefosine. The
extracts with most abundant artemisinin levels are shown bold.
Table 1 also displays the IC50 values of the extracts
investigated and artemisinin against T. brucei
rhodesiense, T. cruzi and L. donovani. The AAAcetone and AA-Hexane extracts showed the highest
trypanocidal effect against African trypanosome (T.
b. rhodesiense) with IC50 values of 0.30 and 0.455
μg/mL. The next best activity was exerted by AAToluene and AA-DCM, which had almost ten times
higher IC50 values (3.31 μg/mL and 3.82 μg/mL,
respectively). AA-T40 and AA-EtOH extracts
showed comparable but reduced trypanocidal effects
(IC50 values 13.31 and 14.57 μg/mL, respectively),
whereas all remaining extracts exhibited IC50 values
above 20 μg/mL. The potency of artemisinin was
similar (IC50 24.4 μg/mL). It is noteworthy that the
trypanocidal effect of acetone and n-hexane extracts
is much greater than that of artemisinin. This finding
may be due to the presence of other bioactive
compounds in these extracts or compounds having
significant synergistic effect with artemisinin. On the
other hand, none of the extracts or artemisinin
appeared to have the ability to inhibit the growth of
American trypanosome, T. cruzi at the highest
concentrations tested (IC50 values >30 μg/mL). Of
the twelve extracts that were studied for in vitro
antileishmanial activity, only the organic A. annua
extracts proved to have reasonable potential. Notably
AA-Acetone and AA-Hexane extracts had almost
identical activities (IC50 values 5.1 and 5.64 μg/mL),
as did the AA-DCM and AA-Toluene extracts (IC50
values 8.5 and 9.0 μg/mL). All other remaining
extracts, including AA-EtOH were inactive (IC50 >30
μg/mL). Leishmanicidal activity of artemisinin was
also comparable (IC50 8.80 μg/mL) to AA-DCM.
Trypanocidal and leishmanicidal activities of A. annua extracts
Natural Product Communications Vol. 3 (12) 2008 1965
To our knowledge, this is the first time that A. annua
extracts have been studied for trypanocidal and
leishmanicidal properties. Although the results might
appear low in comparison to standard drugs, organic
A. annua extracts, in particular the acetone and nhexane extracts still represent good activity against T.
b. rhodesiense and L. donovani. These two extracts
merit further investigations for the identification of
their active components, which might be different
from artemisinin.
boiling water, then left to cool and filtered through a
filter paper. Sample I2: A 9 g sample of dried aerial
parts of A. annua was extracted with 1 L of boiling
water, covered, then left to cool and filtered through a
filter paper. Sample I3: A 9 g sample of dried aerial
parts of A. annua was extracted with 1 L of boiling
water, then left to cool for 15 min and filtered
through a filter paper. For analytical purposes, all
infusions (filtrates) were freeze-dried and directly
analysed.
Experimental
HPLC-DAD and HPLC-MS systems: The HPLC
analyses were performed using a HP 1100 Liquid
Chromatograph (Agilent Technologies, Palo Alto,
CA, USA) equipped with a HP 1040 Diode Array
Detector (DAD), an automatic injector, an auto
sampler, a column oven and managed by a HP 9000
workstation (Agilent Technologies, Palo Alto, CA,
USA). Separations were performed on a reversed
phase column (Purospher®Star RP-18, namely
Hibar®). The HPLC system was interfaced with a HP
1100 MSD API-electrospray (Agilent Technologies,
Palo Alto, CA, USA). The interface geometry, with
an orthogonal position of the nebulizer with respect
to the capillary inlet, allowed the use of analytical
conditions similar to those of HPLC-DAD analysis.
Mass spectrometry operating conditions were
optimized in order to achieve maximum sensitivity
values: gas temperature 350°C at a flow rate of 10
L/min, nebulizer pressure 30 p.s.i., quadrupole
temperature 30°C, and capillary voltage 3500 V. Full
scan spectra from m/z 100 to 800 in the positive ion
mode were obtained (scan time 1 s). A prepacked
column RT (250 x 4.6 mm) with particle size 5 µm
(Merck, Darmstadt, Germany) was employed. The
eluents were A: water adjusted to pH 3.2 with formic
acid; B: acetonitrile; C: methanol. The system was
operated with the oven temperature at 26oC. Before
HPLC analysis, each sample was filtered through a
cartridge-type sample filtration unit with a polytetrafluoroethylene (PTFE) membrane (d=13 mm,
porosity 0.45 µm, (Lida Manufacturing Corp.) and
immediately injected. Chromatograms were recorded
between 200 and 450 nm. DAD spectra were stored
for all peaks exceeding a threshold of 0.1 mAu.
Plant material: The selected high-yield cultivar of A.
annua [3f] was provided by P.M. Magalhães of the
Universidade Estadual de Campinas (Brazil). Dried
aerial parts of the plant were used in all analyses.
Chemicals: Artemisinin was purchased from Sigma
(Sigma-Aldrich). All the solvents used for the
extraction and HPLC analysis (EtOH, toluene,
MeOH, n-hexane, dichloromethane, and acetone)
were HPLC grade from Merck (Darmstadt,
Germany); 85% formic acid was provided by Carlo
Erba (Milan, Italy). Water was purified by a MilliQplus system from Millipore (Milford, MA).
Preparation of organic extracts: Small pieces of
dried aerial parts of A. annua (10 g) were
exhaustively extracted at room temperature by
maceration with 100 mL of organic solvent (toluene,
n-hexane, acetone, dichloromethane or EtOH) for 72
h, separately. The filtrates were subsequently
evaporated to dryness under reduced pressure to
obtain the crude organic extract.
Preparation of the tinctures: Small pieces of dried
aerial parts of A. annua (10 g) were extracted at room
temperature by maceration with 100 g of ethanol
[either 40 or 60% v/v (samples T40 and T60)]. The
solutions were concentrated under vacuum and
freeze-dried prior to the analysis.
Preparation of the decoctions: Sample D1: A 9 g
sample of dried aerial parts of A. annua was extracted
with 1 L of boiling water, kept boiling for 5 min, then
left to cool and filtered through a filter paper. Sample
D2: A 9 g sample of dried aerial parts of A. annua
was extracted with 1 L of boiling water, kept boiling
for 5 min and immediately filtered through a filter
paper. For analytical purposes, both D1 and D2
(filtrates) were freeze-dried and directly analysed.
Preparation of the infusions: Sample I1: A 9 g
sample of dried A. annua was extracted with 1 L of
Calibration curves: A calibration curve, obtained
from a methanolic solution of artemisinin (1 mg/mL),
was used to quantify artemisinin in the extracts and
tinctures, while a methanolic solution of artemisinin
(0.5 mg/mL) was employed to determine the
artemisinin content of infusions and decoctions.
1966 Natural Product Communications Vol. 3 (12) 2008
Sample analysis: Samples of 5 mg of the different
extracts were accurately weighed and suspended in
methanol (1.0 mL). The suspensions were sonicated
for 10 min and filtered through a cartridge-type
sample filtration unit before HPLC analysis. The
tinctures were injected as prepared.
In vitro assay for Trypanosoma brucei rhodesiense:
Minimum Essential Medium (50 μL) supplemented
according to [4a] with 2-mercaptoethanol and 15%
heat-inactivated horse serum was added to each well
of a 96-well microtiter plate. Serial drug dilutions
were prepared covering a range from 90 to 0.123
μg/mL. Then 104 bloodstream forms of Trypanosoma
b. rhodesiense STIB 900 in 50 μL were added to each
well and the plate incubated at 37°C under a 5% CO2
atmosphere for 72 h. After addition of 10 μL of
Alamar Blue™ to each well, the plates were
incubated for another 2-4 h and read in a Spectramax
Gemini XS microplate fluorometer using an
excitation wavelength of 536 nm and emission
wavelength of 588 nm. Fluorescence development
was expressed as percentage of the control, and IC50
values determined.
In vitro assay for Trypanosoma cruzi: Rat skeletal
myoblasts (L6 cells) were seeded in 96-well
microtiter plates at 2000 cells/well in 100 μL RPMI
1640 medium with 10% FBS and 2 mM L-glutamine.
After 24 h, the medium was removed and replaced by
100 μL per well containing 5000 trypomastigote
forms of T. cruzi Tulahuen strain C2C4 containing
the β-galactosidase (Lac Z) gene. After 48 h, the
medium was removed from the wells and replaced by
100 μL fresh medium with or without a serial drug
Bilia et al.
dilution. After 96 h of incubation, the substrate
CPRG/Nonidet (50 μL) was added to all wells. A
color reaction, developed within 2-6 h, was read
photometrically at 540 nm. Data were transferred into
a graphic program (MS Excel™); sigmoidal
inhibition curves were determined and IC50 values
calculated.
In vitro assay for Leishmania donovani:
Amastigotes of Leishmania donovani strain
MHOM/ET/67/L82 were grown in axenic culture at
37°C in SM medium [4b] at pH 5.4 supplemented
with 10% heat-inactivated fetal bovine serum under
an atmosphere of 5% C02 in air. Culture medium
(100 μL) with 1 x 105 amastigotes from axenic
culture with or without a serial drug dilution were
seeded in 96-well microtiter plates. Seven 3-fold
dilutions were used covering a range from 30 to
0.041 μg/mL. After 72 h of incubation, 10 μL of
Alamar Blue™ was added to each well. The plates
were incubated for another 2 h and read with a
Spectramax Gemini XS microplate fluorometer
(Molecular Devices Cooperation, Sunnyvale, CA,
USA) using an excitation wavelength of 536 nm and
an emission wavelength of 588 nm. Data were
analyzed using the software Softmax Pro (Molecular
Devices Cooperation, Sunnyvale, CA, USA).
Decrease of fluorescence (= inhibition) was
expressed as percentage of the fluorescence of
control cultures and plotted against the drug
concentrations. From the sigmoidal inhibition curves
the IC50 values were calculated.
Acknowledgments – Authors thanks Prof. P.M.
Magalhães of the Universidade Estadual de
Campinas (Brazil) for providing the plant material.
References
[1]
[2]
[3]
[4]
(a) van Agtmael MA, Eggelte TA, van Boxtel CJ. (1999) Artemisinin drugs in the treatment of malaria: from medicinal herb to registered
medication. Trends in Pharmacological Sciences, 20, 199-205.; (b) Stöger EA. (1991) Arzneibuch der Chinesischen Medizin. Deutscher
Apotheker-Verlag, Stuttgart, 37-45; (c) Räth K, Taxis K, Walz G, Gleiter CH, Li SM, Heide L. (2004) Pharmacokinetic study of
artemisinin after oral intake of a traditional preparation of Artemisia annua L. (annual wormwood). American Journal of Tropical
Medicine and Hygiene, 70, 128-32; (d) The World Health Report (2004) Changing History (World Health Organisation, Geneva). Fact
Sheet Number 116; World Health Organization: Geneva, Switzerland.
World Health Organization 2003. Chagas’ disease, http://www.who.int/tdr/ dw/chagas2003.htm
(a) Berhe N, Wolday D, Hailu A, Abraham Y, Ali A, Gebre-Michael T, Desjeux P, Sonnerborg A, Akuffo H, Britton S. (1999) HIV viral
load and response to antileishmanial chemotherapy in co-infected patients. AIDS, 13, 1921-1925; (b) Mishina YV, Krishna S, Haynes RK,
Meade J.C. (2007) Artemisinins inhibit Trypanosoma cruzi and Trypanosoma brucei rhodesiense in vitro growth. Antimicrobial Agents
and Chemotherapy, 51, 1852-1854; (c) Sen R, Bandyopadhyay S, Dutta A, Mandal G, Ganguly S, Saha P, Chatterjee M. (2007)
Artemisinin triggers induction of cell-cycle arrest and apoptosis in Leishmania donovani promastigotes. Journal of Medical Microbiology,
56, 1213-1218; (d) Bilia AR, Gabriele C, Bergonzi MC, Melillo de Malgalhaes P, Vincieri FF. (2006) Variation in artemisinin and
flavonoid content in differerent extracts of Artemisia annua. Natural Product Communications, 1, 1111-1115; (e) Bilia AR, Melillo de
Malgalhaes P, Bergonzi MC, Vincieri FF. (2006) Simultaneous analysis of artemisinin and flavonoids of several extracts of Artemisia
annua L. obtained from a commercial sample and a selected cultivar. Phytomedicine, 13, 487-493; (f) Magalhães PM, Delabays N,
Sartoratto A. (1997) Ciência e Cultura. Journal of Brazilian Association for the Advancement of Science, 49, 413-415.
(a) Baltz T, Baltz D, Giroud C, Crockett J. (1985) Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei,
T. equiperdum, T. evansi, T. rhodesiense and T. gambiense. EMBO Journal, 4, 1273-1277; (b) Cunningham I. (1977) New culture medium
for maintenance of tsetse tissues and growth of trypanosomatids. Journal of Protozoology, 24, 325-329.
NPC
2008
Vol. 3
No. 12
1967 - 1970
Natural Product Communications
Secondary Metabolites from the Roots of Salvia palaestina
Bentham
Antonio Vassalloa, Ammar Baderb, Alessandra Bracac, Angela Bisiod, Luca Rastrellia,
Francesco De Simonea and Nunziatina De Tommasia,*
a
Dipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo,
84084 Fisciano (SA), Italy
b
Faculty of Pharmacy, Al-Zaytoonah Private University of Jordan, P.O. Box 130, 11733 Amman, Jordan
c
Dipartimento di Chimica Bioorganica e Biofarmacia, Università di Pisa, Via Bonanno 33, 56126 Pisa, Italy
d
Dipartimento di Chimica e Tecnologie Farmaceutiche ed Alimentari, Università di Genova,
Via Brigata Salerno 13, 16147 Genova, Italy
detommasi@unisa.it
Received: July 25th, 2008; Accepted: November 3rd, 2008
Two new sesquiterpenes (1-2), and one diterpene (3) were isolated from the roots of Salvia palaestina Bentham (Lamiaceae),
together with eight known diterpenes and two triterpenes. Their structures were elucidated by 1D and 2D NMR spectroscopy,
including 1D-TOCSY, DQF-COSY, ROESY, HSQC, and HMBC experiments, as well as ESIMS and chemical analysis.
Keywords: Salvia palaestina, Lamiaceae, sesquiterpenes, diterpenes, NMR.
The genus Salvia L. belongs to the Lamiaceae family
and is represented by numerous species, widely
distributed in various regions of the world, used for
many medicinal and pharmaceutical applications,
including as a rich source of essential oils [1a,1b]. S.
palaestina Bentham, known also as Palestine sage, is
a perennial herb, 30-70 cm long, lemon scented
during the flowering season, distinguishable by the
basal leaves, which are pinnatilobed or pinnatisect,
oblong or lanceolate; the upper lip of the corolla is
large, exceeding 10 mm and the corolla has pure
white or rarely purplish colour, while the floral leaves
are membranous, with white or pink colour [1c]. The
plant is widespread in the Eastern Mediterranean area
and in the Western Irano-Turanian regions. In
Turkish folk medicine a preparation made from an
extract of the leaves is commonly used as a wound
healer [2a]. Previous phytochemical studies on the
aerial parts of the plant reported the presence of
modified abietane diterpenoids, sesquiterpenes,
diterpenes, sesterterpenes, triterpenes, and flavonoids
[2a-2d].
As part of a continuing investigation of Salvia
species [3a-3d], we made a phytochemical study of
S. palaestina roots collected in Jordan and herein we
14
H
2
10
H
9
1
3
5
4
8
6
H
H3COCO
O
7
12
H
O
11
15
13
1
2
OH
O
HOOC
3
Figure 1: Structures of compounds 1-3
report the structural characterization of three new
terpenoids (1-3, Figure 1) from the apolar extract of
the title plant, on the basis of extensive spectroscopic
and spectrometric analysis, including 2D NMR and
ESIMS spectra. Eight known diterpenes and two
triterpenes were also isolated and characterized as
ferruginol [4a], 6β-hydroxyferruginol [4b], pisiferic
acid [4c], dehydroabietane-11,12-diol [4d], carnosic
acid
[4e],
12-O-demethylcryptojaponol
[4f],
12-deoxy-6-hydroxy-6,7-dehydroroyleanone
[4g],
aethiopinone
1968 Natural Product Communications Vol. 3 (12) 2008
Table 1: 1H NMR spectroscopic data of compounds 1 and 2 (CD3OD,
600 MHz)a.
position
1
2a
2b
3a
3b
5
6
7
8a
8b
9a
9b
12
13
14a
14b
15
COCH3
3’
4’
5’
Vassallo et al.
Table 2: 13C NMR spectroscopic data of compounds 1 and 2 (CD3OD,
150 MHz)a.
compounds
1
2
δH
δH
2.22 ddd (10.0, 9.6, 8.8)
2.25 ddd (10.0, 9.6, 8.8)
1.96 m
1.98 m
1.58b
1.58b
1.78 ddd (10.0, 3.5, 2.0)
1.80 ddd (10.0, 3.5, 2.0)
1.58b
1.61b
1.41 dd (11.5, 9.5)
1.44 dd (11.3, 9.5)
0.53 dd (11.5, 9.5)
0.52 dd (11.3, 9.5)
0.72 ddd (11.5, 9.5, 6.0)
0.70 ddd (12.0, 9.5, 6.0)
2.00 m
2.00 m
1.06 m
1.08 m
2.43 ddd (14.0, 7.2, 1.0)
2.40 ddd (14.0, 7.0, 1.0)
2.07 m
2.07 m
1.04 s
1.04 s
1.04 s
1.04 s
4.70 br s
4.70 br s
4.66 br s
4.66 br s
1.25 s
1.27 s
1.99 s
6.10 br q (6.5)
2.00 d (6.5)
1.87 s
δ values were established from the 1D-TOCSY, DQF-COSY, HSQC
and HMBC experiments.b overlapped signals.
position
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
COCH3
COCH3
1’
2’
3’
4’
5’
a
[4h], lupeol [2c], and ursolic acid [2c], respectively,
by comparison of their spectroscopic data with those
reported in the literature.
Compound 1 was isolated as an amorphous powder
and yielded a pseudo-molecular ion in the positive
HR ESIMS at m/z 263.1912. The NMR (Tables 1 and
2) and mass spectral data of 1 indicated it to have the
molecular formula C17H26O2, thus having five
elements of unsaturation. Analysis of its 13C NMR
spectrum showed the presence of one acetyl group
(carbonyl signal at δC 170.0 and methyl signal at δC
21.0 and δH 1.99) and one 1,1-substituted C-C double
bond (δC 153.5 and 105.0) as the only multiple bonds
within the molecule, permitting the recognition of 1
as a tricyclic sesquiterpene. Identifiable from 13C
NMR spectroscopic data was also a resonance
consistent with one carbinolic carbon (δC 84.0). The
1
H NMR spectrum of 1 exhibited two methyl singlets
and one double intensity singlet at δ 1.04, 1.04, 1.25,
and 1.99 and a cyclopropyl moiety [δ 0.53 (1H, dd,
J = 11.5 and 9.5 Hz) and δ 0.72 (1H, ddd, J = 11.5,
9.5, and 6.0 Hz)]. Analysis of 1D-TOCSY and
DQF-COSY experiments allowed the sequential
assignments of hydrogens from H-1 to H-3 and from
H-5 to H-9. HSQC and HMBC experiments provided
unambiguous assignments of all the proton and
carbon resonances. This information, together with
the HMBC spectral data, finally led to the assignment
of an aromadendrane-like skeleton for 1 [5a]. HMBC
compounds
1
δC
54.0
26.2
41.4
84.0
53.8
30.0
27.1
24.4
39.8
153.5
20.4
28.0
28.0
105.0
23.6
170.0
21.0
2
δC
54.2
26.3
41.5
82.0
54.0
31.1
27.0
24.5
39.8
153.0
20.5
28.2
28.2
105.5
23.8
169.0
127.7
139.0
15.6
20.6
δ values were established from the 1D-TOCSY,DQF-COSY, HSQC
and HMBC experiments.
a
correlations
between
H-3a⎯C-5,
H-5⎯C-4,
Me-15⎯C-3, H-6⎯C-4 Me-15⎯C-5 substantiated
the presence of an acetoxy group at C-4. The relative
stereochemistry of 1 was ascertained by ROESY
experiments. By irradiation of the signal for H-1
[δ 2.22 (1H, ddd, J = 10.0, 9.6, 8.8 Hz)], a ROE
effect with the signal of H-6 was observed, indicating
them to be on the same side of the molecule and on
the opposite side to H-5. Further ROEs were detected
between H-5 and Me-13, H-6 and H-7, and H-7 and
Me-12. Therefore, compound 1 was determined as
4-O-acetylspathulenol.
The HR ESIMS of 2 gave a pseudo molecular ion at
m/z 303.2285 (C20H30O2). The NMR data of 2 (Tables
1 and 2) were very similar to those of 1, suggesting
the same skeleton. The NMR spectra revealed
the absence of the acetyl group in 2 replaced by a
2-methyl-2-butenoyl moiety [δ 6.10 (1H, br q, J = 6.5
Hz), δ 2.00 (3H, d, J = 6.5 Hz); δ 1.87 (3H, s)].
The stereochemistry of 2 was supported by
NMR experiments and deduced to be the same
as compound 1. Consequently, the new compound
2
was
characterized
as
4-O-(2-methyl-2butenoyl)spathulenol.
The molecular formula of 3 was determined as
C20H34O4 by the HR ESIMS ion at m/z 339.2438
[M+H]+, as well as from its 13C and 13C DEPT NMR
spectroscopic data.
Terpenoids from Salvia palaestina
Natural Product Communications Vol. 3 (12) 2008 1969
Table 3: 1H (600 MHz) and 13C NMR (150 MHz)spectroscopic data of
compound 3 (CD3OD)a.
position
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
3
δH
1.69b
0.98
1.66b
1.50b
1.80b
1.50b
1.84 dd (11.0, 4.2)
1.67b
1.70 ddd (12.0, 10.0, 4.0)
1.46b
1.20 dd (12.0, 3.2)
1.44b
1.32b
1.90b
1.52b
1.68b
1.52b
3.60 d (4.0)
3.54 d (4.0)
1.26 s
1.31 s
1.15 s
0.79 s
δC
37.8
18.6
38.2
48.2
52.4
20.8
42.7
75.0
56.9
37.0
21.8
29.2
75.0
44.7
62.0
26.4
25.4
185.0
16.0
15.0
δ values were established from 1D-TOCSY, DQF-COSY, HSQC and
HMBC experiments. b overlapped signals.
a
Comparison of NMR data (Table 3) of 3 with those
of 13-epi-manoyloxide-18-oic acid indicated that 3 is
an epi-manoyl oxide derivative [5b]. In particular,
protons and carbons due to rings A-C resonated at
almost the same frequencies as the corresponding
signals in 13-epi-manoyloxide-18-oic acid, while the
side chain NMR signals were the point of difference.
Particularly, NMR spectral data revealed the absence
of the signals for the –CH=CH2 group in 3 in respect
to those of 13-epi-manoyloxide-18-oic acid and
the presence of signals for a –CH2CH2OH group.
These data, together with mass spectral analysis
were consistent with 3 being 15-hydroxy-8,13epoxylabdan-18-oic acid.
A literature survey indicates that the aerial parts of
Salvia species contain flavonoids, triterpenoids, and
monoterpenes, particularly in the flowers and leaves,
while diterpenoids are found mostly in the roots.
Some American Salvia contained also diterpenoids in
the aerial parts, and in a few Salvia species,
triterpenoids and flavones are present in the roots
[5c]. The present investigation of S. palaestina roots
is completely in agreement with our results obtained
from the aerial parts of the plant [2d]. Sesquiterpenes
with an aromadendrane-like skeleton, diterpenes,
sesterterpenes, and triterpenes are characteristic
constituents of the plant; also hypogeal organs lacked
sesterterpenes.
Experimental
General experimental procedures: Optical rotations
were measured on a Perkin-Elmer 241 polarimeter
equipped with a sodium lamp (589 nm) and a 1 dm
microcell. All the NMR spectra were acquired in
CD3OD in the phase-sensitive mode with the
transmitter set at the solvent resonance and TPPI
(Time Proportional Phase Increment) used to achieve
frequency discrimination in the ω1 dimension. The
standard pulse sequence and phase cycling were used
for all 2D experiments. The NMR data were
processed on a Silicon Graphic Indigo2 Workstation
using UXNMR software. Column chromatography
was performed on silica gel (63-200 μm, Merck,
Darmstadt, Germany); high resolution mass spectra
were acquired on a Q-Tof Premier instrument
(Waters, Milford, MA), equipped with a nanospray
ion source; to achieve high accuracy mass
measurements, both external and internal calibrations
of the spectrometer were performed using quercetin
(molecular mass 302.0427) or amentoflavone
(molecular mass 538.0900) as standards. HPLC
separations were conducted on a Waters 590 system
equipped with a Waters R401 refractive index
detector, and with a Waters μ-Bondapak C18 column
(Waters, Milford, MA). TLC was performed on
precoated Kieselgel 60 F254 plates (Merck,
Darmstadt, Germany); compounds were detected
with Ce(SO4)2/H2SO4 solution.
Plant material: The whole plant of Salvia palaestina
Bentham was collected in April 2005 in Amman,
Jordan, and identified by Dr Ammar Bader. A
voucher specimen, number Jo-It-2003/2, has been
deposited in the Herbarium of the Laboratory of
Pharmacognosy and Phytochemistry at Al-Zaytoonah
Private University of Jordan. The roots were
separated from the aerial parts; the latter were
investigated previously [3a].
Extraction and isolation: The dried roots of S.
palaestina (290 g) were finely powdered and
exhaustively extracted by maceration at room
temperature with acetone to give 3.0 g of residue.
This was subjected to column chromatography using
silica gel and eluting with n-hexane followed by
increasing concentrations of CHCl3 (between 50%
and 100%), and by increasing concentrations of
MeOH in CHCl3 (between 1% and 100%). Fractions
1970 Natural Product Communications Vol. 3 (12) 2008
of 40 mL were collected, analysed by TLC (silica gel
plates, in CHCl3-n-hexane 1:1, CHCl3 or mixtures
CHCl3-MeOH 99:1, 98:2, 97:3, 9:1, 4:1), and
grouped into 13 fractions. Fraction 2 contained
pure aethiopinone (30 mg). Fraction 3 (76 mg)
was rechromatographed over silica gel eluting with
n-hexane followed by increasing concentration of
CHCl3 (between 1% and 100%) to obtain pure
pisiferic acid (7.5 mg) and 12-deoxy-6-hydroxy-6,7dehydroroyleanone (20 mg). Fraction 4 (573 mg) was
subjected to silica gel column chromatography,
eluting with n-hexane followed by increasing
concentration of CHCl3 (between 50% and 100%), to
give six major fractions (A-F). Fractions B (40 mg),
C (80 mg), and D (35 mg) were separately subjected
to RP-HPLC on a C18 μ-Bondapak column (30 cm x
7.8 mm, flow rate 2.0 mL min-1) with MeOH-H2O
(4:1) to yield pure 6β-hydroxyferruginol (10 mg, tR =
16 min) from fraction B, carnosic acid (2 mg, tR = 22
min), compound 1 (2 mg, tR = 24 min), and
compound 2 (5 mg, tR = 30 min), from fraction C,
and dehydroabietane-11,12-diol (4 mg, tR = 16 min),
carnosic acid (2.5 mg, tR = 22 min), and 12-Odemethylcryptojaponol (3 mg, tR = 23 min) from
fraction D, respectively. Fractions 7 (32 mg) and 9
Vassallo et al.
(52 mg) were separately chromatographed over RPHPLC on a C18 μ-Bondapak column (30 cm x 7.8
mm, flow rate 2.0 mL min-1) with MeOH-H2O
(68:32) to give pure compound 3 (6 mg, tR = 12 min)
and ferruginol (2 mg, tR = 14 min) from fraction 7,
and ursolic acid (4 mg, tR = 34 min) and lupeol (3
mg, tR = 38 min) from fraction 9, respectively.
Compound 1
[α]D: +23 (c 1.00, MeOH).
1
H and 13C NMR: Table 1 and Table 2.
HR ESIMS: m/z 263.1912 [M+H]+, calcd for
C17H26O2 262.1933.
Compound 2
[α]D: +27 (c 1.00, MeOH).
1
H and 13C NMR: Table 1 and Table 2.
HR ESIMS: m/z 303.2285 [M+H]+ calcd for
C20H30O2 302.2246.
Compound 3
[α]D: +25 (c 1.00, MeOH).
1
H NMR and 13C NMR: Table 3.
HR ESIMS: m/z 339.2438 [M+H]+ calcd for
C20H34O4 338.2457.
References
[1]
[2]
[3]
[4]
[5]
(a) Foster S, Tyler VE. (2000) Tyler’s Honest Herbal. The Haworth Press, Binghamton, New York, 327-330; (b) Steinegger E,
Hansel R. (1988) Lehrbuch der Pharmakognosie. Springer Verlag: Berlin; (c) Al-Eisawi D. (1998) Field Guide to Wild Flowers of
Jordan and neighbouring countries. Jordan Press Foundation Al-Rai, Amman, 175.
(a) Miski M, Ulubelen A, Johansson C, Mabry TJ. (1983) Antibacterial activity studies of flavonoids Salvia palaestina. Journal of
Natural Products, 46, 874-875; (b) Ulubelen A, Miski M, Johansson C, Lee E, Mabry TJ, Matlin SA. (1985) Terpenoids from
Salvia palaestina. Phytochemistry, 24, 1386-1387; (c) Hussein AA, de La Torre MC, Rodriguez B, Hammouda FM, Hussiney HA.
(1997) Modified abietane diterpenoids and a methoxylupane derivative from Salvia palaestina. Phytochemistry, 45, 1663-1668; (d)
Hussein AA, Rodriguez B. (2000) A diterpenoid with a new modified abietane skeleton from Salvia palaestina. Zeitschrift fuer
Naturforschung, B: Chemical Sciences, 55, 233-234.
(a) Cioffi G, Bader A, Malafronte A, Dal Piaz F, De Tommasi N. (2008) Secondary metabolites from the aerial parts of Salvia
palaestina. Phytochemistry, 69, 1005-1012; (b) Malafronte A, Dal Piaz F, Cioffi G, Braca A, Leone A, De Tommasi N. (2008)
Secondary metabolites from the aerial parts of Salvia aethiopis L. Natural Product Communications 3, 877-880; (c) De Felice A,
Bader A, Leone A, Sosa S, Della Loggia R, Tubaro A, De Tommasi N. (2006) New polyhydroxylated triterpenes and antiinflammatory activity of Salvia hierosolymitana. Planta Medica, 72, 643-649; (d) Bisio A, De Tommasi N, Romussi G. (2004)
Diterpenoids from Salvia wagneriana. Planta Medica, 70, 452-457.
(a) Connolly JD, Hill RA. (1991) Dictionary of Terpenoids, vol. 2 Di- and higher terpenoids. Chapman & Hall: London; (b) Kuo
YH, Yu MT. (1996) Diterpenes from the heartwood of Juniperus formosana Hay. var. concolor Hay. Chemical & Pharmaceutical
Bulletin, 44, 1431-1435; (c) Lin TC; Fang JM, Cheng YS. (1999) Terpenes and lignans from leaves of Chamaecyparis formosensis.
Phytochemistry, 51, 793-801; (d) Sanchez AJ, Konopelski JP. (1994) The first synthesis of (±)-taxodone. Synlett 5, 335-336 (e)
Bruno M, Savona G, Piozzi F, De la Torre MC, Rodriguez B, Marlier M. (1991) Abietane diterpenoids from Lepechinia meyeni and
Lepechinia hastata. Phytochemistry, 30, 2339-2343; (f) Rodriguez B. (2003) 1H and 13C NMR spectral assignments of some natural
abietane diterpenoids. Magnetic Resonance in Chemistry, 41, 741-746; (g) Nagy G, Gunther G, Mathe I, Blunden G, Yang MH,
Crabb TA. (1999) 12-Deoxy-6,7-dehydroroyleanone, 12-deoxy-6-hydroxy-6,7-dehydroroyleanone and 12-deoxy-7,7-dimethoxy-6ketoroyleanone from Salvia nutans roots. Phytochemitsry, 51, 809-812; (h) Lin LZ, Blasko G, Cordell GA (1989) Diterpenes of
Salvia prionitis. Phytochemistry, 28, 177-181.
(a) Inagaki F, Abe A. (1985) Analysis of proton and carbon-13 nuclear magnetic resonance spectra of spathulenol by twodimensional methods. Journal of the Chemical Society, Perkin Transactions 2: Physical Organic Chemistry, 1773-1778; (b)
Zetadero C, Bohlmann F, King RM. (1992) Ent-labdanes manoyloxide and elipterol derivatives from Chrysocephalum ambiguum.
Phytochemistry, 31, 1631-1638; (c) Bozan B, Ozturk N, Kosar M, Tunalier Z, Baser KHC. (2002) Antioxidant and free radical
scavenging activities of eight Salvia species. Chemistry of Natural Compounds (Translation of Khimiya Prirodnykh Soedinenii), 38,
198-200.
NPC
Natural Product Communications
Cancer Chemopreventive Potential of Humulones and
Isohumulones (Hops α- and Iso-α-acids): Induction of
NAD(P)H:Quinone Reductase as a Novel Mechanism
2008
Vol. 3
No. 12
1971 - 1976
Gregor Bohra, Karin Klimob, Josef Zappa, Hans Beckera and Clarissa Gerhäuserb,*
a
Fr. 8.2 Pharmakognosie und Analytische Phytochemie der Universität des Saarlandes, Saarbrücken,
Germany
b
Toxikologie und Krebsrisikofaktoren, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg,
Germany
c.gerhauser@dkfz.de
Received: June 10th, 2008; Accepted: August 31st, 2008
Phytochemical analysis and chemopreventive testing of a special “α-/β-acid free” hops extract led to the identification of
isohumulones (hops iso-α-acids) as potent inducers of NAD(P)H:quinone reductase (QR) activity. CD values (concentrations
required to double the specific activity of QR in Hepa1c1c7 cell culture) were in the range of 1.3 to 10.2 µg/mL, with CD value
of trans-isohumulone < cis-isoadhumulone < cis-isocohumulone < cis-isohumulone (+ trans-isoadhumulone). Humulones
(hops α-acids) were equally active with CD values of 3.4 to 7.6 µg/mL. However, these activities were accompanied by
cytotoxicity. Cohumulinone and humulinone, oxidation products of co- and n-humulone, were inactive. We further identified
isohumulones as potent inhibitors of lipopolysaccharide-induced inducible nitric oxide synthase (iNOS) activity in Raw264.7
cell culture, with IC50 values of 5.9 – 18.4 µg/mL. Humulones and humulinones were inactive at concentrations < 20 µg/mL.
These results indicate that isohumulones, which are considered as the most abundant class of polyphenols in beer, should by
further investigated for chemopreventive efficacy in animal models.
Keywords: hops, Humulus lupulus L., cancer chemoprevention, NAD(P)H:quinone reductase, hops α-acids, hops iso-α-acids,
humulone, isohumulone, humulinone.
Hops (Humulus lupulus L.) have been used since
ancient times for brewing [1]. It was soon realized
that they not only added bitterness and aroma to beer,
but also played an important role as a preservative.
Subsequently, hops α- and β-acids (humulones and
lupulones), constituents of the essential bitter resin,
were identified as strong antibiotics against Grampositive bacteria ([2] and literature cited therein).
β-Acids are extremely sensitive to oxidation and do
not survive the brewing process. During wort-boiling,
the poorly water-soluble humulones are isomerized to
isohumulones (iso-α-acids), which are better soluble;
this process is involved in the generation of the bitter
flavor of beer [3a]. Isohumulones also play an
important role in foam stabilization [3b]. Overall,
they represent one of the most abundant classes of
polyphenols in beer; concentrations of up to 100
mg/L have been reported in very bitter English ales
[3c].
Isohumulones are optically active molecules which
occur as cis- and trans-isomers. In analogy to the
chemical structures of humulones, three isoforms
indicated by the prefix “co-“, “-n-“ and “ad-“ are
present in beer, which differ only in their acyl side
chain (summary in Figure 1). Interestingly, Hughes
reported that cis-isohumulones were more bitter than
their trans-isomers. In particular, bitterness of the
compounds was described as cis-isohumulone >
trans-isohumulone ≈ cis-isocohumolone > transisocohumulone [3b].
In recent years, hops have gained considerable
interest because of the biological and potential cancer
chemopreventive activities of some of their
constituents (reviewed in [4a-c]). As an example, the
α-acid n-humulone was described as a potent
antioxidant and anti-inflammatory agent capable of
inhibiting the induction of cyclooxygenase-2 (Cox-2)
in cell culture and mouse skin [5], and displayed
1972 Natural Product Communications Vol. 3 (12) 2008
O
OH
R=
Co-
R
O
HO
R=
O
n-
HO
O
R=
Humulones (α-acids)
Ad-
R= Acyl-side chain of humulones
and isohumulones
O
H
H
O
R
HO
OH
O
cis-Isohumulones
(cis-iso-α-acids)
OH
HO
O
O
O
OH
Cohumulinone
R
HO
OH
O
trans-Isohumulones
(trans-iso-α-acids)
OH
HO
O
O
O
OH
n-Humulinone
Figure 1: Chemical structures of humulones, cis- and transisohumulones, co- and n-humulinone.
anti-proliferative activity by induction of cell
differentiation and apoptosis in HL-60 human
promyelocytic leukemia cells in vitro. It also
inhibited angiogenesis in the chick embryo
chorioallantoic membrane (CAM) assay, with a halfmaximal
effective
concentration
ED50
of
1.5 µg/CAM. Topical application of n-humulone
(1 mg) very potently suppressed tumor incidence
induced by 12-O-tetradecanoylphorbol-13-acetate
(TPA) in the two-stage mouse skin model by 93%
and tumor multiplicity by 99%. In addition to these
cancer preventive effects, humulone was described as
a very potent inhibitor of bone resorption and a
candidate therapeutic agent for osteoporosis, with a
half-maximal inhibitory concentration of 5.9 nM
(2.1 ng/mL) in an in vitro model of pit formation.
Cohumulone was inactive at a concentration of 1 µM
(reviewed in [4c]).
Little information is available regarding potential
cancer chemopreventive activities of isohumulones.
Nozawa et al. demonstrated that freeze-dried beer at
a dose of 1%, and isomerized hops extract (IHE) at
0.01 or 0.05% in the diet significantly reduced
azoxymethane-induced
preneoplastic
precursor
lesions in rat colon. IHE also potently reduced levels
of prostaglandin E2 (PGE2) in colonic mucosa,
Bohr et al.
indicating anti-inflammatory potential by inhibition
of Cox-2 expression [6a]. Several reports have
suggested that isohumulones may have beneficial
effects for the treatment of diabetic symptoms by
inhibition of aldose reductase and reduction of insulin
resistance, hyperlipidemia and obesity by activation
of peroxisome proliferator-activated receptor (PPAR)
α and γ [6b-f]. They were also shown recently to
reduce renal injury in salt-sensitive rats by
antioxidant activity [6g].
In continuation of our studies on hops
prenylflavonoids
[7]
and
acylphloroglucinol
derivatives [8], we here describe results of the
phytochemical analysis and chemopreventive testing
of a special hops extract which led to the separation
of four isohumulones and humulinone, an oxidation
product of n-humulone. The chemopreventive
potential of these compounds was compared with
that of the α-acids cohumulone, n-humulone and
adhumulone.
For the isolation of isohumulones we fractionated a
commercially available “α-/β-acid free” ethanolic
hops extract [9] by size exclusion column
chromatography into 18 fractions. Bitter-tasting
fraction X08 was separated by semi-preparative
HPLC to yield five subfractions X08.A to X08.E.
Comparison of NMR and ESI mass spectra
with those published [10a-f] led to the following
peak assignment: Peak 1 was identified as
“n-humulinone”, peak 2 as “cis-isocohumulone”,
peak 3 as “trans-isohumulone”, peak 4 as
“cis-isohumulone” (maybe plus “trans-isoadhumulone), and peak 5 as “cis-isoadhumulone”. For
comparison of potential cancer chemopreventive
activities, co-, n-, and adhumulone were isolated
from a hops CO2-extract by counter-current
chromatography (modified from [11]). Co- and
n-humulinone were synthesized starting from co- and
n-humulone according to [10b]. Identities were
confirmed by comparison with published spectral
data (see Experimental).
Cancer chemoprevention has been defined as the use
of chemical agents, natural products or dietary
components to block, inhibit, or reverse the
development of cancer in normal tissue and
preneoplastic lesions [12]. Carcinogenesis is a
multi-stage process, which is generally divided into
initiation, promotion and progression phases and
could be regarded as a continuous accumulation of
biochemical and genetic cell damage. The cascade of
QR induction by humulones and isohumulones
Natural Product Communications Vol. 3 (12) 2008 1973
Table 1: Summary of potential chemopreventive activities.
Fractions/
Compds
X08
X08.A
X08.B
X08.C
X08.D
X08.E
n-Humulinone
Cohumulinone
n-Humulone
Cohumulone
Adhumulone
DPPHa
SC50
>200
132.3
75.2
112.5
74.9
95.5
>200
>200
5.0
7.2
11.9
QR
CD
2.6
5.0
7.0
1.3
10.2
5.6
>20
>20
3.4
6.7
7.6
IC50
>20
>20
>20
>20
>20
>20
>20
>20
4.0
9.4
11.5
iNOS
IC50
>20
18.1
18.4
5.9
>20
9.8
>20
>20
>20
>20
>20
a
Test systems: DPPH: DPPH scavenging (SC50 in µg/mL); QR: QR
induction (CD, concentration required to double the specific activity of
QR in µg/mL, IC50 for toxicity in µg/mL); iNOS: iNOS inhibition (IC50 in
µg/mL).
events resulting in tumor formation offers a variety of
targets for intervention at every stage. Accordingly,
fraction X08, its five subfractions X08.A – X08.E
containing isohumulones, as well as the purified
humulones and humulinones were tested in a series
of test systems indicative of cancer chemopreventive
potential.
Manifestation of oxidative stress by infections,
immune diseases and chronic inflammation has been
associated with carcinogenesis in the initiation and
promotion phase. Antioxidants may prevent the
formation of highly reactive oxidation products,
activation of carcinogens, formation of oxidized
DNA bases and DNA strand breaks, which have been
associated with overproduction of reactive oxygen
species (ROS) and are involved in the carcinogenic
process [13]. We determined radical scavenging
potential by reaction with 1,1-diphenyl-2picrylhydrazyl (DPPH) free radicals. Consistent with
an earlier report [14], the α-acids n-, co- and adhumulone were identified as potent radical
scavengers
with
half-maximal
scavenging
concentrations (SC50) of 5.0 µg/mL (13.7 µM),
7.2 µg/mL (20.6 µM) and 11.9 µg/mL (32.9 µM),
respectively, as indicated in Table 1. These activities
were attributed to the presence of a hydroxyl-group
in position C-5 [14] and were only slightly lower than
those of ascorbic acid (SC50: 8.5 µM) and the watersoluble Vit. E analog Trolox (SC50: 9.7 µM) [15]. In
contrast, fractions containing isohumulones were
about 10-fold less potent in scavenging DPPH
radicals than the humulones, with SC50 values in the
range of 75 – 132.3 µg/mL. Oxidation to n-humulinone and cohumulinone further reduced antioxidant
activities. Both compounds scavenged DPPH radicals
less than 50% at the maximum test concentration of
200 µg/mL.
Xenobiotics, including carcinogens, are metabolized
and generally detoxified during phase 1 and 2
metabolism. Phase 2 enzymes, such as glutathione Stransferases (GST), conjugate phase 1 metabolites with
endogenous ligands and thus enhance their excretion in
the form of these conjugates. NAD(P)H:quinone
reductase (QR) is not a conjugating enzyme. However,
it contributes to detoxification of reactive quinones by
2-electron reduction, thereby preventing the formation
of reactive semiquinones and ROS formation by redox
cycling [16]. QR activity is induced coordinately with
other phase 2 enzymes, making it a well established
marker for potential chemopreventive activity [15].
Using QR induction in murine Hepa1c1c7 cell culture
as a test system, we identified isohumulones as very
potent inducers of QR activity (Table 1). All fractions
dose-dependently induced QR activity in a
concentration range of 1.25 to 20 µg/mL (Figure 2).
Fraction X08.C containing trans-isohumulone was
identified as the most active fraction followed by
fractions X08.A and X08.E. Humulones also
demonstrated good QR-inducing potential with CD
values (concentration required to double QR activity)
in the range of 3.4 to 7.6 µg/mL. In contrast to
isohumulones, these compounds were toxic to the
utilized murine hepatoma cells with IC50 values of
4.0 to 11.5 µg/mL. The ratio between IC50 values and
CD values, previously defined as Chemopreventive
Index, was close to 1, indicating that these compounds
may stimulate their own detoxification [15]. Oxidation
of humulones to humulinones completely abrogated
QR-inducing potential, but also cytotoxicity. Induction
of QR activity by humulones and isohumulones may be
explained by activation of the transcription factor
Nrf2/Keap-1 pathway similar to other natural products
containing “Michael acceptor” functionality [17].
Chronic infections and inflammation lead to nuclear
factor κB (NF-κB)-dependent induction of proinflammatory enzymes, such as Cox-2 and inducible
nitric oxide synthase (iNOS). (Over)production of
NO has been linked to early steps in carcinogenesis
via nitrosative desamination of DNA bases,
accumulation of reactive nitrogen oxide species and
DNA adduct formation [18]. We and others have
shown previously that induction of QR activity is
often related to inhibition of iNOS induction [19a-c].
It was, therefore, of interest to analyze whether
humulones and isohumulones would inhibit iNOS
induction, using the murine macrophage cell line
Raw264.7 stimulated with bacterial lipopolysaccharides (LPS) as a model.
1974 Natural Product Communications Vol. 3 (12) 2008
Bohr et al.
QR induction (T/C)
6
4
1.25 µg/mL
2.5 µg/mL
5.0 µg/mL
10.0 µg/mL
20.0 µg/mL
2
0
X08
X08.A X08.B X08.C X08.D X08.E
n-
coadhumulone
Figure 2: Induction of NAD(P)H:quinone reductase (QR) activity in Hepa1c1c7 cell culture. QR induction was computed by comparison with a solvent treated
control (T: treated/ C: control).
In correlation with QR induction, fraction X08.C was
most potent in inhibiting LPS-induced iNOS activity
with an IC50 value of 5.9 µg/mL. These data were in
agreement with previous findings of Nozawa et al.,
who reported that isomerized hops extract and
isohumulone inhibited PGE2 production by Cox-2 in
LPS/interferon-γ-stimulated Raw264.7 macrophages
[6a]. Neither humulones nor humulinones inhibited
iNOS induction in our test system at concentrations
up to 20 µg/mL (Table 1). In contrast, TNF-αmediated Cox-2 expression was potently inhibited by
humulone in the murine osteoblastic MC3T3-E1 cell
line [20]. Mechanistic investigations indicated that
transcription factors NF-κB and NF-IL6 were targets
of humulone action. The observed discrepancy of
results obtained with humulone in the MC3T3-E1
and Raw264.7 cell lines may be due to differences in
the utilized inducers and variations in the signal
transduction machinery in both cell lines. Humulone
was also reported to inhibit Cox-2 enzymatic activity
with an IC50 of 1.6 µM [20]. We were not able to
reproduce this result at concentrations up to 5 µM
using human recombinant Cox-2 as an enzyme
source (data not shown, method as described in [7]).
In addition to these studies on humulone and
isohumulones, Zhao et al. have investigated the
potential of other hops constituents, including the
β-acid lupulone, xanthohumol, and a series of
derivatives of both compounds, to inhibit NO
production in the Raw macrophage system [21]. Only
chalcones such as xanthohumol were identified as
potent inhibitors in this study, whereas lupulone and
the β-acid derivatives were inactive.
In conclusion, the present report provides first
evidence that induction of phase 2 metabolizing
enzymes could contribute to humulone- and
isohumulone-mediated cancer chemoprevention. We
have identified humulones and isohumulones as
novel potent inducers of QR activity. Taking into
consideration the relatively high concentrations of
isohumulones in beer compared with other bioactive
hops components, such as xanthohumol [7], further
investigations on potential cancer chemopreventive
efficacy and their influence on phase 2 metabolizing
enzymes in animal models are warranted. A first
indication may be seen in the dose-dependent
reduction of carcinogen-induced mammary carcinogenesis by freeze-dried beer [22]. In that study,
Nozawa et al. also demonstrated that feeding rats
with freeze-dried beer (4% in the diet) increased
hepatic GST activity and reduced carcinogen-DNA
adducts in mammary tissue. So far, the beer
components responsible for these preventive effects
have not been analyzed.
Experimental
Plant material: An ethanolic hops extract, as well as
a CO2-hops extract, was produced, as described in
[23], from Humulus lupulus, var. Taurus
(Cannabinaceae) and supplied by Hallertauer
Hopfenveredlungsgesellschaft (HHV) mbH, Mainburg, Germany.
General experimental conditions: NMR spectra
were recorded on Bruker Avance 500 and Bruker
Avance DRX 500 spectrometers in CD3OD. Mass
spectra were measured on a Finnigan MAT 90 mass
spectrometer.
Extraction and fractionation: An ethanolic hops
extract was treated with supercritical carbon-dioxide
to remove hops α- and β- acids. This “α-/β-acid free”
fraction is a commercially available hops extract that
has recently been introduced into the brewing
industry for producing xanthohumol-enriched beers
[9]. From this extract, 20 g was separated by size
exclusion column chromatography using Sephadex
LH-20 (column: Ø 5.5 x 120 cm). A step gradient
from methanol/dichlormethane 50:50 (v/v), to 70:30
(v/v) to 90:10 (v/v) was performed to obtain the
following fractions X01 (5.80 g), X02 (2.75 g), X03
QR induction by humulones and isohumulones
Natural Product Communications Vol. 3 (12) 2008 1975
(2.64 g), X04 (1.41g), X05 (0.39g), X06 (0.67g), X07
(0.95 g), X08 (1.23 g), X09 (0.05 g), X10 (0.64 g),
X11 (0.38 g), X12 (0.47 g), X13 (0.18 g), X14 (0.21
g), X15 (0.16 g), X16 (0.06 g), X17 (0.03 g), and
X18 (0.01 g).
Synthesis of humulinones: n-Humulinone and
cohumulinone were synthesized by partial synthesis,
as described in [10b], starting from pure cohumulone
and n-humulone, respectively. Structure elucidation
was performed as described above. Spectra were in
agreement with published literature [10a,b,e].
An intense bitter taste indicated the presence of bitter
acids in fractions X07 and X08. Because of higher
yield, fraction X08 (330 mg) was further separated by
semi-preparative HPLC, which was performed on a
RP-18ec column (VP 250/4 Nucleosil 100-5
C18Hop, Macherey–Nagel, Düren, Germany) using
acetonitrile/water 56:44 (v/v) with 0.05% TFA. The
solvent delivery system was a Waters M-45 (Waters,
Milford USA). Peaks were detected with a RIDetector (RI-Detector 8110, Bischoff, Leonberg,
Germany) and, after 7 min, collected to yield 5.1 mg
of X08.A, 7.1 mg of X08.B, 5.0 mg of X08.C, 4.6 mg
of X08.D and 3.9 mg of X08.E. Structures were
determined by NMR spectroscopy (1H, 13C, HSQC,
HMBC) and ESI mass spectrometry in comparison
with literature data [10b-d,f].
Isolation of humulones as reference compounds:
Cohumulone, n-humulone and adhumulone were
isolated from a hops CO2-extract by a modified
counter-current separation, as described previously
[11]. Identity was confirmed by NMR and mass
spectrometry in comparison with literature data [10b,
24a,b].
Determination of potential cancer chemopreventive
activities: Experimental details of most test systems
utilized in this study are summarized in [7,15].
Briefly, radical scavenging potential was determined
photometrically by reaction with 1,1-diphenyl2-picrylhydrazyl (DPPH) free radicals in a microplate
format [7]. Induction of NAD(P)H:quinone reductase
(EC 1.6.99.2) activity in cultured Hepa1c1c7 cells
was assayed as described in [25], monitoring the
NADPH-dependent menadiol-mediated reduction of
MTT [3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide] to a blue formazan. Inhibition of
lipopolysaccharide-induced inducible nitric oxide
synthase (iNOS) activity (EC 1.14.13.39) in murine
Raw 264.7 macrophages was quantified via the
Griess reaction, as described previously [15,19b].
Acknowledgments - We thank Hallertauer
Hopfenveredelungs Gesellschaft mbH (HHV), Mainburg for providing the extracts and financial support
for this work. We thank Dr M. Biendl for fruitful
discussion and Dr S. Boettcher for LCMSmeasurements.
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skin by blocking activation of NF-κB and AP-1: I-κB kinase and c-Jun-N-terminal kinase as respective potential upstream targets.
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Shindo S, Tomatsu M, Nakda T, Shibamoto N, Tachibana T, Mori K. (2002) Inhibition of aldose reductase activity by extracts
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Tsuboyama-Kasaoka N, Ezaki O, Oikawa S, Kondo K. (2004) Isohumulones, bitter acids derived from hops, activate both
peroxisome proliferator-activated receptor α and γ and reduce insulin resistance. Journal of Biological Chemistry, 279, 3345633462; (d) Yajima H, Noguchi T, Ikeshima E, Shiraki M, Kanaya T, Tsuboyama-Kasaoka N, Ezaki O, Oikawa S, Kondo K. (2005)
Prevention of diet-induced obesity by dietary isomerized hop extract containing isohumulones, in rodents. International Journal of
Obesity (London), 29, 991-997; (e) Miura Y, Hosono M, Oyamada C, Odai H, Oikawa S, Kondo K. (2005) Dietary isohumulones,
the bitter components of beer, raise plasma HDL-cholesterol levels and reduce liver cholesterol and triacylglycerol contents similar
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Bohr et al.
to PPARalpha activations in C57BL/6 mice. British Journal of Nutrition, 93, 559-567; (f) Shimura M, Hasumi A, Minato T,
Hosono M, Miura Y, Mizutani S, Kondo K, Oikawa S, Yoshida A. (2005) Isohumulones modulate blood lipid status through the
activation of PPAR alpha. Biochimica et Biophysica Acta, 1736, 51-60; (g) Namikoshi T, Tomita N, Fujimoto S, Haruna Y, Ohzeki
M, Komai N, Sasaki T, Yoshida A, Kashihara N. (2007) Isohumulones derived from hops ameliorate renal injury via an antioxidative effect in Dahl salt-sensitive rats. Hypertension Research, 30, 175-184.
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[8]
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M, De Keukeleire D. (1991) Chemistry and analysis of hop and beer bitter acids. Elsevier Science Publishers B.V., Amsterdem,
New York, 1-417; (c) Nord LI, Sorensen SB, Duus JO. (2003) Characterization of reduced iso-α-acids derived from hops
(Humulus lupulus) by NMR. Magnetic Resonance in Chemistry, 41, 660-670; (d) Vanhoenacker G, De Keukeleire D, Sandra P.
(2004) Analysis of iso-α-acids and reduced iso-α-acids in beer by direct injection and liquid chromatography with ultraviolet
absorbance detection or with mass spectrometry. Journal of Chromatography A, 1035, 53-61; (e) Chadwick LR, Nikolic D,
Burdette JE, Overk CR, Bolton JL, van Breemen RB, Frohlich R, Fong HH, Farnsworth NR, Pauli GF. (2004) Estrogens and
congeners from spent hops (Humulus lupulus). Journal of Natural Products, 67, 2024-2032; (f) Khatib A, Wilson EG, Kim HK,
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NPC
2008
Vol. 3
No. 12
1977 - 1980
Natural Product Communications
A Polar Cannabinoid from Cannabis sativa var. Carma
Giovanni Appendinoa*, Anna Gianaa, Simon Gibbonsb, Massimo Maffeic, Giorgio Gnavic,
Gianpaolo Grassid and Olov Sternere*
a
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Via Bovio 6,
28100 Novara, Italy
b
Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London,
29-39 Brunswick Square, London WC1N 1AX, UK
c
Dipartimento di Biologia Vegetale e Centro di Eccellenza CEBIOVEM, Università di Torino,
Viale Mattioli 25, 10125 Torino, Italy
d
CRA-CIN Centro di Ricerca per le Colture Industriali, Sede Distaccata di Rovigo, Via Amendola 82,
45100 Rovigo, Italy
e
Department of Organic Chemistry, Lund University, P.O. Box 124, 221 00 Lund, Sweden
appendino@pharm.unipmn.it; Olov.Sterner@organic.lu.se
Received: July 29th, 2008; Accepted: October 15th, 2008
The aerial parts of Cannabis sativa var. Carma afforded a novel polar cannabinoid whose structure was established as
rac-6’,7’-dihydro,6’,7’-dihydroxycannabigerol (carmagerol, 1) on the basis of spectroscopic data and semisynthesis from
cannabigerol (2a). The dihydroxylation of the ω-double bond was detrimental to the anti-bacterial activity.
Keywords: Cannabis sativa, Cannabinaceae, carmagerol, cannabigerol, antibacterial activity.
The successful development of Sativex, a
combination of Cannabis extracts, for the
management of multiple sclerosis and cancer pain [1]
has rekindled interest in the phytochemistry of
Cannabis sativa L. Over 70 natural cannabinoids are
known [2], most of them characterized in the 1960s
and 70s in the wake of the identification of Δ9tetrahydrocannabinol (THC) as the psychotropic
constituent of marijuana [3]. Recently, a series of
very apolar terpenyl esters of pre-cannabinoids was
reported from a THC-rich chemotype of marijuana
[4], suggesting that the earlier investigations, focused
on a defined range of polarity, might have missed
minor compounds with higher or lower polarity than
the major cannabinoids. We report here the isolation
of the novel polar cannabinoid 1 from the Carma
variety of hemp. This hemp is named after the
Piedmontese town of Carmagnola, where the
cultivation of the celebrated pest- and stress resistant
homonymous fibre hemp thrived for centuries [5].
9'
HO
8'
10'
7'
3'
5'
6'
OH
4'
OH
1'
2'
2
1
HO
6
4
1
4''
2''
5
3
1''
3''
5''
OR
RO
R
2a H
2b Ac
The minor (0.0045% isolation yield) cannabinoid 1
was named carmagerol because of its plant origin and
its structrual relationship with cannabigerol (CBG,
2a). It was obtained from the polar fraction of an
acetone extract from the aerial parts of Carma hemp.
Its isolation involved filtration over RP-18 silica gel
to eliminate fats and pigments, partition between
aqueous methanol and light petroleum to remove
most of CBG (2a), its major cannabinoid, and gravity
1978 Natural Product Communications Vol. 3 (12) 2008
column chromatography on silica gel. The 1H NMR
spectrum of a fraction more polar than those
containing the prenylated flavonoids cannflavins [6]
showed a series of signals in the aliphatic region that
suggested the presence of a compound with a gross
cannabinoid structure. A pure sample was eventually
obtained after preparative HPLC on silica gel.
Carmagerol (1) was obtained as an optically inactive,
colorless gum, and had molecular formula C21H34O4
(MS). Its 1H NMR spectrum was similar to that of
CBG (2a) [7], except for the replacement of the
terminal olefinic double bond with two oxygenated
functions, as evidenced by the observation of an
oxymethine resonance at δ 3.35, and by the upfield
shift of the allylic geminal methyls, resonating as
sharp singlets at δ 1.19 and 1.15. While the chemical
shift of the oxymethine was compatible with the
epoxidation of the ω-double bond of the geranyl
moiety of CBG, the 13C chemical shift of the
corresponding carbon (δ 78.2) and of that of the
adjacent quaternary oxygenated carbon (δ 73.2)
showed that, in accordance with the molecular
formula, the terminal double bond of the geranyl
residue had undergone dihydroxylation and not
epoxidation.
To confirm the structure elucidation of carmagerol
and obtain further amounts of the compound
necessary to investigate its biological profile, a
semisynthesis from CBG was undertaken. Racemic
dihydroxylation with the Upjohn protocol gave a
complex mixture, but, after acetylation, the
asymmetric version of the reaction with AD-mix-α
[8] afforded, in excellent yield, a compound identical,
apart from the optical rotation, to the natural product.
A compound with the gross formula of carmagerol
was mentioned in a study on the mammal metabolism
of cannabigerol. The major metabolic pathway was
the hydroxylation of the allylic methyls, but
epoxidation of the ω-double bond was also observed,
without, however, detecting its hydrolysis product,
namely carmagerol [9]. The racemic nature of
carmagerol is puzzling, since biological oxidations
are generally enantioselective. On the other hand,
auto-oxidation of geranylated phenols shows a strong
bias toward the proximal, and not the terminal,
double bond [10], and carmagerol was clearly
detectable by HPLC in crude extracts of Carma
hemps (see Experimental), in accordance with its
natural origin.
Appendino et al.
While cannabigerol (2a) is a potent antibacterial
agent, especially against the so called super-bugs
(IC50 = 1 μg/mL against methicillin-resistant
Staphylococcus aureus SA1199B) [11], the activity
of carmagerol (1) was modest (IC50 = 32 μg/mL),
showing that dihydroxylation of the ω-double bond is
detrimental to antibiotic activity, an important
observation that points to the existence of strict
structure-activity
relationships
within
the
antimicrobial cannabinoid chemotype.
The characterization of a novel polar cannabinoid
from Cannabis sativa suggests that, despite studies
spanning almost 50 years and the identification of
over 500 different constituents [2], modern
phytochemical techniques can still lead to the
isolation of new minor compounds missed by earlier
studies and worth investigation in terms of
bioactivity.
Experimental
Plant material: Cannabis sativa var. Carma came
from greenhouse cultivation at CRA-CIN, Rovigo
(Italy), where a voucher specimen is kept, and was
collected in November 2006. The isolation and
manipulation of all cannabinoids was in accordance
with their legal status (Licence SP/101 of the
Ministero della Salute, Rome, Italy).
Isolation of 1: The powdered plant material (500 g)
was distributed as a thin layer on cardboard, and
heated at 120°C for 2 h in a ventilated oven to affect
decarboxylation, and then extracted with acetone
(ratio solvent: plant material 3:1, x 3). The residue
(20.5 g) was dissolved in methanol, adsorbed onto a
pad of RP-18 silica gel (100 g), and washed with
methanol (400 mL). The yellowish filtrate was
evaporated and then partitioned between 10% aq.
methanol (150 mL) and light petroleum (150 mL).
The lower phase was separated, washed again with
light petroleum (100 mL), and evaporated The
residue (5.1 g) was purified by gravity column
chromatograpy on silica gel (50 g), using a light
petroleum-EtOAc gradient. Fractions eluted with
light petroleum-EtOAc 4:6 were further purified by
prep HPLC on a silica gel column (250 x 21.2 mm
Chromasyl column) using light petroleum-EtOAc 3:7
as eluant to give 21 mg (0.0042%) 1a as a colorless
foam.
Cannabinoids from Cannabis sativa
Natural Product Communications Vol. 3 (12) 2008 1979
Carmagerol
nabigerol, 1)
petroleum-EtOAc 7:3, and filtered over silica gel (10
mL). The filtrate was evaporated, dissolved in THF
(10 mL), and treated with pyrrolidine (3 mL, 24
mmol, 12 mol. equiv.). After heating at 50°C for 16
h, the reaction was cooled to room temp. and worked
up by partition between 2N H2SO4 and EtOAc. The
organic phase was separated, washed with brine,
dried (Na2SO4), evaporated, and the residue purified
by gravity column chromatography on silica gel (10
g, light petroleum-EtOAc 5:5 as eluant) to afford 700
mg 1a (overall 63% from 2a) as a colorless foam.
[α]D = +51 (c 0.8, MeOH).
(6’,7’-Dihydro-6’-7’-dihydroxycan-
Colorless gum.
Rf : 0.35 (light petroleum-EtOAc 4:6).
IR (KBr): 3293, 3199, 1703, 1601, 1425, 1372, 1155,
1029, 833 cm-1.
1
H NMR (500 MHz, CDCl3): 0.89 (3H, t, J = 7.0 Hz,
H-5’’), 1.15 (3H, s, H-10’), 1.19 (3H, s, H-8’), 1.29
(2H, m, H-3’’a,b), 1.31 (2H, m, H-4’’a,b), 1.44
(1H, m, H-5’a), 1.56 (2H, m, H-2’’a,b), 1.60 (1H, m,
H-5’b), 1.82 (3H, s, H-9’), 2.13 (1H, m, H-4’a), 2.27
(1H, m, H-4’b), 2.44 (2H, t, J = 7.8 Hz, H-1’’a,b),
3.35 (1H, dd, J = 10.2, 2.1 Hz, H-6’), 3.38 (2H, t,
J = 7.2 Hz, H-1’), 5.32 (1H, br t, J = 7.2 Hz, H-2’),
6.24 (2H, s, H-4 and H-6).
13
C NMR (125 MHz, CDCl3): 14.0 (t, C-5’’), 16.1
(q, C-9’), 22.2 (t, C-1’), 22.5 (t, C-4’’), 23.3 (q,
C-10’), 26.3 (q, C-8’), 29.4 (t, C-5’), 30.8 (t, C-2’’),
31.4 (t, C-3’’), 35.5 (t, C-1’’), 36.9 (t, C-4’), 73.2
(s, C-7’), 78.2 (d, C-6’), 108.2 (d, C-4 and C-6),
110.9 (s, C-2), 122.8 (d, C-2’), 139.4 (s, C-3’), 146.2
(s, C-5), 154.8 (s, C-1 and C-3).
CI-EIMS: m/z [M]+ 373 (M + Na)+, (C21H34O4 + 23)+.
Synthesis of carmagerol (1) from cannabigerol (2a):
To a stirred soln of AD-mix-α (3.5 g) in tert-ButOHwater (1:1, 10 mL), N-methylmorpholine oxide (875
mg, 7.5 mmol, 3 mol. equiv.), methansulfonamide
(250 mg, 2.5 mmol, 1 mol. equiv.), and a soln of
cannabigerol diacetate (2b, 1.0 g, 2.5 mmol; obtained
from the treatment of 2a (1 g) with excess Ac2O (10
mL) in pyridine (10 mL) in tert-ButOH (7 mL) were
added. After stirring at room temp. overnight, the
reaction was worked up by the addition of sat.
Na2SO3, and stirred at room temp. for 30 min. EtOAc
was then added, and the reaction mixture was
extracted with EtOAc. The organic phase was
washed with brine, dried with Na2SO4, and
evaporated. The residue was taken up in light
Analysis of carmagerol in Cannabis sativa var.
Carma: A 5 g sample of plant material was extracted
with acetone (3 x 30 mL) by stirring at room temp.
(10 min. for each extraction). The pooled extracts
were evaporated, and a portion of the residue (100
mg) was dissolved in 1 mL methanol and filtered in a
Pasteur pipette over Celite (50 mg). The filtration pad
was washed with 0.5 mL methanol, and the pooled
filtrates were diluted with 0.15 mL water. After
washing twice with light petroleum (2 x 2.5 mL) to
remove most of the apolar cannabinoids, the lower
methanol phase was evaporated, taken up in 200 mL
methanol, and analyzed by HPLC (C-18 column,
detection at 210 nm). The following conditions were
employed: Solvent A: 0.5% orthophosphoric acid in
water; Solvent B: acetonitrile. Gradient: from 0 to 8
min, 60% A, 40% B; from 9 to 14 min, 50% A, 50%
B, from 15 to 24 min 10% A, 90% B, from 25 to 30
min 1% A and 99% B. The Rt of 1 was 13.8 min, and
its concentration was in the range of 56-98 mg/Kg
depending on the sample analyzed
Acknowledgments – We are grateful to Dr Lucia
Maxia (Università del Piemonte Orientale, Faculty of
Pharmacy) for her help in the isolation of carmagerol.
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assignments of the major cannabinoids and cannabiflavonoids isolated from flowers of Cannabis sativa. Phytochemical Analysis,
15, 345-354.
[8]
For a related example, see: Vidari G, Di Rosa A, Castronovo F, Zanoni G. (2000) Enantioselective synthesis of each stereoisomer
of the pyranoid linalool oxides: the geraniol route. Tetrahedron Asymmetry, 11, 981-989.
[9]
Harvey, D, Brown NK. (1990) In vitro metabolism of cannabigerol in several mammalian species. Biomedical & Environmental
Mass Spectrometry, 19, 545-553.
[10]
A series of cannabigerol derivatives epoxidized on the proximal double bond have recently been reported (Radwan MM, Ross SA,
Slade D, Ahmed SA, Zulfiquar F, Elsohly MA. (2008) Isolation and characterization of new Cannabis constituents from a high
potency variety, Planta Medica, 74, 267-272.
[11]
Appendino G, Gibbons S, Giana A, Pagani A, Grassi G, Stavri M, Smith E, Mukhlesur Rahman M. (2008) Antibacterial
cannabinoids from Cannabis sativa. A structure-activity study. Journal of Natural Products, 71, 1427-1430.
NPC
2008
Vol. 3
No. 12
1981 - 1984
Natural Product Communications
HPLC-DAD-MS Fingerprint of Andrographis paniculata
(Burn. f.) Nees (Acanthaceae)
Sabrina Arpinia, Nicola Fuzzatia*, Andrea Gioria, Emanuela Martinob, Giacomo Mombellia,
Luca Pagni a and Giuseppe Ramaschi a.
a
Indena S.p.A. Research and Development Laboratories, Via Don Minzoni 6, 20090 Settala (MI), Italy
b
Dipartimento di Ecologia del Territorio, Università di Pavia, Via S. Epifanio 14, 27100 Pavia, Italy
nicola.fuzzati@indena.com
Received: July 1st, 2008; Accepted: October 24th, 2008
An HPLC-UV fingerprint analysis was developed for the quality evaluation of Andrographis paniculata aerial parts.
HPLC-DAD-MS experiments allowed the identification of eleven diterpenes and five flavonoids. Plant material of Indian and
Chinese origin was evaluated employing the developed method. The chemical fingerprints of the plant material of different
origins do not show significant differences.
15
13
13
13
13
13
11
12
12
12
12
7
8
9+ 10
6
5
3
2
12
16
14
A
0.20
1
AU
0.40
15
11
7
8
6
5
3
2
16
9+10
B
0.20
1
AU
0.40
14
4
0.00
11
7
8
9+ 10
5
3
2
16
C
0.20
1
AU
0.40
14
15
4
0.00
5
6
3
2
15
11
7
8
9+10
16
D
0.20
1
AU
0.40
14
4
0.00
4
0.00
11
7
8
6
2
3
1
5
9+ 10
E
0.20
14
15
16
0.40
AU
The aerial parts of Andrographis paniculata
(Acanthaceae) have been used widely in Indian folk
medicine and Ayurveda for the treatment of different
diseases such as dysentery, cholera, diabetes,
consumption, influenza, bronchitis and gonorrhoea
[1]. Chinese medicine employs this herb mainly for
its bitter properties as a treatment for digestive
problems and for a variety of febrile illnesses [2].
More recently, A. paniculata standardized extracts
have become very popular in Europe for the
treatment of upper respiratory infection and influenza
[3]. Among these extracts, the product developed by
the Swedish Herbal Institute and called Kan Jang is
the most widely tested. Kan Jang is standardized to
contain 5.25 mg of a mixture of andrographolide and
deoxyandrographolide per tablet. Andrographolide is
a diterpene lactone which is considered to be the
active principle. Hence all the published analytical
methods are focused on the determination of
andrographolide [4-10]. However, phytochemical
investigations of A. paniculata have shown the
presence of more than twenty labdane diterpenoids,
and over ten 2’-oxygenated flavonoids have been
reported. In the present paper a new HPLC
method, which allows the detection of both
classes of compound in plant material of different
origins, is described. Since the greater part of the
4
Keywords: Andrographis paniculata, fingerprint, HPLC-DAD-MS.
0.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
Minutes
Figure 1: HPLC-UV chromatograms of A. paniculata aerial parts
A: batch # 117302 China; B: batch # 114817 India; C: batch # 111364
India; D: batch # 112927 China; E: batch # 112928 China.
commercialized A. paniculata aerial parts comes
from China and India, plant material of Chinese and
Indian origin was analyzed.
The HPLC-UV chromatograms, recorded at 220 nm,
showed several peaks (Figure 1), but no significant
differences between the two origins.
1982 Natural Product Communications Vol. 3 (12) 2008
Fuzzati et al.
Table 1: HPLC-DAD-MS data of peaks 1-16.
Peak
1
2
Rrt
0.65
0.79
UV max
nm
Molecular ions
m/z
Fragments
m/z
227
1047 [2M+Na+].
551 [M+K+]
535[M+Na+]
351 [M +H-Glc]+,
333 [M+H-Glc-H2O]+,
315 [M+H-Glc-2H2O]+,
297, 285, 257
200
739 [2M+K+]
701 [2M+H+].
389 [M+K+]
351[M+H+]
+
3
4
5
0.94
1.00
1.03
200
225
237 sh
286 325
735 [2M+K ]
697 [2M+H+].
387 [M+K+]
349[M+H+]
739 [2M+K+]
701 [2M+H+]
389 [M+K+]
351[M+H+]
+
963 [2M+K ]
501 [M+K+]
O
O
HO
O
O
O
O
OH
333 [M+H -H2O]+,
315 [M+H -2H2O]+,
297, 285, 257
HO
HO
OH
OR
1 R= glucose
4 R= H
andrographiside
andrographolide
OGlucose
2, 6 14-deoxy-11(or 12)-hydroxyandrographolide
16 neoandrographolide
O
O
O
O
O
OH
O
O
+
331 [M+H -H2O] ,
313 [M+H -2H2O]+,
301
HO
HO
HO
333 [M+H -H2O]+,
315 [M+H -2H2O]+,
297, 285, 257
OR
OH
OH
7 andropanolide
3 14-deoxy-11-oxoandrographolide
8 R= glucose andropanoside
15 R= H
14-deoxy-andrographolide
R3
O
O
R2
O
O
O
O
O
O
+
301 [M +H-Glc] ,
OGlucose O
5 andrographidine A
OR1
O
HO
+
6
7
8
9
1.09
200
739 [2M+K ]
389 [M+K+]
+
333 [M+H -H2O] ,
315 [M+H -2H2O]+,
297
226
739 [2M+K+]
701 [2M+H+].
389 [M+K+]
351[M+H+]
1.19
200
1031 [2M+K+]
993 [2M+H+].
535 [M+K+]
497[M+H+]
1.22
991[2M+K+]
975 [2M+Na+].
515 [M+K+]
499 [M+Na+]
477[M+H+]
315 [M +H-Glc]+,
260,
341
333 [M +H-Glc]+,
315 [M+H-Glc-H2O]+,
297 [M+H-Glc-2H2O]+,
285, 257
1.16
333 [M+H -H2O]+,
315 [M+H -2H2O]+,
297, 285, 257
335 [M +H-Glc]+,
317 [M+H-Glc-H2O]+,
299 [M+H-Glc-2H2O]+,
287, 259
10
1.23
255
1027 [2M+K+]
989 [2M+H+].
533 [M+K+]
495[M+H+]
11
1.63
266, 344
959 [2M+K+]
499 [M+K+]
299 [M +H-Glc]+
12
1.43
263,
335
529 [M+K+]
513 [M+Na+]
329 [M +H-Glc]+
13
1.49
263,
355
1079 [2M+K+]
559 [M+K+]
543 [M+Na+]
359 [M +H-Glc]+,
252
703 [2M+K+]
687 [2M+Na+]
665 [2M+H+].
371 [M+K+]
333[M+H+]
315 [M+H-H2O]+,
297 [M+H -2H2O]+,
285, 257
204
707 [2M+K+]
691 [2M+Na+]
669 [2M+H+].
373[M+K+]
357[M+Na+]
317 [M+H-H2O]+,
299 [M+H -2H2O]+,
287, 257
999 [2M+K+]
983 [2M+Na+].
519 [M+K+]
503[M+Na+]
319 [M +H-Glc]+,
301 [M+H-Glc-H2O]+,
289
14
15
16
1.59
1.61
1.66
201
OR
10 R= glucose 14-deoxy-11,12-didehydroandrographiside
14 R= H
14-deoxy-11,12-didehydroandrographolide
9
11
12
13
R1= H
R2= glucose
R1= glucose R2= H
R1= glucose R2= OCH3
R1= glucose R2= OCH3
R3= H skullcapflavone I 2'-O-glucoside
R3= H
andrographidine C
R3= H
andrographidine E
R3= OCH3 andrographidine D
Figure 2: Structures of compounds 1-16.
The HPLC-DAD-UV analysis of A. paniculata
extracts allowed differentiation betweeen diterpenes
(peaks 1-4, 6-8, 10 and 14-16) and flavonoids
(peaks 5, 9 and 11-13). In addition, the ESI-MS of
the detected peaks exhibited clusters signals at
[2M+K+], [2M+Na+], [2M+H]+ and adduct ions at
[M+K+] and [M+Na+] allowing the determination of
the molecular weights (Table 1, Figure 2).
Furthermore, several fragments attributable to the
loss of glucose (Glc) and to successive losses of
water were observed.
Peaks 1, 4 and 7 exhibited a maximum at
approximately 225 nm, indicating the presence of an
α,β-unsaturated-γ-lactone group with an exo-cyclic
double bond. Peaks 4 and 7 showed the same
ESI-MS spectra indicating that they are isomers.
Peak 4 was identified as andrographolide by injection
of the reference compound. A number of isomers of
andrographolide were previously described [8,11-13],
such as 14−epi-andrographolide, isoandrographolide
and andropanolide, all possessing the α,βunsaturated-γ-lactone group with an exo-cyclic
double bond.
In a previous work [8], isoandrographolide exhibited
the same HPLC behaviour as peak 7. However,
isoandrographolide was described with two different
structures: A [8,12] and B [11,13] (Figure 3). Only
recently the correct structure and stereochemistry of
HPLC-UV fingerprint analysis of Andrographis paniculata
O
O
16
14
OH
O
OH
O
12
20
11
1
3
HO
17
9
5
6
HO
18
19
OH
OH
14-epi-andrographolide
isoandrographolide structure A
O
O
O
O
OH
Natural Product Communications Vol. 3 (12) 2008 1983
The ESI-MS of the flavonoids (peaks 5, 9 and 11-13)
showed signals due to the loss of a glucose unit
allowing the identification of the aglycons.
Comparison of the UV and MS data with those of the
literature allowed the identification of peak 5 as
andrographidine A [15], peak 9 as skullcapflavone I2’-glucoside [16], peak 11 as andrographidine C [15],
peak 12 as andrographidine E [15] and peak 13 as
andrographidine D [15].
H
H
O
HO
HO
OH
OH
andropanolide
isoandrographolide structure B
Figure 3
isoandrographolide (structure B) was determined by
X-ray analysis [13]. In this paper, the authors isolated
also a compound which exhibits the same 13C NMR
signals as the isoandrographolide structure A
described in the work of Li and Fizloff [8].
Furthermore,
they
determined
the
correct
stereochemistry of the compound and named it
andropanolide. Hence peak 7 should be identified as
andropanolide.
The ESI-MS of peak 1 exhibited the loss of a
glucose unit and fragments attributable to the
andrographolide moiety. Peak 1 was identified as
andrographiside [12]. The ESI-MS of peaks 2 and 6
suggested that these compounds are isomers of
andrographolide. However, their UV spectra
showed no absorption at 220 nm indicating the
presence of an α,β-unsaturated-γ-lactone group with
an endo cyclic double bond. Peaks 2 and 6 were
identified as the compounds previously described by
Matsuda et al. [12] and named 14-deoxy-11(or 12)hydroxyandrographolide. Peaks 3, 8, 15 and 16
exhibited UV spectra similar to those of peaks 2
and 6. The study of their ESI-MS allowed the
identification of peak 3 as 14-deoxy-11oxoandrographolide [14], peak 8 as andropanoside,
peak 15 as 14-deoxyandrographolide and peak 16 as
neoandrographolide [12]. Peaks 10 and 14 exhibited
a maximum at approximately 250 nm indicating the
presence of two conjugated double bonds. The study
of their ESI-MS allowed the identification of peak 10
as 14-deoxy-11,12-didehydroandrographiside [12],
and peak 14 as 14-deoxy-11,12-didehydroandrographolide [12].
Experimental
Plant material: A. paniculata aerial parts (batches #
111364 and 114817) were collected in November
2005 in Maredehalli (India). Batches # 112927,
112928 and 117302 were purchased from Yee Po
International Co. (Hong Kong, China). Voucher
specimens are kept at Indena R&D Laboratories,
Settala, Italy.
Plant material extraction: About 2.5 g of A.
paniculata plant material, milled through a 6 mm
screen, was weighted into a 250 mL Pyrex flask.
After addition of 120 mL ethanol, 80% v/v, the
samples were shaken in a mechanical shaker for 3 h
at 60°C. The suspension was filtered through paper
and the extraction procedure repeated. The obtained
extracts were filtered and pooled in a 250 mL
volumetric flask and finally diluted to volume with
ethanol 80% v/v.
Reference material: Andrographolide 98% (catalog
number # 365645) was purchased from SigmaAldrich (Milano, Italy).
HPLC-DAD-MS analyses: The HPLC-DAD-MS
analyses were conducted on a Finnigan Surveyor
HPLC system equipped with a SpectraSYSTEM
DAD UV6000LP and a Finnigan MAT LCQ ion trap
mass spectrometer fitted with a Microsoft® Window®
XP™ data system and an ESI interface. The
chromatographic separation was performed on a
column of Zorbax SB-C18 (5 µm) (250 x 4.6 mm
I.D.) from Agilent Technologies, employing water
(solvent A) and methanol (solvent B). The linear
gradient applied started from 60% A going to 30% A
in 30 min, then in 1 min to 10% A, finally remaining
9 min at 10% A. The column temperature was set at
20°C and the flow rate at 1 mL/min. The injected
volume was 10 μL. The wave length range of the
DAD detector was set at 210-400 nm. Mass
spectrometer conditions were optimized in order to
1984 Natural Product Communications Vol. 3 (12) 2008
achieve maximum sensitivity. ESI conditions: source
voltage 6.0 kV, sheath gas flow rate 70 au, source
current 80 μA, capillary voltage 34 V and capillary
temperature 240°C. Full scan spectra from 150 to
Fuzzati et al.
1500 u in the positive ion mode were obtained (scan
time 1 s). Ion trap conditions: acquisition in
automatic gain control with a max-inject time of 200
msec.
References
[1]
(a) Gupta AK, Neeraj Tandan. (2004) Review on Indian Medicinal Plants Vol. 2. Indian Council of Medical Research, New Delhi,
283-306; (b) Perumal Samy R, Thwin MM, Gopalakrishnakone P. (2007) Phytochemistry, pharmacology and clinical use of
Andrographis paniculata. Natural Product Communications, 2, 607-618.
[2]
Pharmacopoeia of the People’s Republic of China 2005 (English Edition). People’s Medical Publishing House, Beijing, 121.
[3]
Kligler B, Ulbricht C, Basch E, De Franco Kirkwood C, Rae Abrams T, Miranda M, Khalsa KPS, Giles M, Boon H, Woods J.
(2006) Andrographis paniculata for the treatment of upper respiratory infection: a systematic review by the natural standard
reserch collaboration. Explore, 2, 25-29.
[4]
Sharma A, Lal K, Handa SS. (1992) Standardization of the Indian crude drug Kalmegh by high pressure liquid chromatographic
determination of andrographolide. Phytochemical Analysis, 3, 129-131.
[5]
Jain DC, Gupta MM, Saxena S, Kumar S. (2000) LC analysis of hepatoprotective diterpenoids from Andrographis paniculata.
Journal of Pharmaceutical and Biomedical Analysis, 22, 705-709.
[6]
Li W, Fitzloff JF. (2002) Determination of andrographolide in commercial andrographis (Andrographis paniculata) products using
HPLC with evaporative light scattering detection. Journal of Liquid Chromatogrophy & Related Technologies, 25, 1335-1343.
[7]
Pholphana N, Rangkadilok N, Thongnest S, Ruchirawat S, Ruchirawat M, Stayavivad J. (2004) Determination and variation of
three active diterpenoids in Andrographis paniculata (Burm.f.) Nees. Phytochemical Analysis, 15, 365-371.
[8]
Li W, Fitzloff JF. (2004) HPLC-PDA determination of bioactive diterpenoids from plant materials and commercial products of
Andrographis paniculata. Journal of Liquid Chromatogrophy & Related Technologies, 27, 2407-2420.
[9]
Srivastava A, Misra H, Verma RK, Gupta MM. (2004) Chemical fingerprinting of Andrographis paniculata using HPLC, HPTLC
and densitometry. Phytochemical Analysis, 15, 280-285.
[10]
Akowuah GA, Zhari I, Norhayati I, Mariam A. (2006) HPLC and HPTLC densitometric determination of andrographolides and
antioxidant potential of Andrographis paniculata. Journal of Food Composition and Analysis, 19, 118-126.
[11]
Cava MP, Chan WR, Stein RP, Willis CR. (1965) Andrographolide. Further transformations and stereochemical evidence; the
structure of isoandrographolide. Tetrahedron, 21, 2617-2632.
[12]
Matsuda T, Kuroyanagi M, Sugiyama S, Umehara K, Ueno A, Nishi K. (1994) Cell differentiation-inducing diterpenes from
Andrographis paniculata Nees. Chemical Pharmaceutical Bulletin, 42, 1216-1225.
[13]
Pramanick S, Banerjee S, Achari B, Das B, Sen Sr. AK, Mukhopadhyay S, Neuman A, Prangé T. (2006) Andropanolide and
isoandrographolide, minor diterpenoids from Andrographis paniculata: structure and X-ray crystallographic analysis. Journal of
Natural Products, 69, 403-405.
[14]
Balmain A, Connoly JD, (1973) Minor diterpenoids constituents of Andrographis paniculata Nees. Journal of Chemical Society
Perkin Transactions I, 1247-1251
[15]
Kuroyanagi M, Sato M, Ueno A, Nishi K. (1987) Flavonoids from Andrographis paniculata. Chemical and Pharmaceutical
Bulletin, 11, 4429-4435.
[16]
Gupta KK, Taneja SC, Dhar KL. (1996) Flavonoid glycoside of Andrographis paniculata. Indian Journal of Chemistry, 35B,
512-513.
NPC
Natural Product Communications
Diterpenoid Alkaloids and Phenol Glycosides from
Aconitum naviculare (Brühl) Stapf.
2008
Vol. 3
No. 12
1985 - 1989
Stefano Dall’Acquaa*, Bharat B. Shresthab , Mohan Bikram Gewalic, Pramod Kumar Jhab ,
Maria Carrarad and Gabbriella Innocenti a
a
Department of Pharmaceutical Sciences, University of Padova, Padova, Italy
b
Central Department of Botany, Tribhuvan University, Kirtipur, Kathmandu, Nepal
c
Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu, Nepal
d
Department of Pharmacology and Anesthesiology, University of Padova, Padova, Italy
stefano.dallacqua@unipd.it
Received: June 20th, 2008; Accepted: October 28th, 2008
Phytochemical investigation of the aerial parts of Aconitum naviculare, a medicinal plant used in traditional Nepalese
medicine, led to the isolation and characterization of two new diterpenoid alkaloids, navirine B (1), and navirine C (2), along
with
(+) chellespontine (3), kaempferol-7-O-β-D-glucopyranosyl(1→3)α-L-rhamnopyranoside (4), kaempferol-7-O α-Lrhamnopyranoside,3-O-β-D-glucopyranoside (5), p-coumaric-4-O-β-D-glucopyranoside acid (6), and ferulic-4-O-β-Dglucopyranoside acid (7). The structures of the isolated compounds were elucidated on the basis of extensive analyses of 1D
and 2D NMR spectra (HMQC, HMBC, COSY, ROESY) and HR-MS data. The antiproliferative activity of alkaloids 1-3
against human tumor cell lines (LoVo and 2008) was also evaluated.
Keywords: Aconitum naviculare, diterpenoid alkaloids, antiproliferative activity, traditional medicine.
Aconitum species are well-known for their contents
of C19 and C20 diterpenoid alkaloids. Some of these
(e.g., aconitine) are highly toxic and some exhibit
powerful biological activities, for example
antiarrhythmic [1,2], analgesic [2], antiinflammatory
[2], antiepileptic [2] and antiproliferative [3], making
them potential new pharmaceutical entities. Aconitum
naviculare (Brühl) Stapf (Ranunculaceae) is a
biennial medicinal herb of Alpine grassland (>4000
m a.s.l.) found in the trans-Himalayan region of
Nepal, i.e., the Manang, Mustang and Dolpa districts.
The aerial parts are used in Nepalese and Tibetan folk
medicine against cold, fever and headache, as well as
for sedative and analgesic remedies [4,5]. In the
Manang region, the aerial parts are collected during
flowering. The local inhabitants usually dry the
whole plant and prepare a bitter decoction, which is
used for various medicinal purposes. Although
Manangis living in and outside Manang commonly
use A. naviculare, it has not yet become an item of
trade. Due to its supposed high effectiveness in
traditional healthcare, A. naviculare may become a
potential source of income for mountain people.
Little information is available regarding the
phytochemical composition of A. naviculare. Gao et
al. [4] reported the isolation of a new diterpenoid
alkaloid and some atisine-like alkaloids. We recently
reported three novel glycosylated flavonoids from
this plant [6]. Here we report the isolation and
characterization of two new diterpenoid alkaloids,
navirine B (1) and navirine C (2), and five known
compounds (3-7) from A. naviculare.
The isolated diterpenoid alkaloids 1-3 were also
studied for their ability to affect tumor cell
proliferation. In particular, antiproliferative activity
against ovarian (2008 cells) and colon (LoVo cells)
adenocarcinoma was tested.
Compound 1 had a molecular formula of C30H40N2O3
on the basis of the protonated molecular ion [M+H]+
displayed at m/z 477.3122 in the HRAPITOFMS. The
IR spectrum showed absorption bands supporting the
presence of an imino group (1670 cm-1).
1986 Natural Product Communications Vol. 3 (12) 2008
Dall’Acqua et al.
12
20
2
N
3
19
11
1
14
9
10
5
4
H
OH 6
18
13
13
13
12
17
OH
11
O
16
1'
2'
15
2
6'
8
3'
5'
7
4'
1
7'
N
3
1
4
19
8'
N
15
10
5
H
18
H
8
1'
1
2'
H
5'
4'
7'
2
3
O
10
5
N
22
4
19
H
18
16
14
9
2
6'
3'
7
6
11
O
16
14
9
17
12
17
H8
15
OH
7
6
3
8'
N
9'
9'
9'
9'
Figure 1: Structure of isolated compounds 1-3.
The 1H NMR spectrum showed signals ascribable to
one methyl group (δ 1.05 s, 3H), an N,N-dimethyl
group (δ 2.40 s, 6H), and one olefinic proton (δ 5.67
brs, 1H). Two ortho coupled doublets (J = 8.0) in the
aromatic region (δ 6.85 and 7.12, 2H each) also
indicated the presence of an o-p disubstituted
aromatic ring. The structure of compound 1, a
diterpenoid alkaloid, was obtained by exhaustive
analysis of COSY, HMQC and HMBC data. Various
spin systems were detected when COSY and HMQC
data were compared, establishing the connectivity
between CH2 in positions 1,2,3 and 6,7 and between
positions 9,11,12,13. Further connectivity between
the highly deshielded CH-19 (δH 7.43 s, 1H; δC
169.9) and CH-20 (δH 3.57 brs, 1H; δC 80.6) and also
between the olefinic CH-15 (δH 5.67 brs, 1H; δC
130.8) and CH2-17 (δH 4.55 brs, 1H; δC 68.8) were
seen in the COSY spectrum. Diagnostic long-range
(HMBC) correlations were observed from the
methyl group 18 and carbon resonances at δ 30.9
(C-3), 44.9 (C-4), 72.6 (C-5) and 169.9 (C-19).
HMBC correlations, observed from the proton signal
of CH-20 (δ 3.57) with carbon resonances C-19 and
C-5, supported the presence of a six-member ring
containing an imino group. Diagnostic long-range
correlations were observed from the same proton
signal (H-20) with C-1 (δ 27.9), C-9 (47.1), C-5
(72.6) and C-8 (43.8). HMBC correlations of
the proton at δ 5.67 (H-15) with C-9 (δ 47.1), C-12
(δ 31.7) and C-17 (68.8) were also seen. These
observations, as well as analysis of COSY and
HMQC data, suggested two more hexa-atomic rings
in the compound. Long-range correlations observed
from proton signals at δ 1.91-1.56 (CH2-13) with
carbon resonances at δ 146.8 (C-16) and 80.6 (C-20)
supported the linkage between positions 20 and 14.
Long-range correlations observed from the aromatic
doublet δH 7.12 (H-3’ 5’) with the carbon at δC 33.3
(C-7’) and from the methyl group at δH 2.59 (CH3-9’)
with the carbon at δC 52.5 (C-8’) suggested the
presence of one hordenine moiety in the molecule.
NOESY data and comparisons with the spectral
data of Gao et al. [4] for navirine evidenced the
relative stereochemistry of the molecule, supporting
an α orientation for groups at positions 10, 8 and 12.
NOESY cross-peaks were also obtained between the
exchangeable proton signal at δ 3.48 (OH-5) and H-9
(δ 1.64), which were assigned as β on the basis of
previous references [4,7], suggesting a β orientation
for the hydroxy group. With all this evidence,
compound 1 was established as a new alkaloid, called
navirine B (5β-hydroxy navirine).
The HRAPITOFMS of compound 2 displayed a
protonated molecular ion [M+H]+ at m/z 461.3532,
corresponding to a molecular formula of C31H45N2O.
The 1H NMR spectrum of compound 2 was quite
similar to that of compound 1, but there were some
differences. It lacked the signal for the imino proton,
and the proton signal of H-20 was shifted downfield
to δ 2.53 (δ 3.57 for 1). In addition, a singlet signal
integrating for nine protons was observed at δ 2.50,
supporting the presence of three nitrogen linked
methyl groups. Extensive analyses of COSY, HMQC
and HMBC data revealed a similar diterpenoid
skeleton, as well as the same hordenine moiety as
found in compound 1. In the HMBC spectrum,
long-range correlations were observed between the
methyl group at δ 1.00 (CH3-18) and carbon
resonances at δ 34.1 (C-3), 41.7 (C-4), 53.5 (C-5) and
58.0 (C-19), which supported the presence of aminolinked CH2 at position 19 (δH 2.30; δC 58.0), as well
as a CH group at position 5 (δH 1.26; δC 53.5). The β
configuration for H-5 was established on the basis of
the NOESY correlation observed from the methyl
group at δ 1.00 and the proton signal at δ 1.26 (H-5).
Complete analyses of 1D and 2D NMR data afforded
a novel structure, called navirine C.
Diterpenoid alkaloids and glycosides from Aconitum naviculare
Natural Product Communications Vol. 3 (12) 2008 1987
Compound 3 showed a protonated molecular ion
[M+H]+ at m/z 344.2590, establishing its molecular
formula as C22H33NO2. The 1H NMR spectrum of 3
displayed one aldehydic proton signal (8.71 s, 1H),
one exocyclic methylene (δ 5.02 d, 5.12 d, 1H each;
J = 1.0 Hz), one methyne signal linked to an
electronegative atom (δ 3.70 m, 1H), and one methyl
group (δ 1.06 s, 3H). Its COSY, HMQC and HMBC
data revealed a diterpenoid skeleton. Compound 3
possessed a hydroxyl group at position 15, as
supported by the HMBC correlation between H-17
(δ 5.05 and 5.12) and the carbon resonance at δ 74.7
(C-15). The β orientation of the OH group at position
15 was deduced on the basis of NOESY cross-peaks
observed between H-15 and H-13 and H-14. The
proton and carbon resonances at position 15 were
at δH 3.70 and δC 74.7 respectively, due to the β-OH
group. The H 9 configuration in compound 3 was
established by observing the NOESY correlations
from the methyl group 18 (δ 1.06) and H-5 (δ 1.38),
and from this latter and the H-9 signal (δ 2.19). On
the basis of the spectral data, compound 3 was
established as chellespontine [8].
whereas, in 2008 cells, only compound 1 induced a
marked inhibition of cell growth, as shown in Table 1
as IC50 values.
To our knowledge alkaloid 3, flavonol glycosides 4
and 5, and phenylpropanoid glycosides 6 and 7 have
been isolated and characterized from A. naviculare
for the first time.
Recently some Aconitum alkaloids have been
evaluated for their antiproliferative activity against
A172 malignant cells [3]. For this reason, we decided
to study this activity for the isolated diterpenoid
alkaloids 1-3.
Our study on the phytochemical composition of A.
naviculare may be useful in improving scientific
knowledge of this Nepalese medicinal plant.
Experimental
Optical rotations were measured on a Jasco P2000
digital polarimeter, IR spectra on a Perkin Elmer
1600 FT-IR spectrometer, and NMR spectra, in
CDCl3 or CD3OD, on a Bruker AMX-300
spectrometer, operating at 300.13 MHz for 1H NMR
and 75.03 MHz for 13C NMR. 2D experiments, 1H-1H
DQF-COSY, and inverse-detected 1H-13C HMQC
and HMBC spectra were performed with UXNMR
software.
HRMS were obtained on an API-TOF spectrometer
(Mariner Biosystems). Samples were diluted in a
mixture of H2O-AcCN (1:1), with 0.1% formic acid
for positive ion mode, and directly injected at a flow
rate of 10 µL/min. Sephadex LH 20 and silica gel 60
were used for column chromatography. Silica gel
plates were used for preparative and analytical TLC
(Merck cat. 5717 and 5715). Compounds on TLC
were detected with a UV lamp (254 nm) and by
treating plates with Dragendorff’s reagent.
Semi-preparative HPLC was performed on a Gilson
series 305 liquid chromatograph equipped with a
LiChrosphere 100 RP-18 column (particle size 10
μm, 250 x 10 mm ID, E. Merck).
Table 1: Antiproliferative activity of compounds 1-3.
LoVo cellsa
IC50 (confidence limits) μM
22 (19-25)
2008 cellsa
IC50 (confidence limits) μM
33 (30-37)
2
nd
nd
3
38 (33-41)
nd
33.3 (28.7-36.1)
13.8 (11.5-!6.5)
Compound
1
Cisplatin
b
a
LoVo: colon cell line; 2008: ovarian cell line.
Cisplatin was used as a reference compound.
nd: IC50 is not determined.
b
Compounds 1 and 3 showed a significant
antiproliferative activity, whereas compound 2 was
inactive. The capacity of the compounds to affect
tumor cell growth revealed a dose-dependent effect.
In particular, colon cell line LoVo was more sensitive
than ovarian cells 2008. In fact, compounds 1 and 3
were able to decrease cell proliferation in LoVo cells,
Plant material: Aerial parts, including stems, leaves,
flowers and immature fruits of Aconitum naviculare
were collected from Ladtar (4100 m a.s.l., upper
Manang) during the last week of September 2004. A
voucher specimen was collected during a field survey
and deposited at Tribhuvan University Central
Herbarium (TUCH; n° ANV904); identification was
confirmed by Prof. Ram Prasad Chaudhary of the
Central Department of Botany, Tribhuvan University,
Kathmandu.
Extraction and isolation: Air-dried powdered aerial
parts (100 g) were exhaustively extracted in a Soxhlet
apparatus with MeOH. The solvent was evaporated
under reduced pressure (230–250 mbar) and a
semisolid MeOH extract was obtained (8 g). About
1988 Natural Product Communications Vol. 3 (12) 2008
5.0 g of this extract was suspended in a mixture of
9:1 H2O–MeOH (200 mL), and the pH was adjusted
to 2 with 5% aq HCl. The solution was then
partitioned first with CHCl3 (5 x 50 mL) and then
with EtOAc (5 x 50 mL). Solvents were removed
under vacuum, yielding the CHCl3 fraction CL-I (850
mg) and EtOAc fraction EA-I (330 mg). The pH of
the residual aqueous layer was then adjusted to 8 with
diluted NH3. It was then partitioned with CHCl3 (5 x
50 mL) and EtOAc (5 x 50 mL). Solvents were
removed under vacuum, yielding the CHCl3 fraction
CL-II (250 mg) and EtOAc fraction EA-II (133 mg).
The pH of the aqueous layer was then adjusted to 7.0,
and the solvent was removed by freeze-drying, giving
fraction AQ (3.3 g).
TLCs of the fractions CL-II and EA-II in several
solvents
(toluene/diethylamine/EtOAc
80:5:20;
v/v CHCl3/MeOH/NH3 85:15:0.5; v/v) showed
spots that gave a positive reaction to Dragendorf
reagent. Fraction CL-II was repeatedly subjected
to preparative thin layer chromatography (PTLC)
with (toluene/diethylamine/EtOAc 80:5:20 v/v,
CHCl3/MeOH/NH3 85:15:0.5 v/v). as eluents,
yielding compounds 1 (10.1 mg) and 2 (6.5 mg).
Fraction EA-II was subjected to silica plate
chromatography using chloroform/methanol 4:1 as
eluent. Further purification was achieved by
semi-preparative HPLC with aqueous 0.1% HCOOH
(A) and AcCN (B) as eluents. Gradient elution was
used from 90% (A) to 85% (A) in 4 min, and then to
40% (A) in 22 min, yielding compound 3 (5.2 mg).
The AQ fraction was subjected to several semipreparative HPLC steps (Spherisorb C-18, 1 x 300
mm, 10 μm) with aqueous 0.1% HCOOH (A) and
methanol (B) as eluents. Gradient elution used for the
isolation of compounds 4-7 was as follows: from
90% (A) to 85% (A) in 10 min, and then to 70% (A)
in 15 min, yielding compounds 4 (11.0 mg) and 5
(13.0 mg); from 75% (A) to 45 % A in 25 min for the
isolation of compounds 6 (8.0 mg) and 7 (6.5 mg).
Navirine B (1)
amorphous solid.
[α]D: +12.0 (c 0.083, CH3OH).
IR (KBr): 3350, 3030, 3020, 1670, 1650, 1610, 1508,
820 cm-1.
1
H NMR (300 MHz, CDCl3): 1.65-1.78 m (H-1), 1.30
m (H-2), 1.75-1.98 m (H-3), 1.87-2.02 m (H-6), 1.261.58 m (H-7), 1.64 m (H-9), 1.42-1.88 m (H-11), 2.54
m (H-12), 1.56-1.91 m (H-13), 5.67 brs (H-15), 4.55
brs (H-17), 1.05 s (H-18), 7.43 brs (H-19), 3.57 brs
Dall’Acqua et al.
(H-20), 6.85 d (8.0) (H-2’/6’), 7.12 d (8.0) (H-3’/5’),
2.99 brt (H-7’), 3.02 brt (H-8’), 2.40 s N-(CH3)2-9’.
13
C NMR (75.03 MHz, CDCl3): 27.9 (C-1), 30.1
(C-2), 30.9 (C-3), 44.9 (C-4), 72.6 (C-5), 30.9 (C-6),
20.9 (C-7), 43.8 (C-8), 47.1 (C-9), 41.0 (C-10), 43.2
(C-11), 31.7 (C-12), 43.5 (C-13), 69.8 (C-14), 130.8
(C-15), 146.8 (C-16), 68.8 (C-17), 19.1 (C-18), 169.9
(C-19), 80.6 (C-20), 158.2 (C-1’), 114.9 (C-2’/6’),
129.2 (C-3’/5’), 125.0 (C-4’), 33.3 (C-7’), 52.5
(C-8’), 34.3 (N-(CH3)2-9’).
HRAPITOFMS: m/z [M + H+] calcd for C30H40N2O3:
477.3117; found: 477.3122.
Navirine C (2)
amorphous solid.
[α]D: +7.5 (c 0.070, CH3OH).
IR (KBr): 3350, 3030, 3018, 1650, 1605, 1508,
820 cm-1.
1
H NMR (300 MHz, CDCl3): 1.74-2.08 m (H-1), 1.62
m (H-2), 1.25-1.78 m (H-3), 1.26 m (H-5), 1.83-2.14
m (H-6), 1.80 m (H-7), 1.34-1.68 m (H-11), 2.87 m
(H-12), 1.33 m (H-13), 5.63 brs (H-15), 4.52 brs
(H-17), 1.00 s (H-18), 2.30 m (H-19), 2.53 m (H-20),
6.84 d (8.0) (H-2’/6’), 7.10 d (8.0) (H-3’/5’), 2.89 m
(H-7’), 2.89 m (H-8’), 2.55 s (N-(CH3)2-9’).
13
C NMR (75.03 MHz, CDCl3): 21.8 (C-1), 28.5
(C-2), 34.1 (C-3), 41.7 (C-4), 53.5 (C-5), 33.9 (C-6),
34.1 (C-7), 44.4 (C-8), 44.5 (C-9), 44.0 (C-10), 28.1
(C-11), 31.1 (C-12), 44.1 (C-13), 45.0 (C-14), 132.4
(C-15), 145.8 (C-16), 68.7 (C-17), 27.8 (C-18), 58.0
(C-19), 76.7 (C-20), 158.8 (C-1’), 115.1 (C-2’/6’),
129.7 (C-3’/5’), 128.0 (C-4’), 33.2 (C-7’), 60.5
(C-8’), 44.3 (N-(CH3)2-9’).
HRAPITOFMS: m/z [M + H+] calcd for C31H44N2O:
461.3532; found: 461.3512.
Chellespontine (3)
amorphous solid.
[α]20D: +5.5 (c 0.052, CHCl3).
IR (KBr) νmax: 2915, 1730, 990, 892 cm-1.
1
H NMR (300 MHz, CDCl3): 1.68 m (H-1), 1.31 m
(H-2), 1.48-1.74 m (H-3), 1.38 m (H-5), 1.70 m
(H-6), 1.61-2.05 (H-7), 2.19 m (H-9), 1.16-1.93 m
(H-11), 2.40 m (H-12), 1.80-1.97 m (H-13), 1.301.93 m (H-14), 3.70 m (H-15), 5.05-5.12 d (J = 1.0)
(H-17), 1.06 s (H-18), 3.77 m (H-19), 4.00 m (H-20),
3.75 m (H-21), 8.71 s (H-22).
13
C NMR (75.03 MHz, CDCl3): 25.7 (C-1), 19.6 ( C2), 40.5 (C-3), 33.5 (C-4), 45.6 (C-5), 19.7 (C-6),
34.2 (C-7), 37.2 (C-8), 39.8 (C-9), 46.0 (C-10), 27.9
(C-11), 36.1 (C-12), 25.3 (C-13), 27.9 (C-14), 74.7
Diterpenoid alkaloids and glycosides from Aconitum naviculare
Natural Product Communications Vol. 3 (12) 2008 1989
(C-15), 154.8 (C-16), 109.7 (C-17), 24.0 (C-18), 59.5
(C-19), 56.5 (C-20), 65.0 (C-21), 183.3 (C-22).
HRAPITOFMS: m/z [M + H+] calcd. for C22H33NO2:
344.2590; found: 344.2550.
Cell growth was determined by the MTT reduction
assay [12] after 72 h of incubation. Briefly, 20 μL of
MTT solution (5 mg/mL in PBS) was added to each
well and plates were incubated for 4 h at 37°C.
DMSO (150 μL) was added to all wells and mixed
thoroughly to dissolve the dark blue crystals.
Absorbance was measured on a micro-culture plate
reader (Titertek Multiscan) at a test wavelength of
570 nm and a reference wavelength of 630 nm.
Experiments were performed at least in triplicate, and
results were statistically evaluated using Student's
t-test [13]. IC50 95% confidence limits were estimated
with the Litchfield and Wilcoxon method.
Compounds 4-7 were characterized on the basis of
reported data [9-11].
Antiproliferative activity: Human ovarian carcinoma
(2008) and human intestinal adenocarcinoma(LoVo)
cell lines were used. The 2008 cells were maintained
in RPMI 1640, and LoVo cells in Ham’s F 12, in
both cases supplemented with 10% heat-inactivated
FCS, 1% antibiotics and 1% 200 mM L-glutamine
(all products of Biochrom KG Seromed). Cells were
seeded on 96-well tissue plates (Falcon) at 5 x 104
cells/mL, and treated 24 h later with various
concentrations of the compounds 1-3.
Acknowledgment - The authors are grateful to
MIUR for financial support.
References
[1]
Amiya T, Bando H. (1988) Aconitum alkaloid, in The Alkaloids: Chemistry and Biology, Brossi A. (Ed) Academic Press Inc, San
Diego, CA, Vol. 34, Chapter 3, 95-177.
[2]
Ameri A. (1998) The effects of Aconitum alkaloids on the central nervous system. Progress in Neurobiology, 56, 211-235.
[3]
Wada K, Hazawa M, Takahashi K, Mori T, Kawahara N, Kashiwakura I. (2007) Inhibitory effects of diterpenoid alkaloids on the
growth of A172 human malignant cells. Journal of Natural Products, 70, 1854-1858.
[4]
Gao L, Wei X, Yang L. (2004) A new diterpenoid alkaloid from a Tibetan medicinal herb Aconitum naviculare Stapf. Journal of
Chemical Research, 307-308.
[5]
Shrestha BB, Jha PK, Gewali MB. (2007) Ethnomedicinal use and distribution of Aconitum naviculare (Bruhl) Stapf in upper
Manang, central Nepal. In: Local Effects of Global Changes in the Himalayas: Manang, Nepal. Chaudhary RP, Aase TH, Vetaas
OR, Subedi BP. (Eds.) Tribhuvan University, Nepal, and University of Bergen, Norway, 171-181.
[6]
Shrestha BB, Dall’Acqua S, Gewali MB, Jha PK, Innocenti G. (2006) New flavonoid glycosides from Aconitum naviculare (Bruhl)
Stapf, a medicinal herb from the trans-Himalayan region of Nepal. Carbohydrate Research, 341, 2161-2165.
[7]
Pelletier SW, Keith LH. (1970) In The Alkaloids: Chemistry and Physiology, Manske RHF. (Ed.) Academic Press, New York, Vol.
12, Chapter 2, 143-155.
[8]
Desai HK, Joshi BS, Pelletier SW, Sener B, Bingol F, Baykal T. (1993) New alkaloids from Consolida hellespontica. Heterocycles,
36, 1081-1089.
[9]
Du M, Xie J. (1995) Flavonol glycosides from Rhodiola crenulata, Phytochemistry, 38, 809-810.
[10]
Pauli GF. (2000) Higher order and substituent chemical shift effects in the proton NMR of glycosides. Journal of Natural Products,
63, 834-838.
[11]
Galland S, Mora N, Abert-Vian M, Rakotomanomana N, Dangles O. (2007) Chemical synthesis of hydroxycinnamic acid
glucosides and evaluation of their ability to stabilize natural colors via anthocyanin copigmentation, Journal of Agricultural and
Food Chemistry, 55, 7573-7579.
[12]
Mosmann T. (1983) Rapid colorimetric assay for cellular growth survival: application to proliferation and cytotoxic assay. Journal
of Immunoogical Methods, 65, 55-63.
[13]
Tallarida RJ, Murray RB. (1987) Manual of Pharmacological Calculations with Computer Programs. Springer-Verlag.
NPC
Natural Product Communications
Inhibition of PGHS-1 and PGHS-2 by Triterpenoid Acids
from Chaenomelis fructus
2008
Vol. 3
No. 12
1991 - 1994
Eveline Reiningera and Rudolf Bauerb,*
a
Institute of Pharmaceutical Biology, Heinrich-Heine University, 40225 Düsseldorf, Germany
b
Institute of Pharmaceutical Sciences, Department of Pharmacognosy, Karl-Franzens-University,
Universitätsplatz 4, 8010 Graz, Austria
rudolf.bauer@uni-graz.at
Received: July 14th, 2008; Accepted: October 28th, 2008
The dichloromethane extract of the dried fruits of Chaenomeles speciosa (Sweet) Nakai (Rosaceae) showed strong inhibitory
activity against both prostaglandin-H-synthase isoenzymes [IC50 (PGHS-1) = 5.1 µg/mL; IC50 (PGHS-2) = 2.3 µg/mL]. The
lipophilic portion of the extract was mainly responsible for the inhibitory effect. Several triterpenoid acids were isolated and
identified as contributing to this inhibitory activity (oleanolic, pomolic, 3β-O-acetylursolic and 3β-O-acetylpomolic acids).
Comparison of their inhibitory potential with their selectivity to PGHS-2 showed that 3β-O-acetylursolic acid had the highest
potency in the inhibition of PGHS-1 and PGHS-2 enzymes, whilst pomolic and 3β-O-acetylpomolic acid, with a hydroxyl
group at position 19α, showed selectivity for PGHS-2. The inhibitory effect of the extract seems to be the result of the activity
of the mixture of these different triterpenoid acids.
Keywords: Chaenomeles speciosa, Rosaceae, anti-inflammatory activity, prostaglandin-H-synthase, PGHS-1, PGHS-2,
triterpenoid acids.
Prostaglandin-H-synthase (PGHS) catalyses the first
two steps of the formation of prostaglandins (PG)
from arachidonic acid, with a fatty acid cyclooxygenase activity (catalyzing the reaction from
arachidonic acid to PGG2) and a PG hydroperoxidase
activity (catalyzing the reaction from PGG2 to PGH2).
The enzyme exists in two isoforms: the constitutive
PGHS-1, which is responsible for ‘housekeeping’
purposes, like the formation of protective PGs in the
stomach and the kidneys, while PGHS-2 is mainly
induced at sites of inflammation to form
inflammatory PGs, mainly PGE2 [1]. PGE2 is a
common marker for the determination of
inflammation [2]. Therefore, PGHS is an important
target of anti-inflammatory drug research. While
common non-steroidal anti-inflammatory drugs act as
unspecific inhibitors of both isoenzymes, the
selective inhibition of PGHS-2 is the goal of new
NSAIDs. They are expected to have less side effects
on the stomach and the kidneys [3], even if it is
known that long-term treatment with COX-2selective drugs (rofecoxib) for cancer prevention
suggested an elevated incidence of myocardial
infarction after long term use [4].
The dried fruits of Chaenomeles speciosa (Sweet)
Nakai (“Mugua”), Rosaceae, are used as an antiinflammatory and antirheumatic drug in traditional
Chinese medicine. The dichloromethane-extract of
Mugua was tested for its inhibitory potential on
prostaglandin synthesis in PGHS-1/-2 screening
assays. We now report on the bioassay-guided
fractionation and the identification of the PGHSinhibitory active principles.
The fruits of C. speciosa were powdered and
extracted with dichloromethane. The extract, when
tested in PGHS-1 and -2 microtiter assays with EIA
evaluation [5], showed very strong inhibition of both
isoenzymes. The IC50 values were determined to be
5.1 µg/mL for PGHS-1 and 2.3 µg/mL for PGHS-2.
The extract showed slight preferential inhibition of
PGHS-2. When fractionated by vacuum liquid
chromatography (VLC) with different mixtures of
n-hexane/ethylacetate of increasing polarity there was
a clear preference of inhibitory potential by the
lipophilic part of the extract (Figure 1).
1992 Natural Product Communications Vol. 3 (12) 2008
Reininger et al.
Table 1: PGHS-1/ -2 inhibition of triterpenoid acids from Chaenomelis
fructus.
100
80
Conc.
Inhibition [%]
60
Compounds
Oleanolic acid
Pomolic acid
Maslinic acid
Pirolonic acid
Euscaphic/Tormentolic acid
40
20
0
I
II
III
IV
V
VI
VII
VIII
IX
X
-20
3β-O-Acetylursolic acid
3β-O-Acetylpomolic acid
-40
-60
Figure 1: Inhibition (%) of PGHS-1/-2 in vitro by fractions from VLCseparation of the dichloromethane extract of Mugua (tested conc.: 50
µg/mL)
HO
HO
COOH
O
Euscaphic acid
HO
HO
HO
44.9 ± 8.8
48.2 ± 18.4
63.1 ± 6.9
56.7 ± 12.3
Table 2: IC50 values of the positive controls and the major triterpenoid
acids of Mugua.
Compound
HO
100
97
* means from enriched fractions
Fractions
HO
110
106
106
103
102
Inhibition
PGHS-1
PGHS-2
23.8 ± 27.7
10.4 ± 8.4
51.6 ± 8.2
49.4 ± 8.4
14.3 ± 7.4
11.8 ± 3.7
47.4 ± 4.1
43.4/21.4*
82.7/55.4*
[µM]
COOH
Pirolonic acid
Oleanolic acid
Pomolic acid
3β-O-Acetylursolic acid
3β-O-Acetylpomolic acid
Indometacin
NS-398
IC50 PGHS-1
[µM]
IC50 PGHS-2
[µM]
ratio PGHS-1/
PGHS-2
204
68
34
165
0.9
50.7
348
36
39
90
0.8
2.6
0.6
2.0
0.9
1.8
1.1
19.5
COOH
3β-O-Acetylpomolic acid
3β-O-Acetylursolic acid
HO
HO
Tormentolic acid
From this part we isolated and identified different
triterpenoid acids as the major constituents. The
structures have been established on the basis of their
spectral data (IR, EI-MS, DCI-MS, 1H NMR, 13C
NMR) and by comparison with literature data [6-15]
as 3β-O-acetylursolic acid, 3β-O-acetylpomolic acid,
oleanolic acid, pomolic acid, euscaphic acid,
tormentolic acid and pirolonic acid. The acids,
reported for the first time from C. speciosa, were
tested for their inhibitory activity of PGHSisoenzymes (Table 1). Most exhibited inhibition of
both enzymes. Only euscaphic acid showed high
inhibitory potential for PGHS-2. Separation of
euscaphic and tormentolic acid was not achieved, so
their inhibition values are derived from enriched
fractions (ca. 90% purity). The IC50 values of the
major triterpenoid acids are listed in Table 2.
3β-O-acetylursolic acid turned out to be the most
potent inhibitor, while oleanolic acid showed only
weak inhibition of both isoenzymes. When
calculating the inhibition ratio PGHS-1/PGHS-2,
pomolic acid and 3β-O-acetylpomolic acid showed
more pronounced inhibitory activity against PGHS-2
than on PGHS-1. Both constituents possess a
hydroxyl moiety in ring E. Figure 2 illustrates the
selectivity by comparison of the ratios of PGHS1/PGHS-2 inhibition. Ratios > 1 are considered as
preferential PGHS-1 inhibitors, ratio < 1 as
preferential PGHS-2 inhibitors, a ratio = 1 as nonselective inhibitors.
COOH
COOH
AcO
AcO
HO
COOH
COOH
HO
Pomolic acid
Oleanolic acid
HO
PGHS-2 selective
PGHS-1
3
2
1
2
3
Figure 2: Selectivities of the main triterpenoid acids of Mugua by
comparison of the ratios of IC50 values (the ratios are calculated as
PGHS-1/PGS-2 when preferential for PGHS-1, and PGHS-2/PGHS-1
when preferential for PGHS-2).
Oleanolic and ursolic acid have already been
described as potent inhibitors of PGHS [16,17]. In
other test systems using microsomal preparations for
PGHS-1 vs. pure enzyme for PGHS-2 [18], oleanolic
and ursolic acid exhibited selectivity for PGHS-2.
However, in the series of triterpenoid acids isolated
from Mugua, oleanolic acid did not show the best
inhibitory effect against either pure PGHS-1 or
PGHS-2. Oleanolic and 3β-O-acetylursolic acid were
more inhibitory on PGHS-1 than on PGHS-2. The
selectivity was dependent on the 19α−hydroxyl
moiety, found in pomolic acid and its derivatives.
This observation could be confirmed by all isolated
triterpenoid acids: all 19α-hydroxylated acids showed
better inhibitory potential towards PGHS-2 than
towards PGHS-1. A second hydroxyl moiety in ring
A seems to intensify the preference.
Triterpenoids as prostaglandin-H-synthase inhibitors
Natural Product Communications Vol. 3 (12) 2008 1993
For high inhibitory potential, the α-amyrine-structure
seems to be especially important. 3β-O-Acetylursolic
acid turned out to be the most potent inhibitor.
Acetylation at position 3β did not seem to influence
the inhibitory potential.
dissolved in EtOH p.a. for pharmacological testing at
a concentration of 1 mg/mL.
Interestingly, it was shown recently that oleanolic
acid induced up-regulation of COX-2 via early
phosphorylation of p38 MAPK and p42/44 MAPK
[19], as was also found for alkamides of Echinacea
and for the selective COX-2 inhibitor NS-398 in
high concentration [20]. Oleanolic acid also induces
rabbit platelet aggregation through a phospholipase
C-calcium dependent signaling pathway [21].
Therefore, oleanolic acid may be considered as a
modulatory compound for inflammation.
Since about 70% of the dichloromethane extract of
Mugua consists of triterpenoid acids, the very good
inhibitory effect of the extract seems to be the result
of the activity of these different acids. It will be
interesting to demonstrate whether the inhibitory
potential of triterpenoid acids can be further
increased by structural modification.
Experimental
Extraction and fractionation: The plant material was
provided by the TCM Hospital, Kötzting, Germany,
and was identified in the Institute of Pharmaceutical
Biology, Munich. A voucher specimen (12/1998) has
been deposited there.
The dichloromethane extract (17.5 g) was prepared
by exhaustive Soxhlet extraction of 925 g powdered
Chaenomelis fruits. This extract was fractionated in
two portions by vacuum liquid chromatography
(VLC) with n-hexane/ethyl acetate mixtures on
180 g Silica gel (Kieselgel 60, Merck) into fractions
I - X. The fractions were evaporated to dryness and
Table 3: Fractions obtained by VLC from the dichloromethane extract of
Mugua.
Fraction
I
II
III
IV
V
VI
VII
VIII
IX
X
Eluent: n-Hexane/Ethyl acetate [mL]
300 : 0
280 : 20
270 : 30
250 : 50
230 : 70
210 : 90
190 : 110
170 : 130
150 : 150
130 : 170
120 : 180
90 : 210
60 : 240
30 : 270
0 : 300
Yield [g]
1.22
0.20
1.24
1.23
1.44
1.57
1.32
3.39
1.49
1.09
Isolation and structure elucidation of tested
compounds: VLC fraction VI was further separated
on Sephadex LH20 (100 g; column 90 x 2 cm ID)
with a mixture of cyclohexane and ethyl acetate (6:4)
as eluent, which resulted in a fraction of 550 mg
phytosterols (sitosterol, campesterol) and ca. 55 mg
triterpenoid acids. The purification of 3β-O-acetylpomolic acid (27.1 mg) and 3β-O-acetylursolic acid
(28.0 mg) was achieved by MPLC on RP-18 (23 g
LiChroprep RP 18, 25 – 40 µm, Merck; column
47 x 1 cm ID) with a water / acetonitrile gradient
(10 – 40% in 10 min, 40 – 100% in 40 min). Fraction
IX and X were first separated on Sephadex LH20
(100 g; column 90 x 2 cm ID) with ethyl acetate.
Fraction IX was further separated by MPLC on
RP-18 (8 g LiChroprep RP 18, Merck, 15 - 25 µm;
column 40 x 0.8 cm ID) with a water/acetonitrile
gradient (30 – 65% in 16 min, 65 – 90% in 34 min,
90 – 100% in 10 min). The final purification of
oleanolic, pomolic and pirolonic acid was achieved
by semipreparative HPLC on RP-18 (Hibar RT 250 x
10 mm ID, LiChrosorb RP 18, 7 µm, Merck) with a
water/acetonitrile gradient (30 – 65% in 12 min,
65 – 78% in 14 min, 78 – 98% in 4 min, 98%
10 min). The identity of the compounds was
confirmed on the basis of IR, DCI- and EI-MS, 1H
and 13C NMR spectra in comparison with literature
data [6-15]. The purity was checked by TLC and
HPLC.
Pharmacological assays: The PGHS-1 and PGHS-2
assays were performed on a microtiter scale with
purified PGHS-1 from ram seminal vesicles and
purified PGHS-2 from sheep placental cotyledones
(both Cayman Chemical Company), as previously
described [5]. The incubation mixture contained 180
µL 0.1 M tris-buffer (pH 8.0), 5 µM hematin, 18 mM
epinephrin-hydrogentartrate, 0.2 U of enzyme
preparation and 50 µM Na2EDTA (only PGHS-2
assay). Each compound solution (10 µL) was added
and pre-incubated for 5 min at room temperature. The
reaction was started by adding 10 µL of 5 µM
arachidonic acid in EtOH p.a. and subsequent
incubation at 37°C. The reaction was terminated after
20 min by adding 10 µL formic acid 10%.
Determination of PGE2: The concentration of PGE2,
the main metabolite of arachidonic acid in this
reaction, was determined by a competitive PGE2EIA-kit (R&D Systems), which was used as
described by Reininger et al. [5]. Because of the use
1994 Natural Product Communications Vol. 3 (12) 2008
of alkaline phosphatase, the procedure was
shortened to 2 h incubation time and 45 min for
the development process, which involves pNPP
(p-nitrophenyl phosphate) as a substrate. The
development was stopped by adding 2N NaOH. All
samples were diluted 1:15 in EIA-buffer. The EIA
was evaluated by an ELISA reader ‘rainbow’ (Tecan
Deutschland GmbH, Crailsheim, Germany) and
determined as previously described [5]. Inhibition
refers to reduction of PGE2 formation in comparison
with a blank without inhibitor.
NS-398 and indomethacin (both from Cayman
Chemical Company) were used as positive controls.
Reininger et al.
For testing, they were dissolved in EtOH p.a. at a
concentration of 1 mM and further diluted with
EtOH p.a..
Statistics: All IC50 values were determined for both
enzymes by measuring at least three concentrations;
all inhibition values are means of at least three
experiments.
Acknowledgments - We are grateful to the TCM
Hospital, Kötzting, Germany, for supplying plant
materials and for financial support, and we thank Mr
Jansen for technical assistance.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
Hinz B, Brune K. (2002) Cyclooxygenase-2 – 10 years later, Journal of Pharmacology and Experimental Therapeutics, 300,
367-375.
Vane JR, Botting RM. (1998) Anti-inflammatory drugs and their mechanism of action. Inflammation Research, 47 (Suppl. 2),
S78-S87.
Vane JT, Botting RM. (1995) New insights into the mode of action of anti-inflammatory drugs. Inflammation Research, 44, 1-10.
Simmons DL, Botting RM, Hla T. (2004) Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition.
Pharmacological Reviews, 56, 387-437.
Reininger EA, Bauer R. (2006) Prostaglandin-H-synthase (PGHS)-1 and -2 microtiter assays for the testing of herbal drugs and in
vitro inhibition of PGHS-isoenzyms by polyunsaturated fatty acids from Platycodi radix. Phytomedicine, 13, 164-169.
Guo X, Zhang L, Quan S, Hong Y, Sun L, Liu M. (1998) Isolation and identification of triterpenoid compounds in fruit of
Chaenomeles lagenaria (Loisel.) Koidz.. Zhungguo Zhongyao Zazhi, 23, 546-547.
Mahato SB, Kundu AP. (1994) 13C NMR spectra of pentacyclic triterpenoids – a compilation and more important features.
Phytochemistry, 36, 1517-1575.
Kakuno T, Yoshikawa K, Arihara S. (1992) Triterpene saponins from fruit of Ilex crenata. Phytochemistry, 31, 2809-2812.
Santos GG, Alves JCN, Rodilla JML, Duarte AP, Lithgow AM, Urones JG. (1997) Terpenoids and other constituents of Eucalyptus
globulus. Phytochemistry, 44, 1309-1312.
Yamaguchi K. (1970) Spectral Data of Natural Products, Vol. 1, Elsevier PC, Tokyo.
Guang-YL, Gray A, Waterman PG. (1989) Pentacyclic triterpenes from fruits of Rosa sterilis. Journal of Natural Products, 52,
162-166.
Takahashi K, Kawaguchi S, Nishimura KI, Kubota K, Tanabe Y, Takani M. (1974) Studies on the constituents of medical plants:
XIII. Constituents of the pericarps of the capsules of Euscaphis japonica Pax. (1). Chemical & Pharmaceutical Bulletin, 22,
650-653.
De Tommasi N, Rastrelli L, Cumanda J, Speranza G, Pizza C. (1996) Aryl and triterpenic glycosides from Margyriecarpus setosus.
Phytochemistry, 42, 163-167.
Brieskorn CH, Süss HP. (1974) Triterpenoide der Birnen- und Apfelschale. Archiv der Pharmazie, 307, 949-961.
Yagi A, Okamura N, Haraguchi Y, Noda K, Nishioka I. (1978) Studies on the constituents of Zizyphi Fructus. II. Structure of new
p-coumaroylates of maslinic acid. Chemical & Pharmaceutical Bulletin, 26, 3075-3079.
Liu J. (1995) Pharmacology of oleanolic acid and ursolic acid. Journal of Ethnopharmacology, 49, 57-68.
Díaz AM, Abad MJ, Fernández L, Recuero C, Villaescusa L, Silván AM, Bermejo P. (2000) In vitro anti-inflammatory activity of
iridoids and triterpenoid compounds isolated from Phillyrea latifolia L. Biological & Pharmaceutical Bulletin, 23, 1307-1313.
Ringbom T, Segura L, Noreen Y, Perera P, Bohlin L. (1998) Ursolic acid from Plantago major, a selective inhibitor of
cyclooxygenase-2 catalyzed prostaglandin biosynthesis. Journal of Natural Products, 61, 1212-1215.
Martínez-González J, Rodríguez-Rodríguez R, González-Díez M, Rodríguez C, Herrera MD, Ruiz-Gutierrez V, Badimon L. (2008)
Oleanolic acid induces prostacyclin release in human vascular smooth muscle cells through a cyclooxygenase-2-dependent
mechanism. Journal of Nutrition, 138, 443-448.
Hinz B, Woelkart K, Bauer R. (2007) Alkamides from Echinacea inhibit cyclooxygenase-2 activity in human neuroglioma cells.
Biochemical and Biophysical Research Communications, 360, 441-446.
Lee JJ, Jin YR, Lim Y, Yu JY, Kim TJ, Yoo HS, Shin HS, Yun YP. (2007) Oleanolic acid, a pentacyclic triterpenoid, induces rabbit
platelet aggregation through a phospholipase C-calcium dependent signaling pathway. Archives of Pharmacal Research, 30,
210-214.
NPC
2008
Vol. 3
No. 12
1995 - 1997
Natural Product Communications
Preparative Isolation of Antimycobacterial Shoreic Acid
from Cabralea canjerana by High Speed Countercurrent
Chromatography
Gilda G. Leitãoa*, Lisandra F. Abreua, Fernanda N. Costaa, Thiago B. Bruma,
Daniela Fernandes Ramosb, Pedro Eduardo A. Silvab, Maria Cristina S. Lourençoc and
Suzana G. Leitãod
a
Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro,
Av. Carlos Chagas Filho, 373, Bl. H, CCS. Ilha do Fundão, Rio de Janeiro, RJ, Brazil, 21.941-590
b
Universidade Federal do Rio Grande, FURG, Departamento de Patologia,
Laboratório de Micobactérias, Rua General Osório S/N Área Acadêmica da Saúde,
CEP: 96200-190 Rio Grande/RS, Brazil
c
Instituto de Pesquisa Clínica Evandro Chagas, Fiocruz Laboratório de Bacteriologia e
Bioensaios em Micobactérias, Plataforma de Bioensaios II, FIOCRUZ, 21045-900,
Rio de Janeiro, Brazil
d
Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho,
373, Bl. A, CCS. Ilha do Fundão, Rio de Janeiro, RJ, Brazil, 21.941-590
ggleitao@nppn.ufrj.br
Received: July 14th, 2008; Accepted: November 11th, 2008
High speed countercurrent chromatography (HSCCC) was used to isolate the dammarane type triterpene shoreic acid from the
dichloromethane extract of leaves of Cabralea canjerana, which showed activity against Mycobacterium tuberculosis. A
preparative scale-up of the process was also developed.
Keywords: High-speed countercurrent chromatography, HSCCC, Cabralea canjerana, tuberculosis, antimycobacterial
activity, Meliaceae.
The separation of bioactive secondary metabolites
from crude plant extracts has always been a challenge
to natural products researchers and countercurrent
chromatography (CCC) offers many advantages
compared
with
traditional
phytochemical
techniques of purification, especially those where
chromatography with solid supports is used.
The main advantage of CCC is that it is a form of
liquid-liquid chromatography, which does not
use a solid support and, therefore, there can be no
loss of compounds or bioactivity due to interactions
between the solid phase and the target compounds
[1]. In the course of our investigation of
bioactive plants from the Brazilian Atlantic forest,
the dichloromethane extract of leaves of Cabralea
canjerana (Meliaceae) showed antimycobacterial
activity (minimal inhibitory activity, MIC of
OH
H
O
HO2C
Figure 1: Shoreic Acid.
100 μg/mL) and was fractionated by high-speed
countercurrent chromatography (HSCCC). The
success of any HSCCC fractionation is dependent on
the correct choice of solvent system, as this form of
chromatography is based on the partition of solutes
1996 Natural Product Communications Vol. 3 (12) 2008
Table 1: Amount of dichloromethane extract injected into the HSCCC
equipment (sample size) and corresponding amount of shoreic acid
obtained
in
the
isolation
process.
Solvent
system
n-hexane:EtOAc:MeOH:H2O 1:1.5:2.5:1.
a
Sample size (g)
Fraction no.a
Fraction size (mL)
0.8
1
1.5
17-25
14 -24
17-26
4
4
5
Shoreic acid
(mg)
213
321
642
fractions where shoreic acid was eluted.
between two immiscible liquid phases. Compounds
are separated according to their distribution constants
(KD), expressed as the ratio of their concentration in
the stationary phase to their concentration in the
mobile phase [2]. The test-tube partitioning test [3] is
a good way of predicting the distribution ratios of
target compounds to be separated and was used here
for the choice of the appropriate solvent system. As
the bioactive extract of C. canjerana contained
medium polarity compounds, the solvent system nhexane-ethyl acetate-methanol-water was chosen.
Various ratios of the solvents in the biphasic solvent
system were tested: n-hexane-ethyl acetate-methanolwater 1:2:1:2, 1:1.5:2.5:1, 1:2:2:1; 1:2:2.5:0.5,
1:2:2.5:1, (v:v:v:v). The best solvent system is that
when the KD of the target compounds remains around
1. Also, the volumes of upper and lower phases
should be equivalent. When this is not the case (as in
some of the solvent systems tested), the chosen
system should be that where the mobile phase has a
larger volume. Bearing this in mind, the final solvent
system chosen for this fractionation was n-hexaneethyl acetate-methanol-water 1:1.5:2.5:1 (v:v:v:v),
with the upper organic layer acting as the stationary
phase and the lower aqueous layer as the mobile
phase. In this mode of CCC fractionation (reversed
phase mode), the more polar compounds in the
extract elute first. The separation was initially run
with 820 mg of the dichloromethane extract of leaves
of C. canjerana, affording ca. 213 mg of a
dammarane triterpene, shoreic acid (KD approx. 1)
(Fig. 1), the major compound in this extract. The
structure of this compound was confirmed by 1H and
13
C NMR spectroscopic data, which were in
accordance with those in the literature [4]. The
activity of this compound against Mycobacterium
tuberculosis was tested by the MABA as well as the
REMA assays, showing a MIC of 100 μg/mL. Less
polar triterpenes were retained in the stationary
organic phase, which was also fractionated, affording
other triterpenes (structures under investigation).
CCC is particularly useful in the preparative range
(mg to grams) and the time required for preparative
Leitão et al.
separations is no more than a few hours. The
previous separation took about 3 h and consumed
about 1.5 L of solvent. The isolation of shoreic acid
was scaled-up from 820 mg to 1.5 g, using the same
column volume, with good reproducibility (Table 1).
This method proved to be fast, economic and highly
effective in the scaled-up isolation of shoreic acid
directly from a crude plant extract.
Experimental
General procedures: The NMR spectra were
recorded using a Bruker Avance DRX400
spectrometer (Karlsruhe,
Germany)
at
25oC,
1
operating at 400.13 MHz for H and 100.61MHz for
13
C. NMR spectra were recorded in CDCl3 using
TMS as internal standard. TLC analyses were carried
out on pre-coated silica gel plates GF254 from Merck,
and visualized by UV (254 nm) and reaction with
vanillin in sulfuric acid (2%), followed by heating.
For HSCCC separations a P.C. Inc countercurrent
chromatograph equipped with a triple multi-layer coil
equilibrated by a counterweight was used. Solvents
were pumped into the coil with a Rainin Dynamax
Model SD-200 pump. A Rainin Dynamax FC-1
fraction collector was also used.
Plant material and extraction: Leaves of Cabralea
canjerana (Vell.) Mart. were collected in May 2003
at Mata Boa Vista Farm, Levy Gasparian, Rio de
Janeiro State, Brazil. The plant was identified by
Sebastião José Silva Neto, from the Instituto de
Pesquisas Jardim Botânico do Rio de Janeiro, and a
voucher specimen is deposited at the herbarium of
the Federal University of Rio de Janeiro. The dried
and ground leaves (890 g) were exhaustively
extracted with ethanol 96oGL. The resulting dried
ethanolic extract was suspended in water-methanol
70:30 (v:v) and extracted with n-hexane,
dichloromethane, ethyl acetate and n-butanol, in this
order.
Choice of solvent system by test tube experiments:
Small amounts of the dichloromethane extract from
leaves of C. canjerana were dissolved in separate test
tubes containing n-hexane-ethyl acetate-methanolwater 1:2:1:2, 1:2:2.5:0.5, 1:2:2:1; 1:2:2.5:1, and
1:1.5:2.5:1 (v:v:v:v). The test tubes were shaken and
the compounds allowed to partition between the two
phases. Equal aliquots of each phase were spotted
beside each other separately on silica gel TLC plates
and developed with CHCl3:MeOH 6:0.5 (v:v). The
results were visualized by spraying with vanillin in
sulfuric acid (2%), followed by heating. The final
HSCCC isolation of shoreicacid from Cabralea canjerana
Natural Product Communications Vol. 3 (12) 2008 1997
solvent system was set as n-hexane-ethyl acetatemethanol-water 1:1.5:2.5:1.
Antimycobacterial
tests:
Samples
were
simultaneously screened by both microbiology
laboratories (FURG and FIOCRUZ), using the
MABA and REMA bioassays, respectively, as
described previously [5]. Final concentration of
plant extracts/substances was either 200 μg/mL or
100 μg/mL. Media plus bacteria with and without
rifampicin were used as controls. The strain H37Rv
(ATCC - 27294) was used for all methodologies.
MABA
(Microplate
Alamar
Blue
Assay)
susceptibility testing was performed at FIOCRUZ
according to the method described in [6]. The
REMA - Resazurin Microtiter Assay Plate [7]
method was used for the determination of the
antimycobacterial activity at FURG. In brief, the
assay is accomplished in microplates (96 wells) using
resazurin as indicator of cellular viability. Medium
7H9 enriched with
10% OADC was used. The
minimal inhibitory concentration (MIC) was
determined (starting at
200 µg/mL in 1:2 serial
dilutions).
HSCCC separation: The volume of the coil used in
the experiments was 80 mL. The CCC column
was filled with the organic stationary phase of the
solvent system n-hexane-ethyl acetate-methanolwater 1:1.5:2.5:1. After the coil had been filled with
the stationary phase, rotation started and the
aqueous mobile phase was pumped into the head to
tail direction at 2 mL/min until hydrodynamic
equilibrium was achieved. In these conditions, the
organic stationary phase, VS, initially retained in the
CCC column was 67 mL (Sf = 75 %; VM = 20 mL).
Eight hundred and twenty milligrams of the
dichloromethane extract of leaves of C. canjerana
was dissolved in 2.5 mL of each phase of the solvent
system. The 5 mL was injected in the 80 mL coil
using a Rheodyne injection valve at a flow rate of
2 mL/min., 850 rpm; 57 fractions of 4 mL were
collected. Rotation stopped at tube 40 (which
corresponds to KD = 2). Shoreic acid (213 mg) was
obtained from fractions 17 – 25, corresponding to a
KD of approximately 1. Further fractionations were
carried out with 1 g and 1.5 g of the dichloromethane
extract (Table 1). The same 80 mL coil was used,
but the injection volumes were now 10 mL for
both experiments. All other conditions were the
same, except for the volume of fractions collected in
the 1.5 g separation, which was 5 mL.
Acknowledgments - The authors wish to thank
CNPq
(Edital
MCT-CNPq/MS-SCTIE-DECIT
25/2006, Process no. 410475/2006-8), FAPERJ
(E-26/170.442/03) for financial support. We are also
indebted to Centro Nacional de Ressonância
Magnética Nuclear Jiri Jones, UFRJ, Rio de Janeiro,
for the use of NMR equipment. Collaborative work
was performed under the auspices of the
Iberoamerican Program for Science and Technology
(CYTED), Project X.11:PIBATUB.
References
[1]
Alvi KA. (2001) Screening natural products:bioassay-directed isolation of active components by dual mode CCC, Journal of
Liquid Chromatography & Related Technologies, 24, 1765-1773.
[2]
Conway WD, Chadwick LR, Fong HHS, Farnsworth NR, Pauli GF. (2005) Extra column volume in CCC, Journal of Liquid
Chromatography & Related Technologies, 28, 1799-1818.
[3]
Berthod A, Carda-Broch S. (2004) Determination of liquid-liquid partition coefficients by separation methods. Journal of
Chromatography A, 1037, 3-14.
[4]
Roux D, Martin MT, Adeline MT, Sevenet T, Hadi H, Pais M. (1998) Foveolins A and B, dammaranes triterpenes from Aglaia
foveolata. Phytochemistry, 49, 1745-1748.
[5]
Leitão SG, Castro O, Fonseca EN, Julião LS, Tavares ES, Leo RRRT, Vieira RC, Oliveira DR, Leitão GG, Martino V, Sülsen V,
Barbosa YAG, Pinheiro DPG, Silva PEA, Teixeira DF, Neves-Junior I, Lourenço MCS (2006) Screening of Central and South
American plant extracts for antimycobacterial activity by the Alamar Blue test. Brazilian Journal of Pharmacognosy, 16, 6-11.
[6]
Franzblau SG, Witzig RS, Mclaughlin JC, Torres P, Madico G, Hernandez A, Degnan MT, Cook MB, Quenzer VK, Ferguson RM,
Gilman RH. (1998) Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the
Microplate Alamar Blue Assay. Journal of Clinical Microbiology, 36, 362-366.
[7]
Palomino JC, Martin A, Camacho M, Guerra H, Swings J, Portaels F. (2002) Resazurin microtiter assay plate: Simple and
inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy, 46,
2720-2722.
NPC
Natural Product Communications
Antiplasmodial Effects of a few Selected Natural Flavonoids
and their Modulation of Artemisinin Activity
2008
Vol. 3
No. 12
1999 - 2002
Anna Rita Biliaa,*, Anna Rosa Sannellab, Franco Francesco Vincieria, Luigi Messoric, Angela Casinic,
Chiara Gabbiani c, Carlo Severini b and Giancarlo Majori b
a
Department of Pharmaceutical Sciences, University of Florence, Via U. Schiff 6,
50019 Sesto Fiorentino, Florence, Italy
b
Department of Infectious, Parasitic and Immunomediated Diseases, Vector-Borne Diseases and
International Health Section, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
c
Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino,
Florence, Italy
ar.bilia@unifi.it
Received: May 16th, 2008; Accepted: July 26th, 2008
The direct antiplasmodial effects of five structurally-related flavonoids, namely quercetin, rutin, eriodictyol,
eriodictyolchalcone and catechin, were analyzed in vitro on P. falciparum. Notably, all these flavonoids, with the only
exception of rutin, caused relevant inhibition of P. falciparum growth when given at 1 mM concentration. In addition, they
were found to affect greatly the potent antiplasmodial activity of artemisinin, leading to significant additive and even
synergistic effects. In particular, quercetin induced a pronounced synergistic effect. The observed synergisms might be
conveniently exploited to design new and/or more effective combination therapies.
Keywords: Flavonoids, quercetin, rutin, eriodictyol, eriodictyolchalcone and catechin, artemisinin, Plasmodium falciparum,
additive and synergistic effects.
Artemisinin, a sesquiterpene endoperoxide isolated
from Artemisia annua L., is today a leading drug for
the treatment of malaria. It is very effective against
severe forms of malaria and, in particular, against
chloroquine-resistant, primaquine-resistant and some
other life-threatening forms of malaria. It is a drug of
high efficacy and rapid action, with a good
tolerability, leading to clearance of parasites from the
blood within two days [1].
Artemisinin and its semi-synthetic derivatives induce
a very rapid reduction of parasitaemia, starting
almost immediately after administration and are
potent blood schizontocides [2]. These compounds
are also gametocytocidal [3] and, interestingly, block
the early sexual-stage (gametocyte) development
[4,5], but they do not kill the hepatic stages of the
parasite. In addition, they inhibit important
pathophysiological processes, such as cytoadherence
of Plasmodium falciparum-infected erythrocytes to
microvasculature, more effectively
antimalarial drug classes [6].
than
other
The mechanism of action of artemisinin and its
derivatives is still a matter of intense debate [7,8].
Their activity is strictly associated to the presence of
a unique structural feature, the endoperoxide bridge;
their primary target, however, is yet not known.
Meunier and coworkers demonstrated that heme is
alkylated by artemisinin in meso positions and an
extensive characterization of such adducts was
carried out accordingly [9-11]. Heme alkylation was
recently shown to occur also in vivo [12]. Similar
heme adducts are formed in vitro with some
derivatives of artemisinin, as found in our previous
investigation [13]. A significant reactivity of
artemisinin with hemoglobin has been described as
well [14]. However, more recently, it was proposed
that artemisinin and its derivatives most likely act
through a mechanism similar to thapsigargin. This
2000 Natural Product Communications Vol. 3 (12) 2008
Bilia et al.
latter drug potently inhibits PfATP6, a key parasite
Ca2+ transporter with a particular selectivity for the
SERCA of malarial PfATP6 rather than for
mammalian pumps [15].
Within the frame of a larger research project aimed at
evaluating the possible synergism/antagonism of
antimalarial drugs with a variety of widespread
natural products, we have recently shown that green
tea extracts, as well as their main catechin
constituents, markedly inhibit P. falciparum growth
in vitro and potentiate the antimalarial effects of
artemisinin [17]. These results are nicely consistent
with the concept that complex mixtures of
artemisinin with other natural products, as those that
are usually found in the crude plant extracts, are often
more effective than the purified drug itself. The
higher antimalarial potency of the extract compared
to the pure drug has thus been ascribed to the
concomitant and independent biochemical actions of
the other constituents as well as to the occurrence of
appreciable synergistic interactions. A number of
additional experimental and clinical observations
support these ideas.
In view of this background and of the known
antimalarial properties of other flavonoids [18], this
article describes the effects against P. falciparum
3D7 strain, sensitive to chloroquine, of a group of
structurally-related flavonoids, namely the flavonol
quercetin (2), its diglycoside rutin (3), the flavanone
eriodictyol (4), the chalcone eriodictyolchalcone
((E)-3-(3,4-dihydroxy-phenyl)-1-(2,4,6-trihydroxyphenyl)-propenone) (5), and catechin (6). These
flavonoids were assayed either alone or in
combination with artemisinin.
The first step of our investigation was the evaluation
of the effects of the above flavonoids, administered
alone, on P. falciparum growth, using two typical and
well separated drug concentrations, namely 10 μM
and 1 mM, after 48 hours incubation. Growth
inhibition was evaluated by a well established
90
10 m icroM
1mM
80
70
% inhibition
Due to the evident lack of conclusive ideas
concerning the mechanism of action, together with
the fact that artemisinin kills parasites rapidly, but is
also rapidly excreted, the WHO Expert Consultative
Group in 2001 strongly recommended the
development of combination therapies since such
drug associations have the potential to reduce the
appearance of resistance and to be more effective in
eradicating malaria [16].
100
60
50
40
30
20
10
0
Rutin
Quercetin
Catechin Eriodictyol Eriodictyol
chalcone
Figure 1: Percentage inhibitions of 3D7 P. falciparum strains against two
different concentrations (1mM and 10 μM) of flavonoids. The data are
expressed assuming no inhibition of untreated controls.
enzymatic method based on lactate dehydrogenase
activity of P. falciparum (pLDH). Results are
reported in Figure 1. From inspection of Figure 1, it
is evident that all these flavonoids as such, produce
rather modest growth inhibition of Plasmodium 3D7
strain, if given at 10 μM concentration. Indeed, only
quercetin, eryodictiol and its chalcone produced
some measurable effect, causing growth decreases of
~ 5-15% (see Figure 1). In contrast, large inhibitory
effects were seen when flavonoids were given at 1
mM concentration. In this latter case, eryodictiol
chalcone and catechin turned out to be the most
active with 88% and 86% inhibition, respectively.
Using the same 1 mM concentration, eryodictiol
caused 68% inhibition and quercetin 51% inhibition.
In contrast, rutin, the diglycoside of quercetin, had
only a modest activity causing just 10% inhibition. It
is well known that these substances are usually safe
and that their concentrations in many plant extracts
may be very high; this observation implies that even
relatively moderate antimalarial activities, such as
those reported above, may have, nonetheless,
important practical consequences.
Afterwards, possible synergism between artemisinin
and the single flavonoid constituents 2-6 on parasite
growth was investigated by monitoring artemisinin
effects in the presence (or in the absence) of either 10
µM or 1 mM concentrations of the individual
compounds. In this second set of experiments, ART
concentration ranged between 0.625 to 40 nM, as
administration of such concentrations resulted in
partial inhibition of P. falciparum growth (Figure 2).
Notably, we found that the effects of artemisinin,
given at different concentrations, and 10 µM
quercetin are more than additive; this effect becomes
still more evident when quercetin was tested at 1 mM
Antiplasmodial effects of flavonoids
quercetin
1
artemisinin
100
90
In particular, the observed synergism between
quercetin and artemisinin might be conveniently
exploited to design new and/or more effective
combination therapies. Moreover, we believe that
valuable mechanistic information on the individual
antimalarial agents may be extracted from a careful
comparative analysis of the established positive and
negative drug-drug interactions [20].
artemisinin plus quercetin
1
80
% inhibition
Natural Product Communications Vol. 3 (12) 2008 2001
70
60
50
40
30
Experimental
20
10
0
0
0.625 1.25
2.5
5
10
20
40
concentration artemisinin (nM )
Figure 2: Percentage inhibition of 3D7 P. falciparum strain against
quercetin 1 mM, increasing concentrations (from 0.625 nM to 40 nM) of
artemisinin and increasing concentrations (from 0.625 nM to 40 nM) of
artemisinin plus quercetin 1 mM. The data are expressed assuming no
inhibition of untreated controls.
concentration, implying some significant synergism
between these two substances. The largest synergistic
effects are detected for (sublethal) ART doses
ranging from 0.625 to 10 nM (Figure 2). The other
flavonoids did not show evident synergistic effects;
however, sizable additive effects were found when
eriodictyol and eriodictyolchalchone 1 mM were
added to 5, 2.5, 1.25 and 0.625 nM of artemisinin.
These flavonoids at 10 μM concentrations did not
show additive effects.
Overall, the results reported above point out that the
intrinsic antiplasmodial activities of the five tested
flavonoids are rather moderate or even modest.
However, they cannot be neglected in view of the
usually high concentrations of these substances in
plant extracts and of their safety. In addition, we have
shown that all these flavonoids generally manifest
additive effects with artemisinin, leading to a
substantial potentiation of its antimalarial actions.
Most remarkably, considerable synergism was found
in the case of quercetin. This latter finding is not
surprising as quercetin is known to be an inhibitor
of thioredoxin reductase and a prooxidant [19]. As
artemisinin is thought to work through induction of
oxidative stress on P. falciparum, the observed
synergism might be straightforwardly ascribed to
exacerbation of oxidative stress caused by quercetin.
Reagents: Artemisinin was obtained from Sigma
(Milan, Italy). Flavonoids 2-6 were from
Extrasynthese (Genay, France).
Parasite maintenance: Cultures of P. falciparum,
3D7 drug-sensitive to chloroquine (CQR), were
grown in vitro in human red blood cells (O+), as
formerly described [21].
In vitro determination of antimalarial activity of
flavonoids: The selected flavonoids were prepared as
stock solutions in 95% ethanol and then diluted in
complete RPMI medium (containing 10% human
serum). In all tests, the concentration of ethanol was
maintained at 0.02% and did not inhibit the growth of
control cultures. The individual flavonoids were
presented at concentrations of 10 μM and 1 mM.
Growth inhibition was evaluated by a well
established enzymatic method based on lactate
dehydrogenase activity of P. falciparum (pLDH)
[22]. The parasite strain of P. falciparum, 3D7 drugsensitive to chloroquine, was used for the in vitro
tests after culture syncronization by sorbitol [23] .
The effects of flavonoids on cultured parasites were
determined by light microscopy and pLDH activity,
as previously described in detail [24]. Each assay was
performed in triplicate, on 3 separate occasions.
Monitoring flavonoid modulation of artemisinin
activity: P. falciparum growth inhibition was
analysed as a function of added artemisinin, at
sublethal doses, ranging from 0.625 to 40 nM, either
in the presence or absence of either 10 μM or 1 mM
of the flavonoid constituents.
Acknowledgments - The Ente Cassa di Risparmio di
Firenze is gratefully acknowledged for generous
financial support.
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Messori L, Piccioli F, Eitler B, Bergonzi MC, Bilia AR, Vincieri FF. (2003) Spectrophotometric and ESI-MS/HPLC studies reveal
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Messori L, Gabbiani C, Casini A, Siragusa M, Vincieri FF, Bilia AR. (2006) The reaction of artemisinins with hemoglobin: a
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Eckstein-Ludwig U, Webb RJ, Van Goethem ID, East JM, Lee AG, Kimura M, O'Neill PM, Bray PG, Ward SA, Krishna S. (2003)
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NPC
Natural Product Communications
Comparative Analysis of Antimalarial Principles in
Artemisia annua L. Herbal Drugs from East Africa
2008
Vol. 3
No. 12
2003 - 2006
Silvia Lapenna§, Maria Camilla Bergonzi, Franco Francesco Vincieri and Anna Rita Bilia*
Department of Pharmaceutical Sciences, via Ugo Schiff,6, Universit of Florence, Sesto Fiorentino
(FI), Italy 50019
§
Present address: Centro di Ricerche Oncologiche “Fiorentino Lo Vuolo” (CROM), Via Ammiraglio
Bianco, 83013, Mercogliano (AV), Italy
ar.bilia@unifi.it
Received: July 25th, 2008; Accepted: October 23rd, 2008
Malaria mortality continues to increase across the world and represents the most important parasitic disease of man. Artemisia
annua L. (Asteraceae) has been used to treat fevers in China for over two millennia and recently the clinical efficacy of teas
and decoctions derived from this species have been demonstrated, using high artemisinin-yielding plants. Therefore, it is
important to verify the artemisinin levels in local cultivations in areas where malaria is endemic and to assess how different
geographical and climatic conditions may affect the efficacy of traditional treatments. In this study, samples of the aerial parts
of A. annua (ANAMED 3 hybrid) cultivated in three different locations in Burundi were compared for their content of active
principles. Artemisinin levels in the plant materials ranged from 0.20% to 0.35%, while total flavonoid contents ranged from
0.32% to 0.80%.
Keywords: Artemisia annua L., Burundi, different cultivations, artemisinin, flavonoids, HPLC/DAD/MS.
Malaria is one of the oldest and most important lifethreatening parasitic diseases in the tropical regions
of the world. It causes more than 300 million acute
illnesses and at least 1-2.7 million deaths annually.
The majority of these deaths are due to cerebral
malaria and other complications resulting from
malaria-related anaemia, and the cost in human life,
incapacity for work, programs of control and medical
treatments are enormous [1a-1c]. Ninety per cent of
those who die are in Africa, where malaria accounts
for about one in five of all childhood deaths, mainly
children under the age of five in sub-Saharan Africa.
Burundi is among the African countries with a high
incidence of malaria, which is probably the leading
cause of death in this as well as other East African
countries [1d]. The 2000-2001 epidemic of
Plasmodium falciparum in Burundi, with an attack
rate peak of 109% in the northern provinces of
Kayanza, Karuzi, and Ngozi, is well documented
[1e,1f].
Some
non-governmental
organizations
and
international agencies working in Burundi have
offered to introduce to these regions the use of the
plant Artemisia annua L. (sweet or annual
wormwood) because its active constituent,
artemisinin, has proven suitable for the control of
malaria epidemics, including chloroquine- and
quinine-resistant strains, and has shown a low
propensity to induce resistance [2].
A. annua is an annual herb native to the northern
parts of Chahar and Suiyuan provinces in China,
where it is called “quinghao” and has been used as a
remedy for chills and fevers for more than 2000 years
[3,4].
In the most recent literature, clinical trials using teas
or decoctions of A. annua leaves from high
artemisinin-yielding plants (> 0.5% dried weight)
grown in Central Africa, have shown a rapid
disappearance of malaria parasites from the blood of
patients treated with doses corresponding to the
Chinese Pharmacopoeial recommendations [5-7].
Artemisinin plasma concentrations after intake of
these A. annua traditional preparations were lower
2004 Natural Product Communications Vol. 3 (12) 2008
than those achieved with modern artemisinin drugs
used in malaria therapy, but still above 10 μg/L, the
threshold for parasite growth inhibition.
Therefore, the locally grown herb may offer an
additional tool for the control of malaria, especially
in poor countries with scarce or no access to modern
medicines or medical services. However, it is known
that levels of artemisinin in A. annua may vary
considerably (0.01-1.4% plant dry weight) with
growing conditions, particularly climate and
geographical location [8]. Furthermore, A. annua
flavonoids have been shown to enhance the
antiplasmodial activity of artemisinin in vitro [9a-9c].
Therefore, these components should be monitored in
locally prepared A. annua herbal drugs in order to
assess the quality of the drug used to treat malaria.
In this regard, we report herein the HPLC-DAD-MS
analyses of different extracts of the aerial parts of A.
annua, cultivated in malarial-endemic regions in East
Africa. Finally, quantification of the active
constituents in these plant materials offered an
opportunity to assess the possible role of
environmental conditions, such as altitude, in their
biosynthesis, as the same A. annua cultivar, Artemis
[8], was planted in three different locations in
Burundi with distinct geographical and climatic
conditions. The applied HPLC-DAD-MS method
[9d] was specific for the detection of artemisinin and
A. annua flavonoids, even if present only in trace
amounts.
The plant material used was the cultivar Artemis
[8], grown in different locations in Burundi, East
Africa, namely Kyenzi, a prairie at 2300 m altitude
in central Burundi, J1, a wooded area at ca. 1800 m
near the border with Ruanda, and in a field near the
hospital of Bubanza, a city situated in north-west
Burundi, at 950 m altitude.
The n-hexane and dichloromethane extracts of each
herbal drug sample were prepared, because it was
known from previous investigations [9d] that the
extraction efficiency for artemisinin and flavonoids is
maximised using these solvents. Samples were
analysed by HPLC-DAD-MS.
In the extracts, all polymethoxyflavonoids related
to the antimalarial activity, such artemetin,
chrysoplenetin, casticin, cirsilineol and eupatin were
detected. However, as shown in Table 1, yields of
each of these constituents varied significantly among
Lapenna et al.
the different samples. In the n-hexane extracts
artemisinin yields ranged from 10.7% to 5.7% (w/w),
while in the dichloromethane extracts the total
flavonoid content ranged from 12.8% to 5.2%. Table
2 shows the flavonoid variability in the herbal drugs
from different cultivations. Finally, table 3 reports the
artemisinin and total flavonoid levels expressed as
percentage, w/w, of herbal drug. We found that the A.
annua plant materials obtained from the different
cultivation areas of Burundi contained artemisinin the
range 0.20%-0.35% of the herbal drug. These values
are inferior to those reported for the same original
cultivar (Artemis) after professional cultivation
(0.5%-0.75%) [9d].
The lower yields of artemesinin found in the analysed
herbal drugs could be a consequence of altered
agricultural and collection practices operated at the
local sites of production with respect to the
established methods for attainment of high-yielding
plants [10]. Furthermore, the diverse geographical
and climatic conditions, or soil composition of the
different fields in Burundi could be responsible for
the observed yield variation in A. annua antimalarial
constituents. In particular, we noticed that plants
grown at higher altitude (i.e. at 2300 m in the Kyenzi
region) were richer in artemisinin (0.35%, w/w, of
herbal drug) than plants produced at either 1800 m
(0.24%) or 950 m (0.20%), in the J1 area and in
Bubanza, respectively. The beneficial influence of
altitude on artemisinin yields in A. annua plants
cultivated at tropical latitudes had been suggested
previously [10] and the data collected in this work
seems to indicate an analogous relationship. The total
flavonoid percentage in the analysed samples ranged
from 0.80% to 0.32%, w/w, of herbal drug, with the
highest amount present in the samples produced in
the fields at higher altitude.
In conclusion, we analysed samples of A. annua
herbal drug obtained from cultivation at three
different sites in Burundi, East Africa. All three
samples analysed possessed detectable levels of
artemisinin and flavonoids. However, the quantitative
profiles of the antimalarial active compounds varied
significantly in the different samples. Our results
suggest that there could be a correlation between the
content of artemisinin and flavonoids and the altitude
of the growing site in the East African territory.
Further investigations will need to be undertaken in
order to assess the best conditions for growing high
artemisinin and flavonoid-yielding A. annua plants in
these conditions.
Comparative analysis of antimalarial principles in Artemisia annua Natural Product Communications Vol. 3 (12) 2008 2005
Table 1: Extraction yield and percentage of artemisinin and flavonoids in the Artemisia annua extracts (dm: dichloromethane, hx: n-hexane) from the Burundi
cultivations of J1, Bubanza (Bb) and Kyenzi (Ky).
Yield (mg)a
1
2
3
4
5
6
a
J1_dm
J1_hx
Bb_dm
Bb_hx
Ky_dm
Ky_hx
622.0
287.6
762.0
351.8
627.0
329.2
% total flavonoidsb
average value
5.2
2.4
6.1
2.1
12.8
3.3
stand. dev.
0.025
0.040
0.026
0.026
0.042
0.042
% artemisininb
average value
3.9
8.3
2.6
5.7
4.8
10.7
stand. dev.
0.396
1.048
0.355
0.807
0.632
1.261
Dried extract obtained from 10,0 g herbal drug; bPercentage content in the dried extract.
Table 2: Percentage content (w/w) of individual flavonoids in the Artemisia annua herbal drugs from the Burundi cultivations of J1, Bubanza (Bb) and
Kyenzi (Ky). Percentage content in the dried extract (w/w); n=3 samples.
Extract
1
2
3
4
5
6
J1_dm
J1_hx
Bb_dm
Bb_hx
Ky_dm
Ky_hx
% Eupatin
average value stand. dev.
2.49
0.010
0.25
0.003
4.35
0.033
0.19
0.003
2.13
0.027
0.23
0.002
% Cirsilineol
average value stand. dev.
0.06
0.003
0.20
0.003
0.10
0.004
-
Table 3: Percentage content (w/w) of artemisinin and flavonoids in the
Artemisia annua herbal drugs from the Burundi cultivations at J1,
Bubanza (Bb) and Kyenzi (Ky).
J1
Bb
Ky
% total flavonoidsa
average value
stand. dev.
0.32
0.000
0.46
0.000
0.80
0.000
% artemisininb
average value
stand. dev.
0.24
0.029
0.20
0.023
0.35
0.044
a
Percentage of the dichloromethane extract; n=3 samples. bPercentage of
the n-hexane extract; n=3 samples.
Experimental
Chemicals: Artemisinin (98%) was purchased from
Sigma (Sigma-Aldrich S.r.l., Milan, Italy) and rutin
(99.5%) from Merck (Darmstadt, Germany). Solvents
for extraction and HPLC analysis (n-hexane,
dichloromethane, MeOH and acetonitrile) were
HPLC grade and were purchased from Merck. 85%.
Formic acid was purchased from Carlo Erba (Milan,
Italy). Water for HPLC analysis was purified by a
Milli-Qplus system from Millipore (Milford, MA).
Plant material: The seeds of Artemisia annua cv.
Artemis were provided by Anamed in Germany
(seeds “ANAMED A3”). Cuttings of germinated
plants were planted in June 2006 by local farmers at
three different locations in Burundi, namely Kyenzi,
J1 and Bubanza, in areas exposed to sunlight. Leaves
were harvested in November 2006 and dried in the air
at temperatures below 40°C. Samples of dry aerial
parts were sent to Europe in packages of 100 g. Plant
material was cultivated and collected under the
supervision of Paolo Monti, working for the
Artemisia Project of the Medical Foundation for
Africa and supported by the Department for the fight
against malaria of the Ministry of Health of Burundi.
% Casticin and Chrysoplenetin
average value
stand. dev.
2.52
0.018
1.95
0.032
7.60
0.050
2.63
0.036
3.55
0.041
1.58
0.055
% Artemetin
average value stand. dev.
0.13
0.006
0.25
0.003
0.62
0.016
0.47
0.007
0.28
0.011
0.27
0.010
Preparation of extracts: Artemisia annua dried aerial
parts were cut into small pieces and the leaves were
separated from the branches and stems. Only leaves
and flowering tops (herbal drug) were used for the
analyses (Figure 1). Samples of 10 g herbal drug
were exhaustively extracted at room temperature by
maceration with 100 mL of either n-hexane or
dichloromethane for 72 h. The eluates were
subsequently dried under vacuum to obtain the crude
extracts.
HPLC sample preparation: Five mg or each
n-hexane or dichloromethane dried extracts were
accurately weighed and suspended in acetonitrile
(1.0 mL) in a volumetric flask. The suspensions were
sonicated for 20 min, then filtered through a
cartridge-type
filtration
unit
with
a
polytetrafluoroethylene (PTFE) membrane (d = 13
mm, porosity 0.45 µm, Lida manufacturing Corp.,
Kenosha, WI) and immediately injected.
HPLC analyses: Artemisinin and flavonoid
(Figure 2) contents of the dried extracts were
determined
by
high
performance
liquid
chromatography (HPLC) coupled with mass
spectrometer (MS), according to [9d]. HPLC
analyses were performed using a HP 1100 Liquid
Chromatograph (Agilent Technologies, Palo Alto,
CA, USA) equipped with a HP 1040 Diode Array
Detector (DAD), an automatic injector, an auto
sampler and a column oven, and managed by a HP
9000 workstation (Agilent Technologies, Palo Alto,
CA, USA). The HPLC system was interfaced with a
HP
1100 MSD
API-electrospray
(Agilent
Technologies).
2006 Natural Product Communications Vol. 3 (12) 2008
Separations were performed on a reversed-phase
column of Purospher® Star RP-18, namely Hibar®
pre-packed column RT (250 x 4.6 mm), with particle
size 5 µm (Merck, Darmstadt, Germany). Eluents
were: water adjusted to pH 3.2 with formic acid (A)
and acetonitrile (B). The mobile phase was isocratic
50% A and 50% B for 15 min, following by gradient
from 50% to 100% B in 5 min, at a flow-rate of 1.0
mL/min. The system was operated with an oven
temperature at 26oC; the injection volume was 20 μL.
Chromatograms were recorded both at 350 nm to
detect the flavonoids and at 210 nm to detect
artemisinin and any other constituents, with a peak
threshold of 0.1 mAu. The following mass
spectrometry operating conditions were used: gas
temperature 350°C at a flow-rate of 10 L/min,
nebulizer pressure 30 psi, quadrupole temperature
30°C, and capillary voltage 3500 V. Full scan spectra
from m/z 100 to 800 in the positive ion mode were
recorded (scan time 1 s).
Calibration and quantitative analyses: Calibration
curves of artemisinin and rutin were obtained from
Lapenna et al.
stock solutions of each standard in acetonitrile
(artemisinin 0.170 mg/mL and rutin 0.092 mg/mL)
and used to quantify the artemisinin and flavonoid
contents, respectively, in the samples of A. annua
extracts. For artemisinin calibration, HPLC injection
volumes of 10, 15, 20, 25 and 30 μL of the
artemisinin stock solution were used and the peak
areas in the MS were recorded. For flavonoid
calibration, HPLC injection volumes of 2, 4, 6, and 8
μL of the rutin stock solution were used and the peak
areas in the UV chromatogram at 350 nm were
measured. Linear regression was used to establish the
calibration curve. Each HPLC sample of A. annua
extract was injected three times and the artemisinin
and flavonoid contents were calculated on the basis
of the peak areas in the mass spectra (for artemisinin)
or in the UV spectra at 350 nm (for the flavonoids).
Acknowledgments - We are thankful to Mr Paolo
Monti, responsible for the Artemisia Project in
Burundi, for sending us the plant materials. The
financial support of Ente Cassa di Risparmio di
Firenze is gratefully acknowledged.
References
[1]
(a) Trigg PI, Kondrachine AV. (1998) Commentary: malaria control in the 1990s. Bulletin of the World Health Organization, 76,
11-16; (b) World Health Organization. (2000) Severe falciparum malaria. Transactions of the Royal Society of Tropical Medicine
and Hygiene, 94, 1-90; (c) Winstanley P. (2000) Chemotherapy for P. falciparum malaria: the armoury, the problems and the
prospects. Parasitology Today, 16, 146-153; (d) Guthmann J-P, Bonnet M, Ahoua L, Dantoine F, Balkan S, Van Herp M, Tamrat
A, Legros D, Brown V, Checchi F. (2007) Death rates from malaria epidemics, Burundi and Ethiopia. Emerging Infectious
Diseases, 13, 140-143; (e) Checchi F, Cox J, Balkan S, Tamrat A, Priotto G, Alberti KP, Guthmann J-P. (2006) Malaria epidemics
and interventions, Kenya, Burundi, southern Sudan, and Ethiopia, 1999-2004. Emerging Infectious Diseases, 12, 1477-1485; (f)
World Health Organization. (2001) Weekly Epidemiological Report, 5th January 2001.
[2]
Van Agtmael MA, Eggelte TA, Van Boxtel CJ. (1999) Artemisinin drugs in the treatment of malaria: from medicinal herb to
registered medication. Trends in Pharmacological Sciences, 20, 199–204.
[3]
Hien T, White N. (1993) Qinghaosu. Lancet, 341, 603-608.
[4]
Klayman DL. (1985) Qinghaosu (artemisinin): an antimalarial drug from China. Science, 228, 1049-1055.
[5]
Mueller MS, Karhagomba IB, Hirt HM, Wemakor E. (2000) The potential of Artemisia annua L. as a locally produced remedy for
malaria in the tropics: agricultural, chemical and clinical aspects. Journal of Ethnopharmacology, 73, 487–494.
[6]
Mueller MS, Runyambo N, Wagner I, Borrmann S, Dietz K, Heide L. (2004) Randomized controlled trial of a traditional
preparation of Artemisia annua L. in the treatment of malaria. Transactions of the Royal Society of Tropical Medicine and Hygiene,
98, 318-321.
[7]
Räth K, Taxis K, Walz G, Gleiter CH, Li S-M, Heide L. (2004) Pharmacokinetic study of artemisinin after oral intake of a
traditional preparation of Artemisia annua L. American Journal of Tropical Medicine and Hygiene, 70, 128-132.
[8]
Delabays N. (1997) Biologie de la reproduction chez l’Artemisia annua L. et génétique de la production en artémisinin.
Contribution à la domestication et à l'amélioration génétique de l'espèce. Université de Lausanne.
[9]
(a) Bilia AR, Lazari D, Messori L, Taglioli V, Temperini C, Vincieri FF. (2002) Simple and rapid physico-chemical methods to
examine action of antimalarial drugs with hemin. Its application to Artemisia annua constituents. Life Sciences, 70, 769-778; (b)
Elford B, Roberts M, Phillipson J, Wilson R. (1987) Potentiation of the antimalarial activity of Qinghaosu by methoxylated
flavones. Transactions of the Royal Society of Tropical Medicine and Hygiene, 81, 434-436; (c) Liu KCS, Yang SL, Roberts MF,
Elford BC, Phillipson JD. (1989) The contribution of flavonoids to the antimalarial activity of Artemisia annua. Planta Medica, 55,
654-655; (d) Bilia AR, De Malgalhaes PM, Bergonzi MC, Vincieri FF. (2006) Simultaneous analysis of artemisinin and flavonoids
of several extracts of Artemisia annua L. obtained from a commercial sample and a selected cultivar. Phytomedicine, 13, 487-493.
[10]
Ferreira JFS, Laughlin JC, Delabays N, De Magalhães P.M. (2005) Cultivation and genetics of Artemisia annua L. for increased
production of the antimalarial artemisinin. Plant Genetic Resources-Characterization and Utilization, 3, 206-229.
NPC
Natural Product Communications
In vitro Apoptotic Bioactivity of Flavonoids from
Astragalus verrucosus Moris
2008
Vol. 3
No. 12
2007 - 2012
Joseph A. Buhagiara, Alessandra Bertolib*, Marie Therese Camilleri-Podestac and Luisa Pistellib
a
Department of Biology, Faculty of Science, University of Malta, MSIDA MSD 06, Malta
b
Dipartimento di Chimica Bioorganica e Biofarmacia (DCBB), University of Pisa, Via Bonanno 33,
Pisa I-56126, Italy
c
Department of Anatomy, Faculty of Medicine and Surgery, University of Malta, MSIDA MSD 06, Malta
bertoli@farm.unipi.it
Received: July 30th, 2008; Accepted: November 17th, 2008
Six aglycone flavonoids and their corresponding glycosides: genistein and genistin, quercetin and rutin, apigenin and apigenin
7-O-β-D-(6-p-coumaroyl) glucoside, as well as the aglycone daidzein isolated from Astragalus verrucosus Moris, were tested
for their apoptosis-inducing potential. In vitro techniques that monitor bioactivity through morphological and biochemical
changes were carried out on HCT116 (human colon carcinoma) and MCF7 (human Caucasian breast adenocarcinoma) cancer
cell lines. Dose-dependent cytotoxic effects were monitored through changes in mitochondrial dehydrogenase activity using
the standard MTT assay. The median inhibitory concentration (GI50) determined from the dose-response curves showed that
the aglycones apigenin and quercetin were the most bioactive (low GI50), whilst daidzein and genistein, which had not been
previously tested on these cell lines, showed a smaller cytotoxic effect (high GI50). The remaining flavonoids, mostly
glycosides, showed negligible cytotoxicity. Morphological changes were monitored by microscopic observation with a
photographic record. Results showed important hallmarks of apoptosis, including cell rounding with reduction of cell volume,
small condensed nuclei, membrane blebbing and formation of apoptotic bodies.
Keywords: Flavonoids, aglycones, glycosides, apoptosis, cancer cell lines, HCT116, MCF7.
Astragalus verrucosus Moris, a very rare perennial
endemic plant belonging to the family Fabaceae
(Leguminosae), grows in a restricted area of southwestern Sardinia, Italy [1]. The genus Astragalus is
well known in Chinese folk medicine because its
properties are somewhat similar to those of the more
expensive herb ginseng (Panax ginseng) and have
been used as a substitute for this species [2]. The
roots of various Astragalus species have been used to
increase body resistance against viral infections, to
re-balance the immune system and for their tonic
action on the liver. Extracts from Astragalus species
have been used as antiperspirant, diuretic and general
tonic agents. The roots have been applied in the
treatment of diabetes mellitus, nephritis, and
bacterial infection, as well as against leukaemia and
uterine cancer [3,4]. Phytochemical studies of A.
verrucosus have shown the presence of several
classes of flavonoids, such as flavonols, isoflavones,
and flavones, both as aglycones and glycosides
(Table 1). Over 6000 different flavonoids
(aglycones) have been identified to date, and this
figure increases considerably if their corresponding
glycosides are included [6-8]. Flavonoids are plant
secondary metabolites, especially widespread in the
plant kingdom and ubiquitous in photosynthesising
cells.
Flavonoids represent important dietary
constituents because of their antioxidant activity.
They are accumulated in different plant parts
including fruits, vegetables, nuts, seeds, and flowers,
as well as in products derived from them, such as
propolis, honey and tea [5].
Flavonoids are well known for their different
pharmacological properties, including: anti-allergic,
immunoregulatory, antioxidant, anti-inflammatory,
hypotensive, antibacterial, antifungal, antiviral,
cytotoxic and osteogenic activities [6-10].
Preparations containing these compounds have been
used to treat different diseases for centuries. Some
are borrowed directly from nature, as for example
propolis, which has been used since antiquity to heal
2008 Natural Product Communications Vol. 3 (12) 2008
Table 1: Typical flavonoids of Astragalus verrucosus
FLAVONES
apigenin
apigenin 7-O-β-D(6”-p-coumaroyl)glucoside
FLAVONOLS
kaempferol 3-O-rutinoside
kaempferol 3-robinobioside
rutin
quercetin
ISOFLAVONES
daidzein
daidzin
ononin
calycosin
pseudobaptigenin
genistein
genistin
pratensein
REF.
[19]
[19]
[19]
[24]
[15]
[4]
[19]
[19]
[25]
[17]
[15]
[19]
[19]
[19]
sores and wounds. Several flavonoid-rich foods
(such as quercetin-rich apples) have been reported to
lower the risk of different types of cancer, including
hepatoma, lung and breast cancer [8]. Flavonoids
such as quercetin have been reported to inhibit the
growth of various cell lines derived from human
cancers [6,9]. They have been shown to be selective
cytotoxic agents to a range of cancer cell lines,
including Jurkat, PC-3, colon 205 and HepG2. The
mode of cell death in the human promyelocytic
leukaemia HL-60 cell line treated with the
structurally related flavonoids apigenin, quercetin,
myricetin, and kaempferol has been established as
being apoptotic through the release of cytochrome C,
as well as caspase-9 and caspase-3 into the cytosol
[7,11]. Other flavonoids have been studied and
shown to be capable of inducing cell death by
apoptosis [12]. Interestingly, quercetin was found to
restore apoptosis sensitivity in cell lines usually
resistant to apoptosis [13]. Terminal differentiation
has also been identified as another possible
mechanism of flavonoid action and, therefore, these
compounds can serve as potential chemotherapeutic
agents [14]. Most plant-derived natural products are
often produced as a mixture of related compounds
that have a synergistic mode of action, either among
themselves or with other compounds, and flavonoids
are no exception. The synergistic effects of three
isoflavones: genistein, biochanin-A and daidzein
have also been demonstrated in cancer cell lines
through cell growth inhibition, cell cycle changes,
and induction of apoptosis. Enhanced expression of
pro-apoptotic caspase 3 and downregulation of antiapoptotic Bcl-2 was also demonstrated [8,11,14].
Of increasing interest also is the potential role of
flavonoids in reducing the problems of drug
resistance to antimicrobials and in cancer therapy.
This has also been extensively investigated and holds
Buhagiar et al.
some promise, especially with the increasing
problems associated with β-lactamase resistance and
MRSA [6,7].
This current work was addressed to test the bioactive
potential and apoptosis-inducing activity of some
characteristic flavonoids isolated from A. verrucosus
on HCT-116 and MCF-7 tumor cell lines. These
compounds include two aglycones (daidzein and
genistein) that have not been previously tested on
these cell lines. Although work on the induction of
apoptosis in cancer cells by flavonoids appears to be
gaining momentum, there is still insufficient
published data on the action of the various
flavonoids on different cell types. The NCI database
gives the average GI50 values for its 50 cancer cell
line panel as 27 μM for apigenin and 59 μM for
quercetin. Though the values for the individual cell
lines are not available, these average values confirm
the trend that we obtained for the same test
compounds in this work. This research has also
confirmed the trend in the published literature that
the aglycone flavonoids are generally more potent
than their corresponding glycosides. What seems to
be omitted in the literature is a reference to the
paradoxical increase in cell proliferation on exposure
to low concentrations of the aglycones, something
which has also been consistently observed with
another group of natural compounds, the monosesqui- and di-terpenoids. Flavonoid molecules have
structural similarities and comparable molecular
sizes to the diterpenoids. Like diterpenoids, aglycone
flavonoid molecules are planar and have been shown
capable of interacting, to various degrees, with the
phospholipid bilayer. The degree of flavonoid
interaction has been shown to vary according to
whether hydroxyl groups are present or absent, the
number of hydroxyl groups and the position of their
attachment to the A and B ring [5,15]. Apart from
the chemical interactions of the side groups, the
small size of the aglycones allows for a greater
chance of interacting deep within the inner layers of
the phospholipid bilayer and brings about changes in
membrane fluidity. Such changes in membrane
fluidity as a result of flavonoid interactions have
been reported both for prokaryotic and eukaryotic
cells, as a result of which cells lose important
capabilities. For instance, flavonoid action on the
inner membrane of Gram-positive bacteria has been
reported to lead to dissipation of membrane potential
electrochemical gradient with consequent reduction
of ATP synthesis, membrane transport and motility.
In other experiments, the action of flavonoids leads
Bioactivity of flavonoids from Astragalus verrucosus
Natural Product Communications Vol. 3 (12) 2008 2009
to increased permeability of the inner membrane
with loss of important cell constituents such as K+
ions [5]. A similar effect is reported for a number of
flavonoids, including quercetin; this has been shown
to interact with the membrane of the mitochondria,
decreasing its fluidity and, as a result, either
inhibiting the respiratory chain or inducing
membrane permeability transition [15]. The latter is
equivalent to a state where the mitochondria lose
their function and release apoptosis-inducing factors
that lead to cell death by apoptosis.
membranes by natural products such as flavonoids
could explain why apoptosis results when cancer
cells are treated with these compounds. A change in
membrane fluidity (especially of the mitochondrial
membrane) could lead to the release of pro-apoptotic
signals from the mitochondria, such as cytochrome c,
and trigger the apoptotic cascade which eventually
results in cell death.
Experimental
Extraction, flavonoid isolation and identification:
Astragalus verrucosus dried aerial parts were
extracted in a Soxhlet apparatus using different
solvents with increasing polarity (n-hexane,
chloroform, and methanol) [4]. The methanolic
extract was purified by gel permeation
(Sephadex LH-20, MeOH-Water 8:2) and medium
pressure liquid chromatographic steps (SiO2 RP-9,
MeOH-water 7:3). Seven flavonoids: apigenin,
apigenin
7-O-β-D-(6p-coumaroyl)
glucoside,
quercetin, rutin, daidzein, genistein and genistin
were isolated and identified by NMR and MS
experiments, and by comparison with authentic
samples and literature data [4,18,19]. Table 1 gives
additional data on other flavonoids isolated from A.
verrucosus [21,22].
The aglycone flavonoids that have been shown to be
the most bioactive represent relatively small
molecules that can easily interact with the cell
membrane. Conversely, since the glycoside
flavonoids represent a bulkier molecule, of which
only a part can interact with the phospholipid
bilayers, they are unable to interact deeply enough
inside the membrane to cause drastic changes in
fluidity. This hypothesis could neatly explain the
differences observed in the action of different
aglycone and glycoside flavonoids where for the
glycosides, a relatively flat dose-response curve was
maintained, even at high concentrations, indicating
that interaction does not increase, even under an
appreciable concentration gradient.
Cell lines and medium: Two adherent cell lines,
namely human colon carcinoma cell line HCT 116
and human Caucasian breast adenocarcinoma cell
line MCF 7, were obtained from ECACC Porton
Down, Salisbury, UK. RPMI-1640 medium with 2
mM L-glutamine and 1mM sodium pyruvate (Gibco
BRL, Life Technologies) was supplemented with
10% fetal bovine serum (Gibco) and 25 IU/mL
penicillin G and 25 µL/mL streptomycin (PenStrep,
Gibco). All cell lines were kept in exponential
growth phase by twice weekly subculture in T25 cell
culture flasks (Nunc, Kampstrum, Denmark) in 6 mL
of medium using a split ratio of 1:5.
The relevance of the role of aglycone flavonoids in
the induction of apoptosis in cancer cells has to be
considered in the light of emerging research as to the
role of mitochondria in cancer. Formerly, it was
thought that cancer cells are predominantly glycolytic
because their mitochondria were defective and
incapable of generating the vast quantities of ATP
needed to sustain growth of cancer cells. However,
the evidence points to fully functional mitochondria
that are pushed into an inactive state so as to suppress
the apoptotic cascades that are normally initiated in
mitochondria [16,17]. Thus, this perturbation of
Table 2: Results of apoptotic activity in HCT116 and MCF7 cancer cell lines for the flavonoids isolated from the methanolic extract of Astragalus
verrucosus. All median inhibitory concentration values (GI50) shown are an average of at least three replicates.
COMPOUNDS
Quercitin
Daidzein
Apigenin
Genistein
Rutin
apigenin -7-O-β-D-(6’’-pcoumaroyl)-glucoside
genistin
replicate
1
9.0
76.8
5.9
33.2
>100
>100
HCT116
2
20.9
>100
3.3
>100
>100
>100
(GI50)
3
28.8
>100
2.8
53.9
>100
>100
Average (µg/mL)
19.6
76.8 (>100)
4.0
43.5 (>100)
>100
>100
>100
>100
>100
>100
replicate
1
>100
66.2
5.7
60.2
>100
>100
MCF7 (GI50)
2
3
47.2
9.8
>100
>100
4.2
4.0
37.8
64.9
>100
>100
>100
>100
>100
>100
>100
Average (µg/mL)
28.5 (>100)
66.2 (>100)
4.6
54.3
>100
>100
>100
2010 Natural Product Communications Vol. 3 (12) 2008
Buhagiar et al.
Figure 1: Dose-response curves for 48 h exposure of HCT116 (top) and MCF7 (bottom) cancer cell lines treated with various concentrations of aglycone
flavonoids (left) and their corresponding glycosides (right). All values are plotted as a percentage of control absorbance values. Absorbance values were
measured at 550 nm using the standard MTT photometric assay.
MTT assay: Cell cultures in exponential growth
phase were trypsinised with 0.25% trypsin in EDTA,
and after a viable cell count diluted to give a seeding
density of 5000 cells per 180 µL of culture medium.
The cells were then plated in flat-bottomed 96-well
micro titer plates (Nunc) and incubated overnight at
37°C in a 5% CO2 humidified atmosphere. A time
zero (T0) reference plate was also set up to check cell
viability at the point of first drug exposure. The
appropriate dilutions of the flavonoids in culture
medium were prepared from a stock solution of
40µg/µL in DMSO and 20µL aliquots of the drug
were added per well to give a final concentration
range from 0.01 μg/mL to 100 μg/mL, plus solvent
controls.
Plates were further incubated for a
maximum of 44 h, with inspection at regular
intervals to check growth progress. Cell viability was
determined by the addition of a 50 µL aliquot of
MTT (2.0 mg/mL) per well, and insoluble formazan
crystals allowed to develop by incubating for a
further 4 h. Formazan was dissolved by adding 125
mL of solubilization Sorensen’s buffer (DMSO :
glycine 4:1) to each well and the plates were agitated
for 5 minutes. The absorbances were read using a
microplate reader (BioTech ELx 808) at 550 nm and
650 nm wavelength. Dose-response curves were
generated using a Delta soft 3 software package and
in combination with the T0 value, the median growth
inhibition (GI50) was determined for three
independent trials. Absorbance values derived from
the MTT assays were used to generate dose-response
curves, from which the median inhibitory
concentration (GI50) was calculated. The average
GI50 values (μg/mL) for three independent trials are
shown in Table 2. The cell growth was also plotted
as a percentage of the control, and typical graphs are
shown in Figure 1.
Study of morphological changes: These were
restricted to a photographic record of the changes
that were observed in the overall cell structure when
visualised under an inverted microscope at high
magnification. Photos were taken after 24 and 44 h
exposure before adding the MTT. The doseresponse curves for the aglycone flavonoids were
typical of cytotoxic drugs with GI50 values in the
range of 4-76 µg/mL. The highest activity of the
aglycones was for apigenin. Furthermore, its GI50
values are very similar in the two cell lines tested
with an average GI50 for HCT116 of 4.0 µg/mL and
Bioactivity of flavonoids from Astragalus verrucosus
Natural Product Communications Vol. 3 (12) 2008 2011
Figure 2: HCT 116 and MCF7 cells after 48 h treatment with quercitin (10 and 50 µg/mL) with solvent controls (top).
for MCF7 of 4.6 µg/mL. The bioactivity of quercetin
was the next best with an average GI50 value in the 20
µg/mL range for HCT116 and 28.5 µg/mL for MCF7.
The largest GI50 values for the aglycones and,
therefore, the least bioactive were for genistein and
daidzein. Conversely, the dose-response curves of
their corresponding glycosides were in most cases
rather flat and showed large GI50s in excess of 100
µg/mL, indicating that there is little cytotoxic effect
on these two cell lines. The poor bioactivity of the
glycoside derivatives was also corroborated from the
morphological observations. One interesting feature
of the dose-response curves of some of the aglycones
and glycoside flavonoids was that at very low
concentrations, a paradoxical stimulation of growth
occurred, sometimes reaching 130% of the control.
This is usually followed by a steep decrease in
activity as the concentration increases. This
paradoxical increase in cell activity for very low
concentrations has been reported for other natural
products including mono- and diterpenoids and may
be related to their as yet unexplained mode of action
[23].
Morphological changes resulting from the exposure
of cells to different types of flavonoids and different
time periods demonstrated that the cells undergo
apoptosis with increasing concentration and with
increased exposure time. Figure 2 shows the two
cell lines treated at the two concentrations (10 and 50
µg/mL) with solvent controls after 48 h exposure,
although the full exposure included a concentration
range from 1-100 µg/mL. These morphological
changes were clearly demonstrated under the high
power objective (x400) without the need of further
staining since these changes were comparable to
those obtained in previous works [23]. The major
hallmarks were the loss of cellular extensions and
substrate contact resulting in the formation of round,
free floating cells, some with extensive membrane
blebbing and formation of apoptotic bodies. A
reduction in overall cell volume resulting from
cytoskeletal disruption and the formation of a small
pycnotic nucleus from DNA condensation were also
noted. For the active flavonoids, changes such as
partial loss of contact with substrate and rounding off
were initially observed at 10 µg/mL after 24 hour
2012 Natural Product Communications Vol. 3 (12) 2008
exposure, progressing to pronounced hallmarks of
apoptosis, such as membrane blebbing and
nuclear condensation as concentration increased. At
Buhagiar et al.
50 µg/mL the cells were completely spherical and
lost all attachment to the substrate after the same
exposure time.
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Cui B, Sakai Y, Takeshita T, Kinjo J, Nohara T. (1992) Four new oleanene dervatives from the seeds of Astragalus complanatus.
Chemical & Pharmaceutical Bulletin, 40, 136-138.
[23]
Buhagiar JA, Camilleri Podesta MT, Wilson AP, Micallef MJ, Ali S. (2000) The induction of apoptosis in human melanoma, breast
and ovarian cancer cell lines using an essential oil extract from the conifer Tetraclinis articulata. Anticancer Research, 19,
5435-5444.
[24]
Nawwar MAM, El-Mousallamy AMD, Barakat HH, Buddrush J, Linscheia M. (1989) Flavonoid lactates from leaves of
Marrubium vulgare. Phytochemistry, 28, 3201-3206.
[25]
Rao LJM, Kumari GNN, Rao NSP (1985) Flavonoid glycosides from Anisomeles ovata. Journal of Natural Products, 48, 150-151.
NPC
Natural Product Communications
Qualitative Profile and Quantitative Determination of
Flavonoids from Crocus sativus L. Petals by LC-MS/MS
2008
Vol. 3
No. 12
2013 - 2016
Paola Montoroa, Carlo I. G. Tuberosob, Mariateresa Maldinia, Paolo Cabrasb and Cosimo Pizzaa
a
Dipartimento di Scienze Farmaceutiche, Università di Salerno, Via Ponte Don Melillo,
84084 Fisciano (SA), Italy
b
Dipartimento di Tossicologia, Università di Cagliari, via Ospedale 72, 09124 Cagliari, Italy
pmontoro@unisa.it
Received: June 10th, 2008; Accepted: October 16th, 2008
From the methanolic extract of Crocus sativus petals nine known flavonoids have been isolated and identified, including
glycosidic derivatives of quercetin and kaempferol as major compounds (1-2), and their methoxylated and acetylated
derivatives. Additionally, LC-ESI-MS qualitative and LC-ESI-MS/MS quantitative studies of the major compounds of the
methanolic extract were performed. The high content of glycosylated flavonoids could give value to C. sativus petals, which
are a waste product in the production of the spice saffron.
Keywords: Crocus sativus, LC-ESI-MS, LC-MS/MS, quercetin, kaempferol.
Saffron, the dried stigmas of Crocus sativus L.
(Iridaceae), is a very expensive spice, and is used as a
herbal medicine, for food coloring and as a flavoring
agent in different parts of the world [1a]. Saffron
originally grew in India, Iran, Europe and other
countries, and it has been successfully cultivated in
different countries, including Europe. The most
important European production areas are Sardinia
and Abbruzzo (Italy), Castile-la Mancha (Spain) and
western Macedonia (Greece). For saffron, the flowers
are cultivated to produce the stigmas. After
harvesting, the flowers are subjected to a delicate
treatment which will give the saffron spice. This
procedure is performed the same day of harvest. One
of the most traditional procedures is the separation of
petals from stigmas. A large amount of petals is
discarded for obtaining a small amount of stigmas.
Earlier investigation reported the isolation of
carotenoids, crocins, monoterpenoids and flavonoids
from the stigma, leaves, petals and pollen of C.
sativus [1b,1c].
Considering the large amount of petals that are waste
products in this production procedure, we have
undertaken a study to recover chemical compounds
from this matrix. Only one previous paper concerning
petals is reported in the literature, oriented towards
the biological activity of some new phenols isolated
from this part of the flowers [2a].
Flavonoids are polyphenolic compounds with
antioxidant properties [2b,2c]. Several studies have
shown that a high intake of flavonoids has been
correlated to a decrease in heart disease; in addition,
biological effects of this class of compounds have
been described in several in vivo and in vitro studies
[3a-3d]. These compounds are largely used for
chemotaxonomic surveys of plant genera and
families because of their almost ubiquitous presence
in vascular plants and of their structural variety.
A phytochemical study was undertaken with the aim
of identifying and determining quantitatively the
major compounds in the petals. In this study
flavonoid compounds were isolated; the major
compounds were glycosidic derivatives of quercetin
and kaempferol, including their methoxylated and
acetylated derivatives (1-9).
The study provided a method to define the flavonoids
fingerprint by LC-ESI- IT MS/MS (liquid chromatography electrospray tandem mass spectrometry with
ion trap analyser), with full scan acquisition in data
dependent scan mode, and a method to quantify
the content of the major compounds by LC-ESI- TQ
2014 Natural Product Communications Vol. 3 (12) 2008
MS/MS (liquid chromatography electrospray tandem
mass spectrometry with triple quadrupole analyser)
by using a MRM (multiple reaction monitoring)
mode. The quantitative method, performed by using
internal and external standards, was validated in
agreement with EMEA note guidance on validation
of analytical methods [4].
By using tandem in time mass spectrometry it was
possible to reveal the compounds on the basis of their
specific fragmentation. The specific fragmentation
pattern was used for developing the selective MS/MS
method for the two major compounds (1, 2). The use
of tandem mass spectrometry in quantification of
secondary metabolites from plants has led to
sensitive, selective and robust methods for quality
control and standardisation of plant extracts [5a-5c].
Phytochemical investigation of the methanolic extract
of C. sativus led to the isolation of flavonoid
compounds 1-9. The compounds, identified by
comparing their NMR data with those reported
in the literature, were quercetin-3,7-di-O-β-Dglucopyranoside (1) [6], kaempferol-3,7-di-O-β-Dglucopyranoside (2) [2a], isorhamnetin-3,7-di-O-β-Dglucopyranoside
(3)
[7], kaempferol-3-O-β-Dglucopyranoside (4) [2a], quercetin-3-O-β-Dglucopyranoside (5) [8], isorhamnetin-3-O-β-Dglucopyranoside (6) [9], kaempferol-7-O-β-Dglucopyranoside (7) [2a], kaempferol-3-O-β-D-(2-Oβ-D-glucosyl) glucopyranoside (8) [2a], and
kaempferol-3-O-β-D-(2-O-β-D-6-O-acetylglucosyl)
glucopyranoside (9) [2a]. The high content of
glycosylated flavonoids could give value to C.
sativus petals, which are a waste product in the
production of saffron spice.
In order to realise a qualitative analysis for the
flavonoid derivatives in C. sativus extracts, MS
experiments were performed by using an LC-MS
system equipped with an ESI source and an Ion Trap
analyser. Positive ion electrospray LC/MS analysis,
total ion current (TIC) profile and reconstructed
ion chromatograms (RICs) of extract are shown in
Figure 1. Flavonoid derivatives were identified by
comparing retention times and m/z values in the
total ion current chromatogram with those of the
selected standards, obtained in the isolation step.
Reconstructed ion chromatograms were obtained
for each value of m/z observed for the standard
compounds (m/z 627, 1; m/z 611, 2; m/z 641, 3; m/z
449, 4, 7; m/z 465, 5; m/z 479, 6; and m/z 653, 9) in
order to improve the separation and identification of
Montoro et al.
single compounds. The chromatographic profile
obtained in Total Ion Current revealed two very
major compounds, respectively 1 and 2, in C. sativus
extracts, whereas the other compounds were present
in lesser amounts. Quantitative analysis was focused
on compounds 1 and 2, which could potentially be
recovered from discarded petals as economic
secondary products.
In order to obtain an accurate quantitative
determination of compounds 1-2, a quantitative LCMS/MS method was developed. Since the sugar loss
is the most representative fragmentation for
glycosidic flavonoids, ESI-MS/MS analyses were
recorded for the two major compounds by using an
LC-MS equipped with an ESI source and a triple
quadrupole analyser. Analyses were performed by
direct introduction and both the spectra showed the
characteristic fragment resulting from sugar loss.
Thus an MRM method was developed. Transition
from the specific pseudomolecular ion [M+H]+ of
each compound to the corresponding aglycon ion
[A+H]+ was selected to monitor the flavone
glycosides in C. sativus using as internal standard
(I.S.), rutin (m/z 611) (I.S.).
Compound 1: precursor ion m/z 627.0, product ion
m/z 303.0, collision energy 30%; compound 2:
precursor ion m/z 611.0, product ion m/z 287.0,
collision energy 30%; I.S. precursor ion m/z 611.0,
product ion m/z 303.0, collision energy 30%.
The MRM analyses of C. sativus methanolic extract,
spiked with I.S., contained the peaks corresponding
to the compounds under investigation, with
appreciable intensity for quantitative purposes.
Validation of the method was realised in agreement
with EMEA note guidance on validation of analytical
methods [4].
Validation of the LC/MS/MS method included intra
and inter-day precision and accuracy studies on three
days. Accuracy and precisions were calculated by
analysing five samples of each extract (MeOH and
water). Standard deviations calculated in this assay
were < 7% for the two compounds under
investigation. Specificity is usually reported as the
non interference with other substances detected in the
region of interest; the present method, developed by
using a characteristic fragmentation of flavone
glycosides 1, 2, was specific with no other peak
interfering in the MS/MS detection mode.
Flavonoids from Crocus sativus petals
1.13
10
5
26.45
3.22
0
8.65
12.8
21.9
17.18
25.55
36.09 37.8
Base Peak
ESI Full ms
[ 200.00-1000.00]
Equipment: Semi-preparative HPLC was performed
using an Agilent 1100 series chromatograph,
equipped with a G-1312 binary pump, a G-1328A
rheodyne injector and a G-1365B multiple wave
detector. The column was an RP C18 column
μ-bondapak 300 mm x 7.6 mm (Waters, Milford,
MA). HPLC–ESI-MS analysis was performed using a
Thermo Finnigan Spectra System HPLC coupled
with an LCQ Deca ion trap. Chromatography was
performed on an RP C18 column Symmetry Shield
(Waters, Milford, MA). HPLC–ES-MS/MS for
quantitative analysis was performed on a 1100 HPLC
system (Agilent, Palo Alto, CA) coupled with a triple
quadrupole
instrument
[API2000
(Applied
Biosystems, Foster City, CA, USA)]. The instrument
was used in the tandem MS mode, with multiple
reaction monitoring (MRM).
1.13
10
1
21.9
5
20.32
0
1.13
10
m/z= 626.50-627.50
26.45
5
0
1.16
10
21.1
5
m/z= 640.50-641.50
26.4
1.13
10
m/z= 610.50-611.50
2
3
0
7
5
4
m/z= 448.50-449.50
19.5
0
1.13
10
5
5
0
21.9
m/z= 464.50-465.50
20.29
0.99
10
21.1
5
10
30.73
Natural Product Communications Vol. 3 (12) 2008 2015
m/z= 478.50-479.50
6
1.16
0
m/z= 652.50-653.50
9
26.3
5
0
0
5
10
15
20
Time (min)
25
30
35
Figure 1. HPLC-MS qualitative analysis
Table 1: Quantitative results and quantification data.
n
1
2
mg/g
MeOH ext.
41.8±6.2
31.1±4.7
mg/g
water ext.
8.1±0.2
5.5±0.1
mg/g
petals
27.6±6.0
20.2±4.6
Calibration
equation
y = 0.183x-0.74
y = 0.094x-0.39
r2
0.998
0.997
The calibration graphs, obtained by plotting area ratio
between external and internal standards versus the
known concentration of each compound, were linear
in the range of 1-100 μg mL-1 for all compounds.
Correlation values (r2) are reported in Table 1.
Five aliquots of methanol and water extracts,
respectively, obtained from C. sativus were analysed
in order to quantify the flavonoid contents. Table 1
reports the quantitative data for compounds 1-2,
regression of calibration curves, and quantitative
values. Quantitative analyses results confirmed that
waste petals of C. sativus can represent an interesting
source of such phenolic compounds, with respect to
the high content showed.
Experimental
Reagent and standards: Standards of pure
compounds 1-2 were isolated in our laboratory and
their structures were elucidated by NMR
spectroscopy (Bruker DRX-600). Each standard was
dissolved in methanol. HPLC grade methanol
(MeOH), acetonitrile (ACN) and trifluoroacetic acid
(TFA) were purchased from Merck (Merck KGaA,
Darmstadt, Germany). HPLC grade water (18mΩ)
was prepared using a Millipore Milli-Q purification
system (Millipore Corp., Bedford, MA). The reagents
used for the extractions, of analytical grade, were
purchased from Carlo Erba (Rodano, Italy). Column
chromatography was performed over Sephadex LH20 (Pharmacia, Uppsala, Sweden).
LC-ESI-MS and LC-ESI-MS/MS analysis: The
mass spectrometer was operated in the positive ion
mode under the following conditions: capillary
voltage 3 V, spray voltage 5 kV, tube lens offset 40
V, capillary temperature 260°C, and sheath gas
(nitrogen) flow rate 60 arbitrary units. Data were
acquired in the MS1 scanning mode with scan ranges
of 200 – 1000 m/z: the maximum injection time was
50 ms, and the number of microscans was 3. In order
to tune the LCQ for flavonoids, the voltages on the
lenses were optimised using the TunePlus function of
the Xcalibur software in the positive ion mode whilst
infusing a standard solution of quercetin (1 μg mL-1
in methanol) at a flow rate of 3 μL min-1. For
qualitative LC-ESI-MS analysis, a gradient elution
was performed on a RP C18 column Symmetry
shield (Waters, Milford, MA), 2mm x 150 mm, by
using a mobile phase A represented by water
acidified with trifluoroacetic acid (0.05%) and a
mobile phase B represented by water: acetonitrile
50:50 acidified with trifluoroacetic acid (0.05%). The
gradient started from 20% of eluent B, to achieve the
33% of solvent B in 18 min. After another 12 min the
percentage of B became 40%, and remained at this
value for 10 min, then became 50%. The flow (250
μL min-1) generated by chromatographic separation
was directly injected into the electrospray ion source.
MS were acquired and elaborated using the software
provided by the manufacturer.
For quantitative LC-ESI-MS/MS a gradient elution
was performed by using a mobile phase A
represented by water acidified with trifluoroacetic
acid (0.05%) and a mobile phase B represented
by acetonitrile acidified with trifluoroacetic acid
2016 Natural Product Communications Vol. 3 (12) 2008
Montoro et al.
Plant material: Petals of C. sativus, discarded by
production companies of saffron spice, were
collected in Sardinia (Italy) in November 2004.
lyophilized to give 3 g of crude extract. Part of the
methanolic extract (3.7 g) was fractionated initially
on a 100 cm×5.0 cm Sephadex LH-20 column, using
CH3OH as mobile phase, and 56 fractions (10 mL
each) were obtained. Fractions 28-29 (24.5 mg) (a),
19-20 (250 mg) (b), 31-33 (31.4 mg) (c) and 36-40
(40.4 mg) (d) were chromatographed by HPLC-UV.
The mobile phase was a linear gradient of
water/acetonitrile (50:50) with trifluoroacetic acid
0.1% (solvent B) in water acidified with
trifluoroacetic acid 0.1% (solvent A), at a flow rate of
2.000 mL min-1. From sample a, compounds 1 (3.3
mg, tR=26.8), 2 (2.9 mg, tR=29.7) and 3 (1 mg,
tR=28.7) were obtained; from sample c, compounds 4
(1.6 mg, tR=36.4) and 6 (0.9 mg, tR=37.8); and from
sample d, compounds 5 (0.6 mg, tR=32) and 7 (1.1
mg, tR=36.9) using the following gradient: 0 min,
10% B, 0-5 min, 10-20% B, 5-25 min, 20-40% B;
25-40 min, 40% B; 40-50 min, 40-70% B, 50-60 min,
70-100% B.
Extraction and isolation: Dried and powdered petals
(23 g) of Crocus sativus were extracted for 3 days,
3 times, at room temperature with methanol to give
9.7 g of crude methanolic extract. This was extracted
for one day with water. The filtered extract was
From sample b, compounds 8 (5.6 mg, tR=29.8) and 9
(1.2 mg, tR=37.9) were obtained using the following
gradient: 0 min, 20% B; 0-18 min, 20-33% B; 18-30
min, 33-40% B; 30-40 min, 40% B; 40-45 min,
40-85% B; and 45-55 min, 85-100%.
(0.05%). The gradient started from 5% of eluent B,
remained isocratic for 5 minutes, to achieve the 80%
of solvent B in 15 min. The flow (250 μL min-1)
generated by chromatographic separation was
directly injected into the electrospray ion source.
The mass spectrometer was operated in the positive
ion mode under the following conditions:
declustering potential 200 eV, focusing potential
155 eV, entrance potential 10 eV, collision energy
30 eV, and collision cell exit potential 15 eV, ion
spray voltage 5000, temperature 250°C. The
instrument was used in the tandem MS mode with
multiple reaction monitoring (MRM). For all
flavonoids analyzed, the selected fragmentation
reaction was the loss of the glycoside moiety.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
(a) Fernandez, JA. (2004) Biology, biotechnology and biomedicine of saffron. Recent Research Developments in Plant Science, 2
127-159; (b) Rios JL, Recio MC, Giner RM, Manets S. (1996) An update review of saffron and its active constituents. Phytotheapy
Research, 10, 189-193; (c) Xi L, Qian Z. (2006) Pharmacological properties of crocetin and crocin (digentiobiosyl esters of
crocetin) from saffron, Natural Product Communications, 1, 65-75.
(a) Li CY, Lee EJ, Wu TS. (2004) Antityrosinase principles and constituents of the petals of Crocus sativus. Journal of Natural
Products, 67, 437-440; (b) Rice-Evans CA, Miller NJ, Paganga G. (1997) Antioxidant properties of phenolic compounds. Trends in
Plant Science, 2, 152-159.
(a) Cao G, Sofic E, Prior RL. (1997) Antioxidant and pro-oxidant behavior of flavonoids: structure-activity relationships. Free
Radical Biology and Medicine, 22, 749-760; (b) Rice-Evans CA, Miller NJ, Panaga G. (1996) Structure-antioxidant activity
relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine, 20, 933-956; (c) Lien EJ, Ren S, Bui H, Wang
R. (1999) Quantitative structure-activity relationship analysis of phenolic antioxidants. Free Radical Biology and Medicine, 26,
285-294; (d) Montoro P, Braca A, Pizza C, De Tommasi N. (2005) Structure-antioxidant activity relationships of flavonoids
isolated from different plant species. Food Chemistry, 92, 349-355.
ICH Q2B, International Conference on Harmonisation, London, 1995.
(a) Li X, Xiong Z, Ying X, Cui L, Zhu W, Li F. (2006) A rapid ultra-performance liquid chromatography-electrospray ionization
tandem mass spectrometric method for the qualitative and quantitative analysis of the constituents of the flower of Trollius
ledibouri Reichb. Analytica Chimica Acta, 580, 170-180; (b) Benavides A, Montoro P, Bassarello C, Piacente S, Pizza C. (2006)
Catechin derivatives in Jatropha macrantha stems: Characterisation and LC/ESI/MS/MS quali-quantitative analysis. Journal of
Pharmaceutical and Biomedical Analysis, 40, 639-647; (c) Montoro P, Tuberoso CIG, Perrone A, Piacente S, Cabras P, Pizza C.
(2006) Characterisation by liquid chromatography -electrospray tandem mass spectrometry of anthocyanins in extracts of Myrtus
communis L. berries used for the preparation of myrtle liqueur. Journal of Chromatography A, 1112, 232-240.
Merfort I, Wendisch D. (1992) New flavonoid glycosides from Arnicae flos DAB 9. Planta Medica, 58, 355-357.
Grouiller A, Pacheco H. (1967) Flavonoid compounds. VI. Nuclear magnetic resonance spectra of some O-glucosylflavonals,
their aglycons and three synthetic mono- and di- O –glucosylflavanones. Bulletin de la Societe Chimique de France, 6, 1938-1943.
Nawwar MAM, El-Mousallamy AMD, Barakat HH. (1989) Quercetin 3-glycosides from the leaves of Solanum nigrum.
Phytochemistry, 28, 1755-1757.
Senatore F, D'Agostino M, Dini I. (2000) Flavonoid glycosides of Barbarea vulgaris L. (Brassicaceae). Journal of Agricultural
and Food Chemistry, 48, 2659-2662.
NPC
Natural Product Communications
HPLC/DAD/ESI-MS Analysis of Non-volatile Constituents of
Three Brazilian Chemotypes of Lippia alba (Mill.) N. E. Brown
2008
Vol. 3
No. 12
2017 - 2020
Patrícia Timóteoa*, Anastasia Kariotia, Suzana G. Leitãob, Franco Francesco Vincieri a and
Anna Rita Biliaa
a
Department of Pharmaceutical Sciences, University of Florence, Via Ugo Schiff, 6, 50019,
Sesto Fiorentino (FI), Italy
b
Faculty of Pharmacy, Federal University of Rio de Janeiro, Bloco A, 2° andar, Ilha do Fundão,
21941-590, Rio de Janeiro, Brazil
patimoteo@gmail.com
Received: August 27th, 2008; Accepted: November 3rd, 2008
Aqueous preparations and ethanolic extracts of three Brazilian chemotypes of Lippia alba (Mill.) N.E. Brown (Verbenaceae)
were investigated for the chemical variability of their non volatile constituents by HPLC/DAD/ESI-MS analysis. The main
class of compounds in all the extracts investigated was phenylpropanoids, mainly verbascoside, followed by the flavonoids
tricin-7-O-diglucuronide (present in Lippia alba chemotypes 2 and 3), luteolin-7-O-glucuronide (present in L. alba chemotype
1) and mono-and di-O-glucuronic derivatives of apigenin and tricin. Four iridoids, geniposidic acid, theveside, 8-epi-loganin
and mussaenoside were also identified.
Keywords: Lippia alba, Verbenaceae, Brazilian chemotypes, flavonoid glycosides, phenylpropanoid glycosides, iridoids.
Lippia alba (Mill.) N. E. Brown (Verbenaceae), is a
very common herb in Brazil, where is popularly
known as ‘Erva-cidreira’. It occurs in all regions of
the country as a spontaneous or cultivated plant. In
Brazilian folk medicine infusions and decoctions of
its leaves are traditionally used as a sedative and for
gastrointestinal disorders. Ethanolic preparations are
also popularly used for fever, coughs and asthma
[1,2].
There are approximately eighteen chemotypes of
L. alba, mainly based on the composition of their
essential oil. Brazilian chemotypes have been
classified by Matos and coworkers according to the
percentage of citral (chemotype 1), carvone
(chemotype 2) and linalool (chemotype 3). However,
the last is hardly found in Brazil as a spontaneous
plant [3,4].
Several studies have been carried out regarding the
characterization of volatile compounds, but to the
best of our knowledge nothing has been reported
yet concerning the distribution of non-volatile
constituents by chemotypes, which would be useful
for pharmacological purposes. Since the chemical
variability of the three Brazilian chemotypes seems to
be important for both the volatile and non-volatile
constituents, the present study aimed to investigate
the polar extracts of these chemotypes in order to
establish differences between them.
Infusions, decoctions and ethanolic extracts of the
leaves of three chemotypes of L. alba were
investigated according to their traditional uses [4,5].
The samples were submitted to HPLC/DAD/ESI-MS
analyses in order to obtain a complete
characterization of these preparations. In Fig. 1 the
HPLC/DAD profiles of the infusions of the three
chemotypes of L. alba at different wavelengths are
presented: 240 nm (monitoring of iridoids), 330 nm
(monitoring of phenylpropanoids) and 350 nm
(monitoring of flavonoids).
Results are shown in Table 1, where the identified
constituents in the extracts of the three chemotypes
are presented.
2018 Natural Product Communications Vol. 3 (12) 2008
Timóteo et al.
Table 1: Identified compounds in the three extracts of the Lippia alba
compared by chemotypes.
Cpds
1
2
3
4
5
6
7
8
9
10
11
12
13
LA1I
+
+
+
t
t
t
+
+
+
+
+
+
LA2I LA3I LA1D LA2D LA3D LA1E LA2E LA3E
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
t
+
t
t
t
t
t
t
+
t
+
+
t
+
+
t
t
t
t
t
t
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
t
t
t
-
Cpds: compounds; LA: Lippia alba; 1, 2, 3: chemotypes: 1 (citral), 2
(carvone), 3 (linalool);
I: infusion; D: decoction; E: ethanolic extract.
Iridoid compounds: (1) theveside; (2) geniposidic acid; (3) 8-epi-loganin;
(4) mussaenoside; Flavonoid derivatives: (5) apigenin-7-O-glucuronide;
(6) apigenin-7-O-diglucuronide; (7) tricin-7-O-diglucuronide; (8) tricin-7O-glucuronide:
(9)
luteolin-7-O-glucuronide;
Phenylpropanoid
derivatives: (10) calceolarioside E; (11) verbascoside; (12)
isoverbascoside; (13) β-OH-acteoside diastereisomers; t: traces; +
presence of the compound; -: absence of compound.
The constituents of the extracts from these
chemotypes belong mainly to three classes of
compounds: iridoids, flavonoids and phenylpropanoids. This finding is in good agreement with
literature data [5]. Four iridoids, theveside (1),
geniposidic acid (2), 8-epi-loganin (3) and
mussaenoside (4), were detected in infusions and
decoctions of all chemotypes studied, in accord with
literature data [6-8].
Concerning the phenylpropanoid content of the
samples, four phenylpropanoids were detected,
namely, calceolarioside E (10), verbascoside (11),
isoverbascoside
(12)
(isobaric
isomer
of
verbascoside), and a pair of diastereoisomeric forms
of β-OH-acteoside (13). Compound 13 was detected
in infusions and decoctions, but not in the ethanolic
extracts. Phenylpropanoids 10-12 have been
previously reported in the literature for L. alba
whereas β-OH-acteoside was detected for the first
Table 2: Positive and negative MS fragmentation and Uv-vis absorption data of the compounds detected in the three chemotypes of Lippia alba.
tR (min)
UV-vis
Λmax
(MeOH)
eV
MW
ESI-MS+ m/z (rel. intensity, %)
ESI-MS- m/z (rel. intensity, %)
3.8
236
80
390
413 (100) [M+Na+]+; 429 (20) [M+K+] +
389 (100) [M-H]-; 227 (20)
[Aglycone-H]-
4.4
238
80
390
413 (72) [M+Na+]+; 429 (24) [M+K+] +
389 (100) [M-H]-; 227 (14) [Aglycone-H]-
Geniposidic acid (3)
4.8
234
80
374
397 (100) [M+Na+]+; 413 (25) [M+K+] +;
787 (5) [M+K+] +
373 (73) [M-H]-; 211 (30) [Aglycone-H]-
Mussaenoside (4)
12.6
238
80
+ +
+ +
390 413 (50) [M+Na ] ; 429 (36) [M+K ] ; 229
(20) [Aglycone +H]+
Apigenin 7-O-glucuronide (5)
25.1
342
180
446
447 (100) [M+H]+; 271 (12)
[Aglycone+H]+
445 (35) [M-H]-; 285 (100) [Aglycone-H]-
Apigenin 7-O- diglucuronide (6)
18.4
350
180
622
623 (100) [M+H]+
621 (45) [M-H]-; 269 (38) [Aglycone-H]-; 351 (100)
[M-H-Aglycone]- or [(2x glucuronic acid)-H]-
Tricin 7-O-diglucuronide (7)
19.4
350
180
682
683 (100) [M+H]+
681 (78) [M-H]-; 329 (12) [Aglycone-H]-; 351 (100)
[(2x glucuronic acid)-H]-
Tricin 7-O-glucuronide (8)
25.6
348
180
506
507 (100) [M+H]+; 331 (10) [Aglycone]+
505 (100) [M-Hx]-; 329 (12) [Aglycone-H]-; 351
(100) [(2 glucuronic acid)-H]-
21.8
350
180
462
463 (100) [M+H]+
461 (35) [M-H]-; 285 (100) [Aglycone-H]-
21.2
330
180
610
-
609 (100) [M-H]-; 447 (20) [M-H-162]-
Verbascoside (11)
22.3
330
180
624
-
623 (100) [M-H]-; 461 (9) [M-H-caffeic acid]-; 161
(20) [caffeic- acid-H2O-H]-
Isoverbascoside (12)
23.9
330
180
624
-
623 (100) [M-H]-; 461 (9) [M-H-caffeic acid]-; 161
(20) [caffeic- acid-H2O-H]-
16.7/17.3
330
180
640
-
Compound
Theveside (1)
8-epi-loganin (2)
Luteolin 7-O-glucuronide (9)
Calceolarioside E (10)
β-OH-acteoside diastereoisomers (13)
389 (100) [M-H]-; 227 (18) [Aglycone-H]-
639 (100) [M-H]-; 621 (30) [M-H-H2O]-;
459 (16) [M-H-caffeic acid- H2O]-; 179 (20)
[caffeic acid-H]-
Constituents of three Brazilian chemotypes of Lippia alba
mAU
240 nm
120
100
80
60
40
20
0
1
3
0
mAU
30
25
20
15
10
5
0
-5
Infusion Lippia alba chemotype I: LA1I
2
5
4
10
15
20
330 nm
13 13
0
mAU
20
15
10
5
0
-5
5
0
15
15
20
9
5
240 nm
40
30
20
10
0
10
2
1
mAU
40
30
20
10
0
mAU 0
15
25
30
min
5 8
20
25
5
15
20
25
30
min
11
13 13
5
10
10
15
20
25
6
10
15
30 min
5 8
20
25
30 min
240nm
Infusion Lippia alba chemotype III: LA3I
mAU
2
70
60
50
40
30
20
10
0
1
1
2
50
40
30
20
10
0
350nm
5
1
1
20
6
0
5
10
15
2
30 min
11
12
10
13 13
0
mAU
1
330nm
40
30
20
10
0
To the best of our knowledge, this is the first time
that β-OH-verbascoside (13) has been found in L.
alba, and tricin-7-O-diglucuronide (7) in the Lippia
genus.
For each chemotype, only small differences were
observed among the three preparations. However,
differences were detected between the chemotypes,
especially regarding the flavonoids (Figure 1). These
differences should be taken into consideration when
pharmacological studies are carried out.
4
3
0
mAU
Table 2 reports the UV-vis absorptions and MS
fragmentation profiles of all the compounds detected
in L. alba infusions, decoctions and ethanolic
extracts.
12
7
5
In addition, a pair of diastereoisomeric forms of
β-OH-acteoside (13) was detected. These are an
analogue of verbascoside with a hydroxyl group in
the β position: 3,4-dihydroxyl-phenyl-ethanol moiety.
min
4
10
330 nm
0
30
11
350 nm
60
50
40
30
20
10
0
min
Infusion Lippia alba chemotype II: LA2I
3
0
30
12
10
350 nm
mAU
25
11
Natural Product Communications Vol. 3 (12) 2008 2019
25
30
min
7
5 8
20
25
30
Figure 1: Chromatographic profiles at 240, 330 and 350 nm of a
representative samples (infusions) of the three chemotypes of Lippia alba
considered. Compound 7 was only detected in chemotypes 2 and 3, and
compound 9 in chemotype 1.
time in this plant. Its presence was confirmed by,
comparing UV, MS and retention time data with
those reported previously for this compound [9,10].
Finally, five flavonoids, namely apigenin-7-Omonoglucuronide (5), apigenin-7-O-diglucuronide
(5); tricin-7-O-monoglucuronide (8) tricin-7-Odiglucuronide (7) and luteolin-7-O-glucuronide (9),
were detected, in accordance with literature data
[11,12].
As shown in Table 1, and as expected, the main class
of compounds of L. alba was phenylpropanoids,
represented mainly by verbascoside (11), followed by
the flavonoids tricin-7-O-diglucuronide (7) and
luteolin-7-O-glucuronide (9). From the results
described above, the flavonoids 7 and 9 could be
used as markers to distinguish the three Brazilian
chemotypes of L. alba, since 7 seems to be present
only in chemotypes 2 and 3, and compound 9, only in
chemotype 1.
Experimental
Chemicals: All solvents used were HPLC grade;
CH3CN and MeOH for HPLC were purchased from
Merck (Darmstadt, Germany). Formic acid (85 %)
was provided by Carlo Erba (Milan, Italy). Water
was purified by a Milli-Qplus system from Millipore
(Milford, MA, USA). A 0.45 mm PTFE membrane
filter was purchased from Waters Co. (Milford, MA).
Plant material: Dried leaves of three cultivated
chemotypes of L. alba (Mill.) N. E. Brown, were
collected in 2008 from the Herbarium CESJ (Federal
University of Juiz de Fora), MG, Brazil.
Herbal preparations: Infusions: Dried powered
leaves of each chemotype of L. alba (1 g) were
extracted with 20 mL of boiling water. The mixture
was cooled for 20 min and then filtered. Decoctions:
Dried powered leaves of three chemotypes of the
plant (1 g) were put in 20 mL of water and both were
boiled for 2 min before filtration. Ethanolic extracts:
Dried powered leaves of each chemotype of L. alba
(1 g) were extracted three times successively with
EtOH for 24 h each time. All preparations were
lyophilized and then freeze-dried. For HPLC-DADMS analysis, the samples were obtained by
dissolving and filtering the solid residues (1 mg
exactly weighed) in 1 mL of MeOH.
2020 Natural Product Communications Vol. 3 (12) 2008
General experimental procedures: HPLC/DAD/ESIMS analysis was performed on a HP 1100 L
instrument with DAD and managed by a HP 9000
workstation interfaced with a HP 1100 MSD APIUSA. The column used was a Varian Polaris TM
C18-E (250 x 4.6 mm i.d., 5 μm maintained at 26°C.
Eluents were H2O adjusted to pH 3.2 with formic
acid (A), and acetonitrile (B). A multi-step linear
gradient was applied from 87% A to 85% in 10 min;
in 10 min to 75% B and a plateau for 3 min; 2 min to
95% CH3CN and a final plateau for 3 min. Total time
analysis was 28 min, and equilibration time 10 min.
Flow rate: 0.8 mL min -1. Oven temperature: 26°C.
UV-vis spectra were registered between 220-500 nm
and the chromatographic profiles were registered at
240, 330 and 350 nm. Mass spectrometry conditions
were optimized in order to achieve sensitive values:
negative and positive ionisation mode, scan spectra
from m/z 100 to 800, gas temperature: 350°C,
Timóteo et al.
nitrogen flow rate: 10L min-1, nebulizer pressure 30
psi, quadrupole temperature: 30°C, capillary voltage:
3500 V. Applied fragmentors range: 80-180 V.
Identification of constituents was carried out by
HPLC/DAD/ESI-MS analysis. UV-vis and mass
spectra of the peaks were compared with those of
authentic standards, previously isolated compounds
and literature data.
Acknowledgments – Supported by the Programme
Alβan, the European Union Programme of High
Level Scholarships for Latin America, scholarship
no: (E06M104124BR). The authors would also like
to thank Professor Lyderson F. Viccini and his
undergraduate students (Laboratory of Genetic,
Department of Biology/ICB), University of Juiz de
Fora (MG, Brazil) for providing the L. alba
chemotypes.
References
[1]
Lorenzi H, Matos FJA. (2002) Plantas Medicinais no Brasil: Nativase Exóticas Cultivadas. Instituto Plantarum (Ed.) São Paulo,
Brazil, 488-489.
[2]
Matos FJA. (1996) As ervas cidreiras do Nordeste do Brasil. Estudo de três quimiotipos de Lippia alba (Mill.) N.E.Brown
(Verbenaceae) Parte II – Farmacoquimica. Revista Brasileira de Farmácia, 77, 137-141.
[3]
Tavares ES, Julião LS, Lopes L, Bizzo HR, Lage CLS, Leitão SG. (2005). Análise do óleo essencial de folhas de três quimiotipos
de Lippia alba (Mill.) N. E. Brown (Verbenaceae) cultivados em condições semelhantes. Revista Brasileira de Farmacognosia. 15,
1-15.
[4]
Gilbert B, Ferreira JP, Alves LF. (2005) Monografias de Plantas Medicinais Brasileiras e Aclimatadas. Abifito (Ed), Curitiba,
61-77.
[5]
Hennebelle T, Sahpaz S, Joseph H, Bailleul F. (2008) Ethnopharmacology of Lippia alba. Journal of Ethnopharmacology, 116,
211-222.
[6]
Hennebelle T, Sahpaz Joseph H, Bailleul F. (2006) Phenolics and iridoids of Lippia alba. Natural Product Communications, 1,
727-730.
[7]
Alipieva K, Kokubun T, Taskova R, Evstatieva L, Handjieva N. (2007) LC-ESI-MS analysis of iridoid glycosides in Lamium
species. Biochemical Systematics and Ecology, 35, 17-22.
[8]
Filho JGS, Duringer JM, Uchoa, DEA, Xavier HS, Filho JMB, Filho, RB. (2007) Distribution of iridoid glycosides in plants from
the genus Lippia (Verbenaceae): An investigation of Lippia alba (Mill.) N.E. Brown. Natural Product Communications, 2,
715-716.
[9]
Bilia AR, Giomi M, Innocenti M, Gallori S, Vincieri FF. (2008) HPLC-DAD-ESI-MS analysis of the constituents of aqueous
preparations of verbena and lemon verbena and evaluation of the antioxidant activity Journal of Pharmaceutical and Biomedical
Analysis, 46, 463-470.
[10]
Li L, Tsao R, Yang R, Liu C, Young JC, Zhu H. (2008) Isolation and purification of phenylethanoid glycosides from Cistanche
deserticola by high-speed counter-current chromatography. Food Chemistry, 108, 702-710.
[11]
Kowalska I, Stochmal A, Kapusta I, Janda B, Pizza C, Piacente S, Oleszek W. (2007) Flavonoids from barrel medic (Medicago
truncatula) aerial parts. Journal of Agricultural and Food Chemistry, 55, 2645-2652.
[12]
Stochmal A, Simonet AM, Macias FA, Oleszek W. (2001) Alfalfa (Medicago sativa L.) flavonoids. 2. Tricin and chrysoeriol
glycosides from aerial parts. Journal of Agricultural and Food Chemistry, 49, 5310-5314.
NPC
Natural Product Communications
Optimization and Validation of an HPLC–Method for
Quality Control of Pueraria lobata Root
2008
Vol. 3
No. 12
2021 - 2027
Lidiya Bebrevska, Mart Theunis, Arnold Vlietinck, Luc Pieters and Sandra Apers*
Laboratory of Pharmacognosy and Pharmaceutical Analysis, Department of Pharmaceutical Sciences,
University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium
sandra.apers@ua.ac.be
Received: June 25th, 2008; Accepted: October 19th, 2008
Pueraria lobata Willd. (Fabaceae) is a widely used medicinal plant, known as “Kudzu” and “Ge” in Japanese and Chinese
traditional medicine, respectively. P. lobata is a rich source of isoflavones with phytoestrogenic properties, and its commercial
use is widespread. In this study the optimization and validation of an HPLC-method for quality control of Pueraria root
material is presented. By means of this analytical method the major individual constituents, i.e. the isoflavone 8-C-glycosides
hydroxypuerarin, puerarin, methoxypuerarin and xylosylpuerarin, the 7-O-glycosides daidzin and genistin, and the aglycones
daidzein and genistein could be quantified. The extraction procedure and the extraction solvent composition were optimized in
order to ensure the exhaustive extraction of the plant material. The HPLC - conditions were evaluated and optimized for the
exact quantification of all individual compounds. The HPLC analysis was carried out on a Agilent XDB RP C18 (150 x 4.6)
column eluted with a binary system consisting of water (+ 0.01% formic acid) and methanol (+ 0.01% formic acid) using a
linear gradient; detection was at 262 nm. Daidzin, daidzein, genistin and genistein were used as external standards. Due to the
great difference in content between the C-glycosides on the one hand, and daidzin, genistin and the aglycones on the other, two
separate HPLC runs were necessary for a complete analysis. The final method was fully validated according to the ICH
guidelines in terms of linearity, precision and accuracy. Linear relationships for the responses of all four standards were
proven. The method was shown to be repeatable for the individual compounds, i.e. RSD% between days values were within 2 to
7.5%. The accuracy of the method was demonstrated to be equal to 100% by recovery experiments. These validation results
demonstrated the suitability of the method for the precise and accurate determination of Pueraria root isoflavones.
Keywords: HPLC, quality control, method validation, Pueraria lobata root, isoflavones, phytoestrogenes.
Pueraria lobata (Willd.) Ohwi (Fabaceae) is a
medicinal plant widely used in the Oriental systems
of traditional medicine. It is known as “Kudzu” in
Japanese and as “Ge” in Chinese traditional
medicine. It is recommended for the alleviation of
different conditions and the curative properties of this
drug are known to be due to its isoflavonoid
phytoestrogen content. The major isoflavonoids
present in Pueraria root are the 8-C-glycosides
puerarin, hydroxypuerarin, methoxypuerarin and
xylosylpuerarin, the 7-O-glycosides daidzin and
genistin, and the aglycones daidzein and genistein
(Fig. 1). Numerous commercial preparations of
isoflavone extracts from the root of the plant are
marketed as dietary supplements with different health
promoting properties. Great attention has been paid to
the analysis of isoflavones because of their
importance to human health, especially in age-related
and hormone-dependent diseases, such as cancer,
cardiovascular diseases, menopausal symptoms, and
osteoporosis [1].
Several reports have been published on the
determination of Pueraria isoflavones in different
matrices, i.e. Pueraria plant preparations, blood
and urine samples, using HPLC or capillary
electrophoresis, but these methods have yielded
variable recoveries and/or were designed to
determine only a limited number of phytoestrogens
[2-4]. A narrow-bore HPLC-MS method was
developed for the identification of Pueraria root
constituents, but did not provide sufficient resolution
for their quantification in routine LC-UV analysis [5].
Another method reported for the quantification of
isoflavones in Kudzu root extracts suffered from poor
2022 Natural Product Communications Vol. 3 (12) 2008
R1
R5
4
5
6
O
O
3
7
O
R2
3’-Hydroxypuerarin
Puerarin
3’-Methoxypuerarin
6”-O-Dxylosylpuerarin
Daidzin
Genistin
Daidzein
Genistein
O
8
R4
2
1
R3
R1
H
H
H
H
R2
H
H
H
H
H
OH
H
OH
Glc
Glc
H
H
R3
Glc
Glc
Glc
GlcXyl
H
H
H
H
R4
OH
H
OCH3
H
R5
H
H
H
H
H
H
H
H
H
H
H
H
Figure 1: Structure of isoflavonoid glycosides and aglycones from
Pueraria root.
resolution, and only puerarin was quantified [6]. In
the work of Delmonte et.al., the isoflavone content
was determined in dietary supplements containing
soy, red clover and Kudzu, but since the glycosides
were hydrolyzed during sample preparation the real
composition was not determined [7]. The method
developed in our laboratory to determine the
isoflavone content in soy extracts could not be
applied directly since here we need to extract the
isoflavones from the root material.[8]. The methods
available for quantitative determination of Pueraria
root isoflavones in plant material or dietary
supplements are limited to the detection of one or
only a limited number of compounds, including the
official method in the current edition of the Chinese
Pharmacopoeia, which only determines the puerarin
content [9,10]. Several recent papers dealing with the
use of LC-MS for the analysis of isoflavones in crude
mixtures have recently being published [11-15]. The
aim of these studies was to identify isoflavone
aglycones and major glycosides in Pueraria root by
using LC-MS-MS with different modes of MS, for
example neutral loss-scan mass spectrometry,
selected ion monitoring (SIM), and selected reaction
monitoring (SRM).
From this short review of the literature it appears that
no fully optimized and validated method quantifying
all major constituents for routine quality control of
Pueraria root material or its dry extract is available.
Recently we have published the development and
validation of an HPLC method for quality control of
Pueraria lobata flower [16]. However, the main
constituents of the flower, i.e. the isoflavone
tectorigenin and two of its glycosides, were
completely different from the root constituents. When
developing a quantitative method, it must be
Bebrevska et al.
considered that the therapeutic potency of a
preparation depends not only on the amount of the
active ingredients, but also on their absorption
profile. The pharmacokinetic profiles of the C- and
O-glycosides present in Pueraria root are different,
and therefore special attention should be paid to their
individual determination. The method presented here
was designed to determine separately a maximum
number of compounds, i.e. all major isoflavones
mentioned above.
In our samples of P. lobata root material 3’hydroxypuerarin, puerarin, 3’-methoxypuerarin, 6”xylosylpuerarin, daidzin, genistin, daidzein and
genistein could be identified. Puerarin, 3’-methoxypuerarin, daidzin and daidzein were isolated, and
identified by NMR spectroscopy (1H NMR, 13C
NMR, DEPT-135 and DEPT-90, and 2D NMR when
necessary, including 1H-1H DQF-COSY, HSQC and
HMBC) and mass spectrometry. The NMR
assignments were in agreement with literature data
(Supplementary data: Table 1S) [17-19]. The
presence of the other constituents in the root extract
was confirmed by LC-MS analysis, by comparison
with published data and standards of genistein and
genistin.
For the development of an analytical method, first the
separation conditions were optimized in order to
achieve satisfactory resolution. Because of the
complexity of the mixture this was only possible
when using rather long gradient conditions and run
times. The initial set of separation conditions is
summarized in Table 1. The LiChrosphere column
did not provide the desired separation of the major
peaks with any of the tested eluents or gradients,
which included water / methanol mixtures without
and with addition of 0.01% formic acid (pH ≈ 3 – 4).
Therefore, the separation on a Zorbax Eclipse XDB
column was tested in combination with a linear
gradient of a binary phase: water + 0.01% formic acid
and methanol + 0.01% formic acid. These conditions
provided satisfying resolution of the peaks (Figure 2,
Table 2). The obvious difference in selectivity
between the two column packings might be due to
differences in the amount of bonded phase per unit
surface of the silica particles (density), the way in
which the bonded phase is attached to the silica
surface, and the purity of the silica.
Another problem that had to be overcome in the
design of the procedure was the big difference in
content between the glycosides and the aglycones. If
Quality control of Pueraria lobata root
Table 1: Initial separation conditions.
Separation variable
Column
Length x ID (mm)
Particle size (µm)
Stationary phase
Mobile phase
Detection
Flow rate (ml/min)
Injection volume standards (µl)
Solution
LiChrosphere
250 x 4
5
C 18
5% methanol in water to 100% for
gradient elution over 60 min (broad
linear gradient to survey the mixture)
262 nm
1.0
20 / 100
Sample volume (µl)
Standard solutions
20 / 100
20/100
Table 2: Final separation conditions.
Separation variable
Column
Length x ID (mm)
Particle size (µm)
Stationary phase
Mobile phase
Gradient time (min)
Wash up and equilibration (min)
Run time (min)
Detection
Flow rate (ml/min)
Standard solutions volume
Sample volume (µl)
Solution
Agilent Zorbax Eclipse XDB
150 x 4.6
5.0
C 18 XDB
15% methanol + 0.01% formic acid
to 65% methanol + 0.01% formic
acid for gradient elution
50
10
60
262 nm
1.0
20 / 100
20 / 100
Figure 2: HPLC profile of Pueraria root extract. The glycosides elute
between 5 and 20 min and the aglycones start from 22 min.
20 µL of the final solution was injected, the
aglycones were barely detectable, therefore it was
necessary to measure them in a second separate run
with an injection volume of 100 µL. All peaks of
interest were separated from the adjacent ones
allowing their exact integration. The peaks of
hydroxypuerarin (peak 5 on the chromatogram),
genistin (13), daidzin (11), daidzein (18) and
genistein (21) were well separated from their
neighboring peaks. The critical band couples
were: puerarin (peak 8 on the chromatogram) /
3’-methoxypuerarin (9), 3’-methoxypuerarin (9) / 6”xylosylpuerarin (10), and 6”-xylosylpuerarin (10) /
daidzin (11). However, they were successfully
resolved for the purpose of quantification. All peaks
of interest are symmetric.
Natural Product Communications Vol. 3 (12) 2008 2023
The extraction procedure and the extraction solvent
composition were optimized in order to ensure the
exhaustive extraction of the plant material, in the
same way as described for Pueraria flower [16]. The
final conditions are described in the experimental
section.
Daidzin, daidzein, genistin and genistein were
chosen as external standards. The content of
3’-hydroxypuerarin, puerarin, 3’-methoxypuerarin,
6”-xylosylpuerarin and daidzin were calculated with
respect to daidzin, while the contents of genistin,
daidzein and genistein were calculated with respect to
each individual standard. The standards were
analyzed using the same HPLC conditions as for the
analytes.
This method was validated according to the ICH
guidelines on the validation of analytical methods
[20,21]. The calibration functions for each of the four
standards were investigated by injecting six different
concentrations of reference solution in duplicate. The
concentration level for the isoflavone glycoside
daidzin was within the range of 4.13 – 103.2 µg/mL,
and for the glycoside genistin and the aglycones
daidzein and genistein within the range of 0.20 – 4.0
µg/mL, 0.43 – 8.68 µg/mL, and 0.034 – 0.54 µg/mL,
respectively. In order to describe and investigate the
relationship, least square lines and the correlation
coefficients were calculated. The significance of the
slopes (a≠0) and the intercepts (b=0) of the obtained
functions were tested by means of Student’s t-test. To
check whether the linear model fitted the obtained
data a lack-of-fit (LOF) test was performed and the
resulting residuals were graphically inspected.
The response functions of the four standards were
investigated and the results are shown in Table 3.
Graphical inspection of the residuals, the LOF test
and the correlation coefficients proved that a linear
model response function could be fit to the data in the
inspected range. The t-test of the intercept revealed
that point (0.0) fell within each of the calibration
curves. This implied that for routine analysis in this
range, the isoflavone content could be determined
against a single standard concentration level instead
of constructing a calibration line each time the
analysis is performed.
The repeatability and the time-different intermediate
precision were evaluated by analyzing six
independently prepared samples (100% level,
i.e. performed on 0.600g of plant material) on three
2024 Natural Product Communications Vol. 3 (12) 2008
Bebrevska et al.
Table 3: Overview of response function data for the reference materials used.
Concentration range (µg/mL)
Number of levels (n=2)
Analysis of the residuals
Correlation coefficient
95% CI of the intercept
Intercept ± standard error
Slope ± standard error
ANOVA Lack of fit,
F=MSLOF/MSPE (Fcrit = 5.409)
Daidzin
4.13 – 103.2
6
Randomly scattered
0.9995
[-53879 +23646]
-15117 ± 17397
31747 ± 303
Genistin
0.434 – 8.68
6
Randomly scattered
0.9999
[- 751 +10261 ]
4755 ± 2471
209128 ± 586
Daidzein
0.2012 – 4.024
6
Randomly scattered
0.9998
[-746 +7047]
3150 ± 1749
217842 ± 895
Genistein
0.0335 – 0.5372
6
Randomly scattered
0.9992
[-3317 +1119]
-1099 ± 995
292237 ± 3743
2.1
1.0
2.3
4.8
Table 4: Overview of the precision data.
Parameter
Repeatability /
Time-intermediate
precision
Number of replicates on
each day
Number of days
Mean content (%)
Day 1
Day 2
Day 3
RSD (%)
Day 1
Day 2
Day 3
RSD% between
days < 5%
Linearity / Intermediate
precision on different conc.
levels
Numbers of replicates on
each level
Number of levels
Mean content (%)
50
200
RSD (%)
50
200
Cochran’s test
(C critical = 0.506)
RSD% between levels (%)
< 5%
Hydroxypuerarin
Puerarin
Methoxypuerarin
Xylosylpuerarin
Daidzin
Genistin
Daidzein
Genistein
6
6
6
6
6
6
6
6
3
3
3
3
3
3
3
3
0.86
0.83
0.83
3.06
3.06
2.95
0.80
0.87
0.84
0.67
0.64
0.61
0.58
0.63
0.59
0.11
0.11
012
0.074
0.071
0.067
0.0074
0.0082
0.0074
1.09
0.60
1.47
1.16
0.64
0.78
1.64
0.87
0.83
1.09
0.93
0.81
1.18
0.35
0.82
5.26
2.56
6.10
3.59
2.50
3.60
4.19
5.87
4.29
2.21
2.34
4.30
4.91
5.51
7.07
5.05
7.29
6
6
6
6
6
6
6
6
3
3
3
3
3
3
3
3
0.84
0.80
3.21
3.08
0.83
0.83
0.65
0.62
0.60
0.60
0.11
0.11
0.076
0.068
0.0094
0.0051
1.04
0.62
0.334
1.26
0.34
0.417
2.31
1.44
0.464
1.21
0.64
0.305
1.62
1.63
0.360
4.15
4.73
0.355
4.54
1.30
0.410
3.02
1.82
0.446
4.15
3.18
3.22
4.03
3.43
6.13
5.98
21.46
different days. The mean content and the betweendays RSD (%) for each compound are presented in
Table 4. The mean, the standard deviation and the
relative standard deviation were calculated for each
day. ANOVA single factor was used to compare the
results of the three days. Within- and between-days
relative standard deviations were calculated. To test
the precision of the method in terms of between
concentration levels variation, six samples weighing
twice the normal weight (200%) and six samples
weighing half the normal weight (50%) were
analyzed following the same procedure. The mean,
the standard deviation and relative standard
deviations were calculated for each level. By means
of a Cochran’s test (5 groups, n=6), the homogeneity
of the variances of experiments at the three
concentration levels (50, 100 (3 days), and 200%)
was determined prior to ANOVA single factor
comparison of the results for the three levels. Withinand between-level relative standard deviations were
calculated.
The major isoflavone was the C-glycoside puerarin
present in an amount of 3.03%, followed by
3’-hydroxypuerarin 0.84%, 3’-methoxypuerarin
0.83%, 6”-xylosylpuerarin 0.64%, and daidzin
0.60%. Genistin and daidzein were present in minor
amounts of 0.11% and 0.07%, respectively. Genistein
was present at a trace level of 0.008%.
The precision of the method was acceptable for
the major compounds 3’-hydroxypuerarin, puerarin,
3’-methoxypuerarin, 6”-xylosylpuerarin and daidzin
with an RSD (%) of 2.21, 2.34, 4.30, 4.91 and 5.51%
respectively. The RSD between-days (%) values for
genistin, daidzein and genistein were 7.07, 5.05, and
Quality control of Pueraria lobata root
Natural Product Communications Vol. 3 (12) 2008 2025
7.29%, respectively. They are also acceptable with
respect to the low amount in which these isoflavones
are present.
Standards of daidzin (100 mg, purity 99.2%),
daidzein (25 mg, purity 96.7%), genistin (100 mg,
purity 99.1%) and genistein (50mg, purity 100%)
were purchased from LC Laboratories (Woburn,
Massachusetts, USA).
The precision of the measurements in the range
between 50% and 200% of the expected amount of
each analyte was investigated. The results are also
presented in Table 4. The variances of the
measurements for the three levels were compared by
means of Cochran’s test. Since for each of the
isoflavones the calculated C-values were lower than
the critical value, the precision of the determinations
within this range can be considered equal. The RDS
between-levels (%) were still acceptable and no
trend, i.e. lower values at 200% and higher values at
50%, was detected.
The accuracy of the method was investigated by
performing recovery experiments according to the
method of standard additions (Supplementary data,
Tables 2S – 5S). Daidzin, genistin, daidzein and
genistein stock solutions were added before the
extraction at different concentration levels to half
of the normally analyzed amount of plant material
(300 mg). At each level the samples were prepared in
triplicate and analyzed. Mean recoveries of 99.05%
(RSD% = 3.21), 101.55% (RSD% = 4.32), 99.16%
(RSD% = 1.92), and 105.56% (RSD% = 6.36) were
determined for daidzin, genistin, daidzein and
genistein, respectively. The mean recoveries (%)
were verified not to be significantly different from
100 % for all four isoflavones by means of Student’s
t-test, which proved that our method produces
accurate results.
These results proved that the reported method
correctly quantifies all major isoflavones present in
Pueraria root with acceptable accuracy and
precision. This study proved the suitability of the
method for the quality control of Pueraria root crude
drug.
Experimental
Materials: Distilled water (RiOs) was obtained from
a Millipore water purification system (Millipore,
Brussels, Belgium). Methanol (HPLC-quality) and
silica gel 60 (0.040-0.063) were purchased from
Merck (Darmstadt, Germany), and formic acid and
dimethyl sulfoxide from Acros Organics (Geel,
Belgium). All solvents and reagents were of
analytical grade. Preparative C18 (55-105 µm) was
from Waters (Maliford, MA, USA).
Dried roots of Pueraria lobata were kindly provided
by SINECURA (Ghent, Belgium). The root material
was investigated macro- and microscopically
according to the Chinese Pharmacopoeia in order to
confirm its identity.
Spectroscopy: 1D NMR (1H, 13C, DEPT-135 and
DEPT-90) and 2D NMR spectra (1H-1H DQF-COSY,
HSQC and HMBC) were recorded in methanol-d4 on
a Bruker DRX-400 instrument operating at 400.15
MHz for 1H, using standard software packages.
LC-MS analysis was performed on an Agilent 1100
with a diode array detector, coupled to a Bruker
Esquire 3000 plus ion trap MS (Bruker Daltonics,
Billerica, USA). LC-MS analysis was carried out on a
Surveyor LC system with a diode array detector
coupled to a LXQ linear ion trap (Finnigan, Bremen,
Germany).
Thin layer chromatography: 1) Silica gel 60 F 254
plates (Merck), 20 x 20 cm, and 2) RP 18 plates
(Merck), 20 x 10 cm were used. Mobile phase
systems were: 1) n-butanol : ethanol : water 4 : 1 :
2.2; and 2) acetonitrile : water 1 : 1. Compounds were
detected by: UV light at 254 nm; UV light at 366 nm;
and spraying with p-anisaldehyde / sulfuric acid in
methanol followed by heating the plate (100-105°C)
for about 5 min.
HPLC: The isolation of the marker compounds was
carried out on an Agilent 1100 HPLC-system
equipped with a DA (diode array) detector. The
HPLC analyses during the method development and
validation procedure were carried out using a Gilson
instrument (pump model 322, UV-VIS detector
model 156) equipped with a Gilson automatic
injector. The analytical work was performed on an
Agilent Zorbax Eclipse XDB-C18 column (150 x 4.6
mm, 5µm) and the semipreparative work on a
Macherey-Nagel, Nucleosil 100-7 C18 (10 x 250
mm) semipreparative chromatographic column.
Extraction and isolation: Dried roots of Pueraria
lobata were pulverized and sieved in order to assure
particle uniformity. A 50% ethanol extract was
prepared under constant stirring by adding multiple
portions of fresh extracting solvent till complete
2026 Natural Product Communications Vol. 3 (12) 2008
exhaustion of the material. The extracted portions
were combined and concentrated by evaporation
under reduced pressure to give the crude extract.
After lyophilization, 20 g of the powder was
resuspended in distilled water and partitioned
between ethyl acetate and subsequently n-butanol,
both saturated with water. The resulting subfractions
(ethyl acetate, n-butanol and residual water) were
dried under reduced pressure.
The ethyl acetate fraction (5.0 g) was subjected to
column chromatography on silica gel (350 g), eluted
with n-butanol : ethanol : water 6 : 1 : 2.2. The 52
collected fractions were evaluated by means of TLC,
and according to their profile they were combined in
7 subfractions. Subfraction 4 (3.1 g) was further
fractionated on silica gel (360 g), eluted with
n-butanol : ethanol : water 4 : 1 : 2.2. The resulting
18 fractions were evaluated by TLC and joined into
5 fractions. The fourth fraction was submitted to
column chromatography on silica gel (50 g). The 18
obtained fractions were put together in 3 fractions.
Fraction 2 was subjected to semipreparative HPLC.
An HPLC gradient consisting of methanol / 0.01%
formic acid and water / 0.01% formic acid was
optimized for the purpose of semi-preparative
separation: from 15% methanol (t = 0 min), over 21%
(t = 15 min), 25% (t = 25 min), to 95% (t = 55 min).
The three major compounds isolated were identified
as puerarin, 3’-methoxypuerarin, and daidzin.
Subfraction 3 of the ethyl acetate fraction (250 mg)
was submitted to column chromatography on silica
gel (30 g) and eluted with chloroform : methanol
9 : 1. The resulting 18 fractions were combined in 3
subfractions. The second subfraction was purified by
semipreparative HPLC using a gradient from 15% to
67.8% methanol over 33 min at a flow rate of 2.2
mL/min. One compound was isolated, and identified
as daidzein. The presence of 3’-hydroxypuerarin,
6”-xylosylpuerarin, genistein and genistin was
confirmed by LC-MS analysis, by comparison with
published data and standards of genistein and
genistin.
Preparation of the sample solution: The powdered
plant material was sieved (355 μm) to ensure
particles uniformity. An exactly weighed amount of
0.600 g of plant material was placed in a round
bottomed flask and 10 mL dimethyl sulfoxide was
added. After 15 min, 60 mL of 50% methanol was
added, and refluxed for 30 min. To separate the clear
liquid from the plant material centrifugation at 3000
Bebrevska et al.
rpm was performed for 15 min. A fresh portion of
60 mL 50% methanol was added to the plant
material, repeating the extraction procedure. The
supernatants resulting from the two extractions were
joined and collected in a calibrated flask of 200 mL.
The flask was filled to volume with 50% methanol.
Finally 10 mL of this solution was diluted to 20 mL
with water. This sample solution was used for HPLC
analysis. Two separate analyses were performed for
each sample: one injecting 20 µL to quantify the
glycosides, and another one injecting 100 µL for the
quantification of the aglycones.
Preparation of the standard solutions: About 10 mg
of daidzin was accurately weighed and dissolved in
25 mL of dimethyl sulfoxide. An aliquot of 2 mL of
this solution was diluted to 50 mL with 50%
methanol.
About 10.0 mg of genistin was accurately weighed
and dissolved in 25 mL of dimethyl sulfoxide. An
aliquot of 0.5 mL of this solution was diluted to 100
mL with 50% methanol.
About 10.0 mg of daidzein was accurately weighed
and dissolved in 25 mL of dimethyl sulfoxide. An
aliquot of 0.5 mL of this solution was diluted to 100
mL with 50% methanol.
About 10.0 mg of genistein was accurately weighed
and dissolved in 25 mL of dimethyl sulfoxide. An
aliquot of 0.5 mL of this solution was diluted to 50
mL with 50 % methanol.
Calculation: The percentage content (Cs %) of the
compounds in the plant material was calculated using
the formula:
Cs % = As x Cref x 2 x 200 x 100 / (Aref x m)
where As was the area of the peak of the isoflavone in
the sample, Aref the area of the isoflavone in the
reference solution, Cref the concentration of the
reference solution, and m the weight of the drug in
mg.
For the purpose of exact quantification, loss on
drying was determined according to Ph. Eur. Article
2.2.32.d. The water content was found to be 8.8 %.
Acknowledgements - Melisana (Brussels, Belgium)
and the special Fund for Research of the University
of Antwerp (NOI-BOF) are acknowledged for their
financial support.
Quality control of Pueraria lobata root
Natural Product Communications Vol. 3 (12) 2008 2027
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Prasain JK, Kones K, Kirk M, Wilson L, Smith-Johnson M, Weaver C, Barnes S. (2003) Profiling and quantification of
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Zhang Y, Xu Q, Zhang X, Chen J, Liang X, Kettrup A. (2005) High-performance liquid chromatography-tandem mass
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Zhang Y, Chen J, Zhang C, Wu W, Liang X. (2005) Analysis of the estrogenic compounds in kudzu root by bioassay and highperformance liquid chromatography. Journal of Steroid Biochemistry and Molecular Biology, 94, 375-381.
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Boue SM, Carter-Wientjes CH, Shih BY, Cleveland TE. (2003) Identification of flavone aglycones and glycosides in soybean pods
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Wu Q, Wang M, Simon JE. (2003) Determination of isoflavones in red clover and related species by high-performance liquid
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Tian H, Wang H, Guan Y. (2005) Separation and identification of isoflavonoids in Pueraria lobata extracts and its preparations by
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Bebrevska L, Bravo L, Vandervoort J, Pieters L, Vlietinck A, Apers S. (2007) Development and validation of an HPLC method for
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NPC
Natural Product Communications
Pharmacokinetics of Luteolin and Metabolites in Rats
2008
Vol. 3
No. 12
2029 - 2036
Sasiporn Sarawek, Hartmut Derendorf and Veronika Butterweck*
Department of Pharmaceutics, College of Pharmacy, University of Florida, POBOX 100494,
Gainesville, FL 32610, USA
butterwk@cop.ufl.edu
Received: July 21st, 2008; Accepted: October 27th, 2008
The pharmacokinetic parameters of luteolin and its glucuronide/sulfate conjugates were studied in rats after a single 50 mg/kg
dose of luteolin administered as intravenous bolus or oral solution. Plasma and urine samples were enzymatically hydrolyzed to
determine conjugate concentrations of luteolin. Noncompartmental analysis revealed a half-life of 8.94 h for free
(unconjugated) and 4.98 h for conjugated luteolin following intravenous administration. Following oral administration, plasma
concentrations of luteolin attained a maximum level of 5.5 μg/mL at 5 min and decreased to below LOQ (100 ng/mL) after 1 h.
Ke could not be calculated because the elimination phase was below LOQ. The low bioavailability (F) of luteolin, 4.10% at a
dose of 50 mg/kg, is presumably due to the significant first pass effect. For i.v. administration, the maximum concentration of
luteolin was 23.4 μg/mL at 0 h. The plasma concentration versus time profile of luteolin was biphasic, subdivided into a
distribution phase and a slow elimination phase for oral and intravenous administration. Luteolin was found to have a large
volume of distribution and a high clearance. Double peaks were found after intravenous and oral administration, suggesting
enterohepatic recirculation.
Keywords: luteolin, conjugates, pharmacokinetics.
Flavonoids are polyphenolic compounds widely
distributed in plants and vegetables. They have been
reported to possess numerous biological effects such
as antioxidant, free-radical scavenger, antiinflammatory, antiestrogenic, and antiproliferative
activities [1,2]. These effects are helpful for human
health, such as in the treatment and prevention of
cancer, cardiovascular disease, and other pathologies
[1,2]. The flavone luteolin usually occurs in its
glycosylated forms in celery, green pepper, camomile
tea, and artichoke. It has been reported to
have antitumorigenic [3,4], anticancer [5-8],
antiproliferative [9,10], and antioxidant effects [6]
and has been recognized as an inhibitor of protein
kinase C [11] and xanthine oxidase [12,13]. The
absorption and metabolism of luteolin have been
evaluated in vitro using Caco-2 cells and microsomes
obtained from liver or intestine of rats and
humans [14,15]. In addition, pharmacokinetic
studies in animals and humans of luteolin and
luteolin-rich plants have been reported [16-19].
However, until now, in vivo investigations
of the disposition, absorption, bioavailability, and
metabolism of luteolin are limited. Therefore, in the
present experiments, the pharmacokinetics of luteolin
and its glucuronide/sulfate conjugates after oral and
intravenous (i.v.) injection in rats were determined.
In addition, the elimination of luteolin after oral and
intravenous administration was measured in urine
samples. For the present investigation, we focused on
the pharmacokinetic evaluation of the aglycone
luteolin because recently we demonstrated that this
compound produces strong xanthine oxidase
inhibition in vitro [13].
The
concentration-time
profiles
and
the
pharmacokinetic parameters of free luteolin after
oral and i.v. administration are presented in Table 1
and Figure 1. The plasma concentration of luteolin
attained a maximum level of 5.49 μg/mL 5 min
after oral administration of luteolin to rats at a dose
of 50 mg/kg.
The low bioavailability (F) of luteolin, 4.10%
at a dose of 50 mg/kg, is presumably
due to the significant first pass effect. Following
2030 Natural Product Communications Vol. 3 (12) 2008
Sarawek et al.
Table 1: Pharmacokinetic (PK) parameters of unconjugated luteolin after
oral and i.v. administration of luteolin at a dose 50 mg/kg.
Parameter
ke (1/h)
Cl (L/h/kg)
Vdarea (L)
AUC0-last (h*μg/mL)
AUC0-∞ (h*μg/mL)
F (%)
Luteolin oral
Luteolin i.v.
5
5.49
ND
ND
0
23.42
0.08
8.94
2.14
27.58
20.55
23.39
0.87
0.96
4.10
Table 2: Pharmacokinetic (PK) parameters of luteolin conjugates after
oral and i.v. administration of luteolin at a dose 50 mg/kg.
Parameter
Tmax (h)
Cmax (μg/mL)
ke (1/h)
Luteolin conjugates
oral
0.25
5.77
Luteolin conjugates
i.v.
0.08
4.31
0.10
0.14
t ½ (h)
6.57
4.98
AUC0-last (h*μg/mL)
11.49
12.83
AUC0-α (h*μg/mL)
15.68
15.26
All PK parameters presented in Tables 1 & 2 are mean values calculated
by a normalized dose (50 mg/kg). AUC = area under the curve; Cmax =
maximal plasma concentration; Tmax = time to reach Cmax; Ke =
elimination rate constant; t ½ = elimination half life; Cl/F = clearance
after oral administration; Cl = clearance; Vdarea = distribution volume;
AUC0-last = area under the moment curve extrapolated to Tlast; AUC0-∞ =
area under the curve extrapolated to infinity; F = oral bioavailability; ND
= not determined
i.v. administration, the maximum concentration of
luteolin was 23.42 μg/mL at 0 h. The plasma
concentration versus time profile of luteolin was
biphasic, subdivided into a distribution phase and a
slow elimination phase for oral and intravenous
administration. The concentration-time profiles and
the pharmacokinetic parameters of luteolin
conjugates after oral and i.v. administration are
presented in Table 2 and Figure 1. Plasma
concentrations of luteolin conjugates after oral and
i.v. administration of luteolin attained maximum
levels of 5.77 μg/mL at 0.25 h and 4.31 μg/mL at
0.08 h, respectively, and decreased to below the LOQ
at 24 h. However, the half-life for luteolin was
greater than that of conjugated luteolin following i.v.
injection. Double peaks were found for luteolin
conjugates after oral and i.v. administration at 0.25
and 1 h, respectively, suggesting it might pass
enterohepatic circulation.
Urinary excretion of free luteolin and its conjugates
within 24 h after oral and intravenous administration
of luteolin was very low (0.98 - 4.97% of the dose),
which suggests that these compounds are not
primarily excreted via the urine (Table 3).
Figure 1: Plasma concentration-time curves. A) Luteolin after i.v. oral
administration. B) Luteolin after oral administration. C) Luteolin
conjugates after i.v. administration. D) Luteolin conjugates after oral
administration. Luteolin 50 mg/kg was administered to rats (n = 8-11).
Error bars refers to the standard deviation of concentration data at each
sampling time point.
Table 3: The excretory recovery for 24 h of free luteolin and luteolin
conjugates in urine after oral and i.v administration of luteolin at a dose
50 mg/kg.
Treatment
% Luteolin
% Luteolin conjugates
oral
0.9 ± 0.9
3.9 ± 0.5
i.v.
2.0 ± 0.9
4.9 ± 1.7
Data expressed as mean ± SD (n = 8-11)
Free luteolin and luteolin conjugates were present in
rat plasma and urine after oral and intravenous
administration.
However,
the
conjugates
(glucuronides or sulfates) could not be further
identified in this study since the analytical method
was developed only for the parent compound. The
presence of free luteolin suggested that some of it can
escape intestinal and hepatic conjugation.
The pharmacokinetic profiles of free luteolin and
luteolin conjugates in rat plasma are presented in
Figure 1. When luteolin was administered orally, the
maximum concentrations of luteolin and luteolin
conjugates were 5.49 and 5.77 μg/mL at 5 min and
15 min, respectively. The total concentration of
luteolin in rat plasma 5 min after dosing was
9.25μg/mL. Shimoi et al. [18] reported 15.5 ± 3.8
nmol/mL (4.4 ± 1.09 μg/mL) of total luteolin in rat
plasma 30 min after administration of one single dose
of luteolin (50 μmol/kg, 14.3 mg/kg) in propylene
glycol. In dogs, the maximum concentration of
luteolin was about 450 ng/mL 3 h after a single oral
Pharmacokinetic parameters of luteolin
Figure 2: Fitted luteolin concentrations after
Experimental points represent the means of 8-11 rats.
Natural Product Communications Vol. 3 (12) 2008 2031
i.v.
injection.
dose of a Chrysanthemum morifolium extract
(102 mg/kg containing 7.60% luteolin, 7.75 mg/kg
luteolin) [16]. In humans, peak plasma concentrations
of total luteolin were reached within 0.5 h with a
maximum level of 156.5 ± 92.29 ng/mL after a single
oral dose of an artichoke leaf extract (153.8 mg
containing luteolin-7-O-glucosides; equivalent to
35.2 mg luteolin) [19]. The differences observed in
these studies could be explained by the initial dose
administration, the solvents used to dissolve luteolin
and the source of intake flavonoids (extract versus
pure compound).
Figure 2 illustrates the fitted luteolin concentrations
versus time profiles with the two compartment body
model. It can be seen that the model describes the
luteolin data very well. The AIC and SC were -11.18
and -9.59, representing a good fit. The PK parameters
obtained by fitting the mean concentrations versus
time profiles of luteolin concentrations after i.v.
treatment are presented in Table 4.
The rapid absorption of several flavonoids has been
reported in previous literature. For example, in
humans, the main peak appeared approximately 2.9 h
after quercetin administration [20,21]. In rats,
flavonoids seem to appear more rapidly. When
luteolin was administered via gastric intubation, the
compound was detected in rat plasma after 15 min
[18], whereas quercetin (given orally) appeared after
5 min [22]. Apigenin given intraperitoneally,
appeared in plasma after 30 min [23]. Based on these
literature data, the presence of flavonoids in the blood
occurs within a few minutes to a few hours, which
correlates with our results.
The low bioavailability of luteolin (F = 4.1%) and
the high metabolite concentrations indicate a first
pass metabolism. Absorbed luteolin undergoes
biotransformation (methylation, glucuronidation or
sulfation), as has been shown in previous studies. For
example, in vitro experiments demonstrated that 74%
of luteolin was conjugated to glucuronic acid after
incubation with microsomal samples from the human
intestine [14]. The most common binding sites of the
molecule were the hydroxyl groups in the 3′- and
4′- position (51% and 44%) [14]. Boersma et al.
[14] found three glucuronosyl conjugates of luteolin,
7-O-, 3′-O- and 4′-O-glucuronosyl luteolin, after
incubation with microsomes from rats and humans.
Shimoi et al. [18] investigated the absorption of
luteolin by rat inverted small intestine. Luteolin was
recovered in rat plasma as two metabolites, the
glucuronidate or sulfate forms of the O-methylate
conjugate. Only a small part of the compound
remained unconjugated. Murota et al. [15] reported
the uptake and transport of flavonoid aglycones by
human intestinal Caco-2 cells. The flavonoids
quercetin, kaempferol, luteolin and apigenin were
converted to their glucuronide/sulfates by Caco-2
cells, and the level of the intact aglycone was lower
than those of the glucuronide/sulfates in the
basolateral solution.
In addition to the first-pass metabolism, the role of
efflux transporters in contributing to the low oral
bioavailability of drugs has been well acknowledged
[24]. Among the efflux transporters located in the
intestine and the liver, the ATP-binding cassette
(ABC) superfamily, including several members such
as P-glycoprotein (P-gp), multidrug resistance
associated proteins (MRPs), and breast cancer
resistance protein (BCRP/MXR), has been well
investigated for its roles in intestinal and hepatic
disposition of drugs [24]. Increasing evidence has
demonstrated the interactions of flavonoid aglycons
and glycosides with ABC transporters. For example,
ginkgo flavonols, quercetin, kaempferol, and
isorhamnetin were demonstrated to be the substrates
of P-gp in the Bacap37/MDR1 transfected cell model
[25]. The interaction of flavonoid aglycones and
glycosides with ABC transporters may greatly
influence their absorption, disposition and excretion
in the body, and also alter pharmacokinetic profiles
of the concurrently administrated drugs acting on the
same transporters, thereby probably leading to
significant impacts in the therapeutic outcomes.
2032 Natural Product Communications Vol. 3 (12) 2008
However, the bioavailability of luteolin is similar to
that of quercetin [15]. Chen et al. [22] reported the
systemic bioavailability of quercetin and quercetin
conjugates as 5.3% and 55.8%, respectively, in rats.
Moreover, after oral administration of quercetin,
about 93.3% of it was metabolized in the gut, with
only 3.1% metabolized in the liver. Interestingly, the
half-life for free luteolin was greater than that for
conjugated luteolin following i.v. injection. This is
unusual since in theory the metabolites should not
have a shorter half-life than the parent drug.
However, similar results were reported by Cave et al.
[26], who also reported a longer half-life for the
parent compound genistein than for the genistein
metabolites after i.v. injection.
Therefore, the pharmacokinetic variables reported for
the conjugates should be interpreted with caution,
since they describe the appearance and disposition of
endogenously synthesized compounds, each of which
may possess distinct kinetics [26]. It also can be
suggested that the terminal half-life has not been
reached yet and that by using a more sensitive assay
one would find a little longer half-life of the
conjugates. Thus, a more complete analysis of the
metabolism of luteolin will require separation into
individual metabolites and kinetic analysis of the
individual compounds.
Only small amounts of free luteolin and luteolin
conjugates were found to be eliminated in the urine in
our study. This is consistent with the observations by
others. Shimoi et al. [18] reported an excretory
recovery of unmodified luteolin in rat urine of 4%.
Luteolin conjugates were recovered only by 1.99 ±
1.50% after intake of luteolin-7-O-glucoside [19].
Comparably, only 0.58% of apigenin was recovered
in urine samples within 24 h after parsley ingestion in
humans [27]. Gugler et al. [28] found that after
intravenous administration of quercetin (100 mg),
7.4% of the dose was excreted in the urine as a
conjugated metabolite and 0.65% of the dose was
recovered in the form of unchanged quercetin.
Therefore, it seems that the urinary elimination of
luteolin is not the main excretion route in rats. At
present, it can be speculated that excretion via feces
may be the main route of elimination of luteolin and,
in particular, its metabolites [29].
Since in the present study multiple peaks were
detected in the plasma after oral and intravenous
Sarawek et al.
Table 4: Pharmacokinetic parameters of free luteolin after i.v.
administration of 50 mg/kg luteolin. Data were fitted to a twocompartment model.
Parameter
Luteolin i.v.
A (μg/mL)
9.6
B (μg/mL)
1.3
α (1/h)
1.9
β (1/h)
0.08
K12 (1/h)
1.2
K21 (1/h)
0.3
Ke (1/h)
0.48
Vc (L/kg)
4.5
Vt (L/kg)
18.2
Cl (L/h/kg)
2.2
t½α (h)
0.3
t½β (h)
9.1
t½ke (h)
1.4
AUC (h * µg/mL)
22.9
Cmax(µg/mL)
11.0
administration of luteolin, an enterohepatic
recirculation of luteolin can be suggested; this is
similar to other studies. Liu et al. [30] observed a
rapid absorption and metabolism of aglycones
such as apigenin and quercetin into phase II
conjugates following enteric and enterohepatic
recycling. Ma et al. [31] reported an
enterohepatic recirculation of naringenin in rat
plasma. However, our data did not show multiple
peaks in the plasma concentration-time profile of
free luteolin after intravenous and oral
administration, presumably due to the limited
number of data time points.
In conclusion, after oral administration of luteolin,
the compound was rapidly absorbed and metabolized
in plasma. Moreover, plasma-concentration-time
curves of luteolin metabolites revealed secondary
peaks. The bioavailability of luteolin was low and the
urinary excretion of luteolin and its conjugates did
not dominate. Further studies are needed to confirm
that the luteolin Cmax and AUC data reported
here are genuinely associated with the threshold
of its reported antitumorgenic, antiproliferative
and xanthine oxidase inhibitory effects. In addition,
Pharmacokinetic parameters of luteolin
Natural Product Communications Vol. 3 (12) 2008 2033
further investigations are required to clarify if
luteolin conjugates demonstrate pharmacological
activity.
at 5, 10, 15, 30, 45 min, and 1, 2, 4, 6, 12, and 24 h
for oral administration. Prior to blood collection, the
rats were anaesthetized with halothane and the blood
loss was replaced with an equal volume of isotonic
saline. The blood sample was centrifuged for 15 min
at 4,000 rpm at 4oC. The supernatant, in aliquots of
0.20 mL, was transferred into tubes and 10 μL of
0.58 M acetic acid was added to each aliquot for
stabilization. The plasma samples were stored at
-80oC until analysis. Urine was collected over 24 h
and an aliquot of 50 mL was mixed with 1 g ascorbic
acid as antioxidant and stored at -80oC until analysis.
Experimental
Materials: Luteolin (99%) was purchased from
Indofine Chemical Company, Inc. (Somerville, NJ,
USA) and naringenin (≥ 96%) (internal standard)
from Roth Carl Roth GmbH+Co. (Germany).
Acetone, acetonitrile (CH3CN), acetic acid,
dimethylsulfoxide
(DMSO),
methanol,
and
orthophosphoric acid (85% p.a.) were obtained
from Fisher Scientific (Fair Lawn, NJ, USA). L (+)ascorbic acid (≥ 99.9%) was obtained from Acros
Organics (New Jersey, USA). Trifluoroacetic acid
was purchased from Fluka (Milwaukee, WI, USA)
and β-glucuronidase/sulfatase (type HP-2, Helix
pomatia), polyethylene glycol 200, and sodium
dihydrogenphosphate monohydrate (NaH2PO4.H2O),
from Sigma Chemical Company (St. Louis, MO,
USA). All buffers and aqueous solutions were
prepared with purified water obtained from a
NANOPure® system from Barnstead (Dubuque, IA,
USA).
Animals: Male Sprague-Dawley rats, weighing
300-400 g were purchased from Harlan (IN, USA)
and divided into the experimental groups, each
containing 8-11 rats. The animals were housed in
plastic cages and were allowed to adapt to their
environment for one week before used for
experiments. All the animals were maintained on a
12h/12h light/dark cycle. They received a standard
chow and water ad libitum during the duration of the
experiment. All animal experiments were performed
according to the policies and guidelines of the
Institutional Animal Care and Use Committee
(IACUC) of the University of Florida, Gainesville,
USA (NIH publication # 85-23).
Methods: The pharmacokinetic studies were carried
out by the sparse sampling approach wherein blood
samples were collected from 8-11 rats. Luteolin was
administered to two groups of rats (n = 8-11 in each
group). Group one received a single i.v. dose of 50
mg/kg of luteolin via the tail vein. Group two
received the same dose orally by gavage. Luteolin
was dissolved in 30% DMSO and 70%
polyethylenglycol (PEG) 200. Plasma samples (250
μL per blood sample) were collected from the
sublingual vein into heparinized tubes at 3, 5, 10, 30
min, and 1, 2, 4, 6, 12 , and 24 h for i.v. injection, and
Analytical methods: The plasma concentrations of
unchanged free and conjugated luteolin in rat plasma
and urine were determined by the method published
earlier, with slight modifications [19]. Plasma and
urine samples were analyzed using a Shimadzu VP
series HPLC system (Kyoto, Japan) equipped with an
SPD-M10Avp diode array detector. A Lichrospher®
100 RP-18 (5 μm. Merck KgaA) was used for the
separation of luteolin. The column was kept at 25oC.
The eluents were (A) 50 mM phosphate buffer
(NaH2PO4, pH 2.1) and (B) CH3CN. The following
solvent gradient was applied: 20% B (6 min) and
20-50% B (21 min).The gradient was followed by
10 min column flushing and post-run equilibration,
respectively. Total run time was 40 min. The flow
rate was 1 mL/min. Forty μL of each sample was
injected into the RP-HPLC system. Chromatograms
were acquired at 330 nm. For the determination of
total luteolin, 10.0 μL internal standard (naringenin,
1.05 mg/mL), 10.0 μL 0.5% (m/v) ascorbic acid and
20.0 μL acetic acid (0.58 M) were added to 200.0 μL
of the plasma sample, followed by the addition of
12.0 μL β-glucuronidase/sulfatase solution. The
mixture was incubated at 37oC for 1 h. Protein was
precipitated by adding 240 μL of acetone. The
mixture was vortexed for 1 min and centrifuged for
15 min at 4000 rpm at 4oC. The supernatant was
transferred to tubes containing 4.0 μL 0.5% (w/v)
ascorbic acid and 8.0 μL 1 M trifluoroacetic acid, and
evaporated to dryness in a vacuum centrifuge. The
residue was reconstituted in 60 μL of methanol:
water (1:1, v/v), centrifuged for 10 min at 13,200
rpm, and 40 μL was injected into the HPLC. For the
determination of unchanged luteolin in plasma, the
sample was extracted in the same manner as
described above, without adding the enzyme. The
concentrations of luteolin and its conjugates in urine
samples were measured using the same method as
described for the plasma, except urine samples were
2034 Natural Product Communications Vol. 3 (12) 2008
centrifuged for 15 min at 13,200 rpm after adding
240.0 μL acetone, and then 40.0 μL of the
supernatant was injected into the HPLC.
The conjugates (glucuronides or sulfates) of luteolin
were calculated by subtracting total luteolin from
unchanged luteolin. The calibration curve was linear
in the examined concentration range of 0.1 to 50
μg/mL with (R2 ≥ 0.99), with the detection limit of
0.1 μg/mL. The inter-day and intra-day precisions
were less than 15%. The extraction recoveries were
more than 90% for all samples.
Data analysis: Mean concentration of luteolin and its
conjugates versus time curves were generated in
Grapad Prim® (version 4.0, San Diego, CA). The
pharmacokinetic parameters were determined by noncompartmental and compartmental analysis using
WinNonlin software package, version 4.1, (Pharsight
Corporation, USA). The mean data were used for the
analysis. Non-compartmental PK: The PK
parameters determined were the AUC, maximum
concentration in plasma (Cmax), time to reach Cmax
(Tmax), the elimination rate constant (ke), the
elimination half life (t1/2), the volume of distribution
(Vd) and the clearance (CL). AUC 0→last was
calculated using a linear/log trapezoidal method from
time zero to last sampling point equal to or above the
lower limit of quantification. Both Cmax and Tmax
were obtained from the plots of plasma concentration
versus time. The ke was obtained by linear regression
of the terminal log linear phase of the concentrationtime curve. The elimination half-life (t1/2) was
determined as 0.693/ke. The volume of distribution
area (Vdarea) was calculated using CL/ke. The
clearance (Cl) was calculated as Dose/AUC and the
systemic bioavailability (F %) was calculated as F %
= (AUC p.o. /AUC i.v..) × 100. Compartmental PK:
Luteolin concentrations showed a better fit in a two
compartment body model compared to a one
compartment body model. The equation for the two
compartment model (Figure 2) is as followed:
C = A.e-αt + B.e-βt (i.v.), where C is the
concentration of drug in plasma at time t; A and B are
the mathematical coefficient; α is the distribution rate
Sarawek et al.
constant; β is the elimination rate constant; and t is
time. After i.v. administration, AUC 0-∞ was
calculated using following equation: AUC 0-∞ i.v. =
A/α + B/β. The elimination half life was calculated as
ln (2/ke). The volume of distribution of central
compartment (Vc) was calculated as Dose/Ke ×
AUC. The volume of distribution of peripheral
compartment (Vt) was calculated as Vc × k12/k21.
The clearance (Cl) was calculated as Dose/AUC.
Goodness of fit was determined by the AIC (Akaike
Criteria) and SC (Schwartz Criteria). The lower the
AIC and SC, the more appropriate is the selected
model.
Validation: The method was validated over the range
of concentration of luteolin present in plasma and
urine. The validation parameters of linearity,
sensitivity, specificity, precision, accuracy and
stability were determined. Plasma: The calibration
curve (n = 9) operating in the range of 100-10000
ng/mL for luteolin in rat plasma was linear (r2 >
0.99). The limit of quantification (LOQ) of luteolin
in plasma was 100 ng/mL. The precisions intra- and
inter-day for luteolin were satisfactory with CV
values between 1.3 and 12.3%. Similarly, the
accuracy of the assay obtained with quality control
samples containing 300, 800, and 3000 ng/mL
luteolin was between 94.2 and 106.3% of the nominal
values. The mean recovery assessed at three distinct
levels of concentration (100, 500 and 10000 ng/mL)
ranged from 95.7 to 106.4% of the expected values.
Urine: The calibration curve (n = 9) operating in the
range of 500-50,000 ng/mL for luteolin in rat urine
was linear (r2 > 0.99). The LOQ of luteolin in urine
was 500 ng/mL. The precisions intra- and inter-day
for luteolin were satisfactory with CV values between
0.30 and 13.25%. Similarly, the accuracy of the assay
obtained with quality control samples containing 500,
3,000, and 10,000 ng/mL luteolin was between 98.21
and 109.28% of the nominal values. The mean
recovery assessed at three distinct levels of
concentration (500, 3,000 and 10,000 ng/mL) ranged
from 99.56 to 112.23% of the expected values.
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NPC
Natural Product Communications
Complete Characterization of Extracts of
Onopordum illyricum L. (Asteraceae) by HPLC/PDA/ESIMS
and NMR
2008
Vol. 3
No. 12
2037 - 2042
Luisella Verottaa*, Laura Belvisia, Vittorio Bertaccheb and Maria Cecilia Loic
a
Dipartimento di Chimica Organica e Industriale, Universita’ degli Studi di Milano, via Venezian 21,
20133 Milan, Italy
b
Istituto di Chimica Organica “A. Marchesini”, Universita’ degli Studi di Milano, via Venezian 21,
20133 Milan, Italy
c
Dipartimento di Scienze Botaniche, Università degli Studi di Cagliari, Viale Sant'Ignazio da Laconi,
13, 09123 Cagliari, Italy
luisella.verotta@unimi.it
Received: August 5th, 2008; Accepted: November 17th, 2008
The aerial parts of Onopordum illyricum L. (Asteraceae) are eaten raw in salad in the Mediterranean area, representing a food
of good nutritional value. Extracts of different parts of this plant have been analyzed by HPLC/DAD/ESIMS and the major
compounds identified by NMR spectroscopy. Fatty acids, sesquiterpene lactones, triterpenes and polyphenols (flavones and
caffeoyl quinic acids) fully describe the plant metabolism during the vegetation year. All the metabolites are non toxic
nutrients, and are reported in the literature to possess biological activities positive for health, confirming the beneficial use in
the diet of this thistle
Keywords: Onopordum illyricum, Asteraceae, HPLC/PDA/ESIMS analysis, polyphenols, caffeoylquinic acids.
The
development
of
dietary
agents
for
chemoprevention is a highly attractive anticancer
strategy, because of the long-standing exposure of
humans to compounds of this type, their relative lack
of toxicity, and the existence of encouraging
epidemiological clues [1a,1b]. Polyphenols present in
the diet, for example, are receiving increasing interest
as chemopreventive agents because of the
epidemiological association between nutrients rich in
polyphenols and the prevention of diseases like
cancer and stroke [1c].
Onopordum illyricum L., Asteraceae, (“cardo
maggiore” or “onopordo maggiore”a type of thistle)
is included in the daily diet of Sardinian people. In
some villages in the Nuoro (Seulo, Torpè) and
Campidano districts it is used as a vegetable (“kàrdu
molentìnu” in campidanese idiom), young scapes
and capitula being eaten raw in salad, as side dishes
[2]. In folk medicine either a decoction or a tea of the
whole plant is used as a digestive, cough sedative and
in biliary diseases [2]. In Tempio Pausania village
(Northern Sardinia) the decoction or the infusion of
flowering tops is used for the alleged treatment of
malarial fever as an antipyretic and for washing
exanthematic skin [3].
Plants of the genus Onopordum are characterized by
the presence of sesquiterpene lactones, flavonoids
and lignans. Although a phytochemical study of this
species was presented a few years ago [4a], we
decided to investigate collections from different
vegetative periods of the phenotype growing in
Sardinia. O. illyricum is a wild plant spread in the
Mediterranean region in Portugal and Albania [4b].
This study was carried out on two different
collections, namely on the plant at full vegetation
(June) and the plant dried in the field (July
collection). Extracts of different parts (scapes and
capitula) were obtained through multiple solvent
extraction techniques (Table 1).
Variations in extraction yields between the
young plants and the old ones are plausible, due
to the residual water content in materials dried in the
2038 Natural Product Communications Vol. 3 (12) 2008
Verotta et al.
Table 1: Yields (g) of extractives from different parts of
Onopordum illyricum, collected at different periods.
Part (w)
Scapes and leaves
(419 g)
Capitula ( 274 g)
OI050603UT
Scapes and leaves
(473 g)
OI310703UI
Capitula (461 g)
OI310703UI
MeOH
14.0%*
n-Hexane
CHCl3
5.0 (1.2%)* 15.5 (3.7%)
n-BuOH
11.5 (2.7%)
11.2%
4.2 (1.5%)
2.7 (1.0%)
7.3 (2.6%)
11.0%
**
14.9 (3.2%)
13.2 (2.7%)
7.2%
**
11.3 (2.4%)
9.5 (2.1%)
* referred to the dry vegetable material
** total methanol extract was directly counter extracted with CHCl3
laboratory. A constant percentage is seen among the
most polar extracts (n-BuOH), while the less polar
ones show differences, mostly related to the presence
of chlorophyll in young material.
The n-hexane fraction contained fatty acids (mainly
oleic and linoleic acids) [5] and triterpenes
(taraxasteryl acetate and pseudotaraxasteryl acetate)
as major components. The dichloromethane extract
afforded onopordopicrin [6a,6b] as the most abundant
constituent,
accompanied
by
8α-sarracinoyl
salonitenolide as a by-product [6c]; these
sesquiterpene lactones represented more than 70% of
the total dichloromethane fraction. In this phenotype,
onopordopicrin alone represents about 1% of the
vegetable material.
The lipophilic extracts of young scapes and capitula
showed identical phytochemical profiles, which were
also identical to those of the aged material, with a
clear difference between the yield of fatty acids and
sterols and the sesquiterpene lactones that are more
abundant in the young vegetable material.
The n-BuOH extract from young scapes was
separated into ten fractions by chromatography on
Sephadex LH20. They were further purified by
preparative reversed phase HPLC to give two
dicaffeoylquinic acids [M+ 516 m/z] and a succinyl
dicaffeoylquinic [M+ 616 m/z] derivative.
Comparison of the spectroscopic data of the isolated
compounds with those reported in the literature
allowed the assignment of the structures as 3,5-di-Ocaffeoylquinic acid (1) [7a,7b], and 1,5-di-Ocaffeoylquinic acid (3) [7c]. The structure of the triester (2) was tentatively assigned as 1-succinyl 3,5dicaffeoylquinic acid by 1D- and 2D- NMR
experiments. The quinic acid ring was shown to be
esterified at positions 3 and 5 (proton chemical
shifts higher than 5.00 ppm), while the proton at
position 4 remained unchanged with respect to the
original value, for example in 5-caffeoyl quinic acid
Compound (2)
Table 2: 1H NMR (400 and 500 MHz) assignments δ, m, J (Hz)
for compounds 2 and 3.
Proton
8”
(2)
Acetone-d6
7.67 d, 15.9
7.60 d, 15.9
7.24 d, 2.0
7.22 d, 2.0
7.10 dd, 8.0,
2.0
7.05 dd, 8.0,
2.0
6.89 dd, 8.0,
3.1
6.88 dd, 8.0,
3.1
6.40 d, 15.9
8’
5
6.30 d, 15.9
5.52 m
6.28 d, 15.9 6.37, d,
6.21 d, 15.9
15.9
6.23 d, 15.9 6.33 d, 15.9 6.21 d, 15.9
5.25 m
5.47 m
5.24 bt, 8.3
3
5.46 dt,
5.27 m
4
3.63 bt, 8.0
6eq
4.04 dd, 8.6, 3.85 dd, 8.5, 3.96 dd,
3.5
3.3
8.5, 3.3
2.60 m
2.46 m
2.60 m
2eq
2.60 m
2.43 m
2.77 m
2.28 m
6ax
2.01 m
2.01bt, 12.1 1.94 bt, 12.0
2ax
2.60 m
1.94 dd,
10.6, 2.0
2.43 m
2’’’
3’’’
2.60 m
2.60 m
2.43 m
2.43 m
2.52 m
2.52 m
7”
7’
2”
2’
6”
6’
5”
5’
(2)
DMSO-d6
7.50 d, 15.9
7.50 d, 15.9
7.07 bs
7.07 bs
7.01 d, 8.0
7.01 d, 8.0
6.78 d, 8.0
6.78 d, 8.0
(2)
CD3OD
7.65 d, 15.9
7.63 d, 15.9
7.10 d, 2.0
7.08 d, 2.0
7.01dd, 8.0,
2.0
7.01 dd,
8.0, 2.0
6.82, d, 8.0
(3)
DMSO-d6
7.48, 15.9
7.48 d, 15.9
7.03 d, 2.0
7.03 d, 2.0
6.97 dd, 8.0,
2.0
6.97 dd, 8.0,
2.0
6.78 d, 8.2,
2.0
6.80 d, 8.0 6.78 d, 8.2
(3)
CD3OD
7.60 d, 15.9
7.60 d, 15.9
7.07 d, 2.0
7.07 d, 2.0
6.98 dd,
8.0, 2.0
6.98 dd,
8.0, 2.0
6.80 d, 8.0
6.32 d, 15.9
5.47 m
2.48 m
4.09 bt, 8.0
2.34 m
2.28 m
6.80 d, 8.0
6.28 d, 15.9
5.41 dt, 8.3,
3.5
4.31 ddd,
8.5, 8.0, 3.7
3.79 dd,
7.9, 3.3
2.59 dd,
13.2, 2.9
2.51, dd,
15.2, 3.4
2.08 dd,
13.2, 8.7
2.44 dd,
15.2, 4.4
(chlorogenic acid), where it resonates below 4.0 ppm.
The diagnostic resonance of carbon 1 (around 80
ppm) indicated esterification of the hydroxyl group
present on this carbon atom. Several attempts to
determine either heteronuclear multiple bond
correlations from the caffeoyl or succinyl side chain
carbons to ring protons, or vice versa were made,
even recording spectra in different solvents (see
Tables 2 and 3), in order to ameliorate chemical
shifts differentiation and solvent effects. However no
information was obtained on the correct ring
substitution, even by reproducing the experimental
conditions reported for 3,5 dicaffeoylquinic acid [7a].
HPLC/DAD/ESIMS analysis of extracts of Onopordum illyricum
Table 3: 13C NMR (100 and 125 MHz) assignments (δ, m)
for compounds 2 and 3.
Carbon
1
2
3
4
5
6
7
1’
1’’
2’
2’’
3’
3’’
4’
4’’
5’
5’’
6’
6’’
7’
7’’
8’
8’’
9’
9’’
1’’’
2’’’
3’’’
4’’’
(2)
DMSO-d6
80.0 s
32.3 t
71.8 d
70.0 d
70.3 d
o*
173.7 s
125.9 s
125.9 s
115.4 d
115.4 d
146.1s
146.1 s
149.0 d
149.0 d
116.3 d
116.3 d
121.8 d
121.7 d
145.7 d
145.7 d
114.6 d
114.6 d
166.5 s
165.6 s
172.1 s
29.1 t
29.4 t
172.2 s
(2)
Acetone-d6
78.9
32.2
71.2
69.9
69.9
36.1
173.5
126.7
126.7
114.4
114.5
146.3
145.6
148.5
148.5
115.5
115.5
121.9
121.7
146.0
145.4
114.6
114.5
165.5
166.1
171.4
29.5
29.5
171.4
(3)
DMSO-d6
79.8
34.6
67.9
71.4
70.5
36.2
173.0
126.0
126.0
114.8
114.8
145.8
145.7
148.9
148.9
115.3
115.3
121.8
121.8
146.0d
146.0d
114.7
114.7
166.4
165.7
o* =overlapped
Figure 2: Lowest minimum energy conformation for compound 2. The
arrow indicates the hydrogen bonding.
Superimposable chemical shifts and shape of the
proton spectra of the aromatic and quinic acid region
of the triester with those of 3,5-di-O-caffeoylquinic
acid (1) isolated in this work and by comparison with
literature data [7a], tentatively assigned compound 2
as 1-succinyl 3,5-di-O-caffeoylquinic acid. Partial
alkaline hydrolysis of 2 [7d] afforded a complex
mixture of compounds where 3,5-di-O-caffeoylquinic
acid was identified by TLC.
It becomes apparent from an unconstrained Monte
Carlo/Energy Minimisation (MC/EM) conformational
search [7e] by molecular mechanics methods that the
lowest minimum energy conformation (the 1C4 chair)
Natural Product Communications Vol. 3 (12) 2008 2039
is that originally reported for known quinic acid diand tri-esters. It accommodates the succinyl moiety
in an axial position, the conformational asset being
further supported by the existence of hydrogen
bonding between the succinyl terminal carboxy group
and the 5-O-caffeoyl carbonyl moiety (Figure 2).
To characterize the complete compositions of the
n-butanol extracts of scapes and capitula collected
at different vegetative periods, they were analyzed
by HPLC/PDA/ESIMS. Accordingly, flavonoid
glycosides are eluted first, followed by the group of
caffeoylquinic acids and the flavonoids at the end [8].
As can be seen in Figures 1A-D and Table 4,
caffeoylquinic acids are the dominant phenolic
compounds accumulating in the young material.
The presence of important nutritional biomarkers like
fatty acids, sesquiterpene lactones, triterpenes,
flavones and their glycosides and caffeoylquinic acid
derivatives justifies the use of O. illirycum as a
beneficial food with chemopreventive potential.
All the identified compounds in fact show interesting
in vitro and in vivo bioactivities. Onodorpicrin, a
sesquiterpene lactone, present in the leaves of
Arctium lappa L. [6b], has an IC50 value of
approximately 15 µM in cellular lineage of
promyelocytic leukemia (HL60) used as a model for
antitumor studies. In classic models of ulcer
induction the compound also presented significant
antiulcerogenic activity. The data also reveal that pretreatment with onopordopicrin is able to reduce
intestinal inflammation in a model of colitis in rats,
suggesting an excellent potential for therapy in the
gastrointestinal area [9]. Taraxasterol inhibits tumor
promotion, invasion of tumor cells and metastasis.
The acetate has weaker activity [10,10b]. Mono- and
di-caffeoylquinic acids play a key-role in the overall
anti-oxidant/health value of globe artichoke [10c].
Caffeoylquinic acids have been implicated in the
inhibition of HIV integrase, a key player in HIV
replication and its insertion into host DNA [10d],
the protection of proteins, lipids and DNA from
oxidative damage caused by free radicals [10e],
hepatoprotective, choleretic, diuretic, bile-expelling,
and antibacterial and antifungal activities [11a,11b].
A full description of the phytochemical profile of
O. illirycum has been reported. On this basis we can
assert that the presence of this plant in the daily diet
can be considered generally beneficial to human
health, thus confirming that people living in the
Mediterranean area still maintain a strong knowledge
of the traditional uses of plants.
2040 Natural Product Communications Vol. 3 (12) 2008
Verotta et al.
Table 4: HPLC/PDA/ESIMS of the n-BuOH extract of Onopordum illyricum aerial parts.
Compound
RT (min)
HPLC-DAD λmax (nm)
[M-H]-, [2M-H]- m/z
Molecular formula
Identification
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
11.75
19.93
20.30
20.90
22.36
22.88
24.25
25.11
26.35
28.22
29.29
31.65
32.78
37.65
38.77
219, 328
245, 335
245, 355
250, 350
245, 325
220,240, 325
240, 325
240, 330
245, 325
245, 325
235, 285
255, 270, 345
255, 265, 345
235, 275, 335
235, 265, 335
353
577
623
461
515
515
515
615
615
515
287, 575
315, 630
285, 571
299, 598
269, 539
C16H18O9
C27H30O14
C28H32O16
C22H22O11
C25H24O12
C25H24O12
C25H24O12
C29H30O15
C29H30O15
C25H24O12
C15H12O6
C16H12O7
C15H10O6
C16H12O6
C15H10O5
Chlorogenic acid
Apigenin rutinoside
Rhamnetin rutinoside
Kaempferide glucoside
1,5-Dicaffeoylquinic acid
3,5-Dicaffeoylquinic acid
Dicaffeoylquinic acid
1-Succinyl, 3,5-dicaffeoylquinic acid
Succinyldicaffeoylquinic acid
Dicaffeoylquinic acid
Dihydrokaempferol
Rhamnetin
Luteolin
4’-OMe apigenin
Apigenin
6
A
8
B
7
9
C
5
1
3+4
2
10
11
14
12
13
D
15
Figure 1: HPLC/PDA/ESIMS analyses of Onopordum illyricum n-butanol extracts (λ = 350 nm). Scapes (A), capitula (B), dry scapes (C), dry capitula (D).
Experimental
Plant material: This was collected at Uta (Cagliari
district) in June (fresh material) and July (field dried
material) 2003, and identified by M.C. Loi,
Department of Botanical Science, University of
Cagliari, Italy, where a voucher specimen was
deposited. The fresh materials were dried at room
temperature.
Analytical and prep. HPLC: A) Waters 515 HPLC
pump, equipped with a Waters 2487 Dual λ
Absorbance Detector (fixed wavelengths 280, 330
nm) and a Waters Symmetry Shield RP18 5μm (4.6
x 250) column, eluted with a gradient of 0.1%
CF3COOH in H2O (solvent A) and 0.1%
CF3COOH/MeOH (solvent B), starting from 55%
solvent A for 16 min, to 100% solvent B, at a flow
rate 1 mL/min. 20 μL of solutions containing
5 mg/mL were injected. On the same instrument, the
preparative separations were obtained through a
Waters Symmetry Prep RP18 7 μm (190 mm x 150
mm), at a flow rate of 4 mL/min, eluted under
isocratic conditions (25 min) with H2O-MeOH 70:30
and 0.1% di CF3COOH. B) Alternatively, a gradient
was used of 0.1% CF3COOH in H2O (solvent A) and
0.1% CF3COOH/CH3CN (solvent B), starting from
90% solvent A to 90% solvent B in 60 min, at a flow
rate of 1 mL/min.
HPLC-PDA-MS analyses: A Thermo Finnigan
instrument LCQ Advantage Series (Thermo
Finnigan, San Jose, CA, USA), equipped with a
quaternary pump (Surveyor), a diode-array detector,
an electrospray ionisation source (ESI) and an ion
HPLC/DAD/ESIMS analysis of extracts of Onopordum illyricum
Natural Product Communications Vol. 3 (12) 2008 2041
trap analyser (negative mode) was used for LC–MS
analyses and to obtain UV–vis and mass spectra of
eluted compounds. The n-BuOH extracts and the
single fractions from its purification (see below) were
carried out using a Symmetry Shield (Waters) RP 18
250 x 4.6 mm I.D. (5 μm) column maintained at 30
°C by a column block heater (model 7970, Hichrom
Ltd., Reading, UK). A linear gradient from 90:10 to
30:70 v/v (H2O:CH3CN) with 0.1% w/v TFA and a
flow rate of 1mL/min was used. Samples were
dissolved in the mobile phase and injected through a
Rheodyne (model 7125) valve equipped with a 20 μL
loop. Spectral data for the wavelength range 190 to
600 nm, using a photodiode-array detector
(Surveyor), and integrated areas under the peaks
detected at 350 nm were acquired. Three dimensional
chromatograms using Thermo Xcalibur software
(Thermo Finnigan) were recorded.
pseudotaraxasteryl acetate and taraxasteryl acetate
(84 mg). Fractions eluted with EtOAc (290 mg)
were further purified on silica gel, eluted with
CHCl3/i-PrOH 9:1 (400 mL), giving a mixture of
oleic and linoleic acids (190 mg). Taraxasteryl
acetate and pseudotaraxasteryl acetate were identified
in a mixture by comparison of their IR, 1H, 13C NMR
spectra with literature data. [11c,12].
Extraction and purification: Scapes and leaves
(OI050603UT, 419 g) were reduced to powder with a
robot mixer and extracted through percolation with
MeOH at room temperature (4 x 1.8 L). After solvent
evaporation, 58.5 g (14%) of total extract was
obtained, which was added to MeOH (60 mL) and
water (300 mL), filtered to remove a sticky material,
and extracted with light petroleum (2 x 213 mL), then
CH2Cl2 (2 x 213 mL), and n-butanol (4 x 300 mL).
The organic phases were dried (Na2SO4) and
evaporated to dryness under vacuum. Yields are
reported in Table 1.
Extractions were controlled through TLC, developed
with CHCl3-MeOH (9:1), and revealed through either
UV absorption or by spraying with methanolic H2SO4
10%. The n-butanol fraction was monitored by TLC
using the organic phase of the solvent system CHCl3MeOH-n-PrOH-H2O 5:6:1:4 and revealed through
either UV absorption or by spraying with FeCl3. The
same procedure was followed to extract capitula
(OI050603UT 274 g). Yields are reported in Table 1.
The dried vegetable material collected (OI310703UI)
followed a slightly different extraction procedure
avoiding the counter extraction with light petroleum,
mainly because of the absence of chlorophyll. Yields
are reported in Table 1.
The light petroleum extract (2 g) was purified by
column chromatography on silica gel (100 g), eluted
with CH2Cl2 (780 mL), CH2Cl2-EtOAC 95:5 (810
mL) and EtOAc (270 mL). Fractions were pooled
according to their composition. One of the fractions
eluted with CH2Cl2 (230 mg) was repeatedly
crystallized from EtOAc, giving a mixture (1:3) of
The dichloromethane extract (5.2 g) was purified by
column chromatography on silica gel (120 g) eluted
with CH3Cl-MeOH 95:5 (720 mL). Fractions 7-15
(3.2 g) were further purified on silica gel (flash
column) eluted with light petroleum-EtOAc (2:3),
yielding 2.4 g onopordopicrin (1) [Rf= 0.22, EtOAclight petroleum (70:30) (42.5%)] and 0.32 g 8αsarracinoyl salonitenolide [Rf= 0.21, EtOAc-light
petroleum (70:30)].
The n-butanol extract (5 g) was purified by
chromatography on Sephadex (2.8 x 100 cm) eluted
with MeOH (1 L), at a flow rate of 15 mL/min.
Fractions were pooled according to their profile on
TLC (CHCl3-MeOH-n-PrOH-H2O 5:6:1:4) and
revealing through either UV absorption or by
spraying with FeCl3): 1-57 (3.13 g), 58-74 (0.323 g),
75-80 ( 0.057 g), 81-99 (0.426 g), 100-105 (0.197 g),
106-119 (0.183 g), 120-136 (0.295 g), 140-143 (0.05
g), 144-167 (0.1192 g), 167-184 (0.043 g)
Fraction 81-99 was submitted to prep HPLC
according to method A. 10 mg each of two
compounds (1) (Rt 20.75 min, method A; 23.25,
method B) identified as 3,5 dicaffeoylquinic acid
[7a,7b], and (2) ([α]25D = - 30.1 (c 0.5, MeOH); UV
λmax (nm) 240, 330) (Rt 26.17 min, method A; 25.20,
method B) were isolated. Fraction 106-119 was
submitted to prep HPLC according to method B. 22
mg of 1,5-di-O-caffeoylquinic acid (3) [7c] (Rt
22.85, method B) was isolated.
Computational studies: Conformational preferences
of
1-succinyl
3,5-di-O-caffeoylquinic
acid
(compound 2) were investigated by molecular
mechanics calculations within the framework of
MacroModel version 9.1 (Macromodel), using the
MacroModel implementation of the MM2 force field
[13] (denoted MM2*) and the implicit water GB/SA
solvation model [14a]. The torsional space of the
compound was randomly varied with the usagedirected Monte Carlo conformational search of
Chang, Guida, and Still [7e]; 1000 starting structures
for each variable torsion angle were generated and
minimized until the gradient was less than 0.05
2042 Natural Product Communications Vol. 3 (12) 2008
kJ/Åmol, using the truncated Newton-Raphson
method [14b] implemented in MacroModel.
Duplicate conformations, and those with energy
greater than 6 kcal/mol above the global minimum
were discarded.
Verotta et al.
Acknowledgments – Mrs Manuela Gilberti is
gratefully acknowledged for technical assistance in
recording NMR experiments.
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NPC
Natural Product Communications
Phenolic Profiles of Four Processed Tropical Green Leafy
Vegetables Commonly Used as Food
2008
Vol. 3
No. 12
2043 - 2048
Sule Ola Salawua, Marzia Innocentib, Catia Giaccherinib , Afolabi Akintunde Akindahunsia and
Nadia Mulinacci*,b
a
Department of Biochemistry, Federal University of Technology, P.M.B. 704, Akure, Nigeria
b
Department of Pharmaceutical Science, and CeRA University of Florence, Sesto F.no, (FI), Italy
nadia.mulinacci@unifi.it
Received: July 23rd, 2008; Accepted: October 28th, 2008
The phenolic profiles are presented of four tropical green leafy vegetables (Ocimum gratissimum, Vernonia amygdalina,
Corchorus olitorius and Manihot utilissima) commonly used as food, after application of traditional treatments, such as boiling
and abrasion. The HPLC/DAD/MS technique was mainly used to carry out this study. Preliminary evaluation of the antioxidant
properties of the vegetables was also performed using the DPPH in vitro test. For the first time, seasonal variations in the
phenolic content of the four investigated vegetables were highlighted. Of the four plants, all showed only quantitative
differences, except for Ocimum graticimum, in which cichoric acid, previously detected as one of the main constituents of this
vegetable collected in November (dry season), was absent in the sample harvested in March. The phenolic constituents are
chemically unmodified after a strong heating process, such as the traditional blanching (about 15 minutes) applied by Nigerian
people prior to consuming these vegetables. Nevertheless, these typical preparations showed a consistent decrease in the total
phenolic compounds with respect to the raw material, particularly for Corchorus olitorius (from 42.3 to 5.56 mg/g dried
leaves) and Vernonia amygdalina (from 40.2 to 4.4 mg/g dried leaves). As expected, when the blanching treatment is reduced
to a few minutes, as for Manihot utilissima leaves, the cooked vegetable maintained almost unaltered its original phenolic
content (around 10 mg/g dried leaves). The unique exception is the blanched Ocimum gratissimum sample that showed a
consistent increment of the total phenols, particularly of rosmarinic acid (from 6.1 to 29.8 mg/g dried leaves) with respect to
the unprocessed vegetable.
Keywords: Ocimum gratissimum, Vernonia amygdalina, Corchorus olitorius, Manihot utilissima, phenolic compounds,
traditional preparations, HPLC/DAD/MS.
Most of the compositional aspects of vegetables
commonly used in the Western diet are well known,
but scant data are available on endemic plants from
African regions. The present study attempts to
improve knowledge of the phenolic content of the
processed leaves of four vegetables commonly used
for food and medicinal purposes in Nigeria.
Ocimum gratissimum L. (Og) (Labiateae), Manihot
utilissima Pohl. (Mu) (Euphorbiaceae), Vernonia
amygdalina L. (Va) (Compositae), and Corchorus
olitorius L. (Co) (Tiliaceae) are considered in the
present work. Among them, Og, Va and Co are
mainly consumed as fresh or pot vegetables. The
tuber of Mu is the part mainly eaten, but the young
leaves are gaining acceptance also as a pot vegetable.
Ocimum gratissimum, African basil, and known as
“efinrin” by Nigerian people, is usually collected
from May to October. With regards to its
composition, the principal data are related to its
flavonoid composition [1], but mainly related to
plants grown in the UK [2]. The authors found that
the profile of the main flavonoids was similar in all
accessions belonging to the same species and they
showed vicenin-2, luteolin 7-O-glucoside, quercetin
3-O-glucoside and quercetin 3-O-rutinoside as
constituents of the leaves, together with
xanthomicrol, cirsimaritin, and kaempferol-3-Orutinoside. For medicinal properties, in south-western
Nigeria, Og is mainly known for its antimicrobial
activities against bacteria causing diarrhea [3].
2044 Natural Product Communications Vol. 3 (12) 2008
Manihot utilissima or cassava, known by the local
population as “ewe ege”, is harvested throughout the
year. This vegetable is widespread in Nigeria, which
is the world's largest producer. Though less popular
in the Nigerian diet compared with other vegetables,
the dietary acceptance of Mu leaves has been
increasing within local populations [4].
Vernonia amigdalina leaves, known as “ewuro” by
the local population, are harvested throughout the
year and are characterized by an intense bitterness.
Previous reports on the composition of this plant
highlighted the presence of luteolin, luteolin 7-O-βglucoside and luteolin 7-O-β-glucuronide as
main flavonoids, together with several dicaffeoyl
derivatives [5]. Also, some saponins and
sesquiterpene lactones have been reported in the
leaves [6]. Due to its documented antimalarial [7-9],
antimicrobial [10-11] and anticancer properties [12],
Va is probably one of the most used medicinal plants
in Nigeria.
Corchorus olitorius or “Tossa Jute”, is quite popular
for its leaves, which are usually collected from May
to December and used as a pot herb. Jute leaves, also
known as Jew's Mallow, are popular in West Africa
and the Yoruba people of Nigeria call it "ewedu".
The leaves are made into a common mucilaginous
soup or sauce in some West African cooking
traditions. C. olitorius leaves contain kaempferol
glycosides, rutin and isoquercitrin [13], together with
chlorogenic acid and several dicaffeoyl derivatives of
quinic acid [5]. With regard to its use as a medicinal
plant, this vegetable is mainly known in Nigeria for
its laxative activity and as a blood purifier [14].
The present study was mainly focused on comparing
the phenolic profile of the four vegetables obtained in
different seasons to determine the qualitative and
quantitative content of these compounds in the
processed vegetables after application of traditional
treatments, and to evaluate some antioxidant
properties by using the DPPH in vitro test.
Phenolic distribution in the unprocessed leaves. To
recover all the main phenolic constituents, the dried
leaves were extracted with ethanol/acidic water (7:3)
at room temperature, and the extracts were then
analyzed
using
a
previously
optimized
chromatographic method [5]. The identification of
the components of the extract was carried out by
comparison with our previous results [5], with the
help of their retention times, and UV-Vis and MS
Salawu Sule et al.
spectra. When necessary, the use of standard
reference compounds and/or laboratory extracts
helped to complete this identification.
Our previous findings, which highlighted for the first
time the phenolic composition of these vegetables,
took into account samples collected in Nigeria during
the dry season (November), while in this study we
analyzed plants collected in March. Given that these
vegetables are consumed throughout the year by the
local population, it is of interest to evaluate if
seasonality affects the phenolic composition of the
vegetables.
Regarding Co, its chromatographic profile at 330 nm
is similar to that obtained for the sample collected in
November, even if an increment of the ratio between
the more polar monocaffeoyl quinic derivative (Co1)
and the 1,5 dicaffeoyl quinic acid (Co 5) is observed.
It is reasonable to hypothesize that these two
molecules are biosynthetically correlated and that
different climatic conditions can modulate their
production in the plant.
The dominant compound detected in the Va sample
was 1,5 dicaffeoyl quinic acid (Va 10), differently
from the findings observed for the leaves collected in
November. At the same time, almost all the
flavonoids, both luteolin and apigenin glycosides,
were minor constituents.
Among the four plants, the chemical profile of Og is
the most complex due to the co-presence of
metabolites belonging to different chemical classes
(Table 1d). Major differences were observed between
the leaves collected in the two seasons. In the March
sample, together with a consistent amount of
rosmarinic acid and the presence of the polar
glycoside, vicenin 2 or apigenin 6,8-di-C-glucoside, a
high concentration of the metoxyflavone, nevadensin,
has again been highlighted [5]. Nevertheless, it is
worth noting that cichoric acid, one of the main
phenolic constituents previously detected and
estimated at nearly 2.5 mg/g in the dried leaves
collected in November [5], was completely absent
in this sample. In the light of this finding, it can be
said that, differently from the other phenolic
constituents, the biosynthesis of this dicaffeoyl
tartaric acid in African basil is particularly sensitive
to seasonal variation. Contrary to the HPLC profiles
observed at 330 nm for Va and Og, that for Mu
seemed to be unaffected by the time of harvest.
In fact, the relative ratios between the four main
Phenolic profiles of four tropical green leafy vegetables
Natural Product Communications Vol. 3 (12) 2008 2045
Table 1a
Compounds of Corcorus olitorius
Raw
Blanched
Co1 - caffeoyl quinic derivative
14.8±0.4
2.8±0.03
Co2 - chlorogenic acid
0.2±0.02
0.07±0.00
Co3+4 - hyperoside+isoquercitrin
2.1±0.06
0.3±0.004
Co5 1, 5 -dicaffeoyl quinic acid
23.2±0.6
2.0±0.03
Co6 - dicaffeoyl quinic acid
0.1±0.004
0.002±0.002
Co7 - dicaffeoyl derivative
1.7±0.03
0.5±0.004
Co8 - quercetin derivative
0.1±0.009
nd
Table 1b
Compounds of Vernonia amigdalina
Raw
Blanched
Va1+2 - caffeoyl quinic and chlorogenic acids
2.3±0.3
0.4±0.02
Abrasion
0.5±0.04
Va4 - rutin
0.2±0.04
nd
nd
Va5 - luteolin 7-O-glu
2.7±0.3
0.3±0.02
0.4±0.02
Va7 - flavonoid
0.3±0.03
0.1±0.009
0.005±0.006
Va9 - luteolin 7-O-glucuronide
4.9±0.5
0.7±0.02
0.7±0.04
Va10 -1,5 dicaffeoyl quinic acid
23.47±3.0
1.4±0.2
6.9±1.5
Va11 - dicaffeoyl quinic acid
2.6±0.1
0.3±0.02
0.5±0.08
Va12 - dicaffeoyl quinic acid
2.5±0.4
0.4±0.03
1.3±0.5
Va13 - apigenin-O-glucuronide
0.7±0.02
0.4±0.02
0.2±0.02
Va14 - luteolin
0.2±0.03
0.1±0.01
0.6±0.06
Va15 - flavonoid
0.1±0.02
0.1±0.01
0.02±0.004
Va16 - flavonoid
0.09±0.01
0.04±0.004
0.3±0.01
Va17 - flavonoid
0.06±0.01
0.1±0.001
0.0015±0.0
Table 1c
Compounds of Manihot utilissima
Raw
Mu1 - rutin
5.1±0.2
Blanched
5.8±1.0
Mu2 - kaempferol 4’-O-rut
0.7±0.05
0.8±0.2
Mu3 - kaempferol 3’-O-rut
2.5±0.1
2.8±0.5
Mu5 - amentoflavone
0.9±0.008
1.1±0.2
Table 1d
Compounds of Ocimum graticimum
Raw
Blanched
Abrasion
Og1 - vicenin-2
1.5±0.002
1.0±0.06
0.9±0.06
Og2 - caffeic acid
0.5±0.01
0.5±0.03
0.3±0.007
Og3 - rutin
0.2±0.003
0.1±0.001
0.02±0.001
Og4 - luteolin-7-O-glucoside
0.3±0.01
0.2±0.02
nd
Og5 - kaempferol 3’-O-rutinoside
0.04±0.006
0.03±0.0
nd
Og6 - rosmarinic acid
6.1±0.6
29.8±1.7
1.0±0.2
Og10 - cirsimaritin
0.7±0.05
0.6±0.01
0.5±0.02
Og11 - nevadensin
5.8±0.4
5.0±0.3
4.6±0.2
Table 1a-d: Phenolic compounds in processed and unprocessed leaves. All the data are a mean of three different determinations and are expressed as mg/g
(SD) dried leaves.
constituents (Mu1-Mu4) was unaltered when
compared with those observed for the material
collected in rainy season [5].
Evaluation of the phenolic content. In Nigeria
several green vegetables, among them Va, Mu, Co
and Og, are usually blanched before consumption
using either hot water or steam. Often indigenous
people apply an abrasion treatment to the fresh leaves
to remove part of the juice in order to reduce
the bitterness and/or acidity of the plant. Among
these selected Nigerian plants, this latter treatment
is traditionally applied only to Va and Og and,
therefore, in this study manually squeezed leaves
2046 Natural Product Communications Vol. 3 (12) 2008
Salawu Sule et al.
extracting the phenolic fraction from the leaves and
consequently the blanched vegetable, used as food by
the local population, remains a good source of
phenolic compounds.
45
40
35
30
25
As highlighted for the unprocessed leaves, the total
absence of cichoric acid in the Og extracts after
blanching and abrasion was confirmed. Moreover, a
peculiar behaviour was revealed for the rosmarinic
acid that increased consistently (from 6.1 to 29.7
mg/g) in the blanched sample (Table 1d).
20
15
10
5
n
ra
si
o
ab
O
g
an
ch
ed
ra
w
O
g
bl
O
g
si
on
ab
ra
Va
ch
ed
ra
w
Va
bl
an
Va
ch
ed
ra
w
bl
an
M
u
d
nc
he
o
o
bl
a
C
C
M
u
ra
w
0
Figure 1: Comparison of the total phenolic content in all the samples
expressed as mg/g dried leaves.
Table 2: DPPH radical scavenging activity.
DPPH results from hydroalchoolic extracts
Og
0.05 mg/mL
82.9 ±1.0
0.1 mg/mL
88.0 ± 0.4
0.15 mg/mL
88.9 ± 1.0
79.0 ±0.4
Va
55.9 ± 3.2
65.7 ±1.9
Mu
53.1 ±1.4
78.4 ±0.2
81.1±1.8
Co
84.2 ±0.1
79.2± 2.2
88.9 ±1.0
were also analyzed. The quantitative distribution of
the different phenolic compounds in the processed
and unprocessed leaf extracts is summarized in
Tables 1a-d and in the histogram of Figure 1.
Comparison of the chromatographic profiles of Co
obtained from the blanched and raw leaves showed
an inversion of the relative contents of the Co1 and
Co5 compounds. Moreover, a consistent decrease of
the total phenolic amount (from 42.3 to 5.6 mg/g) in
the processed leaves was observed (Table 1a and
Figure 1). The greater amount of soluble fiber of this
plant [15,16] is removed during the blanching
process producing a gel that can entrap part of the
more soluble phenols released from the leaves.
A dramatic decrease in the total phenolic content was
observed for Va after blanching and abrasion,
suggesting that these compounds are mainly
localized in the juice and consequently they are
almost completely removed from the vegetable after
these treatments.
With regard to Mu, as confirmed by the quantitative
data (Table 1c), very few changes were observed
between the raw and blanched leaves. Effectively the
young Mu leaves, differently from the other plants,
are traditionally blanched only for a few minutes.
This short time of boiling is not efficacious in
Antioxidant activity by DPPH test. One of the aims
of this study was to make a preliminary evaluation of
the antioxidant potency in terms of free radical
scavenging of the phenolic fractions obtained from
the leafy vegetables of these plants. Most of the
antioxidant activities of vegetables and fruits have
been established to be related to phenolic compounds
[17]. Free radical scavenging is one of the known
mechanisms by which antioxidants inhibit lipid
peroxidation [18,19]. The DPPH radical scavenging
activity has also been used extensively for screening
antioxidants from fruits and vegetables [20].
This activity was measured for the extracts from
unprocessed leaves at three different concentrations.
A summary of the results expressed as % DPPH
radical scavenging activity is reported in Table 2.
Overall, all the tested vegetable materials showed a
relatively high inhibition at the highest tested
concentration and this activity could be attributed to
the presence of flavonoids and cinnamoyl derivatives
in the hydroalcoholic extracts. The results from Va
and Mu seem to be dose dependent. Taking into
account the lower concentration, the best results in
terms of antioxidant potency were obtained for Og
and Co, but no differences among the extracts were
highlighted with the higher concentrations.
Experimental
Materials: The vegetables were harvested from the
teaching and research farm of the Federal University
of Technology, Akure, Nigeria in March 2008 and
voucher specimens were deposited at the Department
of Biochemistry, Federal University of Technology,
Akure,
Nigeria
and
the
Department
of
Pharmaceutical Science, University of Florence,
Italy. About 500 g of fresh green leafy vegetables,
Vernonia amygdalina (Va), Corchorous olitorius
(Co), Ocimum graticimum (Og) and Manihot
utilissima (Mu), were rinsed in water, and the edible
portions separated. The edible portions were chopped
Phenolic profiles of four tropical green leafy vegetables
Natural Product Communications Vol. 3 (12) 2008 2047
into small pieces (300 g) and divided into two (Mu
and Co) or three portions (Va and Og). The first
portion of the chopped vegetables served as the
unprocessed sample, the second portion was blanched
in 300 mL boiling water for 15 min (Va, Og and Co)
and 5 min for Mu, while the third portion, with the
aid of small quantity of water, was manually
squeezed by hand (abrasion) to remove the juice. The
blanched and squeezed portions were subsequently
drained of water.
330 nm (range 0.038-0.3 mg/mL and r2 of 0.9996)
was used to evaluate all the cinnamoyl compounds;
luteolin 7-O-glucoside at 330 nm (range 0.11-0.88
mg/mL and r2 of 0.9999) was selected to evaluate all
the luteolin and apigenin derivatives, together with
nevadensin; rutin at 350 nm (range 0.13-1.02 mg/mL
and r2 of 0.9999) was used to quantify all the
derivatives of quercetin and kaempherol. All the
quantitative data were obtained in triplicate.
All samples were dried in an air oven at 30°C prior to
analysis and treated as described in the next
experimental section.
The standards used to confirm the chemical structure
of some compounds (Table 1a-d) were purchased
from Extrasynthese (Geney-France); rutin was from
Sigma-Aldrich (St. Louis, MO-USA).
Extraction methods for HPLC/DAD analysis of the
processed samples: (1 g each) was extracted with
stirring for 2 h in 40 mL (20 mL x 2) of ethanol/water
7:3 (v/v) with water acidified with formic acid (pH
2.5). The samples were centrifuged (4,400 rpm for 10
min) and the supernatant was centrifuged again
(12,000 rpm for 8 min) to obtain a clear solution
directly analyzed by HPLC/DAD.
HPLC/DAD/MS analysis: Analyses were performed
using a HP 1100 liquid chromatograph equipped with
HP DAD and 1100 MS detectors; the interface was a
HP 1100 MSD API-electro spray. All the instruments
were from Agilent Technology (Palo Alto, CA,
USA). The MS analyses were carried out in negative
mode with a fragmentor range between 80-150 V.
Method 1. A C12 column, 150 × 4 mm (4μm)
Synergi Max (Phenomenex-Torrance CA) maintained
at 30°C and equipped with a 10 × 4 mm pre-column
of the same phase was used with a flow rate of 0.4
mL min-1. The eluents were H2O acidified to pH 3.2
with formic acid (A) and acetonitrile (B). The
following linear solvent gradient was applied: from
95 % A to 85% A in 5 min, to 75% A in 8 min and a
plateau of 10 min, to 55% A in 12 min and a plateau
of 5 min, to 10% A in 3 min, and a final plateau of 2
min to wash the column. The total time of analysis
was 45 min.
Quantitative determination: Chlorogenic acid, rutin
and luteolin 7-O-glucoside were used for the
quantitative evaluation. Three five-point calibration
curves were prepared as follows: chlorogenic acid at
DPPH assay: A dried sample (1 g each) was
extracted, by stirring for 2 h, with 40 mL (20 mL x 2)
of either ethanol or ethanol/water 7:3 (v/v), with
water acidified with formic acid (pH 2.5). The
samples were filtered, concentrated to dryness and
redissolved in 96% ethanol. The clear solutions
were directly analyzed by HPLC/DAD and 3
concentrations (0.05, 0.1 and 0.15 mg/mL) used for
the DPPH assay. The radical scavenging activity of
ethanol and ethanol/water extracts was carried out as
follows: 2 mL of each extract was mixed with 1mL
of 0.125 mM DPPH ethanol solution. After shaking
the mixture, the absorbance was measured at 517 nm
after 5 min of incubation. Radical scavenging activity
is expressed as the inhibition percentage.
Conclusions: For the first time, seasonal variation in
the phenolic content of the four investigated
vegetables was highlighted. Within the four plants,
almost all showed only quantitative differences, with
the exception of Ocimum graticimum. In fact,
cichoric acid, previously detected as one of the main
constituents of this vegetable collected in November
(dry season), was completely absent in the sample
harvested in March. The phenolic constituents are
chemically unmodified after a strong heating process
such as the traditional blanching (about 15 minutes)
applied by Nigerian people prior to consuming these
vegetables. Nevertheless, these typical preparations
showed a consistent decrease in the total phenolic
compounds with respect to the raw material,
particularly Corchorus olitorius (from 42.3 to 5.56
mg/g dried leaves) and for Vernonia amygdalina
(from 40.2 to 4.4 mg/g dried leaves). As expected,
when the blanched treatment is reduced to a few
minutes, as for Manihot utilissima leaves, the cooked
vegetable maintained almost unaltered its original
phenolic content (around 10 mg/g dried leaves). The
unique exception is the blanched Ocimum
gratissimum sample that showed a consistent
increment in total phenols, particularly of rosmarinic
acid (from 6.1 to 29.8 mg/g dried leaves) in
comparison with the unprocessed vegetable.
2048 Natural Product Communications Vol. 3 (12) 2008
Acknowledgments - This research was partially
supported by the Italian M.I.U.R. (Ministero
Istruzione Università e Ricerca) and we are grateful
to Ente Cassa di Risparmio di Firenze for supplying a
Salawu Sule et al.
part of the instrumentation used for this research. We
wish to equally acknowledge the ICTP/IAEA who
supported the stay of S.O. Salawu in Italy through a
PhD Sandwich Training Educational fellowship.
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NPC
Natural Product Communications
(Bio)Sensor Approach in the Evaluation of Polyphenols in
Vegetal Matrices
2008
Vol. 3
No. 12
2049 - 2060
M. Camilla Bergonzia*, Maria Minunnib and Anna Rita Biliaa
a
Dipartimento di Scienze Farmaceutiche, via U. Schiff 6, 50019 Sesto Fiorentino, Firenze, Italy
b
Dipartimento di Chimica, via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze, Italy
mc.bergonzi@unifi.it
Received: August 11th, 2008; October 20th, 2008
Polyphenols are compounds widely distributed in the plant kingdom and have attracted much attention, because of their health
benefits and important properties such as radical scavenging, metal chelating agents, inhibitors of lipoprotein oxidation,
anti-inflammatory and anti-allergic activities. Due to their important role in the diet and in therapy, it is important to estimate
their content in the different matrices of interest. Besides classical analytical methods, new emerging technologies have also
appeared in the last decade aiming for simple and eventually cheap detection of polyphenols. This review focused on the recent
applications of biosensing-based technologies for polyphenol estimation in vegetal matrices, using different transduction
principles. These analytical tools are generally fast, giving responses in the order of a few seconds/minutes, and also very
sensitive and generally selective (mainly depending on the enzyme used). Direct measurements in most of the investigated
matrices were possible, both in aqueous and organic phases.
Keywords: Sensor, polyphenols, tyrosinase, laccase, peroxidase, plant tissue, vegetal matrices.
Polyphenols are widely distributed in the plant
kingdom, including foods of vegetable origin,
contributing to their taste and sensorial properties
(such as olive oil and wine) and constituting an
important role in human diet. These compounds are
mainly represented by simple phenolic acids
(hydroxybenzoic acids and hydroxycinnamic acids),
flavonoids and tannins (hydrolyzable tannins and
condensed tannins) [1,2].
Recently, polyphenols have attracted much attention,
because of their health benefits, being considered
responsible for the majority of the antioxidant
capacity in plant-derived products, with a few
exceptions, such as carotenoids. It is reported that
within the Mediterranean diet, the average daily
intake of polyphenols is about 1 g, which is almost
10-fold the intake of vitamin C, 100-fold the intake
of vitamin E, and 500-fold the intake of carotenoids
[3]. Foods and beverages rich in polyphenols may
have a large potential with respect to prevention of
diseases, and fruits and vegetables are generally
associated with the prevention of stroke [4] and
cancers [5,6], including breast cancer [7,8].
Polyphenols act as free radical scavengers [9,10],
metal chelating agents [11], inhibitors of lipoprotein
oxidation [12] anti-inflammatory agents [13] and
have anti-allergic properties [14].
Due to their important role in food and in therapy, it
is important that there is a simple and fast estimation
of their content in the different matrices of interest.
Several methods for polyphenols detection in plant
sources are described in the literature, the most
known and simplest approach being the FolinCiocalteu spectrophotometric method [15], but this
presents limitations since it can estimate other
different reducing non-phenolic compounds too. As
an alternative to the Folin-Ciocalteu assay, a
fluorimetric evaluation of the total phenol content in
vegetal matrices and extracts was also proposed [16].
Other classic techniques used in the evaluation of
polyphenols are UV-visible [17] and FT-NIR
spectroscopies [18].
One of the most selective analytical methods is based
on high-performance liquid chromatography (HPLC)
combined with different detection methods: UV-Vis
detection [19], chemiluminescence detection [20],
fluorescence detection, DAD-ESI-MS detection, and
2050 Natural Product Communications Vol. 3 (12) 2008
direct electrochemical detection [21]. However, these
instrumental
methods,
although
performing
complete and sensitive analyses, require long
sample processing (i.e. extraction, concentration,
resuspension), massive use of organic solvents, costly
instrumentation and skilled personnel.
In view of the need for simple and easy analytical
methods for rapid estimation of polyphenolic content,
one interesting approach is the electroanalytical
techniques, due to the electrochemical behaviour
shown by phenolic compounds. New emerging
technologies have also appeared in the last decade
aiming to provide simple, fast and eventually cheap
detection of polyphenols. These are mainly based on
electrochemical techniques, but some optical-based
sensing has also been proposed.
In this review, some sensing approaches based on
different transduction principles are reported,
focusing on polyphenol characterization and
quantification.
A chemical sensor is a device responding to a
chemical stimulus, giving a recordable signal. When
the sensor is coupled to a biological molecule, a
biosensor is generated. Following the IUPAC
definition, “A biosensor is a whole and integrated
device providing analytical information (qualitative
or semi/quantitative) by using a biomolecular
recognition element (biochemical receptor) in close
spatial contact with a transducer. The transducer
converts the chemical event into a recordable signal.
Both sensors and biosensors can be considered
innovative analytical devices able to detect different
analytes in a qualitative and quantitative manner
directly in complex matrices, without or with very
little sample pre-treatment. They have been presented
eventually as the “analyst dream” for the
simplification they make in the analysis [22].
The paradigmatic example is represented by the
glucose sensor, which represented a real revolution
for diabetes patients; with this very small device
(dimension of a pen, also called “glucose pen” in
some versions) the glucose content in blood is
estimated in a few seconds. The detection principle is
amperometry, meaning that a current is the nature of
the response, correlated to glucose concentration, and
the selectivity is due to the enzyme glucose oxidase,
which selectively uses glucose as substrate. The
catalysis is at the base of the enzymatic event and for
these reasons enzymatic based sensors are called also
catalytic sensors. We can thus say that we could
Bergonzi et al.
Enzymatic (catalytic)
The biological element (enzyme)
converts the substrate into a
product
S
P
Affinity
The biological element (receptor)
binds specificallythe analyte
leading to a complex
A+B
AB
The transducerreveals S or P
The transducer reveals the
complex
Figure1: Catalytic and affinity-based biosensors.
discriminate between mainly two different classes of
biosensors: catalytic and affinity–based ones
(Figure1).
The recognition element, immobilized on the sensing
surface (i.e. an electrode, an optic fiber, a planar
waveguide) is respectively an enzyme (or a system of
enzymes) or a receptor able to form an affinity
complex with the target analyte. To the affinity
sensor category belongs: immunosensors, where the
receptors are an antigen or an antibody, or a DNA
sensor with suitable probes immobilized on the
sensor surface. In Figure 2 are shown some examples
of receptors employed both in catalytic and affinity
sensors.
Relative to polyphenol analysis, catalytic biosensors
are the most used. Different enzymes have been used
over the years as a catalytic element coupled to
electrochemical analysis, such as tyrosinase (also
called polyphenol oxidase), laccase and peroxidase,
using different electrode materials, flow systems and
sample pre-treatment techniques since phenolic
compounds can act as electron donors for these
enzymes [23-26].
Phenol oxidases and peroxidases have different
enzymatic mechanisms of action in the
electrochemical biosensors. Enzyme molecules at the
surface of the electrode are oxidized by oxygen (for
phenol oxidases) or hydrogen peroxide (for
peroxidase), followed by their re-reduction by
phenolic compounds. The tyrosinase biosensors are
restricted to the monitoring of phenolic compounds
with at least one free ortho-position.
On the other hand, the laccase biosensor can detect
free para- and meta-positions, but its catalytic cycle
is complicated and in its major part is different from
tyrosinase and still now not well understood.
Peroxidases exhibit low specificity for electron
donors as the phenolic compounds and can be used
for phenol detection with certain selectivity and
sensitivity.
Tyrosinase: The catalytic sensors reported in
the literature use mostly the enzyme tyrosinase. The
Sensors and polyphenols
Natural Product Communications Vol. 3 (12) 2008 2051
Enzyme/Substrate
b
Antibody/Antigen
c
Whole cells, tissue
d
Lipid Layer / Gas
e
A
T
G
C
T
A
DNA fragments
a
b
c
d
Signal
a
e
A
T
G
T
C
A
B
A
Figure 2: Examples of receptors.
enzyme catalyzes the oxidation of the phenolic
substrate to a quinonic form that is reduced at the
electrode polarized at a fixed potential. In the
presence of oxygen, this enzyme is capable of
catalyzing ortho-hydroxylations of monophenols and
oxidation of the consequent ortho-dihydroxyphenols
to ortho-quinones. Measurements can be carried out
by recording the signal variation related to the
dissolved oxygen consumption or to the formation of
the relative quinone.
Among electroanalytical techniques, in particular,
voltammetry has been successfully employed to
detect phenols in water media [27-29]. Behaviour of
the tyrosinase enzyme electrode has been
investigated under different experimental conditions
[30-33]. Several authors [34-39] have tested the
performance of the tyrosinase electrode in different
organic solvents and the effect of different additives
has also been investigated [40,41]. Organic phase
enzyme electrodes constitute a new class of biosensor
applicable to the analysis of substrates or matrices
insoluble or scarcely soluble in aqueous media.
Many biosensors have a limited lifetime due to
enzyme inactivation by the bio-catalytically
generated quinone products. For this reason, many
studies were made concerning the development of
good immobilization methods and materials to
improve the biosensor stability. Recently, several
amperometric
biosensors
based
on
the
immobilization of tyrosinase on different electrode
materials have been described in the literature.
Glassy carbon electrodes modified with polymers
[42], sol–gel materials [43], self-assembled
monolayers (SAMs) on gold [44], Clark's electrodes
[45,46], reticulated vitreous carbon [47] screenprinted [48] carbon paste [49,50], Nafion® membrane
[51], hydrogel [52], conducting polymers [53] and
natural material such as chitosan [54], and other
composite electrodes [55,56] have been used to
prepare tyrosinase electrochemical biosensors.
Instead of conventional electrodes presenting the
limitation of being poisoned after a certain use,
recently screen-printed electrodes have been
proposed to evaluate the polyphenol content [57].
Screen-printing technology is used for the production
of disposable sensors which are very useful because
during the oxidation process a polymeric film is
formed on the electrode surface leading to electrode
surface “inactivation” (“electrode fouling”), one of
the main drawbacks of common graphite-based
electrodes. Screen-printed electrodes have also been
used for screening natural products using bare
graphite [58]. The electrochemical device consists of
three independent electrodes placed one next to
another to form a rectangle 3 cm high and 1.5 cm
wide comprising a screen-printed graphite working
electrode, a silver reference, and a counter-electrode.
Different compounds, including flavones, flavonols,
catechins, tannins, and phenylpropanoids were tested
with this system. Calibration was performed in a
range between 20 and 80 µM of catechin. This
method can be useful for a rapid and sensitive
screening for polyphenols in plant matrices from
grape, olive and green tea. Thus, many applications
of biosensors, with different techniques of enzyme
immobilization have been concerned with the
determination of polyphenols in wine.
The use of gold nanoparticles is playing an
increasingly important role for the preparation of
biosensors [59] and recently, Sanz and coworkers
reported the preparation of a tyrosinase biosensor
based on the use of a glassy carbon electrode
2052 Natural Product Communications Vol. 3 (12) 2008
modified with electrodeposited gold nanoparticles
[57]. The enzyme, immobilized by cross-linking with
glutaraldehyde, retains a high bioactivity on this
electrode material, giving rise to fast, stable and
sensitive responses to various phenolic compounds.
The biosensor was applied to the amperometric
estimation of the total content of phenolic compounds
in red and white wines, which is of interest because
of the correlation between wines’ antioxidant
capacity and their polyphenol content. The method
used an extremely simple procedure involving the
direct addition of a sample aliquot to the
electrochemical cell. The response was given in a few
seconds; the polyphenol concentration found in the
wine samples ranged from 16 mg L-1 to 50 mg L-1,
expressed as caffeic acid. There was good correlation
when the biosensor data were plotted versus the
results achieved with the Folin–Ciocalteau method.
A different way to entrap the enzyme using
electropolymerizing polymers was also reported [6062]. In particular, Böyükbayram and coworkers [62]
prepared graft copolymers by electropolymerization
of pyrrole with thiophene capped polytetrahydrofuran
and used these conducting copolymers to immobilize
tyrosinase. The enzyme electrodes were used to
determine the amount of phenolic compounds in two
brands of Turkish red wine and found very useful
owing to their high kinetic parameters and wide pH
working range. Thiophene functionalized menthyl
monomer with pyrrole (MM/ppy/Tyrosinase)
represented another copolymer employed to
immobilize tyrosinase [63]. Immobilization of
enzyme was performed via entrapment in conducting
copolymers during electrochemical polymerization of
pyrrole. Maximum reaction rates, Michaelis–Menten
constants and temperature, pH and operational
stabilities of enzyme electrodes were investigated by
the authors. The application of this sensor was again
to evaluate the total amount of phenolic compounds
in red wines. The immobilized enzyme was
optimized at pH 9. MM/ppy/tyrosinase electrode
showed stability up to 80°C, while free tyrosinase
had an optimal temperature of 40°C and lost its
activity completely at 50°C. The system was able to
detect the total phenolic compounds present in two
different red wines in a range of 3.3-6.0 g/L,
expressed as gallic acid equivalents.
Different applications reported for tyrosinase deal
with the development of artificial senses, such as
electronic tongues, where the systems array of
sensors with different selectivities are generally
integrated to give a response “mimicking” the natural
Bergonzi et al.
counterpart. A lot is known about artificial ‘nose’,
but in the last years some work has appeared dealing
with taste. In this regard, Gutés and coworkers [64]
elaborated a simultaneous determination of different
phenols (phenol, catechol, m-cresol), combining
biosensor measurements with chemometric tools and
artificial Neural Networks (ANN) analysis.
Concentrations of the three phenols ranged from 0 to
130 μM for phenol, 0 to 100 μM for catechol and 0 to
200 μM for m-cresol. As the recognition-detection
part, and working electrode, a tyrosinase-based
biosensor was developed. The biosensor employs the
concept of a graphite-epoxy biocomposite with bulk
incorporation of enzyme.
Tyrosinase sensors are also employed for quantitative
analyses of polyphenols in beer samples. Three
amperometric biosensors are described based on
immobilization of tyrosinase on a new Sonogel–
Carbon electrode for detection of phenols and
polyphenols [65]. The electrode was prepared using
high energy ultrasound directly applied to the
precursors. The first biosensor was obtained by
simple adsorption of the enzyme on the Sonogel–
Carbon electrode. The second and third ones,
presenting sandwich configurations, were initially
prepared by adsorption of the enzyme and then
modified by mean of a polymeric membrane, such as
polyethylene glycol for the second one, and the ionexchanger Nafion in the case of the third biosensor.
The optimal enzyme loading and polymer
concentration, in the second layer, were found to be
285 U and 0.5%, respectively. All biosensors showed
optimal activity under the following conditions: pH
7, −200 mV, and 0.02 mol L−1 phosphate buffer.
Sensing performances and kinetic characterizations
of the developed biosensors were investigated using
some phenolic compounds (catechol, phenol, 4chlorophenol, gallic acid, catechin). In the same
paper, the tyrosinase-based Nafion modified
Sonogel-Carbon electrode was used to quantify the
polyphenol and phenol content of four beers (two
lagers and two black) and four environmental water
samples [65].
The same research group [66] produced a biosensor
based on the bi-immobilization of laccase and
tyrosinase. The biosensor employed as the
electrochemical transducer the Sonogel-Carbon
electrode. The immobilization step was accomplished
by doping the electrode surface with a mixture of the
enzymes,
glutaricdialdehyde
and
Nafion-ion
exchanger, as protective additive. The response of
Sensors and polyphenols
Natural Product Communications Vol. 3 (12) 2008 2053
this biosensor, carrying Trametes versicolor laccase
(Lac) and Mushroom tyrosinase (Ty) based on
Sonogel-Carbon detection, was optimized directly in
beer samples and its analytical performance with
respect to five individual polyphenols was evaluated.
The electrode responds to nanomolar concentrations
of flavan-3-ols, hydroxycinnamic acids and
hydroxybenzoic acids. The limit of detection,
sensitivity and linear range for caffeic acid, taken as
an example, were 26 nM, 167.53 nA M-1, and 0.01-2
μM, respectively. The Lac-Ty/sonogel-carbon
electrode was stable in this matrix, maintaining 80%
of its stable response for at least three weeks (RSD
3.6%). The biosensor was applied to estimate the
total polyphenol index in ten beer samples and a
correlation of 0.99 was obtained when the results
were compared with those obtained using the FolinCiocalteau reagent.
polyphenols in black tea samples. Enzyme membrane
fouling was observed with a number of analyses
with a single immobilized enzyme membrane. The
tyrosinase-based biosensor gave maximum response
to tea polyphenols at 30°C. The optimum pH was 7.0.
This biosensor system can be applied in evaluating
tea polyphenols quality.
Amphiphilic, tyrosinase-modified screen-printed
carbon bioelectrodes were decrypted by Cummings
[67] for the analysis of lager beers and were
compared to the p-dimethylaminocinnamaldehyde
(DAC)
colorimetric
method.
Initially,
the
performances of the biosensors under flowing
conditions were appraised using catechol as a model
substrate. The electrodes displayed rapid response
times and a high degree of sensitivity and
reproducibility upon injection of catechol onto a
single mainfold. In addition, simple flavanols,
separated from barley, were utilized to assess the
sensitivity of the biosensors afforded by the presence
of the enzyme. The bioelectrode sensitivity decreased
upon an increase in molecule size. Finally, using flow
injection analysis, authentic beer samples were
analyzed and compared to the DAC colorimetric
method. A good correlation between the two methods
of analysis was observed but, due to the lack of
enzyme substrate specificity, the biosensor response
did not decrease to the same extent as the
colorimetric method; this can be attributed to the
presence of interferents, for example, ferulic acid and
p-coumaric acid.
An amperometric tyrosinase biosensor has also been
used for detection of polyphenols in tea [68]. The
system could detect tea polyphenols in the
concentration range 10–80 mmol L−1. Immobilization
of the enzyme, by the crosslinking method, gave a
good stable response to tea polyphenols. The
biosensor response reached the steady state within 5
min. The voltage response was found to have a direct
linear relationship with the concentration of
Campo Dall’Orto and coworkers have reported the
polyphenol content, expressed as chlorogenic acid
equivalents, in a variety of commercially available
samples of yerba mate (Ilex paraguaiensis). The
compounds were detected using a tyrosinase
biosensor and comparing the results with a
colorimetric method [69]. The 48% of analyzed
samples presented a 92 ± 8 mg of extracted
chlorogenic acid equivalents per gram of sample. The
extracted chlorogenic acid, expressed as mg/g-1, was
evaluated by three methods in a unique yerba mate
sample: biosensor (89.2 mg/g-1), Folin (90.2 mg/g-1),
and HPLC analysis (21.0 mg/g-1). Biosensing system
validation was performed. Repetitiveness of genuine
replicates was consistent with the nature of the
samples. Discrimination between yerba mate and
other plants can be made using principal component
analysis (PCA) and the corresponding physical and
chemical descriptors. Flavor and taste alterations can
be studied by means of analytical methods that
involve low-cost instrumentation.
Peroxidase: Horseradish peroxidase (HRP) has been
eventually employed as another useful enzyme for
polyphenol detection. In this approach, the reduction
current of oxidized polyphenols, formed during the
enzymatic oxidation of polyphenolic compounds in
the presence of H2O2, is proportional to their
concentration. In other words, the polyphenol content
can be detected as the reduction current of the
oxidized polyphenol generated by the enzyme
reaction cycle of HRP with H2O2. The sensitivity of
the detection of various polyphenols by the present
method depends on both the electron-donating
properties of polyphenols and the electron-accepting
properties of oxidized polyphenols.
With this approach, Imabayashi [70] reports about
the development of a sensor using horseradish
peroxidise covalently immobilized on a selfassembled monolayer of mercaptopropionic acid on
gold-electrode by the formation of the bond between
amino groups on the HRP surface and carboxylic
groups on the self-assembled monolayer. The
electrode allows polyphenol detection down to 2 μM
with a linear relationship up to 25 μM in standard
2054 Natural Product Communications Vol. 3 (12) 2008
solutions. The reduction current of oxidized
polyphenols, formed during the enzymatic oxidation
of polyphenolic compounds in the presence of H2O2,
is proportional to their concentration. The sensitivity
of the detection of various polyphenols by the present
method depends on both the electron-donating
properties of polyphenols and the electron-accepting
properties of oxidized polyphenols. When applied to
real matrices, such as wine and tea, the total amounts
of polyphenols, in the order of μM, were estimated,
correlating well with the results determined by the
Folin-Ciocalteu method.
In another paper [71], the decreased amount of H2O2
caused by the action of peroxidase was sensitively
detected with a semipermeable-membrane-covered,
HRP-entrapped, and ferrocene-embedded carbon
paste electrode. This electrode allows the detection of
(+)-catechin down to 0.3 µM and the response is
linear up to 15 µM. The same linearity was obtained
with other polyphenols found in wine and green tea,
such as (-)-epicatechin, caffeic acid, tannic acid and
gallic acid. The content of total phenolic compounds
in wine [1-8 mM using (+)-catechin as the standard;
2-14 mM using gallic acid as the standard] and tea
samples [1-3.5 mM using (+)-catechin as the
standard; 0.6-6 mM using gallic acid as the standard]
determined by the present method agrees well with
results obtained by the Folin-Ciocalteu method.
The same enzyme has also been employed
immobilized onto silica–titanium and it was applied
to measure the polyphenol content of a plant extract
without sample pretreatment, because no significant
influence of the matrix was observed [72]. Silicabased materials have received greatest interest
because they provide a suitable way for designing
electrochemical biosensors. Among the silicacontaining matrices, the silica gel modified with
metal oxides has been used, not only to improve its
amperometric detection, as increase in the internal
electrical conductivity of the silica matrix, but also by
providing a material with high chemical stability. A
biosensor based on horseradish peroxidase and DNA
immobilized onto silica–titanium is applied for
measuring the polyphenol compounds in plant
samples. In the study, various analytical parameters
influencing the biosensor performance, such as
working potential, type and concentration of the
buffer, pH, response time and response in the
presence of other compounds, have been investigated
as a function of chlorogenic acid (CGA). In the
optimized conditions, the biosensor presented a linear
Bergonzi et al.
response range for CGA from 1 to 50 μmol L−1,
applying a potential of −50 mV versus Ag/AgCl, with
a sensitivity of 181 μmol−1 L nA cm−2 and detection
limit of 0.7 μmol L−1. The biosensor was used to
determine the polyphenol content of extracts of
coffee and mate. The experimental results showed
good agreement with those from the Folin–
Ciocalteau method. The polyphenol content in the
aqueous extracts of coffee and mate tea ranged from
1.0 to 3.6 (mmol L-1)g-1 of sample.
Phenolic compounds are also important factors to be
considered in order to evaluate the quality of an
extra-virgin olive oil since they are partly responsible
for its auto-oxidation stability and organoleptic
characteristics. The phenolic content is correlated
with many quality parameters, such as the oxidation
level or free fatty acidity. Free fatty acids provide an
index of the degree of lipase activity and can produce
undesirable aromas in the oil; a high value for free
fatty acid content indicates a high degree of lipase
activity and hence a reduced antioxidant content.
Moreover, the oxidation level is dependent upon the
composition of the oil and, therefore, upon the degree
of unsaturation and the presence of antioxidants, such
as phenols. Free fatty acids are responsible for
undesirable aromas in the oil. Thus, estimating
polyphenol content could provide some indication of
oil quality.
On the basis of this, some work dealing with olive oil
as matrices for polyphenol content evaluation has
been reported [73]. Monitoring the polyphenol
content (oleuropein derivatives) in an extra-virgin
olive oil with varying storage time and storage
conditions was performed using two rapid procedures
based on disposable screen-printed sensors (SPE) for
differential pulse voltammetric analysis, and on an
amperometric tyrosinase based biosensor operating in
an organic solvent (n-hexane) and using an
amperometric oxygen probe as transducer.
Differential Pulse Voltammetry (DPV) parameters
were chosen in order to study the oxidation of
oleuropein, which was used as reference compound.
A calibration curve of oleuropein was determined in
glycine buffer [10 mM, pH = 2, NaCl 10 mM (D.L. =
0.25 ppm oleuropein, RSD= 7%]. In the case of the
tyrosinase based biosensor, the calibration curves
were realized using flow injection analysis with
phenol as the substrate (detection limit = 4.0 ppm
phenol, RSD = 2%). Both of these methods are easy
to operate, require no extraction (biosensor) or rapid
extraction procedure (Solid Phase Extraction, SPE)
and the analysis time is short (min). The results were
Sensors and polyphenols
comparable with those using
reagent and by HPLC analysis.
Natural Product Communications Vol. 3 (12) 2008 2055
Folin-Ciocalteau
Campanella and coworkers [37] monitored the
rancidification process of extra-virgin olive oil using
a biosensor operating in organic solvent. The
progressive rancidification of the oil was monitored
by simultaneously using two different indicators: the
peroxide number and an innovative one consisting of
the progressive decrease in the content of
polyphenols, the main natural antioxidants contained
in the oil, as determined rapidly by means of a new
organic phase enzyme electrode based on tyrosinase.
The aim of the paper was to evaluate the
‘genuineness' of the oil itself and then, above all, to
check the correlation between the stability of an
olive oil to an artificially induced process of
rancidification. The main result of the research was to
demonstrate the possibility of using the organic phase
enzyme electrode based on tyrosinase to monitor the
rancidification process occurring in any sample of
olive oil. Indeed, a clear inverse correlation was
found throughout the entire oxidation process
between the classic indicator, namely the peroxide
number, and the polyphenol content of the sample.
The simplicity, together with the accuracy and
precision of the polyphenol content measurements
performed on the olive oil samples revealed the
advantages offered by this biosensor. The total
polyphenol content in various olive oils ranged from
15.3 to 114.2 mg kg−1 of oil, expressed as phenol.
Very recently, the thermal oxidative degradation
process of polyphenols was studied, both in a
synthetic mixture of five of the more readily
available polyphenols contained in the extra-virgin
olive oil (EVOO) (tyrosol, vanillin, caffeic acid,
ferulic acid and oleuropein) dissolved in glyceryl
trioleate, and commercial extra-virgin olive oil [74].
To this end, a series of oxidative degradation
experiments was carried out on extra-virgin olive oil
samples under isothermal conditions at 98, 120, 140,
160, and 180°C using a thermostatic silicon oil bath.
The change in polyphenol concentration with time
was monitored at selected temperatures using a
tyrosinase biosensor operating in an organic phase (nhexane). The EVOO rancidification process rate
displayed good inverse correlation between the
variation in the peroxide value, the more traditional
index, and that of a more innovative index
determined by the concentration of the “total
polyphenols” (expressed in mol L−1 of phenol). In
this paper, the authors analyzed the kinetic
degradation process and the kinetic parameters of the
process were determined through an isothermal study
carried out at different temperatures (between 98 and
180°C).
Laccase: Some studies reported the use of laccase as
a possible enzyme for development of biosensors for
phenols and polyphenols. Laccase, a coppercontaining oxidase, is widely distributed in fungi,
higher plants and in some bacteria. The use of a
laccase-modified electrode for detection of
flavonoids was reported by Gorton’s group [75]. In a
recent study, the laccase from Cerrena unicolor, as a
highly active enzyme, coupled to amperometric
transduction was reported for the detection of
flavonoids. The enzyme was adsorptively
immobilized on the surface of a graphite electrode. In
particular, catechin hydrate, epicatechin, epicatechin
gallate, prodelphinidin, and caffeic acid were used as
target compounds. Electrodes modified with laccase
yield responses for both simple compounds and
compounds with three or more phenolic and nonphenolic rings, but with different sensitivities.
Considering wine as a matrix of interest, another
example of a laccase biosensor is given. A biosensor
developed with Laccase Coriolus versicolor
immobilized on derivatized polyethersulfone
membranes and applied to a Pt–Ag, AgCl US
electrode base was applied to evaluate several
polyphenols usually found in red wine (caffeic acid,
gallic acid, catechin, rutin, trans-resveratrol,
quercetin and malvidin) [76,77]. It was observed that
an amperometric response was obtained for catechin
at +100 mV (versus Ag/AgCl) and caffeic acid at −50
mV in acetate buffer solutions (pH 4.5) having 12%
ethanol. At pH 3.5 and +100 mV the biosensor was
sensitive to both substrates and their response was
additive. One limit of this biosensor is the necessity
for a previous solid phase extraction of the matrix for
polyphenol enrichment; large interferences can occur.
Amperometric determination using a biosensor based
on immobilized laccase was applied for the analysis
of tannins of tea at different stages of its production
[78]. The enzymes were from Coriolus versicolor,
Coriolus hirsutus and Cerrena maxima, and
immobilized on threadlike DEAE-cellulose. The time
needed for analysis in the flow injection mode was
below 100 s. A column with immobilized enzyme
could be used for up to 500 determinations of
phenolic compounds (tannin content 100-199 mg/g of
dry substance) without decrease of the enzyme
activity.
2056 Natural Product Communications Vol. 3 (12) 2008
The use of a laccase biosensor, both under batch and
flow injection conditions, for a rapid and reliable
amperometric estimation of the total content of
polyphenolic compounds in wines is also reported
[79]. The enzyme was immobilized by cross-linking
with glutaraldehyde onto a glassy carbon electrode.
Caffeic acid and gallic acid were selected as standard
compounds to carry out such an estimation.
Experimental variables, such as the enzyme loading,
the applied potential, and the pH value, were
optimized, and different aspects regarding the
operational stability of the laccase biosensor were
evaluated. Using batch amperometry at -200 mV, the
detection limits obtained were 2.6 × 10-3 and 7.2 ×
10-4 mg L-1 gallic acid and caffeic acid, respectively,
which compares advantageously with previous
biosensor designs. An extremely simple sample
treatment consisting only of an appropriate dilution
of the wine sample with the supporting electrolyte
solution (0.1 mol L-1 citrate buffer of pH 5.0) was
needed for the amperometric analysis of red, rosé,
and white wines. Good correlations were found when
the polyphenol indices obtained with the biosensor
(in both the batch and FI modes) for different wine
samples were plotted versus the results achieved
with the classic Folin-Ciocalteu method. Application
of the calibration transfer chemometric model
(multiplicative fitting) allowed for the confidence
intervals (for a significance level of 0.05) for the
slope and intercept values of the amperometric index
versus the Folin-Ciocalteu index plots (r = 0.997)
including the unit and zero values, respectively. This
indicates that the laccase biosensor can be
successfully used for the estimation of the polyphenol
index of wines and is comparable with the FolinCiocalteu reference method.
Plant tissue: An alterative approach to the use of
purified enzymes is the employment of whole tissue
which contains different enzymes sets. Whole tissue
materials from plants or animals provide many
advantages for the construction of biosensors.
In some cases, plant tissue containing polyphenol
oxidase (e.g., banana, potato, apple, and burdock) has
been used, coupled to electrodes, for the detection of
catechol-related components, such as flavonols and
catechins in beers and green tea [78,80-83]. The
linear detection range of the plant-tissue electrodes,
depending on the enzyme preparation used, was, on
average, between 2 and 12 µM catechins.
Tissue sensors have been applied to the determination
of flavonols in beers [81]. In particular, different
Bergonzi et al.
plant tissues, banana, potato and apple, containing
tyrosinase have been evaluated for their ability to
detect catechol related components in beers.
Calibration graphs were produced for each plant
tissue with both catechol and (−)-epicatechin as
standards. The response of the banana based sensor
was rather erratic and showed a large zero error,
probably due to flavanols in the banana. Potato, wet
apple and dried apple were all satisfactory. The
response with dried apple was 60% that of wet apple,
but showed greater stability. The response of (−)epicatechin was 1.4 to 1.5 times that of catechol with
either wet or dried apple. Banana and potato based
sensors were used for the determination of total
flavanols in a range of commercial beers and lagers.
Good analytical data were obtained with potato,
comparable to those obtained using colorimetric or
liquid chromatographic analyses. The best biosensors
were from potato and apple. While there was slight
loss of response due to the drying process, dried
apple has greater longevity and excellent response
characteristics. In preliminary experiments reported
elsewhere, the same authors [82] have demonstrated
that the flavanol components in beer can be
determined with a ‘bananatrode’ biosensor based on
the carbon paste electrode using catechol as the
standard.
A burdock (Arctium lappa L., a biennial plant) tissuebased biosensor was applied for measuring total
catechins in green tea infusions [83]. This catechin
biosensor was found to respond to five catechins
(catechin/epicatechin/epigallocatechin/epicatechin
gallate/epigallocatechin gallate), gallic acid, catechol
and ascorbic acid and to total catechins in green tea
infusions. The precision of the measurements was
good (< 3% RSD) and the biosensor showed no
interference from major amino acids or carbohydrates
in the infusions. One limitation of this approach,
however, is that the biosensor is inadequate for
accurate quantitation of total catechins because of the
severe variability in the relative biosensor response to
the different catechins.
Jewell and Ebeler developed a simple tyrosinasebased biosensor composed of 5% banana tissue, 10%
mineral oil, and 85% carbon-containing ruthenium,
mixed together in a small beaker to form a stiff
paste, for the measurement in a winery or food
setting for rapid and simple phenolics detection [84].
This was achieved by the design and construction of
an operational amplifier-based tyrosinase biosensor.
Excellent correlation was shown between the
biosensor and the Folin-Ciocalteu assay for simple
Sensors and polyphenols
Natural Product Communications Vol. 3 (12) 2008 2057
phenolics (gallic acid, catechin, epicatechin, caffeic
acid, quercetin, seed tannins) and for wines. The
simple compounds and the seed tannins were chosen
in order to examine their biosensor response, in
correlation with different structure, OH groups and
complexity of matrix. The varying signals observed
were due to the chemical structure and variable
number of OH groups associated with polyphenols,
which caused different reaction with tyrosinase; this
is also observed with all redox methods. No
interferences were observed in a model wine solution
made according to standard wine phenolic
concentrations due to low pH, tartaric acid or ethanol
present in the model wine. Finally, one white wine
and three red wines, selected to examine the effects
of aging and the different phenolic profiles, were
analyzed with the biosensor. Also in this case, the
response between Folin-Ciocalteau and biosensor
methods was similar, and oak aging and varietal
difference did not appreciably influence the biosensor
response [84].
was also demonstrated on the examples of red and
white wines [85].
Behind electrochemical detection, optical sensing
also represents an interesting feature for phenols
detection. One particular application of this sensor
was reported by Edelmann and Lendl, who developed
an electronic tongue to evaluate the tannin content of
red wine [85]. The interaction of tannins with
proline-rich proteins (gelatin) was studied using an
automated flow injection system with Fourier
transform infrared spectroscopic detection to gain
insight into chemical aspects related to astringency.
The PRP gelatin was selected to mimic the parotid
salivary proteins. In the perception of astringency, an
interaction between proline-rich salivary proteins and
tannins present in the sample takes place. To study
this interaction, agarose beads carrying gelatin
(a proline-rich protein) were placed in the IR flow
cell in such a way that the beads were probed by
the IR beam. Using an automated flow system,
samples were injected in a carrier stream and flushed
over the proteins in a highly reproducible manner.
Simultaneously, any retardation due to tannin-protein
interactions taking place inside the flow cell
were monitored by infrared spectroscopy. Tannins
of different sources (grapes, wooden barrels,
formulations used in wine making) were investigated,
and their flow-through behaviour was characterized.
Significant differences in their affinity toward gelatin
could be observed. Furthermore, because of small but
characteristic differences in the IR spectrum, it is
possible to distinguish condensed from hydrolysable
tannins. The selectivity of the flow-through sensor
Concluding remarks: A review of the literature
concerning the application of biosensor technology to
vegetal matrices has been reported and discussed.
Special attention has been focused on the use of these
devices for the identification and quantification of
polyphenols and, in some cases, for the quality
control of these matrices.
Usually the determination of total polyphenol
content is performed by spectrophotometric or
chromatographic methods, but in recent years, with
the aim to develop methods capable of being
employed also in situ, several biosensors have been
developed to determine phenols in either aqueous or
organic solvents. Organic phase enzyme electrodes
constitute a new class of biosensor applicable to the
analysis of substrates or matrices insoluble or
scarcely soluble in aqueous media.
Many different sensors have been developed in the
last 15 years for polyphenol detection and
electrochemical transduction is the approach most
applied. Different enzymes have been used over the
years as the catalytic element coupled to
electrochemical analysis, such as tyrosinase, laccase,
and peroxidase, since phenolic compounds can act as
electron donors for these enzymes. In some cases,
plant tissue containing polyphenol oxidase (e.g.,
banana, potato, apple and burdock) has also been
used for the detection of polyphenols in vegetal
matrices.
The mostly investigated vegetal matrices are those
with a great commercial interest such as olive oil,
wine, beer and tea. By the use of screen-printed
electrodes, different phenols, including flavones,
flavonols, catechins, tannins, and phenylpropanoids
were tested with these analytical tools. Some
limitations occur with these devices in real matrices,
such as the working conditions (pH, temperature) and
the risk of enzyme inactivation. Many biosensors
have a limited lifetime due to enzyme inactivation
by the biocatalytically generated quinone products.
For this reason, many studies were concerned with
the development of good immobilization methods
and materials to improve the biosensor stability.
Recently, the immobilization of enzymes in
electropolymerized conducting polymers, sol–gel
materials, gold nanoparticles, Clark's electrodes,
screen-printed, carbon paste, hydrogels, has received
a great deal of interest.
2058 Natural Product Communications Vol. 3 (12) 2008
Another limitation of this approach, however, is that
sometime the biosensor is inadequate for accurate
quantification of total polyphenols because of the
severe variability in the relative biosensor response to
the different phenol derivatives.
Nevertheless, biosensors may provide a promising
competitive technology for a simple, fast and
sensitive detection of polyphenolic compounds
without any pre-treatment. In most of the cases
successfully employed for the analysis of real
Bergonzi et al.
samples, comparable results were obtained with
conventional analysis by HPLC.
Thus, the impact of biosensors in food and plant
matrices in the future will be enormous because they
are a label-free screening system, which imparts
flexibility to the process of assay design and
facilitates successful integration with other
technologies thanks to the increasing number of
commercially available instruments having novel
sensor surfaces, immobilization techniques and
attachment chemistries.
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NPC
Natural Product Communications
In vitro Radical Scavenging and Anti-Yeast Activity of
Extracts from Leaves of Aloe Species Growing in Congo
2008
Vol. 3
No. 12
2061 - 2064
Annalisa Romania, Pamela Vignolinia, Laura Isolanib, Sara Tombellic, Daniela Heimlerb,
Benedetta Turchettid and Pietro Buzzinid ,*
a
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Firenze,
I-50019 Sesto Fiorentino, Italy
b
Dipartimento di Scienza del Suolo e Nutrizione della Pianta, Università degli Studi di Firenze,
I-50144 Firenze, Italy
c
Dipartimento di Chimica, Università degli Studi di Firenze, I-50019 Sesto Fiorentino, Italy
d
Dipartimento di Biologia Applicata, Sezione di Microbiologia, Università di Perugia,
I-06100 Perugia, Italy
pbuzzini@unipg.it
Received: September 8th, 2008; Accepted: October 29th, 2008
Extracts obtained from leaves of Aloe barbadensis and A. congolensis, growing in Congo, were analyzed for their in vitro
antiradical and anti-yeast activity. Different leaf tissues (tegument and gel) were analyzed separately. Their phenolic fractions
showed the presence of chromones and anthrones (aloesin, aloin B, aloin A, and isoaloeresin), flavonoids (apigenin and
kaempferol derivatives), and hydroxycinnamic acids. A differential quantitative composition was observed between leaf
tegument and gel: in the first, higher concentrations of the four classes of compounds were observed. The extracts from the
tegument exhibited higher in vitro antiradical and antimycotic activity than gel extracts. In a few cases, extracts from
teguments were active against amphotericin B-insensitive yeasts. Due to the lack of radical scavenging and yeast inhibition
observed when aloin was used, it was possible to postulate that the in vitro activities of the teguments could be related to their
high concentration of flavonoids and hydroxycinnamic acids.
Keywords: Aloe spp., antiradical activity, anti-yeast activity, polyphenols, DPPH• radical, yeasts.
Biological activities (e.g. antioxidant, antiradical,
anticarcinogenic, antimutagenic, antiproliferative and
antimicrobial) expressed by extracts obtained from
plant tissues are well known [1]. The increasing
interest in discovering novel plant extracts exhibiting
biological activities is justified by their potential role
in supporting or even substituting commercial drugs.
The genus Aloe L. (Liliaceae) includes over 300
species of perennial herbs, shrubs and trees. Among
them, A. barbadensis Miller (trivially labeled as A.
vera) is a perennial herb with rosettes of long pointed
leaves coming from a shortly branched, creeping
rhizome. The leaves are brittle and exude a clear
yellowish viscous sap when broken. Although it is
widespread throughout the African continent, this
species is considered to originate from southern and
eastern Africa and from the Mediterranean regions.
Leaves of A. barbadensis are characterized by a high
water content and more than 60% of the dry weight is
composed of polysaccharides [2]. Phenolics of the
anthrone and chromone type have been described as
the main secondary metabolites [3,4].
A. congolensis, which grows in a broad area of
Congo, received its first botanical description in 1899
[5]. Neither the phenolic pattern nor the biological
activity of this species has been reported.
Since antiquity, gel obtained from the succulent
leaves of A. barbadensis has been used in folk
medicine for obtaining preparations (traditionally
known as “medicinal aloe”) for treatment of internal
and external diseases, both in humans and animals
[6]; this has been extensively proposed as a
bactericidal and fungicidal drug [7], as well as for
dermatological applications, especially for radiationcaused skin lesions [8,9]. However, no evidence has
2062 Natural Product Communications Vol. 3 (12) 2008
Romani et al.
so far been published on the antiradical and anti-yeast
activity of extracts from teguments of Aloe spp. Thus
the aim of the present study was the characterization
of the phenolic composition of extracts of the
teguments and gel of A. barbadensis and A.
congolensis, and the in vitro determination of their
antiradical and anti-yeast activity (against species
implicated in human mycoses).
Table 1: General characteristics of Aloe leaves. Data are mean values of
ten determinations (standard deviation within brackets).
Chromones,
anthrones,
flavonoids
and
hydroxycinnamic
acids
were
detected
in
chromatograms of the phenolic fractions of extracts
of A. barbadensis and A. congolensis teguments and
gels. Chromones and anthrones have previously been
detected in the phenolic fractions of Aloe leaves
[3,10], whereas no previous studies have reported the
presence of flavonoids and hydroxycinnamic acids.
On the basis of their UV-Vis and mass spectra,
apigenin diglucoside and three kaempferol
derivatives were detected in A. barbadensis leaves.
The most representative compounds in the tegument
extract of this species were aloesin, aloin B, aloin A,
and isoaloeresin (the last two compounds not being
separated), which were also observed in the gel. On
the contrary, flavonoids and hydroxycinnamic acids
were detected exclusively in the tegument.
Table 2: Quantitative data obtained from HPLC measurements. All data
are expressed as mg/g fresh weight. Standard deviations within brackets.
Kinetic curves of the in vitro antiradical activity of
extracts from A. barbadensis and A. congolensis
teguments and gels are reported in Figure 1.
In the first case, a higher antiradical activity has been
observed. This evidence is in agreement with results
obtained by Hu and co-workers [11]. In addition,
tegument of A. congolensis exhibited a higher
antiradical activity than that of A. barbadensis
(80.7% vs 41.3%) (Figure 1). Since in preliminary
tests we found that the aloin standard gave negative
results in the DPPH test (unpublished data), we might
Length
cm
Weight
g
Gel
%
A. barbadensis
27 (3.5)
61.9 (7.9)
55.7
23.6
3.2
A. congolensis
26 (4.2)
34.1 (6.2)
32.0
33.2
5.0
Samples
Total anthrone
and chromone
Total
flavonoids
A. barbadensis
teguments
A. barbadensis
gel
A. congolensis
teguments
A. congolensis
gel
2.32
(0.35)
0.06
(0.01)
2.50
(0.29)
0.03
(0.01)
0.11
(0.003)
0.004
(0.001)
0.53
(0.064)
0.009
(0.002)
Total
hydroxycinnamic
derivatives
0.014
(0.006)
traces
0.042
(0.097)
traces
0.3
a)
0.25
b)
absorbance (nm)
By comparing the HPLC profile of A. congolensis
with that of A. barbadensis, it was possible to
observe that the composition of the tegument was
characterized by a lesser amount of phenolics; in
particular, an apigenin derivative is one of the main
compounds. On the contrary, the gel of A.
congolensis exhibited a higher amount of phenolics
than that of A. barbadensis. It should, however, be
pointed out that the amount of gel obtained is lower
in the case of A. congolensis (see Table 1).
Quantitative data of total phenolics, obtained from
the chromatograms and calculated on the basis of
aloin, apigenin-7-O-glucoside, and caffeic acid
calibration curves, are reported in Table 2.
Yield of
lyophilised sample
mg/g
Tegument
Gel
Species
0.2
c)
0.15
0.1
d)
0.05
0
0
5
10
15
20
25
30
35
time (min)
Figure 1: Kinetic curves of the reaction with the stable DPPH• radical of
: a) gel barbadensis (2 g/mL); b) gel congolensis (2 g/mL); c) tegument
barbadensis (3 g/mL); d) tegument congolensis (3 g/mL)
suppose that the observed antiradical activity could
be related to the presence of flavonoids and
hydroxycinnamic acids (5.2% and 18.7% of the total
polyphenol content for A. barbadensis and A.
congolensis, respectively) (Table 2). Extracts of
teguments of both species exhibited a broad
antimycotic activity against Candida albicans, C.
glabrata, C. tropicalis, Clavispora
lusitaniae
(former Candida lusitaniae), Issatchenkia orientalis
(teleomorph of Candida krusei), Filobasidiella
neoformans (former Cryptococcus neoformans)
and Pichia guilliermondii (former Candida
giulliermondii) (Table 3). Interestingly, in a
few cases, extracts of teguments were active against
Antiradical and anti-yeast activity of Aloe spp.
Natural Product Communications Vol. 3 (12) 2008 2063
Table 3: Antimycotic activity of extracts of Aloe barbadensis and A.
congolensis. n.a. = no activity; AmB = Amphotericin.
DBVPG
Species
accession
number
Candida
albicans
Candida
glabrata
Candida
parapsilosis
Candida
tropicalis
Clavispora
lusitaniae
Filobasidiella
neoformans
Issatchenkia
orientalis
Pichia
guilliermondii
Diameter of inhibition halos (mm)
A.
A.
barbadensis
congolensis
tegument
tegument
250 mg/mL
250 mg/mL
AmB
100 μg/mL
6133
21.8
20.0
22.5
3828
16.0
12.0
18.7
6150
n.a.
n.a.
n.a.
3982
16.2
12.1
n.a.
6142
14.7
13.0
n.a.
6010
30.5
28.4
15.9
6782
13.2
12.6
n.a.
6140
18.5
17.0
n.a.
amphotericin B-insensitive yeast strains (Table 3). As
reported above, in vitro antimicrobial activity of
extracts of A. barbadensis has been reported
previously [7], but, to the best of our knowledge, this
is the first report of anti-yeast activity of extracts of
teguments of A. congolensis. Likewise with the
antiradical activity, as there was a lack of yeast
inhibition observed when aloin was used
(unpublished data), we might speculate that the
in vitro anti-yeast activity of the Aloe species
teguments could be related to their high concentration
of flavonoids and hydroxycinnamic acids (Table 2).
Experimental
Chemicals and reagents: Methanol, acetonitrile
(HPLC grade), methylene chloride, and formic acid
(ACS reagent) were purchased from Aldrich
Company Inc. (Milwaukee, Wiscosin), and aloin,
aloemodin, caffeic acid and apigenin-7-O-glucoside
from Extrasynthèse (Lyon, Nord-Genay, France).
Plant materials: Three years-old Aloe plants were
sampled at Kimbondo, Mont’ngafula, near Kinshasa
(Democratic Republic of Congo) in June 2005. A.
barbadensis was cultivated in a nursery in Minkoti,
whereas a local ecotype of A. congolensis was
sampled in its natural habitat in Minkoti. Living
plants were transported to the laboratory for further
sampling and analytical procedures. Gel and
tegument of succulent leaves were separately
extracted with ethanol-water (70:30, pH 3.2 with
formic acid) in the dark for 12 h. The extracts were
defatted twice with n-hexane, rinsed with water and
Table 4: Linear solvent gradient system used in HPLC-DAD and HPLCMS analysis of Aloe samples. Analysis was carried out during a 58 min
period at as flow rate of 1 mL/min using a Lichrosorb C18 (250 x 4.6 mm
i.d., 5 μm) column operating at 27°C.
Time
min
H2O/H+
%
CH3CN
%
Flow
mL/min
0
20
100
85
0
15
1
1
25
85
15
1
35
75
25
1
43
75
25
1
53
0
100
1
58
0
100
1
subsequently lyophilized for 12 h. Length and weight
of leaves, weight of gel and the percentage of
lyophilized sample are reported in Table 1. The
lyophilized extracts were dissolved in ethanol-water
(70:30, pH 3.2 with formic acid) and directly
analyzed by HPLC/DAD and HPLC/MS.
HPLC/DAD and HPLC/MS analysis: The
lyophilized extracts were analyzed by reverse-phase
and normal-phase high performance liquid
chromatography. The analysis was carried out using a
HP-1100 liquid chromatograph equipped with a DAD
detector and a HP 1100 MSD API-electrospray
(Agilent-Technologies, Palo Alto, USA) operating in
positive and negative ionization mode. The elution
conditions are reported in Table 4. Identification of
individual compounds was carried out on the basis of
their retention times, spectroscopic and spectrometric
data, using aloin, aloe emodin, luteolin-7-Oglucoside and caffeic acid as reference compounds.
Calibration curves with r2 ≥ 0.998 were considered.
The quantification was performed at the maximum
wavelength of UV-Vis absorbance by applying the
correction for molecular weight, and the reported
values are the means of three determination.
Determination of the in vitro antiradical activity:
Free radical scavenging activity was evaluated with
the DPPH• (1,1-diphenyl-2-picrylhydrazyl radical)
assay. The antiradical capacity of lyophilized extracts
was estimated according to the procedure reported by
Brand-Williams [12] and slightly modified. Two mL
of an ethanol solution of lyophilized extracts were
added to 2 mL of an ethanol solution of DPPH•
(0.0025g/100mL) and the mixture kept at room
temperature. The absorption was measured at 517 nm
with a Lambda 25 spectrophotometer (Perkin-Elmer)
with ethanol as a blank. The percentage of inhibition
was calculated according to the following formula:
2064 Natural Product Communications Vol. 3 (12) 2008
%inhibition = [( At =0 − At = 20 ) / At =0 ]× 100
The absorption of the DPPH• solution was checked
daily.
Determination of in vitro anti-yeast activity: Eight
yeast strains (each one representing the type strain of
8 different pathogenic species, belonging to 5 genera)
[13-16] were used as target microorganisms. All
strains are conserved in the Industrial Yeast
Collection DBVPG, University of Perugia, Italy,
www.agr.unipg.it/dbvpg. Anti-yeast activity exhibited by lyophilized extracts of A. barbadensis and
Romani et al.
A. congolensis (as above prepared) were determined
by using the agar diffusion well bioassay (ADWB)
[16,17]. Amphotericin B (AmB) (Calbiochem Inc.,
USA) was also tested as a control anti-yeast agent.
Acknowledgments – We thank Dr Claudio Aroldi
for his technical assistance and Dr Padre Hugo
Rios, Pediatric Foundation of Kimbondo, for sample
collection. The authors wish to express their sincere
gratitude to the Cassa di Risparmio di Firenze that
contributed to the acquisition of part of the
instrumentation used for this work.
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NPC
Natural Product Communications
Antioxidant Principles and Volatile Constituents from the
North-western Iberian mint “erva-peixeira”, Mentha cervina
2008
Vol. 3
No. 12
2065 - 2068
Matteo Politi, * César L Rodrigues, Maria S Gião, Manuela E Pintado and Paula ML Castro
Universidade Católica Portuguesa, Escola Superior de Biotecnologia, Rua Dr. António Bernardino de
Almeida, 4200-072, Porto-Portugal
mpoliti@esb.ucp.pt
Received: July 5th, 2008; Accepted: October 29th, 2008
Total phenol content determined by the Folin–Ciocalteu assay and total antioxidant capacity measured by the ABTS•+ method
were applied for the first time to analyze the aqueous extract prepared from the dried aerial parts of Mentha cervina, a
Portuguese mint species traditionally used as a culinary herb in river fish-based dishes and currently commercialized to prepare
digestive infusions. LC-MS/MS analysis was performed directly on the crude aqueous extract allowing the identification of
seven phenolic compounds. The overall plant aroma was analyzed by the SPME/GC-MS method; this approach allowed the
characterization of various constituents, as well as the comparison between the fresh and dried plant material. Such a
comparison highlighted several metabolic changes that occur during the drying process of the aerial parts of this plant.
Keywords: Mentha cervina, traditional recipe, infusion, volatiles, antioxidant, LC-MS, SPME/GC-MS.
Mentha cervina (L.) Opiz (Lamiaceae), previously
named Preslia cervina (L.) Fresn., is a mint growing
wild in stony places and on the banks of rivers in
north-western regions of the Iberian peninsula. It is
traditionally used as a culinary herb, especially to
aromatize river fish-based dishes in Portugal, mainly
in the interior regions, such as along the “Sabor”
river rather than along the sea coast. The freshly
caught fish are wrapped with the fresh leaves and
steamed [1a]; this explains its common name “ervapeixeira” that means fisher-herb. Contrary to other
mint species, this plant is not so competitive and it is
currently difficult to find it growing spontaneously in
the natural habitat. The recently renewed interest in
this aromatic species has induced local farmers, such
as the “Cantinho das Aromáticas” in Porto district
and “Ervital” in Viseu district, to cultivate it for
selling either as a living plant or as dried material
(aerial parts). Generic recommendations for its
consumption are about the use of the fresh plant for
culinary purpose while the dried material is generally
used to prepare infusions with digestive properties. In
both cases, the aerial parts of the plants are used.
To the best of our knowledge, the only scientific
article currently available on this species is by
Gonçalves and co-workers [1b], who reported the
variation of the essential oil constituents with the
seasons and the corresponding in-vitro antifungal
activity. The major objective of the present work was
the phytochemical investigation, by LC-MS/MS
analysis, of the aqueous extract (infusion) prepared
from the dried plant, as this extract had not been
studied before. Following our intensive investigation
of
medicinal
and
food
plants
currently
commercialized in Portugal [2a], specific emphasis
was given to total phenol content calculated by the
Folin–Ciocalteu assay [2b], and the total antioxidant
capacity measured by the ABTS•+ method [2c].
Antioxidants are now increasingly required in the
human diet because of their benefits to health and it
is, therefore, relevant to evaluate their content within
plants sold in the market as food ingredients or
medicine.
The second part of our investigation describes the
analysis of the volatile constituents through the
SPME/GC-MS method. Such an approach allowed
the rapid metabolic profile of the plant aroma without
the need of the essential oil extraction by
hydrodistillation, as previously performed by
Gonçalves and co-workers [1b]. This approach was
used here with the aim to compare the fresh and dried
plant material. Such a comparison can highlight the
chemical uniqueness of this traditional culinary
recipe that indicates the preferential use of the fresh
2066 Natural Product Communications Vol. 3 (12) 2008
“erva-peixeira” to aromatize the Portuguese river
fish-based dishes.
The antioxidant capacity of M. cervina was measured
by the ABTS•+ assay as ascorbic acid equivalents,
and the total phenol content was calculated by the
Folin–Ciocalteu method as gallic acid equivalents.
By comparison of such data with those from 48 other
infusions prepared from powdered medicinal plants
currently commercialized in Portugal and which had
been analyzed using the same assays and extraction
procedures [2a], M. cervina presents a medium
antioxidant capacity with a value of 0.16 ± 0.013 g
L-1 of ascorbic acid equivalents (avocado leaves Persea americana Mill.- was the best antioxidant
with 1.43 ± 0.13 g L-1 while the black elder flower Sambucus nigra L.- was the weakest with 0.043 ±
0.003 g L-1).
Using the same comparison, M. cervina showed an
intermediate total phenol content with a value of
0.15 ± 0.00 g L-1 of gallic acid equivalents (avocado
leaves gave a value of 0.55 ± 0.03 g L-1 and black
elder flower 0.040 ± 0.007 g L-1). Moreover,
M. cervina was less active as an antioxidant
compared with Mentha x piperita L. (0.40 ± 0.10 g L1
), but as good as M. spicata L. (0.14 ± 0.07 g L-1),
while the total phenol content was half that of the
two other mints (M. x piperita 0.31 ± 0.11 g L-1;
M. spicata 0.35 ± 0.03 g L-1).
LC-MS/MS analysis was performed directly on the
crude aqueous extract (infusion prepared from
powdered aerial parts of the plant). The aim of such
analysis was to identify as many phenols as possible
contained in this extract; normally, the presence of
this class of compounds is well related with the
antioxidant activity measured by the ABTS•+ method.
The identification of such phenols was achieved by
comparison with pure standards previously injected
using the same chromatographic (LC) and detection
(MS/MS) conditions. Our in house library of phenols
contains 33 compounds but, from these, no more than
7 were identified in the aqueous extract of M. cervina
(Table 1). Among them, chlorogenic and caffeic
acids were the most abundant, followed by cumaric
acid and rutin. To the best of our knowledge, these
constituents are reported for the first time in this mint
species.
Chlorogenic acid is a well known cholagogue and
choleretic derivative [3]; its regular ingestion helps
the flow of bile, facilitating, therefore, the digestive
process. The relatively high amount of such a
Politi et al.
Table 1: LC-MS/MS analysis of M. cervina infusion; % values
are indicative of the relative amounts of the seven identified
compounds calculated by integration of the area under the peaks.
Compound name
Protocatechuic acid
Cumaric acid
Caffeic acid
Epicatechin
Chlorogenic acid
Orientin
Rutin
Retention
time (min.)
9.65
20.16
18.30
22.84
16.75
20.50
[M-1]-
Fragments
153.0
163.0
179.0
289.0
353.0
447.0
25.52
609.0
109.0 (100)
119.0 (100)
135.0 (100)
245.0 (100)
191.0 (100)
357.0 (70);
327.0 (100);
285.0 (20)
301.0 (40)
Relative
amount
3.1%
19.7%
29.1%
1.1%
32.1%
3.3%
11.6%
constituent in M. cervina aqueous extract is in accord
with the claimed digestive properties of this
preparation.
The characteristic fragrance of aromatic plants is the
result of the sum of the single volatile compounds,
and their relative percentages are essential to
determine their characteristic perfumes. In this work,
the use of the SPME/GC-MS method allowed the
comparison of the volatile constituents of the fresh
and dried M. cervina aerial parts; both materials came
from the same plant (see Experimental section).
Biochemical changes attributable to the drying
process of the plant were detected. Such modification
appears to be mostly semi-quantitative rather than
qualitative. The area under the peaks in the
chromatograms is proportional to the amount of the
corresponding compounds. Data on the relative
amount (semi-quantitative data) of the single
chemical entities were, therefore, obtained by
comparison of the area of the corresponding peaks.
Such comparison was performed within the same
chromatogram as well as between both
chromatograms obtained from the fresh and dried
material. This was because, as detailed in the
Experimental section, the same amount of plant (1 g)
was used as starting material in both cases (1 g of
fresh aerial parts was directly analyzed, while another
1 g from the same plant was dried before analysis).
In accordance with the results obtained by Gonçalves
and co-workers [1b] on the essential oil constituents
of M. cervina, the major volatiles detected were
pulegone, isomenthone, and limonene, among others
not identified here. Concerning the major compound
pulegone, it was impossible to derive the area under
the peak because this compound was so abundant that
saturation of the MS detector occurred in both cases,
for the fresh and dried plant. The amount of limonene
and isomenthone were higher in the dried plant
compared with the fresh one; however, the relative
amounts of both constituents changed differently. In
Phytochemical investigation of Mentha cervina
Natural Product Communications Vol. 3 (12) 2008 2067
fact, the amount of limonene was three times higher
in the dried plant with respect to the fresh one, while
isomenthone was only two times more abundant in
the dried material. This indicates that the increase in
certain constituents during the drying process of the
plant can be preferential with respect to others (in this
case limonene augments three times, while
isomenthone only two). Other compounds, such as
those detected at RT 42.01, 42.92, and 61.44 min
were only slightly more abundant in the fresh
material rather than in the dried. Other minor
differences, in this case truly qualitative, were noted
such as, for instance, that a peak at RT 43.70 was
only present in the fresh material, and a peak at RT
44.58 min only in the dried plant. From the analysis
of the corresponding MS spectra (not shown), both
compounds appear to be isomers. Such chemical
modifications, which occur during the drying process
of the plant, produce a characteristic metabolic
fingerprinting of the fresh mint distinguishable from
the fingerprinting of the dried plant. This can be
considered as a chemical reason for the distinctivness
of the Portuguese culinary river fish-based recipe that
recommends the use of the fresh mint.
used to characterise the Portuguese culinary recipe
that recommends the use of the fresh mint. Allowing
direct chemical analysis of the volatile constituents
without any previous treatment of the plant material,
the SPME/GC-MS method is proposed here as the
most adequate technique for the study of other
aromatic plants used in traditional culinary or
medicinal recipes.
The overall aroma of the fresh and dried mint vary,
not only in terms of intensity (dried plant has stronger
aroma compared with the corresponding fresh one),
but also in terms of quality. The overall aroma of the
fresh mint appears in particular much gentler.
Therefore, the fresh plant is possibly more
appropriate for the delicate taste of these fish-based
dishes.
New chemical and biological data on M. cervina are
described in this work. Despite the lack of scientific
data on the aqueous extract of this mint, the dried
plant is currently commercialised in Portugal to
prepare an infusion with claimed digestive properties.
It was, therefore, our wish to describe its chemical
content at least in term of known phenolic
compounds with potential antioxidant capacity; the
presence of seven phenolic derivatives were here
described for the first time in this mint species. Total
antioxidant capacity and total phenol content were
compared with other commercial medicinal plants
currently marketed in Portugal. Concerning the
overall aroma analysis of M. cervina, it was possible
to detect some chemical differences between the
volatile constituents of the fresh and the dried plant
material. Such differences were detected by using the
SPME/GC-MS approach, here applied for the first
time to this mint species. The chemical data acquired
on the dried and fresh material of M. cervina can be
Experimental
Sample preparation: The dried aerial parts of M.
cervina were kindly provided by Ervital (Castro
Daire, Portugal). The dried plant material was milled
prior to the preparation of the infusion that was
obtained as follows: 110 mL of boiling water was
added to 1 g of powdered plant; after 5 min (i.e. the
time period typically used by the consumer) the
extract was filtered through a 0.45 µm filter. The
fresh and dried aerial parts of M. cervina here
compared by SPME/GC-MS analysis were purchased
from “Cantinho das Aromáticas” (Vila Nova de Gaia,
Portugal). Fresh aerial parts (1 g) were directly
analyzed by SPME/GC-MS, while another 1 g from
the same plant was dried at room temperature in the
shade for 6 days obtaining, at the end, 0.25 g of dried
material.
Total antioxidant capacity: This activity was
measured through the ABTS•+ method, as previously
described [2a]. Briefly, the ABTS•+ solution was
prepared by addition, at 1:1 (v/v), of 7 mmol L−1
ABTS
(2,2-azinobis(3ethylbenzothiazoline-6sulfonic acid) diammonium salt (Sigma-Aldrich)) to
2.45 mmol L−1 potassium persulfate (Merck,
Darmstadt, Germany); the reaction took place in the
dark for 16 h. In order to obtain an absorbance of
0.700 ± 0.020, at 734 nm, measured with an UV 1203
spectrophotometer (Shimadzu, Tokyo, Japan), the
ABTS•+ solution was diluted in ultra-pure water. For
analysis of experimental samples, an accurate volume
was used in order to obtain an inhibition percentage
between 20 and 80%, after 6 min of reaction, with
1 mL of ABTS•+ solution; the average of three
replicates was used as a datum point. Total
antioxidant capacity was expressed as percentage of
inhibition (PI), according to the equation PI = (Abs
ABTS•+ − Abs sample)/Abs ABTS•+) × 100, where
Abs ABTS•+ denotes the initial absorbance of diluted
ABTS•+, and Abs sample denotes the absorbance of
the sample after 6 min of reaction. Ascorbic acid
(99.0% pure, from Sigma-Aldrich, Steinheim,
Germany) was used as a standard. Quantitative
results (in g L−1 of ascorbic acid equivalents) were
2068 Natural Product Communications Vol. 3 (12) 2008
obtained through calibration curves produced using
standard solutions of ascorbic acid. Using this
calibration curve, the final result was thus expressed
as an equivalent concentration of ascorbic acid
(in g L−1).
Total phenol content: The amount of phenolic
compounds was determined as described elsewhere
[2a,2b]. To 0.5 mL of sample, 0.5 mL of Folin–
Ciocalteu reagent (Merck), 10mL of 75 g L−1 sodium
carbonate (Sigma-Aldrich) and water were added to a
final volume of 25 mL. Absorbance at 750 nm was
measured on a Heλios α spectrophotometer (Unicam,
Cambridge, UK). Gallic acid was used as a standard
to prepare calibration curves in the ranges 4–80 and
20–400 mg L−1. Total phenol content was reported as
gallic acid equivalents (C, in g L−1), using the
expression C = (Abs sample − 0.0201)/2.1456, where
Abs sample denotes absorbance of the sample after
1 h of reaction. The Pearson correlation coefficient of
the above fit was 0.9991.
LC-MS/MS analysis: The chromatographic system
consisted of a Prostar 210 LC pump (Varian, CA,
USA) coupled with a Varian 1200 triple quadrupole
mass spectrometer (Varian, CA, USA) with
electrospray ionization in positive and negative
modes. A 5 µm C18 column (4.6mm x100 mm,
Merck) was used for the separation at a flow rate of
0.4 mL min-1. The LC-MS/MS method previously
developed by Sun et al. [4] was partially modified in
this work. LC separation was performed in 30
minutes using a gradient elution (eluent A, water with
0.1% formic acid; eluent B, 100% methanol: time 0
min, A 90%; 12.05 min, A 78%; 22.05 min, A 50%;
27.05 min, A 95%; 30 min A, 95%). ESI-MS/MS
detection was obtained using a capillary voltage of
55V. For MS/MS fragmentation, argon atoms were
used (pressure 1.20 mtorr; collision energy of 15 V).
Data were acquired with a Varian LC-MS 1200L
Politi et al.
Workstation. An in-house LC-MS/MS library was
created by injecting 33 pure phenolic standards using
the abovementioned LC-MS/MS conditions. The
identification of the phenolic compounds contained
in the M. cervina extract was achieved by direct
injection of the crude extract and comparison with
the in-house library.
SPME/GC-MS analysis: The conditions chosen
for extraction were as follows: 1 g of fresh and
0.25 g of dried M. cervina were placed in 40 mL
flasks, and capped with a gas-tight seal; the
samples were gently stirred during volatile collection
using a stirring bar, at room temperature, while
a divinylbenzene/carboxen/polydimethylsiloxane
(DVB/CAR/PDMS) fiber was introduced and left for
10 min to trap the volatiles; afterwards, the SPME
fiber was placed in the injector port of a GC-MS
Focus GC Thermo Scientific, and left to desorb
(trapped) the volatiles for 10 min. The conditions
selected for separation were as follows: thermal
desorption at the injector port was at 220ºC, in
splitless mode, with the split valve being opened 30 s
after injection; resolution was through a FFAP
column (50 m x 0.22 mm ID BP21, 0.25 μm), with an
oven temperature held at 40ºC for 1 min, and then
increased at 2ºC min-1 up to 220ºC, which was
eventually held for 30 min; helium C-60 (Gasin,
Portugal) was used as carrier gas, at a volumetric
flow rate of 1 mL min-1.
Acknowledgments - The dried plant material was
kindly provided by “Ervital”, while the fresh plant
was obtained from “Cantinho das Aromáticas”. We
express gratitude, in particular to Luís Alves, founder
of the “Cantinho das Aromáticas”, for his precious
suggestions and continuous encouragement. We
thank the European Union (MRTN-CT-2006036053) for financial support through the INSOLEX
project.
References
[1]
[2]
[3]
[4]
(a) Proença da Cunha A, Alves Ribeiro J, Rodrigues Roque O. (2007) Plantas Aromáticas em Portugal Caracterização e Utilizações.
Fundação Calouste Gulbenkian ed., Lisbon. pp. 1-328; (b) Gonçalves MJ, Vicente AM, Cavaleiro C, Salgueiro L. (2007)
Composition and antifungal activity of the essential oil of Mentha cervina from Portugal. Natural Product Research, 21, 867–871.
(a) Gião MS, Gonzalez-Sanjose ML, Rivero-Perez MD, Pereira CI, Pintado ME, Malcata FX. (2007) Infusions of Portuguese
medicinal plants: Dependence of final antioxidant capacity and phenol content on extraction features. Journal of the Science of
Food and Agriculture, 87, 2638–2647; (b) Singleton VL, Rossi JA. (1965) Colorimetry of total phenolics with phosphomolybdic–
phosphotungstic acid reagents. American Journal of Enology and Viticulture, 16, 144–158; (c) Re R, Pellegrini N, Proteggente A,
Pannala A, Yang M, Rice-Evans C. (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay.
Free Radical Biology and Medicine, 26, 1231–1237.
Proença da Cunha A, Pereira Da Silva A, Rodrigues Roque O. (2006) Plantas e Produtos Vegetais em Fitoterapia. Fundação
Calouste Gulbenkian ed., Lisbon. pp. 1-702.
Sun J, Liang-Bin Y, Li P, Duan C. (2007) Screening non-colored phenolics in red wines using liquid chromatography/ultraviolet
and mass spectrometry/mass spectrometry libraries. Molecules, 12, 679-693.
NPC
Natural Product Communications
Chemical Composition of Thymus serrulatus Hochst. ex
Benth. Essential Oils from Ethiopia: a Statistical Approach
2008
Vol. 3
No. 12
2069 - 2074
Bruno Tirillinia*, Roberto Maria Pellegrinob, Mario Chessac and Giorgio Pintorec
a
Institute of Botany, University of Urbino, via Bramante 28, 61029 Urbino, Italy
b
Department of Chemistry, Laboratory of Organic Chemistry, University of Perugia, via Elce di Sotto 8,
06123 Perugia, Italy
c
Dip. Farmaco-Chimico-Tossicologico, University of Sassari, Via Muroni 23, I-07100 Sassari, Italy
bruno.tirillini@uniurb.it
Received: July 22nd, 2008; Accepted: October 17th, 2008
From the essential oil (EO) obtained from the aerial parts of T. serrulatus collected in Ethiopia, fifty-three compounds were
identified by GC/MS, accounting for more than 99% of the total volatile fraction. Thymol and carvacrol were the main
compounds, ranging from 10.0 + 0.9 to 43.8 + 3.8% and 4.5 + 0.4 to 39.1 + 3.8%, respectively, of the total. o-Cymene, γterpinene, and linalool were the most representative compounds in all the EOs.
Keywords: Thymus serrulatus Hochst. ex Benth., Lamiaceae, essential oil, thymol, carvacrol, statistics.
The genus Thymus (Lamiaceae) includes about 350
species worldwide and is widely distributed in
temperate areas [1]. T. vulgaris contains 0.8-2.6%
(usually ca. 1%) volatile oil, consisting of highly
variable amounts of phenols, monoterpene
hydrocarbons, and alcohols. There are several reports
on the chemical composition of thyme oils; many
indicate either thymol or carvacrol as the major
compounds in the oils [1-23].
The leaves of T. vulgaris are used as an herb in food
preparations, while the essential oil from the leaves is
used in the alimentary, cosmetic and pharmaceutical
industries. Thyme oil is used as an antispasmodic,
carminative, antiseptic, anthelmintic, expectorant,
antimicrobial
(broad-spectrum
antibacterial,
antifungal and antiviral activity), antirheumatic,
antioxidative, and natural food preservative [10,2444]. The strong antimicrobial activity of thyme oil is
ascribed mainly to the high content of phenolic
constituents, such as thymol and carvacrol [24]. The
essential oil of T. vulgaris has potent repellent
activity against Culex pipiens pallens [27].
Two species, T. schimperi Ronninger and T.
serrulatus Hochst. ex Benth., are indigenous to
Ethiopia [16], while T. vulgaris has been recently
introduced.
Chemical polymorphism concerning the essential oils
of the genus Thymus is a widespread phenomenon.
For example, the two Finnish species, T. serpyllum
var. serpyllum and T. serpyllum var. tanaenis, turned
out to form 4 different chemotypes each, with
hedycaryol, germacra-1(10),5-dien-4-ol, germacra1(10),4-dien-6-ol, linalool, and linalyl acetate as
type-characterizing compounds. T. schimperi and
T. serrulatus belong to the thymol-carvacrol
chemotypes [16]. The leaves of T. serrulatus are used
in Ethiopia as spices to flavor food, as well as for
medicines. People in Bale harvest T. serrulatus for
making a tea [16].
The aim of this study was to evaluate the essential
oils of wild plants of T. serrulatus, collected from
seven different areas of the Ethiopian plateaus and to
acquire information on the thyme population by
statistical methods. Table 1 reports the mean
chemical analysis of five populations and includes all
compounds found in the EOs from leaves and
flowers. Fifty-three compounds were identified,
which accounted for more than 99% of the total
2070 Natural Product Communications Vol. 3 (12) 2008
volatile fraction from plants of T. serrulatus. Either
thymol or carvacrol were the main compounds, with
average amounts ranging from 10.0 + 0.9 to 43.8 +
3.8% and from 4.5 + 0.4 to 39.1 + 3.8%, respectively.
O-cymene, γ-terpinene, and linalool were the most
representative compounds in all the EOs.
Cluster analysis of the database, including a
compound selection, put in evidence the presence of
four “natural” groups: area 3 is joined with area 6 and
7, area 2 with area 4, while area 5 and area 1 remain
ungrouped (Figure 1). The analysis shows that areas
3-6-7 join at a distance cluster (ds) = 38.88, while
areas 2 and 4 join at a ds = 36.66. The first clusters
were formed at a distance cluster (da) = 1.03.
Five major compounds listed in Table 2, which
accounted for more than 75% of the essential oils,
were selected to discuss the chemical variability due
to areas of origin. Each area could be characterized
by the chemical composition trend of the five
major compounds. In Z1, o-cymene and carvacrol
have the same percent value, in Z2 carvacrol is
comparable to linalool and achieves its minimum
value, in Z3 linalool achieves the minimum value, in
Z4 carvacrol is comparable with γ-terpinene, in Z5
carvacrol is comparable to o-cymene, in Z6 linalool
and γ-terpinene are comparable, and in Z7 no
one compound is comparable to another. These
remarks were supported by the ANOVA applied to a
database in which the cases were the values of the
five compounds, while the variables were the areas
(Z1-Z7).
According to many authors [1-16], there are two
chemotypes of T. serrulatus. The first one comprises
the oils with a high thymol concentration and was in
accord with the plants collected in Z2, Z4, and Z5
areas. The second one, with a high carvacrol content,
corresponds to the plants collected in Z1, Z6, and Z7
areas. There is a question that might be of interest for
these data: do the measurements of the main
compounds discriminate between the two assumed
groups of oils and can they be used to produce a
useful rule for classifying other oils, such as those
from Z3 areas or other oils that might become
available? The Fisher’s linear discriminant function
analysis (FLDFA) works with data that are already
classified into groups to derive rules for classifying
new (and as yet unclassified) individuals on the basis
of their observed variable values. The within-group
covariance matrices suggest that the sample values
differ to some extent, but according to Box’s test for
Tirillini et al.
Z5
Z1
Z2
Z4
Z3
Z6
Z7
Figure 1: Dendrogram using Average Linkage (between groups)
equality of covariances, these differences are not
statistically significant. The canonical correlation
value is 0.988 so that 97.6% of the variance in the
discriminant function scores can be explained by
group differences. In the Wilk’s Lambda test, the
lambda coefficient was 2.5%, and is the proportion of
the total variance in the discriminant scores not
explained by differences among the groups. The
Fisher’s linear discriminant function is:
z = -4.92 x meas1 – 20.84 x meas2 – 15.39 x meas3 +
79.21 x meas4 –74.99 x meas5.
The threshold against which an oil discriminant score
is evaluated is –5.00. Thus, new oils with
discriminant scores above –5.00 would be assigned to
the thymol type; otherwise, they would be classified
as carvacrol type. Following the derived
classification rule, the oils from Z3 areas would be
assigned to the carvacrol type.
In conclusion, if a representative lot of a plant
population was hydrodistilled, a statistical approach
may be useful for population inference. The
evaluation of essential oils from wild plants living in
different geographic areas could be helpful in the
chemotype classification; the analyzed oils might be
divided into two types according to the relative
amounts of thymol or carvacrol. The “natural” group
of oil did not correspond to the geographical areas as
arbitrary chosen, but it might be possible redefine the
areas in accordance with the cluster analysis. When
the thyme population is distributed in an
heterogeneous way across a large area, it might be
possible to correlate the percent oil composition to
the living sample area.
Essential oils from Thymus serrulatus
Natural Product Communications Vol. 3 (12) 2008 2071
Table 1: Chemical composition (area percent + SD) of T. serrulatusa.
.
Compounds b
Methyl-2-methyl-butyrate
α-Thujene
RI c
778
931
Z1 %
+SD
0.07
2.3
Z2 %
+ SD
Z3 %
+ SD
Z4 %
+ SD
Z5 %
+ SD
Z6 %
+ SD
Z7 %
0.01
0.2
0.1
2.8
0.02
0.26
0.1
3.1
0.01
0.3
0.1
1.7
0.01
0.2
Tr
0.8
0.07
Tr
1.7
0.1
+ SD
0.06
2.0
0.01
0.2
α-Pinene
939
1.0
0.08
0.4
0.03
0.4
0.03
0.2
0.02
0.09
0.01
0.3
0.02
0.4
0.04
Camphene
954
0.07
0.01
Tr
-
Tr
-
Tr
-
Tr
-
Tr
-
0.04
0.01
2,4(10)-Thujadien
960
0.07
0.01
0.08
0.01
Tr
-
0.04
0.01
Tr
-
Tr
-
Tr
-
Sabinene
975
0.06
0.01
0.2
0.02
0.2
0.02
0.06
0.01
Tr
-
0.1
0.01
0.2
0.01
β-Pinene
980
0.2
0.02
0.09
0.01
0.09
0.01
0.06
0.01
Tr
-
0.08
0.01
0.1
0.01
1-octen-3-ol
982
0.1
0.01
0.9
0.06
0.7
0.07
0.5
0.05
0.09
0.01
0.1
0.01
0.2
0.02
3-Octanone
985
3.6
0.3
1.6
0.13
1.8
0.2
1.0
0.09
0.4
0.04
2.7
0.2
3.4
0.3
Myrcene
991
1.7
0.2
1.7
0.15
1.3
0.09
1.0
0.1
0.6
0.05
0.9
0.09
0.8
0.08
3-Octanol
992
1.2
0.1
0.7
0.05
0.6
0.06
0.4
0.04
0.1
0.01
0.8
0.07
0.9
0.08
α-Phellandrene
1003
0.4
0.04
0.4
0.04
0.3
0.03
0.3
0.02
Tr
-
0.1
0.01
0.2
0.02
α-Terpinene
1016
2.6
0.3
3.7
0.27
3.0
0.3
2.4
0.2
1.6
0.1
1.7
0.1
1.9
0.2
o-Cymene
1025
22.5
1.9
28.4
2.36
37.2
2.7
18.7
1.5
16.4
1.5
27.1
2.1
26.0
2.5
Limonene
1031
0.4
0.04
0.5
0.05
0.5
0.05
0.3
0.03
Tr
-
0.3
0.02
0.3
0.02
β-Phellandrene
1032
0.3
0.03
0.3
0.03
0.3
0.03
0.2
0.02
Tr
-
0.2
0.02
0.3
0.02
1,8-Cineole
(Z)-β-Ocimene
1033
1036
0.4
0.4
0.04
0.02
0.1
0.2
0.01
0.02
Tr
0.1
0.02
0.1
0.1
0.01
0.01
Tr
Tr
-
Tr
0.2
0.02
0.06
0.2
0.01
0.02
(E)-β-Ocimene
1051
0.1
0.02
0.1
0.01
Tr
-
0.09
0.01
Tr
-
Tr
-
Tr
-
γ-Terpinene
1061
15.6
1.3
22.8
2.06
8.0
0.7
13.7
1.3
12.0
1.1
4.1
0.3
3.1
0.2
cis-Sabinene hydrate
1069
0.9
0.07
1.0
0.1
1.4
0.1
0.8
0.08
0.8
0.06
1.5
0.1
1.8
0.2
cis-Linalool oxide
1087
0.07
0.01
Tr
-
Tr
-
Tr
-
Tr
-
Tr
-
0.04
0.01
Terpinolene
1089
0.1
0.01
0.2
0.02
Tr
-
0.1
0.01
Tr
-
Tr
-
0.03
0.01
Linalool
1098
6.2
0.6
5.0
0.5
1.7
0.1
3.3
0.3
3.3
0.2
3.6
0.3
4.8
0.4
(3Z)-Hexenyl isobutanoate
1147
0.2
0.02
Tr
-
Tr
-
0.2
0.02
Tr
-
Tr
-
Tr
-
Borneol
1170
Tr
-
Tr
-
Tr
-
Tr
-
Tr
-
Tr
-
0.03
0.01
Terpinen-4-ol
1177
1.04
0.08
0.7
0.05
0.6
0.05
0.2
0.02
0.4
0.03
0.2
0.02
0.1
0.01
p-Cymen-8-ol
1183
Tr
-
Tr
-
Tr
-
0.06
0.01
Tr
-
Tr
-
Tr
-
α-Terpineol
1189
0.7
0.07
0.5
0.05
0.6
0.05
0.6
0.05
0.4
0.02
0.2
0.02
0.4
0.04
cis-Dihydrocarvone
1193
0.1
0.01
Tr
-
Tr
-
0.04
0.01
Tr
-
Tr
-
0.07
0.01
Thymol. methyl ether
1235
0.2
0.02
0.2
0.02
0.2
0.02
0.1
0.02
Tr
-
0.3
0.02
0.2
0.02
Linalyl acetate
1257
0.06
0.01
4.0
0.36
Tr
-
2.0
0.1
2.3
0.2
Tr
-
Tr
-
Thymol
1290
10.1
0.8
16.3
1.3
14.9
1.4
34.7
3.1
43.8
3.8
11.5
1.0
10.0
0.9
Carvacrol
1299
20.6
1.8
4.5
0.37
19.1
1.6
13.9
1.0
15.0
1.2
39.1
3.8
38.0
3.3
Thymol acetate
1352
0.3
0.03
0.3
0.03
Tr
-
0.4
0.04
0.
0.01
Tr
-
0.06
0.01
Carvacrol acetate
1373
2.3
0.2
Tr
-
Tr
-
0.1
0.01
Tr
-
0.1
0.02
0.2
0.02
β-Bourbonene
1388
0.05
0.01
Tr
-
Tr
-
Tr
-
Tr
-
Tr
-
Tr
-
α-Gurjunene
1410
0.07
0.01
Tr
-
Tr
-
Tr
-
Tr
-
Tr
-
Tr
-
(E)-Caryophyllene
1419
1.3
0.1
1.3
0.11
1.7
0.1
1.2
0.1
1.3
0.1
1.2
0.1
1.5
0.1
0.03
α-trans-Bergamotene
1435
Tr
-
0.1
0.01
0.3
0.02
0.1
0.01
Tr
-
0.2
0.02
0.3
Aromadendrene
1441
0.9
0.07
Tr
-
Tr
-
Tr
-
Tr
-
Tr
-
Tr
-
α-Humulene
1455
Tr
-
Tr
-
0.1
0.01
Tr
-
Tr
-
Tr
-
0.08
0.01
allo-Aromadendrene
1460
0.1
0.01
Tr
-
Tr
-
Tr
-
Tr
-
Tr
-
0.05
0.01
γ-Muurolene
1480
0.06
0.01
Tr
-
Tr
-
Tr
-
Tr
-
0.2
0.02
0.0
0.01
Germacrene D
1485
0.07
0.01
0.1
0.01
0.3
0.03
0.04
0.01
Tr
-
Tr
-
0.2
0.02
Viridiflorene
1497
0.07
0.01
Tr
-
Tr
-
Tr
-
Tr
-
Tr
-
Tr
-
Bicyclogermacrene
1500
Tr
-
Tr
-
0.1
0.01
0.06
0.01
Tr
-
0.1
0.01
0.2
0.02
0.02
β-Bisabolene
1506
0.1
0.01
Tr
-
0.08
0.01
0.1
0.01
Tr
-
0.1
0.02
0.2
γ-Cadinene
1512
0.09
0.01
Tr
-
Tr
-
0.08
0.01
Tr
-
Tr
-
Tr
-
Δ-amorphene
1513
0.1
0.01
Tr
-
0.1
0.01
0.1
0.01
Tr
-
Tr
-
0.04
0.01
β-Sesquiphellandrene
1525
0.4
0.04
0.06
0.01
0.3
0.03
0.3
0.02
Tr
-
0.3
0.03
0.4
0.04
Spathulenol
1577
Tr
-
Tr
-
Tr
-
Tr
-
Tr
-
Tr
-
0.06
0.01
Caryophyllene oxide
1585
0.1
0.01
Tr
-
Tr
-
0.1
0.01
Tr
-
0.2
0.02
0.2
0.02
Total
a
99.96
99.68
99.59
99.99
99.79
99.52
99.48
essential oils of plants collected from the Z1-Z7 areas. b Compounds were listed in order of their elution from a DB-5MS column. c RI, retention indices as
determined on DB-5MS column using homologous series of n-alkanes. Tr= trace (< 0.01%).
2072 Natural Product Communications Vol. 3 (12) 2008
Table 2: Chemical variability of the major compounds of T. serrulatus
essential oil from Z1-Z7 areas. Values within a row for each compound
having different letters are significantly different from each other using
Tukey’s LSD test (P<0.05).
Compounds
Z1
Z2
Z3
Z4
Z5
Z6
Z7
o-Cymene
γ-Terpinene
Linalool
thymol
Carvacrol
3.11c
2.75 e
1.80 d
2.30 a
3.02 c
3.30 d
3.07 f
1.60 c
2.80 c
1.48 a
3.59 e
2.01 c
0.51 a
2.73 c
2.93 c
2.96 b
2.56 d
1.21 b
3.50 d
2.63 b
2.79 a
2.49 d
1.16 b
3.75 e
2.65 b
3.26 cd
1.45 b
1.22 b
2.46 b
3.60 d
3.24 cd
1.1 a
1.56 c
2.26 a
3.63 d
Experimental
Plant materials: Leaves and flowers of T. serrulatus
growing in different area of Ethiopia were collected
in May-June 2007 and dried at ambient temperature.
A large number of plants (>5 kg) were randomly
collected over the same area. The plants were
collected in seven areas (Z1-Z7). Voucher specimens
were deposited in the Herbarium of the CAMS –
Univ. of Perugia (IPO-E1-05-07).
Extraction of oil: The plants were subjected to
hydrodistillation using a Clevenger-type apparatus
for 3 h yielding 0.8 + 0.1% (mean value) of a
yellowish oil. The oil was dried over anhydrous
sodium sulfate and stored in sealed vials under
refrigeration prior to analysis.
GC and GC-MS analysis: The GC analyses were
carried out using an Agilent 6890N instrument
equipped with a FID and an HP-InnoWax capillary
column (30 m x 0.25 mm, film thickness 0.17 μm),
working from 60°C (3 min) to 210°C (15 min) at
4°C/min or an DB-5MS capillary column (30 m x
0.25 mm, film thickness 0.25 μm) working from
60°C (3 min) to 300°C (15 min) at 4°C/min; injector
and detector temperatures, 250°C; carrier gas, helium
(1 mL/min); split ratio, 1 : 10.
GC-MS analyses were carried out using an Agilent
5975 GC-MS system operating in the EI mode at
70 eV, using the two above mentioned columns.
The operating conditions were analogous to those
reported in the GC analyses section. Injector and
transfer line temperatures were 220°C and 280°C,
respectively. Helium was used as the carrier gas, flow
rate 1 mL/min. Split ratio, 1 : 10.
Identification of the components: The identification
of the components was made by matching their
Tirillini et al.
spectra with those from mass spectral libraries and
the identity of each component was confirmed by
comparing their retention indices, for both columns,
relative to the C6-C22 n-alkanes with those from the
literature.
When
reported,
co-elution
gas
chromatography with reference compounds was used
for an additional confirmation of the compound
identity. The percentage composition of the essential
oil was obtained by the normalization method from
the GC peak areas, without using correction factors.
Experimental design: The plants collected in each
area were hydrodistilled separately and the ghost
effect was minimized. The handling data was the
percent concentration of the identified compounds. If
we suppose that hydrodistillation of 7 batches of the
same lot gives for each compound values +10%
across the mean, from each percent composition of
the 7 hydrodistilled oils, we might obtain 7 parent
percent oil compositions. A 49% composition (7 oils
for 7 areas (Z1-Z7)) for 53 oil compounds were the
basic data file.
Data file handling: Many problems arose from this
type of database in a statistical analysis. The presence
of compounds above 0.01% (tr) required a valuation:
the addition to the database of 0.01, gave a statistical
improvement and removed the null value.
Compounds scarcely present in the database were
removed. The independence of the data were checked
using a linear or logaritmic coefficient of correlation
between each pair of compounds: one of the two
compounds with R >0.98 was removed (between
limonene and β-phellandrene there was a high
logarithmic correlation and β-phellandrene was
removed). The great data dishomogeneity suggests
the use of the Box-Cox method for the choice of most
useful data transformation. The log-likelihood
function suggests the use of natural logarithmic data
transformation. According to Kolmogorov-Smirnov
tests, the database in each group follows a normal
distribution and according to the Levene test the
variance of the database is the same in the groups
(homoschedasticity assumption).
Acknowledgments – We are grateful to IPO
(Increasing People Opportunities) for providing us
with the essential oils of T. serrulatus.
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NPC
Natural Product Communications
GC MS Analysis of the Volatile Constituents of Essential Oil
and Aromatic Waters of Artemisia annua L. at Different
Developmental Stages
2008
Vol. 3
No. 12
2075 - 2078
Anna Rita Biliaa,*, Guido Flaminib, Fabrizio Morgennic, Benedetta Isacchia and
Franco FrancescoVincieria
a
Department of Pharmaceutical Sciences, via Ugo Schiff,6, University of Florence,
50019 Sesto Fiorentino (FI), Italy
b
Department of Bioorganic Chemistry and BioPharmacy, Università di Pisa, Via Bonanno 33,
56126 Pisa, Italy
c
Officina Profumo Farmaceutica di Santa Maria Novella, SRL, via Reginaldo Giuliani 141,
50141 Firenze, Italy
ar.bilia@unifi.it
Received: July 22nd, 2008; Accepted: October 31st, 2008
Artemisia annua L. (Asteraceae) still represents the only source of artemisinin, considered as one of the most important drugs
for the treatment of malaria and which, more recently, has been shown to be effective against numerous types of tumors. The
foliage and inflorescence of A. annua also yield an essential oil upon hydrodistillation. This oil has been evaluated at different
development stages (pre-flowering and flowering) by GC/MS. The volatile oil from plants at full blooming showed numerous
constituents, with germacrene D (21.2%), camphor (17.6%), β-farnesene (10.2%), β-caryophyllene (9%), and
bicyclogermacrene (4.2%) among the main ones. Aromatic waters, after extraction with n-hexane, showed the presence, among
others, of camphor (27.7%), 1,8-cineole (14%), artemisia ketone (10.1%), α-terpineol (6.1%), trans-pinocarveol (5.4%), and
artemisia alcohol (2%). From plants at the pre-flowering stage, aromatic waters were obtained with camphor (30.7%),
1,8-cineole (12.8%), artemisia alcohol (11.4%), artemisia ketone (9.5%), alpha-terpineol (5.8%), and trans-pinocarveol (3.0%)
as the main constituents. The qualitative and quantitative profiles of the two aromatic waters were similar. These results
permitted the conclusion to be made that A. annua could be harvested a long time before the onset of flowering to obtain higher
yields of artemisinin or could be allowed to attain maturity to obtain valuable yields of volatiles.
Keywords: Artemisia annua, Asteraceae, volatile constituents, essential oil, aromatic waters, pre-flowering and blossom time,
GC-MS analysis.
Artemisia annua L.(family Asteraceae), indigenous to
south east Asia, is an annual herb/shrub, which has
become naturalized or is cultivated as a horticultural
or medicinal plant in many parts of Asia, Africa,
Europe, America and Australia. It has been used in
Traditional Chinese Medicine for many centuries for
the treatment of fever and malaria due to the presence
in the leaves and capitula of a unique sesquiterpene
endoperoxide called artemisinin. Nowadays, A.
annua still represents the only source of artemisinin
[1,2], considered to be one of the most important
drugs for the treatment of malaria and, more recently,
it has been shown to be effective also against
numerous types of tumors, including breast cancer,
human leukemia, colon, and small-cell lung
carcinomas [3]. It is well known that natural
populations and genetic resources of A. annua from
different areas have considerable variability in the
accumulation of artemisinin in the leaves and the
capitula of the plant, with an artemisinin content
ranging from 0.001 to 0.8% [1].
The foliage and inflorescence of A. annua plants also
yield an essential oil upon hydrodistillation, which
could represent another potential commercially
valuable product [4,5]. In this current study
the volatile constituents of both the essential oil
and aromatic waters obtained by hydrodistillation of
2076 Natural Product Communications Vol. 3 (12) 2008
Bilia et al.
Table 1: Identified constituents of the investigated samples.
l.r.i.
(A)
Constituents
(E)-3-Hexen-1-ol
α-Pinene
Camphene
Sabinene
β-Pinene
Myrcene
2,3-Dehydro-1,8cineole
Yomogi alcohol
(E)-3-Hexen-1-yl
acetate
α-Terpinene
p-Cymene
Santolina alcohol
1,8-Cineole
γ-Terpinene
Artemisia ketone
cis-Sabinene hydrate
Artemisia alcohol
Linalool
trans-Sabinene hydrate
Dehydrosabinaketone
α-Campholenal
cis-p-Menth-2-en-1-ol
trans-Pinocarveol
trans-p-Menth-2-en-1ol
Camphor
Camphene hydrate
β-Pinene oxide
Sabinaketone
Pinocarvone
δ-Terpineol
Borneol
Terpinen-4-ol
p-Cymen-8-ol
α-Terpineol
Myrtenol
Verbenone
(E,E)-2,4-Nonadienal
trans-Carveol
Thymyl methyl oxide
cis-Carveol
Carvone
cis-Chrysanthenyl
acetate
Lavandulyl acetate
Isobornyl acetate
Thymol
trans-Carvyl acetate
Eugenol
cis-Carvyl acetate
α-Copaene
β-cubebene
Benzyl valerate
(E)-β-Caryophyllene
(E)-β-Farnesene
α-Humulene
853
941
956
978
982
992
994
998
Table 1 (Contd.)
n-Hexane
extract of
aromatic
waters.
Plants at
preflowering
n-Hexane
extract of
aromatic
waters.
Plants at
full
blooming
Volatile
oil -full
blooming
1373
1027
1071
1116
1108
1145
0.2
tr
0.1
tr
0.1
nd
0.1
0.4
0.2
0.2
0.1
0.5
tr
0.7
1.5
0.2
0.5
0.1
-
0.1
2.5
nd
1.2
tr
nd
l.r.i.
(B)
1401
-
1008
1020
1029
1033
1035
1064
1065
1071
1084
1101
1103
1123
1127
1129
1141
1183
1245
1413
1208
1249
1355
1462
1504
1557
1458
1641
nd
nd
tr
0.4
12.8
nd
9.5
0.7
11.4
1.1
1.0
0.8
tr
0.2
3.0
nd
nd
0.1
0.2
14.0
nd
10.1
0.2
2.0
3.9
0.6
0.9
tr
0.2
5.4
0.1
0.1
1.7
nd
1.4
0.3
nd
0.2
nd
0.2
0.2
nd
tr
nd
nd
1142
1148
1153
1159
1161
1166
1169
1171
1180
1186
1192
1197
1207
1220
1222
1233
1234
1245
1560
1522
1611
1549
1676
1796
1607
1835
1688
1791
1715
1705
1874
1593
1845
1742
nd
30.7
nd
0.7
0.2
3.2
0.9
3.9
2.4
0.2
5.8
0.1
0.2
0.8
0.8
0.1
0.2
0.3
nd
27.7
nd
2.5
0.4
3.8
1.0
1.8
3.4
0.4
6.1
0.2
tr
0.6
0.5
0.7
0.3
0.3
1.3
17.6
0.1
nd
nd
0.3
nd
0.9
0.6
nd
0.5
nd
nd
nd
0.3
nd
tr
nd
1263
1286
1286
1293
1339
1358
1363
1377
1389
1391
1419
1456
1457
1596
1582
2186
1756
2173
1793
1475
1545
1604
1660
1666
nd
nd
0.7
nd
nd
1.1
nd
nd
nd
0.3
tr
nd
tr
nd
nd
0.2
nd
nd
1.1
nd
0.2
nd
0.3
0.6
0.3
tr
0.4
0.9
nd
1.5
1.0
tr
0.4
0.9
0.7
nd
9.0
10.2
0.1
β-Chamigrene
Germacrene D
β-Selinene
Bicyclogermacrene
δ-Cadinene
trans-Nerolidol
Spathulenol
Caryophyllene oxide
Globulol
epi-Cedrol
β-Acorenol
Cubenol
T-Muurolol
Kongol
α-Cadinol
Elemol acetate
(Z)-α-Santalol
Total identified
1476
1483
1489
1496
1524
1563
1578
1583
1586
1599
1640
1643
1645
1662
1655
1663
1680
1757
1709
1713
1736
1731
1996
2136
2070
2054
2139
2045
2150
2236
2187
2310
tr
tr
tr
nd
nd
nd
0.5
0.3
nd
nd
0.2
nd
nd
nd
nd
nd
nd
0.1
0.4
0.3
nd
nd
nd
0.7
0.4
nd
nd
nd
nd
nd
0.1
nd
nd
nd
1.4
21.2
0.8
4.2
0.5
0.4
1.3
0.6
0.2
0.5
nd
2.3
0.7
0.6
0.5
0.5
1.3
97.5
95.0
91.2
(A): linear retention index (l.r.t) obtained with a phenylmethylsilicone
column (apolar)
(B): linear retention index (l.r.t) obtained with a PEG column (polar)
A annua collected at different developmental stages
(pre-flowering and full blooming) have been
evaluated by GC/MS. The aim of this study was the
evaluation of the essential oil and aromatic waters as
ingredients of food, pharmaceutical and cosmetic
products, depending on the composition of the
volatile constituents.
Fresh, wild plant materials were collected near Sesto
Fiorentino (FI, Italy) at pre-flowering and full
blooming in August and September 2007 and
submitted to hydrodistillation. Only samples in full
bloom gave essential oil, which was analysed by
GC/MS. Aromatic waters obtained from both
samples were also analysed after extraction with
n-hexane. Seventy-two compounds were identified in
the different samples, accounting for 91.2%-97.5% of
the total compositions.
Volatile oil from plants at full bloom showed
numerous constituents, with germacrene D (21.2%),
camphor (17.6%), β-farnesene (10.2%), βcaryophyllene (9%), and bicyclogermacrene (4.2%)
among the main ones. Aromatic waters, after
extraction with n-hexane, showed the presence,
among others, of camphor (27.7%), 1,8-cineole
(14%), artemisia ketone (10.1%), alpha-terpineol
(6.1%), trans-pinocarveol (5.4%), and artemisia
alcohol (2%). From plants at the pre-flowering stage,
only aromatic waters were obtained with camphor
(30.7%), 1,8-cineole (12.8%), artemisia alcohol
(11.4%), artemisia ketone (9.5%), α-terpineol
(5.8%), and trans-pinocarveol (3.0%) as their main
GC-MS analysis of volatile constituents of Artemisia annua
Natural Product Communications Vol. 3 (12) 2008 2077
constituents. Artemisinin was never detected in
either the essential oil or aromatic waters. The
qualitative and quantitative profiles of the two
aromatic waters were similar.
Constituents found in our sample of essential oil were
very similar to those reported in the literature, namely
camphor, germacrene D, and artemisia ketone. In the
aromatic waters 1,8 cineole was also found and can
represent a good source of volatiles, to be used for
different applications, including cosmetic, alimentary
and pharmaceutical ones.
In the literature there are several studies reporting the
GC analysis of the essential oil obtained from
different parts of A. annua of different origins [4-20],
but none concerning the analysis of aromatic waters.
A great variability in the qualitative and quantitative
composition has been evidenced and apart from
ecological factors, plant part and development stage,
a main source of variability was the wild plant
material or selected cultivar. The majority of the
studies have been performed on plant material from
India and the main compounds identified in the
essential oils from the aerial parts were camphor
(0-44.4%), 1,8 cineole (1.7-28.6%), artemisia ketone
(0-52.9%), 2,5-dihydro-3-methylfuran (0-68.5%),
camphene (0-28.4%), and germacrene D (0-10.9%)
[4, 6-12].
The principal constituents detected in the essential oil
of plants from Iran, depending on the flowering
stage, were camphor (14.3-48.0%), germacrene D
(2.0-18.5%),1,8-cineole (5.8-17.3%), α-pinene (013.3%), β-selinene (0-10.4%), and β-caryophyllene
(0-9.4%) [13-14]. Two studies from the USA took
into account the two subspecies, the “glanded” and
“glandless” ones. Artemisia ketone was found as the
main component (0-35.6% in leaves, up to 56% in
flowers). Other important volatiles were germacrene
D (0-49.8%), 1,8-cineole (0-28.1%), α-pinene
(0-26.7%), camphor (0-20.5%), and pinocarvone
(0-15.8%) [5,15].
A few studies with European plant material were
performed in France [16], the Netherlands [17] and
Hungary [18-19]. All of them reported that artemisia
ketone was the main constituent (11.9-63.9%), with
the exception of plants grown from Vietnamese
seeds, where it was completely absent. Other main
constituents were artemisia alcohol (11.0-56.0%) and
camphor (21.8%) in Hungarian plant material,
germacrene D (2.0-18.5%) in plants collected in
France and the Netherlands, and 1,8-cineole (5.114.7%) in plants from France. Another study on the
essential oil of the fruits reported that sesquiterpenes
were the most abundant chemicals, i.e. caryophyllene
oxide (9.0%), caryophyllene (6.9%), (E)- β-farnesene
(8.2%) and germacrene D (4.0%). However, this oil
was only partially characterized, with only 52% of
the components being identified [20].
Furthermore, the results obtained permitted us to
conclude that A. annua could be harvested either a
long time before onset of flowering to obtain higher
yields of artemisinin or the crop could be allowed to
attain maturity to obtain valuable yields of the
essential oil.
Experimental
Plant material and samples: Artemisia annua
subspecies “glanded” was identified by Dr Lia
Pignotti of the Department of Vegetal Biology,
University of Florence, where an authentic
specimen is also deposited. About 1 kg fresh, wild
plant material was collected near Sesto Fiorentino
and immediately submitted to hydrodistillation. Only
samples in full bloom gave essential oil (0.5%),
which was analysed by GC/MS. Aromatic waters
obtained from both samples were also analysed after
extraction with n-hexane.
GC-EIMS analysis: GC-EIMS analyses were
performed with a Varian CP-3800 gaschromatograph equipped with a DB-5 capillary
column (30 m x 0.25 mm; coating thickness
0.25 mm) and a Varian Saturn 2000 ion trap
mass detector. Analytical conditions: injector and
transfer line temperatures at 220 and 240°C
respectively; oven temperature was programmed
from 60°C to 240°C at 3°C/min; carrier gas helium at
1 mL/min; injection of 0.2 mL (10% n-hexane
solution); split ratio 1:30. Identification of the
constituents was based on comparison of the
retention times with those of authentic samples,
comparing their Linear Retention Indices relative to
the series of n-hydrocarbons, and by computer
matching against commercial [21] and home-made
library mass spectra built up from pure substances
and components of known essential oils and MS
literature data [21-26]. Moreover, the molecular
weights of all the identified substances were
confirmed by GC-CIMS, using MeOH as CI ionizing
gas.
2078 Natural Product Communications Vol. 3 (12) 2008
Bilia et al.
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Gupta SK, Singh P, Bajpai P, Ram G, Singh D, Gupta MM, Jain DC, Khanuja SP, Kumar S. (2002) Morphogenetic variation for
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Efferth T. (2007) Willmar Schwabe Award 2006: antiplasmodial and antitumor activity of artemisinin-from bench to bedside.
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Ma C, Wang H, Lu X, Li H, Liu B, Xu G. (2007) Analysis of Artemisia annua L. volatile oil by comprehensive two-dimensional
gas chromatography time-of-flight mass spectrometry. Journal of Chromatography A, 1150, 50-53.
Tellez MR, Canel C, Rimando AM, Duke SO. (1999) Differential accumulation of isoprenoids in glanded and glandless Artemisia
annua L. Phytochemistry, 52, 1035-1040.
Goel D, Mallavarupu GR, Kumar S, Singh V, Ali M. (2007) Volatile metabolite compositions of the essential oil from aerial parts
of ornamental and artemisinin rich cultivars of Artemisia annua. Journal of Essential Oil Research, 20, 147-152.
Mukhtar HM, Ansari SH, Ali M, Mir SR, Abdin MZ, Singh P. (2007) GC-MS analysis of volatile oil of aerial parts of Artemisia
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De Magalhaes PM, Pereira B, Sartoratto A. (2004) Yields of antimalarial Artemisia annua L. species. Acta Horticoltura, 629,
421-424.
Zhang Y, Zhang J, Yao J, Wang L, Huang A, Dong L. (2004) Studies on the chemical constituents of the essential oil of Artemisia
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Bagchi GD, Haider F, Dwivedi PD, Singh A, Naqvi AA. (2003) Essential oil constituents of Artemisia annua during different
growth periods at monsoon conditions of subtropical north Indian plains. Journal of Essential Oil Research, 15, 248-250.
Jain N, Srivastava SK, Aggarwal KK, Kumar S, Syamasundar KV. (2002) Essential oil composition of Artemisia annua L. 'Asha'
from the plains of northern India. Journal of Essential Oil Research, 14, 305-307.
Ali M, Siddiqui NA. (2000) Volatile oil constituents of Artemisia annua leaves. Journal of Medicinal & Aromatic Plant Sciences,
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Verdian-Rizi MR, Sadat-Ebrahimi E, Hadjiakhoondi A, Fazeli MR, Pirali HM. (2008) Chemical composition and antimicrobial
activity of Artemisia annua L. essential oil from Iran. Faslnamah-i Giyahan-i Daruyi, 7, 58-62.
Lari yazdi H, Khavarinejad RA, Roustaian AH. (2002) The composition of the essential oil of Artemisia annua L. growing wild in
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Biougne J, Chalchat JC, Garry RP, Lamy J. (1993) Essential oil of Artemisia annua: Seasonal variations in chemical composition.
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Woerdenbag HJ, Bos R, Salomons MC, Hendriks H, Pras N, Malingre TM. (1993) Volatile constituents of Artemisia annua L.
(Asteraceae). Flavour & Fragrance Journal, 8, 131-137.
Hethelyi EB, Cseko IB, Grosz M; Mark G, Palinkas JJ. (1995) Chemical composition of the Artemisia annua essential oils from
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Hethelyi I, Ceseko I, Grosz M, Mark G, Palinkas JJ. (1994) Capillary gas chromatographic investigation of Artemisia annua
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20M phases. Journal of Chromatography, 503, 1-24.
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Carol Stream, Illinois.
NPC
Natural Product Communications
Do Non-Aromatic Labiatae Produce Essential Oil?
The Case Study of Prasium majus L.
2008
Vol. 3
No. 12
2079 - 2084
Claudia Giuliania, Roberto Maria Pellegrinob, Bruno Tirillinic and Laura Maleci Binia,*
a
Department of Vegetal Biology, University of Florence, via La Pira 4, 50121 Florence, Italy
b
Department of Chemistry, Laboratory of Organic Chemistry, University of Perugia,
via Elce di Sotto 8, 06123 Perugia, Italy
c
Institute of Botany, University of Urbino, via Bramante 28, 61029 Urbino, Italy
maleci@unifi.it
Received: July 7th, 2008; Accepted: November 19th, 2008
Prasium majus L. (Labiatae, Lamioideae) is considered a typical non-aromatic plant. In this work we examined the glandular
trichomes present on leaves and inflorescences and the essential oils of plants growing along the Tuscan coast of Italy. The
micromorphological study evidenced different types of trichomes responsible for the essential oil production. The essential oil
compositions of leaves and flowers were analyzed by GC/MS and are here reported.
Keywords: Prasium majus, Labiatae, Lamioideae, glandular trichomes, micromorphology, histochemistry, essential oil,
GC/MS analysis.
The Labiatae family contains about 200 genera of
which 40 per cent possess aromatic properties.
Owing to these features, the family has a wide
economic importance and several species have been
widely studied. Currently the family is divided into
seven different subfamilies [1], among which the
largest ones are Nepetoideae and Lamioideae.
Aromatic plants are mostly included within
Nepetoideae [2], while species belonging to
Lamioideae are usually non-aromatic plants, showing
either scarce or absent essential oil production [2].
The production of essential oil is associated with the
presence of highly specialized secretory structures
known as glandular trichomes. Two types of
glandular trichomes are recognized: peltate and
capitate [3,4]. Peltate hairs are considered the site
of synthesis and storage of essential oil [5], while
capitate hairs present a more complex hydrophilic
secretion, in which mucopolysaccharides prevail
[4,5].
Prasium majus L. (Lamioideae) is considered a
typical non-aromatic plant, lacking peltate hairs and
showing only capitate hairs [3,5].
This species is an evergreen perennial shrub with
white to pale lilac flowers, and leaves with a distinct
glossy green colour [6]; neither leaves nor flowers
give off any scent. The plant grows in maquis,
guarigue, among bushes or rocks, field boundaries
and beside dry-stone walls, mainly facing the sea.
This plant is widespread in the whole Mediterranean
basin and in central and southern Portugal [7]; in
Italy, in particular, it is found in southern regions and
Sardinia and the northern limit of its distribution area
is the Tuscan Archipelago [8]. Concerning the
micromorphology, only the anatomy of nutlets has
been investigated [9].
Few popular uses have been reported for this plant; it
has been employed medicinally in Greece as a
tranquilizer [10], and in Tunisia its leaves are used in
popular medicine for their soothing properties [11].
The plant is also consumed as a raw food in Tunisia
[11], in Crete it is stir-fried or used in traditional
vegetable pies [12], and in Sicily (Palermo and
Trapani provinces) its use is limited to rural
communities [13].
Some non-volatile products have been isolated
and identified in the plant [10,14,15]. Recently the
2080 Natural Product Communications Vol. 3 (12) 2008
Table 1: Distribution of the different types of glandular trichomes present
on vegetative and reproductive organs of P. majus. Results: (-) absent; (±)
scarce; (+) present; (++) abundant.
Stem
A
B
C
+
-
Leaf/ Bract
adax
+
-
abax
+
-
Calyx
adax
-
abax
+
+
++
Corolla
adax
-
abax
±
+
essential oil composition of plants from Greece was
reported [16].
In this current study we examined specimens of
P. majus collected from along the Tuscan coast
(Italy), during the blooming period (April-June), in
order to describe the glandular trichomes present on
leaves and flowers and to determine their type of
secretion, particularly of essential oil. The essential
oils both from leaves and inflorescences were
obtained by hydrodistillation and their compositions
were determined by GC/MS analysis.
Micromorphological analysis
The glandular trichomes included peltate (type A)
and capitate types (types B and C) (Figure 1). Stem
and leaves bear only few (Table 1) short capitate
trichomes (type B), widely diffused and described in
the whole Labiatae family [4,5] (Figure 1). They
consist of one basal epidermal cell, one stalk cell and
a secretory head (25-30 μm in size) of four cells, with
a small subcuticular space in which the secretion is
temporarily stored.
Histochemical staining (Table 2) indicated secretion
of polysaccharides (Figure 2) and a small amount of
essential oil.
The secreting cells ultrastructure shows numerous
Golgi bodies and an abundant rough endoplasmic
reticulum (RER), involved in polysaccharidic
secretion [17], and few electron dense plastids
responsible for essential oil production [17]
(Figure 3).
On the inflorescences, especially on the abaxial
surfaces of the calyx (Figure 1) and corolla, besides
the described type B trichomes, other types of
glandular hairs are observed: several type A and
numerous type C (Table 1). Moreover, short
uniseriate non-glandular hairs (type D) are present
(Figure 1).
Type A trichomes, unlike the typical peltate hairs,
present an elongated basal epidermal cell which
Giuliani et al.
Table 2: Histochemical tests on the different types of glandular
trichomes. Results: (-) negative; (±) scarce; (+) intense; (++) very intense.
Staining
Nile Red
Fluoral Yellow
NADI reagent
Ruthenium Red
Alcian Blue
FeCl3
Target compounds
Neutral lipids
Total lipids
Terpenes
Polysaccharides
Polysaccharides
Polyphenols
A
+
+
++
-
B
±
±
±
+
+
-
C
++
++
+
+
±
+
forms a well developed stalk, so that these trichomes
are raised on the epidermal surface (Figures 1 and 4).
This uncommon feature was already observed in
Salvia officinalis [18] and in several species of
Stachys [19]. The neck cell, the broad glandular head
(40-50 μm in size) of eight secreting cells and the
large subcuticular space present the typical
morphology quoted in the literature [4,5,17]. The
secretion stored in the subcuticular space is
composed of essential oil (Table 2), since it shows a
strong positive reaction only to the Nadi reagent
(Figure 4). The most striking ultrastructural features
observed in the cytoplasm of the secreting cells are
plastids with large starch granules (Figure 5),
associated with smooth endoplasmic reticulum
(SER). These cellular compartments are typically
involved in essential oil production and transfer [17].
Type C long capitate trichomes (Table 1; Figure 1),
observed also in several Stachys species [19], consist
of one basal epidermal cell, a stalk of two-three cells
and a multicellular head (40-60 μm in size) of sixeight cells. Each glandular cell is endowed with a
small subcuticular space; the secretion is extruded to
the outside from the subcuticular space and also
from the whole external wall and flows along the
stalk to the epidermis [19]. The secretion shows a
complex composition (Table 2), since it contains
polysaccharides, essential oil and polyphenols
(Figures 6 and 7).
In young trichomes, the glandular cells ultrastructure
shows mitocondria, Golgi bodies, RER vesicles and
multi-shaped plastids with starch granules (Figure 8).
In mature trichomes, Golgi bodies and RER elements
occur occasionally, while plastids, SER and lipidic
droplets (Figure 9) can be observed. Therefore, these
hairs present different types of secretion according to
the different ages of the trichomes.
Essential oils analysis
Very small amounts of essential oils were obtained
by hydrodistillation of the leaves and inflorescences;
their compositions are reported in Table 3.
Micromorphology and essential oil analysis of Prasium majus
Natural Product Communications Vol. 3 (12) 2008 2081
Figure 1: Trichomes on the abaxial side of the calyx: A. peltate, B. short capitate, C. long capitate and D. simple uniseriate non-glandular trichomes. Bar = 100
μm. Figure 2: Histochemistry of type B trichome: Alcian Blue. Bar = 25 μm. Figure 3: Secreting cell cytoplasm of type B trichome. Bar = 1 μm. Figure 4:
Histochemistry of type A trichomes: Nadi reagent. Bar = 25 μm. Figure 5: Secreting cell cytoplasm of type A trichome. Bar = 1 μm. Figures 6, 7:
Histochemistry of type C trichome: Fluoral Yellow 088 (6) and FeCl3 (7). Bars = 25 μm. Figures 8, 9: Secreting cell cytoplasms of a young (8) and mature (9)
type C trichome. Bars = 1 μm. (Cw) Cell wall; (g) Golgi bodies; (ld) lipidic droplets; (m) mitocondria; (n) nucleus; (p) plastid; (rer) RER; (s) starch; (ser) SER.
The essential oil compositions of flowers and leaves
differ. The volatile compounds of leaves are
characterised by phytol (35.5%), (E)-caryophyllene
(19.1%), and hexadecane (7.0%). Oxygenated
diterpene hydrocarbons (35.5%), sesquiterpene
hydrocarbons (25.0%), and hydrocarbons (22.1%)
are the principal fractions. The essential oil of
flowers is characterised by six main compounds,
(E)-caryophyllene (43.6%), 6,10,14-trimethyl-2pentadecanone (7.5%), 9,12-octadecadienal (5.9%),
germacrene D (5.7%), pentadecane (5.7%), and
tetradecane (5.6%). Sesquiterpene hydrocarbons
(56.9%) and hydrocarbons (19.7%) are the principal
fractions.
In conclusion, P. majus, considered a typical nonaromatic plant [3,5], bears glandular trichomes
which produce small quantities of essential oils, both
in leaves and flowers. The plant presents few
glandular trichomes on its leaves (type B), but
numerous on its inflorescences (types A, B and C).
Histochemical observations indicate that the three
2082 Natural Product Communications Vol. 3 (12) 2008
Table 3: Essential oil compositions of leaves and inflorescences of
P. majus. RI = Retention Index.
Compounds
α-Pinene
β-Pinene
ο-Cymene
γ-Terpinene
Thymol
α-Terpinyl acetate
α-Ylangene
Tetradecane
(E)-Caryophyllene
Germacrene D
Pentadecane
δ-Amorphene
Caryophyllene oxide
Hexadecane
9,12-Octadecadienal
Heptadecane
6,10,14-Trimethyl2-pentadecanone
Octadecane
Hexadecanol
Nonadecane
Methyl hexadecanoate
Hexadecyl acetate
Octadecanol
Heneicosane
Phytol
Docosane
Tricosane
Tetracosane
Pentacosane
Total
RI
Leaves %
Flowers %
938
981
1026
1060
1293
1353
1377
1400
1418
1483
1500
1512
1580
1600
1645
1700
0.1
0.1
0.3
0.2
2.7
0.5
1.7
2.7
19.1
2.5
1.4
1.7
7.0
0.9
3.7
5.6
43.6
5.7
5.7
3.9
1.4
4.0
5.9
2.3
1791
1800
1875
1900
1920
2001
2074
2100
2112
2200
2300
2400
2500
2.6
0.2
0.1
1.5
5.1
1.9
35.5
0.5
0.5
1.0
3.5
7.5
1.0
2.1
1.1
3.5
-
93.3%
97.0%
types of trichomes produce different kinds of
substances (polysaccharides, phenols and essential
oil). The organelles observed in the cytoplasm of the
secreting cells are consistent with these types of
secretion.
Essential oil of leaves is produced by type B
trichomes, the only type present, considered a typical
mucopolysaccharides producer [4]. In this species
they are responsible also for the production of the
terpenoid fraction, as already observed in Stachys
recta [20].
The inflorescences, besides type B hairs, bear other
types of trichomes, already described for the Labiatae
[18,19]. Type A trichomes have a typical essential oil
secretion, while type C trichomes produce a complex
secretion, which contains both hydrophilic and
lipophilic substances.
Essential oil was obtained and analyzed also in
flowering plants from Greece [16], but the yield
of essential oil is not reported. The composition of
Giuliani et al.
our samples differs from those of Greece: only
α-pinene, γ-terpinene, thymol, (E)-caryophyllene,
caryophyllene oxide, and tricosane are present in
plants from both sites. Samples A and B from Greece
are characterized, respectively, by 1-octen-3-ol
(20.7%) and dehydro-aromadendrene (31.8%).
The differences could be ascribed not only to the
different plant material examined (fresh leaves and
flowers in our work, the whole dry plant at flowering
time in Greek samples). However, the essential oil
composition is certainly affected by the different
origin of plant material: the northern part of the
distribution area for our samples and typical
Mediterranean distribution area for Greek samples.
Therefore, the different environmental conditions
could be responsible for different chemotypes. It
would also be interesting to verify if the plants of
southern and warmer regions are richer in essential
oil than those of the northern regions.
Experimental
Plant material: Specimens were collected from two
different localities in Tuscany during the blooming
period: 03.05.2005 Baratti (Livorno) and 12.05.2007
Giglio Island, Campese (Grosseto). They were
determined according to Pignatti [8].
Micromorphological analyses were performed on
fresh material (stems, leaves, bracts, calyces and
corollas) using scanning electron microscopy
(SEM), light microscopy (LM) and transmission
electron microscopy (TEM).
SEM observations: Small pieces of plant material
were fixed in 2.5% glutaraldehyde in 0.1 M
phosphate buffer at pH 6.8, dehydrated in ethanol in
ascending grades up to absolute and then dried using
a critical point dryer apparatus. The samples, coated
with gold, were observed with a Philips XL-20 SEM.
LM observations: Fresh material was frozen,
sectioned and stained using different techniques in
order to evidence the different components of the
secretion. The stains employed were: Fluoral Yellow088 for total lipids [21], Nile Red for neutral lipids
[22], Nadi reagent for terpenes [23], Ruthenium Red
[24] and Alcian Blue [25] for acid polysaccharides,
and Ferric Trichloride for polyphenols [26].
Observations were made with a Leitz DM-RB Fluo
optic microscope.
Micromorphology and essential oil analysis of Prasium majus
Natural Product Communications Vol. 3 (12) 2008 2083
TEM observations: Small pieces of plant material
were fixed in 2.5% glutaraldehyde in 0.1 M
phosphate buffer at pH 6.8 and post fixed in 2%
OsO4, dehydrated in ethanol in ascending grades up
to absolute and embedded in Spurr’s resin. Ultra thin
sections were stained with uranile acetate and lead
citrate. Samples were examined with a Philips EM300-TEM.
capillary columns (30 m x 0.25 mm, 0.25 μm film
thickness), working with the following temperature
programme: 10 min at 60°C, and subsequently up to
220°C at 5°C/min; injector and detector temperatures,
250°C; carrier gas, helium (1 mL/min); split ratio,
1:20. GC/MS analyses were carried out using an
Agilent 5975 GC/MS system operating in the EI
mode at 70 eV, using the same columns. The
identification of the components was made for both
the columns, by comparison of their retention time
with respect to n-paraffin (C6-C22) internal
standards. The mass spectra and Kovats Indices (KI)
were compared with those of commercial (NIST 98
and WILEY) and home-made library mass spectra
built up from pure compounds and MS literature data.
Isolation and identification of the essential oils:
Fresh leaves and inflorescences of the specimen
collected at Giglio Island were separately steam
distilled for 3 h, in a Clevenger-type apparatus. The
essential oil obtained was dried over anhydrous
sodium sulfate and stored in sealed vials under
refrigeration prior to analysis.
Gas
chromatography
(GC)
and
gas
chromatography-mass spectrometry (GC/MS): The
GC analyses were carried out using an Agilent 6890N
instrument equipped with HP-WAX and HP-5
Area percentages were obtained electronically from
the GC-FID response without the use of either an
internal standard or correction factors.
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NPC
Natural Product Communications
Olive-oil Phenolics and Health: Potential Biological Properties
2008
Vol. 3
No. 12
2085 - 2088
Francesco Visiolia*, Francesca Ierib, Nadia Mulinaccib, Franco F. Vincierib and
Annalisa Romanib
a
Laboratory of “Micronutrients and Cardiovascular Disease”, UMR7079,
UPMC University of Paris 06, Paris, France
b
Department of Pharmaceutical Science, University of Florence, 50019 Sesto F.no, Florence, Italy
francesco.visioli@upmc.fr
Received: September 15th, 2008; Accepted: October 29th, 2008
Extra virgin olive oil, the primary source of oil in the Mediterranean diet, differs significantly in composition from dietary
lipids that are consumed by other populations. The several minor constituents of virgin olive oil include vitamins such as alphaand gamma-tocopherols (around 200 ppm) and beta-carotene, phytosterols, pigments, terpenic acids, flavonoids, squalene, and
a number of phenolic compounds, such as hydroxytyrosol, usually grouped under the rubric “polyphenols”. The antioxidant
and enzyme-modulating activities of extra virgin olive oil phenolics, such as their ability to inhibit NF-kB activation in human
monocyte/macrophages has been demonstrated in vitro. There is also solid evidence that extra virgin olive oil phenolic
compounds are absorbed and their human metabolism has been elucidated. Several activities that might be associated with
cardiovascular protection, such as inhibition of platelet aggregation and reduction of plasma rHcy have been demonstrated in
vivo. The biologically relevant properties of olive phenolics are described, although further investigations in controlled clinical
trials are needed to support the hypothesis that virgin olive oil consumption may contribute to lower cardiovascular mortality.
Keywords: extravirgin olive oil, phenolic compounds, Mediterranean diet, hydroxytyrosol.
Numerous epidemiological studies have shown that
the incidence of coronary heart disease (CHD) and
certain cancers, for example breast and colon cancers,
is lowest in the Mediterranean basin [1]. It has been
suggested that this is largely due to the protective
dietary habits of this area [1,2]. The traditional
Mediterranean diet, rich in fruit, vegetables, fish,
and whole grain, is thought to promote good
health and longevity. Olive oil, the primary oil
source of this diet, differs significantly in
composition from dietary lipids that are consumed by
other populations. The formulation of an
antioxidant/atherosclerosis hypothesis stimulated
experimental and epidemiological studies on the
possible role of antioxidants, including olive oil
phenolics, in the protection from CHD observed in
the Mediterranean area. Included among the minor
constituents of virgin olive oil are vitamins such as αand γ-tocopherols (around 200 ppm) and β-carotene,
phytosterols, pigments, terpenic acids, flavonoids,
squalene, and a number of phenolic compounds,
usually grouped under the rubric “polyphenols” [3].
Epidemiological studies: From a nutritional point of
view, the choice of a phenol-rich olive oil contributes
to the dietary intake of biologically-active
compounds in quantities that have been correlated
with a reduced risk of developing CHD [4]. Indeed,
the use of extra-virgin olive oil as the principle
source of dietary oil instead of animal fat, in addition
to providing a considerable amount of oleic acid,
provides an intake of bioactive compounds with
potential healthful effects, as described above. It also
appears that the intake and interaction of several
"micronutrients" provided by a healthy diet, such as
that in use in the Mediterranean area during the mid1940s, is likely to be the link that affords protection
from such pathologies [5]. In turn, the answer to the
current debate on the efficacy of antioxidant
supplements is likely to be found in the adoption of a
Mediterranean-style diet, in which the abundance of
bioactive, functional compounds provided by fruits,
vegetables, wine, and olive oil grants a higher
protection toward reactive oxygen species (ROS)induced diseases.
2086 Natural Product Communications Vol. 3 (12) 2008
Visioli et al.
In vitro studies: The lower incidence of CHD
observed in the Mediterranean area [1] lead to the
hypothesis that olive oil phenolics exert a protective
effect with respect to chemically-induced oxidation
of human LDL, which is one of the initial steps in the
onset of atherosclerosis [6].
μM) and are 40-fold weaker than those of the widelyemployed
reducing
agent
ascorbate
[13].
Interestingly, two human intervention studies [14,15]
confirmed these data in vivo, indicating that extra
virgin olive oil might decrease DNA damage, hence
lessening cancer risk.
Results obtained on human LDL demonstrate that
catechol-like compounds present in virgin olive oil
inhibit the formation of lipid oxidation products in a
dose dependent manner and are effective at a
concentration lower than that of pure tyrosol, a
phenolic component of the oil, and of probucol, used
as reference compounds. This effect is probably due
to the synergistic action of hydroxytyrosol,
oleuropein aglycones, and some flavonoids, such as
quercetin, luteolin, and apigenin present in the virgin
olive oil extract in minute amounts. In addition,
changes in electrophoretic mobility of apo B are also
prevented by the phenols.
Oleuropein increases the functional activity of
immune-competent
cells
(macrophages),
as
demonstrated by a significant increase (58.7 ± 4.6%)
in the lipopolysaccharide (LPS)-induced production
of nitric oxide, a bactericidal and cytostatic agent
[10a]. This increase is consequent to a direct tonic
effect of oleuropein on the inducible form of the
enzyme nitric oxide synthase (iNOS), as
demonstrated by Western blot analysis of cell
homogenates and by coincubation of LPS-challenged
cells with the iNOS inhibitor L-nitromethylarginine
methylester [10b].
Pure hydroxytyrosol (HT) and oleuropein (OE) both
potently and dose-dependently inhibit copper sulfateinduced oxidation of LDL at concentrations of 10-6 to
10-4 M [7,8]. The free radical scavenging activities of
hydroxytyrosol and oleuropein have been further
confirmed [8,9] by the use of metal-independent
oxidative systems and stable free radicals, such as
DPPH [10a], in a series of experiments that
demonstrated both a strong metal-chelation and a
free-radical scavenging action. As far as the
mechanism of action of olive oil phenolics is
concerned, it is well-known that the antioxidant
properties of o-diphenols are related to hydrogendonation, which is their ability to improve radical
stability by forming an intramolecular hydrogen bond
between the free hydrogens of their hydroxyl group
and their phenoxyl radicals [10b]. Although specific
investigations of olive oil phenols are yet to be
carried out, studies performed on the structureactivity relationship of flavonoids indicated that the
degree of antioxidant activity is strictly related to the
number of hydroxyl substitutions [11].
The mutagenic properties of oxidatively-damaged
DNA suggest that antioxidants might have protective
activity toward tumor formation. Low concentrations
of hydroxytyrosol (50 μM) are able to scavenge
peroxynitrite and therefore prevent ONOO-dependent DNA damage and tyrosine nitration
[12,13]; also, in a model of copper-induced DNA
damage, the prooxidant activities of hydroxytyrosol
(due to its copper-reducing properties) become
evident at non-physiological concentrations (>500
A
correlation
between
inflammation
and
cardiovascular diseases has long been established;
monocyte/macrophages and NF-κB play a pivotal
role. The effects of an extra-virgin olive oil extract,
particularly rich in phenolic compounds, were
investigated on NF-κB translocation in monocytes
and monocyte-derived macrophages (MDM) isolated
from healthy volunteers. In a concentrationdependent manner, the extra-virgin olive oil extract
inhibited p50 and p65 NF-kB translocation in both
unstimulated and phorbol-myristate acetate (PMA)challenged cells, being particularly effective on the
p50 subunit. Interestingly, this effect occurred at
concentrations found in human plasma after
nutritional ingestion of virgin olive oil and was
quantitatively similar to that exerted by ciglitazone, a
PPAR-γ ligand. However, the extra-virgin olive oil
extract did not affect PPAR-γ expression in
monocytes and MDM. These data provide further
evidence of the beneficial effects of extra-virgin olive
oil by indicating its ability to inhibit NF-κB
activation in human monocyte/macrophages [16].
In vivo studies: Experimental evidence that
phenolic compounds of different origin are absorbed
from the diet is accumulating. Animal studies in rats
and rabbits demonstrated that LDL isolated from
animals fed virgin olive oil exhibit a higher resistance
to oxidation when compared with animals given a
triglyceride preparation with an equivalent amount of
oleic acid, i.e. triolein [17], or «plain» olive oil [18].
We demonstrated that olive oil phenolics are dosedependently absorbed in humans and that they are
excreted in the urine, mainly as glucuronide
Biological significance of virgin olive oil
Natural Product Communications Vol. 3 (12) 2008 2087
conjugates; it is noteworthy that increasing amounts
of phenolics administered with olive oil stimulated
the rate of conjugation with glucuronide [18]. These
data add to the growing experimental evidence that
indicates absorption and urinary disposition of
flavonoids in humans [19].
The effect of EVOO on platelet aggregation and
plasma concentrations of homocysteine (Hcy) redox
forms, in relation to the phenolic compounds’
concentration, was also investigated in rats. Three
olive oil samples with similar fatty acid, but different
phenolic compound concentrations were used:
refined olive oil (RF) with traces of phenolic
compounds (control oil), native extra virgin olive oil
with low phenolic compounds concentration (LC),
and extra virgin olive oil with high phenolic
compounds concentration (HC) enriching LC with its
own phenolic compounds. Oil samples were
administered to rats by gavage (1.25 mL/kg body
weight) using two experimental designs: acute (24 h
food deprivation and killed 1 h after oil
administration) and subacute (12 d treatment, a daily
dose of oil for 12 d, and killed after 24 h of food
deprivation).
It is noteworthy that HT exists in the brain as an
endogenous catabolite of catecholic neurotransmitters, such as dopamine and norepinephrine [20],
but its presence in urine has never, until recently,
been described. On the other hand, the formation of
homovanillic alcohol (HVAlc), the O-methylated
derivative of HT, was reported by Manna et al [21] in
human Caco-2 cell incubated with HT. We also
reported the urinary excretion of HVAlc, in large
excess over its basal excretion (57 ± 3 µg excreted in
24 hours, means ± SD, n= 6). We also described the
substrate-induced enhancement of HVA formation,
also a product of catecholamines metabolism, in
addition to its basal urinary excretion (1660 ± 350 µg
excreted in 24 hours, means ± SD, n= 6). Indeed, the
results reported suggest that HT increases the basal
excretion of HVA, even at the low doses of phenols
administered. Future investigations will adopt
commercially available virgin olive oils, thus
allowing the further elucidation of the in vivo kinetics
of olive oil phenolics in habitual consumption
quantities.
In terms of biological activities, Covas et al. recently
reviewed approximately 15 human intervention
studies, the vast majority of which indicate that extra
virgin olive oil (rich in phenols) is superior to seed
oils and olive oil devoid of phenols in modulating
selected surrogate markers of cardiovascular disease
[22]. One example is an investigation of the effects of
olive oil phenols on post prandial events. Bogani et
al. evaluated the effects of moderate, real life doses
of two olive oils, differing only in their phenolic
content, on some in vivo indexes of oxidative stress
(plasma antioxidant capacity and urinary hydrogen
peroxide levels) in a post prandial setting. Moreover,
the authors assessed whether phenolic compounds
influence a few arachidonic acid metabolites
involved in the atherosclerotic processes, such as
leukotriene B4 (LTB4) and thromboxane B2 (TXB2).
Six subjects in each group received the three oils [30
mL/day of olive oil (OO), corn oil (CO), or extra
virgin olive oil (EVOO), distributed among meals] in
a Latin square design. The results demonstrate that
EVOO is capable of reducing the post prandial events
that associate with inflammation and oxidative stress
[23].
Platelet aggregation was induced by ADP (ex vivo
tests) and a reduction in platelet reactivity occurred in
cells from rats given LC in the subacute study and in
cells from rats administered HC in both studies, as
indicated by an increase in the agonist half maximal
effective concentration. HC inhibited platelet
aggregation induced by low ADP doses (reversible
aggregation) in cells of rats in both the acute and
subacute studies, whereas LC had this effect only in
the subacute experiment. Moreover, in rats
administered HC in both experiments, the plasma
concentration of free reduced Hcy (rHcy) was lower
and Hcy bound to protein by disulfide bonds (bHcy)
was greater than in RF-treated rats. bHcy was also
greater in rats given LC than in RF-treated rats in the
subacute experiment. Plasma free-oxidized Hcy was
greater in rats given LC and HC than in those
administered RF only in the subacute experiment.
These results show that phenolic compounds in
EVOO inhibit platelet aggregation and reduce the
plasma rHcy concentration, effects that may be
associated with cardiovascular protection [24].
In conclusion, the biologically relevant properties of
olive phenolics described in this article, although still
to be further investigated in other controlled clinical
trials, provide evidence to support the hypothesis that
virgin olive oil consumption may contribute to lower
CHD mortality.
Acknowledgement – This paper celebrates Professor
Vincieri’s birthday. We wish to acknowledge his
important contributions to the field.
2088 Natural Product Communications Vol. 3 (12) 2008
Visioli et al.
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NPC
Natural Product Communications
Traceability of Secondary Metabolites in Eucalyptus
and Fagus Wood derived Pulp and Fiber
2008
Vol. 3
No. 12
2089 - 2093
Aline Lamien-Medaa, Karin Zitterl-Eglseera, Heidrun Fuchsb and Chlodwig Franza,*
a
Veterinärmedizinische Universität Wien, Institut für Angewandte Botanik und Pharmakognosie,
Veterinärplatz 1, A-1210 Wien, Austia
b
LENZING AG, Innovation & Business Development Fiber Science & Entwicklung – TBS,
A-4860 Lenzing - Austria
Chlodwig.Franz@vu-wien.ac.at
Received: July 8th, 2008; Accepted: October 20th, 2008
Industrial pulp and fiber of Eucalyptus and Fagus were investigated for possible identification of secondary metabolites, using
chloroform, ethanol and methanol/HCl extracts. The total phenolics test was positive with all the samples and some phenolic
compounds like vanillin, vanillic acid and syringic acid were identified by HPLC analysis in the ethanol and methanol-HCl
extracts. The extracts had also DPPH radical scavenging activity. Fatty acids like palmitic acid, linoleic acid, oleic acid and
stearic acid, cholestane and its derivatives were found in the different extracts by GC/MS analysis. Squalene was also identified
and quantified by GC/FID in the dichloromethane extracts. The results showed that the industrial pulp and fibers still contain
some secondary plant products comparable to those of the original woods, which confirm the ‘botanical origin’ of the fibers
and enables the natural fibers to possess some biological properties, like DPPH antioxidant activity.
Keywords: Eucalyptus, Fagus, natural fiber, pulp, phenolic content, antioxidant activity.
Natural fibers are those originating directly from
nature, usually from plants, animals, and minerals.
The common natural fibers are cotton, wool, silk,
jute, flax/linen and rock wool. Plant fibers, including
vegetable and wood fibers, are generally used in the
manufacture of paper and cloth. Several methods are
used to convert wood into pulp, including the ground
wood, sulfite, and sulfate processes. Other chemical
treatments are used to transform the pulp into fiber
used for yarn production.
Eucalyptus and Fagus woods are two important raw
materials used in the textile industry, for example for
TENCEL® and Modal® fiber production,
respectively. In the pulp and paper industry,
extractives from both softwoods and hardwoods
cause production and environmental problems. A
number of studies have been undertaken on lipophilic
wood extractives to determine their composition and
to solve pitch problems. Compounds like phenolic
acids (ferulic, ellagic, gallic, syringic, vanillic,
benzoic and cinnamic acids), aldehydes, tannins, fatty
acids, terpenes, sterols, sterol esters, and glycerides
have been detected in different types of Eucalyptus
and Fagus woods [1-5].
In the present study, phytochemical investigations
have been performed on Eucalyptus and Fagus pulp
and fiber samples, to see if some secondary plant
products are still detectable after the above
mentioned industrial processes. HPLC and GC/MS
methods have been used for the different extract
analyses and the antioxidant activity of the extracts
has been tested to evaluate the functionality of the
compounds.
The total phenolic content of both ethanol and
methanol/HCl extracts measured by the FolinCiocalteu assay and expressed in µg caffeic acid
equivalents (CAE)/g of dried fiber or pulp, are given
in Figure 1. The values varied from 6.5 µg/g with
the ethanol extract of Eucalyptus fiber to 16.2 µg/g
for the ethanol extract of Eucalyptus pulp. The
phenolic content of the ethanol extract of Fagus fiber
(9.6 µg/g) was higher than that of Eucalyptus fibers
2090 Natural Product Communications Vol. 3 (12) 2008
Lamien-Meda et al.
remaining in the fiber samples, or could also be
derived from the partial depolymerization of some
residual lignin. Previous studies on Fagus and
Eucalyptus woods have shown, however, that
phenolic acids like ferulic acid, vanillin, vanillic acid,
syringic
acid,
cinnamic
acid
derivative,
syringaldehyde and resorcilic acid could be identified
in Eucalyptus urophylla [3] and E. globulus [1,4,6-8].
Catechin, coniferyl alcohol and sinapyl alcohol have
been isolated from the methanol-water extract of
Fagus wood [9].
Figure 1: Total phenolic content of ethanol and methanol/HCl extracts of
the pulp and fiber samples.
GC/MS analysis of the ethanol extract allowed the
identification of simple phenolic compounds (vanillic
acid, syringic acid), fatty acids (n-tetradecanoic acid,
n-pentadecanoic acid, palmitic acid, heptadecanoic
acid, linoleic acid, oleic acid, stearic acid), cholestane
and its derivatives (cholesterol, cholesta-3,5-dien-7one), and other compounds (xylose, octadecane)
(Table 2). These data are in accordance with the
literature on the direct wood study of Eucalyptus
[1,4,6-8]. The fatty acids identified in Fagus pulp and
fiber were also found in Fagus wood [2]. The
phenolic compound 2,4-ditert-butylphenol was
identified in the ethanol extract of Eucalyptus fiber
only (without silylation reaction). This compound
was also identified in E. urophylla wood [3] and is
well known as an antioxidant compound. 2,4-Ditertbutylphenol has recently been described as an
essential oil component of some plants [10-11]. It
seems to be useful for the distinction between
Eucalyptus and other wood sources, but it is not yet
clear if this substance is genuine or an artifact of
volatile compounds.
(6.5 µg/g). The inverse order was observed with the
methanol/HCl extracts: 13.9 and 11.6 µg/g for the
Eucalyptus and Fagus fibers respectively. The
hydrolyzed extracts (methanol/HCl) had, in general,
higher phenolic contents comparatively than the
ethanol extracts.
Cruz et al. [4] had quantified 1.04 g/100 g (gallic acid
equivalent) of total phenolics in Eucalyptus globulus
wood, using hydrolyzed extracts and the Folin
Ciocalteu assay. This value is thousand times higher
than that of Eucalyptus pulp. The high decrease of
phenolic content from the wood to the pulp is easy to
explain by the industrial process of pulp production.
The HPLC/DAD characterization of phenolic
compounds in the extracts allowed the identification
of vanillin, vanillic acid and syringic acid in both
pulp (from wood digestion with magnesium bisulfite
acid) and fiber (from the pulp cleaning) extracts
(Table 1). In the chromatograms of the ethanol
extracts of Eucalyptus pulp and fiber, vanillin,
vanillic acid and syringic acid were identified and
quantified, but only vanillic and syringic acids were
identified in the Fagus fiber. Vanillic acid and
syringic acid could be identified in each sample. All
the identified phenolic compounds were present in
small amounts only (0.3 – 56 µg/10g of pulp or
fiber).
Squalene was identified and quantified by GC/FID in
the dichloromethane extracts of the pulp and fiber of
both Eucalyptus and Fagus. The squalene content
varied from 2 – 9 µg/10g, with the fiber samples
having higher squalene contents than the pulp
samples (Figure 2).
Squalene was also identified and quantified in E.
globulus wood [6,7] at variable concentrations. The
The identified and quantified phenolics could be
residual compounds from the original wood
Table 1: HPLC quantitative results of the pulp and fiber samples.
Samples
Eucalyptus pulps
Fagus pulps
Vanillin (µg/10g)
Vanillic acid (µg/10g)
ETOH
MeOH/HCl
ETOH
MeOH/HCl
Syringic acid (µg/10g)
ETOH
MeOH/HCl
0 – 0.9
0 – 0.6
0.0 – 4.0
0.5 – 1.5
1.1 – 56.0
0.3 – 05.2
0.0 – 2.2
0.4 – 3.8
0.4 – 1.0
0.2 – 1.2
01.9 – 14.2
10.6 – 41.3
Eucalyptus fibers
0 – 0.1
0.0 – 0.2
0
1.3 – 1.7
0.0 – 1.6
43.3 – 52.7
Fagus fibers
0
0.0
0
2.3
0.0
25.0
Phytochemical analysis of wood derived pulp and fiber
Table 2: Compounds identified by GC-MS in the ethanol extracts of the
Eucalyptus pulp (EP), Fagus pulp (BP), Eucalyptus fiber (EF) and Fagus
fiber (BF).
RT(mn)
4.675
5.214
7.335
8.254
10.006
11.443
12.329
14.635
16.592
16.799
17.604
17.680
18.063
26.727
31.125
31.243
31.333
31.841
Compounds
Butanoic acid
Octadecatrienoic acid
D-Xylose
Vanillic acid
Tetradecanoic acid
Syringic acid
n-Pentadecanoic acid
Palmitic acid
Heptadecanoic acid
Octadecane
Linoleic acid
Oleic acid
Stearic acid
Stearic acid derivative
Cholesterol
Cholestane
Cholestane, 2,3-epoxy
Cholesta-3,5-dien-7-one
EP
+
+
+
+
+
+
+
+
+
+
+
+
+
+
BP
+
+
+
+
+
+
+
+
+
+
+
+
+
EF
+
+
+
+
+
+
+
+
+
+
+
+
+
BF
+
+
+
+
+
+
+
+
+
+
+
+
+
Figure 2: Squalene quantity (GC-FID) in dichloromethane extracts of the
pulp and fibers of Eucalyptus and Fagus.
Natural Product Communications Vol. 3 (12) 2008 2091
protective activity of squalene against ultraviolet
radiation [16] and radiation-induced injury in a
mouse model has been demonstrated [17]. The
presence of squalene in the final industrial textile
should have some protective effect on skin.
The presence of active compounds in the pulp and
fiber samples was confirmed by testing the
antioxidant activity of the ethanol and methanol
extracts using DPPH radical scavenging activity. All
the extracts had DPPH antioxidant activity and the
values ranged from 4.3 – 17.1 µg Trolox
equivalents/g sample (Figure 3).
A correlation of 0.5 was observed between the total
phenolic content and the DPPH antioxidant activity.
This positive correlation indicates that the phenolic
compounds contributed partly to the antioxidant
activity. Indeed, the identified phenolic compounds
in the samples (vanillic acid, vanillin, syringic acid)
are known to have antioxidant activity. Syringic acid
is a more potent DPPH radical-scavenger than BHA
and BHT and comparable with ferulic acid, vanillic
acid and coumaric acid [18], and presents also antiinflammatory activity [19]. The fatty acids (palmitic,
linoleic, oleic, and stearic acids) are known to have
antioxidant activity [20] and could contribute to the
antioxidant results of the extracts.
The present study showed that pulps and fibers
originating from wood materials still contain some
(functional) secondary plant compounds after the
different industrial processing steps. The industrial
Eucalyptus and Fagus fibers and pulps contain, for
example, small amounts of vanillin, vanillic acid,
syringic acid, fatty acids and squalene, which confirm
the ‘botanical origin’ of the fibers and enables the
natural fibers to possess some biological properties,
like DPPH antioxidant activity.
Experimental
Figure 3: DPPH antioxidant activity of ethanol and methanol/HCl
extracts of the pulp and fiber samples.
squalene contents (acetone extract followed by a
solid phase extraction fractionation) were 1.6 mg/kg
[7] and 38.5 mg/kg [6]. Considering the data of [6],
the Eucalyptus fiber squalene content was <2 fold
lower. Squalene is an isoprenoid molecule with
cardioprotective [12], antilipidemic, antioxidant and
membrane-stabilizing properties
[13-15]. The
Fiber samples and sample extractions: Thirteen
wood material samples were provided by Lenzing
fibers industry, Lenzing/Austria. Lenzing textile
general process consists of the transformation of
wood to pulp, fiber, yarn and fabric, respectively.
The pulp is derived from wood chemical digestion to
remove lignin and hemicelluloses, and the fiber
derived from the pulp cleaning. The samples
analyzed were bleached Eucalyptus pulp (6),
produced in craft and sulfite pulping processes, this
being the basic material for the production of
2092 Natural Product Communications Vol. 3 (12) 2008
TENCEL®, Fagus pulp (4) from Lenzing Mg-sulfite
pulping process, this being the basis of Lenzing
Modal®, and further commercial Eucalyptus (2) and
Fagus fiber (1) samples. The pulp samples were cut
into small pieces using a paper cutter, and normal
scissors were used to cut the fiber samples.
Three different extracts were prepared from each
sample: cold-ultrasonic dichloromethane, reflux with
ethanol at 80ºC for 6 h, and reflux with methanol/HCl
2N (1:1) extracts. For each type of extraction, 10 g of
sample was extracted with 100-170 mL of solvent.
The methanol/HCl extract was neutralized with 9-10
g CaCO3 and partitioned with ethyl acetate (3 x 30
mL), the ethyl acetate part being used. All the
extracts were evaporated to dryness under pressure at
40ºC, and dissolved in 1.5 mL methanol for analysis,
except those with dichloromethane, which were
dissolved in 1.5 mL of dichloromethane for squalene
quantification. A blank extract was prepared with
each solvent for correction in the different analyses.
Spectrophotometric
determination
of
total
phenolics: The Folin Ciocalteu reagent was used to
determine the total phenolic content [21]. The
extracts (40 µL) in 2 mL H2O were mixed with 100
µL of 2N Folin Ciocalteu reagent (Merck, Darmstadt,
Germany). This mixture was allowed to stand at
room temperature for 3 min and then 200 µL of
sodium carbonate (Carl Roth & Co) solution (35 g in
100 mL H2O) was added, and the final volume was
completed to 5 mL. After 1 h of incubation in the
dark, the absorbance was measured at 725 nm against
a water blank using a spectrophotometer (HITACHI
150-20, Ltd. Tokyo, Japan). A calibration curve was
plotted using caffeic acid (Sigma-Aldrich Chemie,
Steinheim, Germany) (0-40 µg). Determination was
performed in duplicate and results were expressed as
mg of caffeic acid equivalents (CAE)/ g of dried
wool or pulp weight.
HPLC determination of phenolic compounds: The
wool and pulp extracts were analyzed by HPLC in a
Waters instrument fitted with a PDA996 detector, a
626 pump, a 717 plus autosampler and a Symmetry
C18 column (5.0 µm, 4.6 x 150 mm) with a column
oven temperature at 25°C. Gradient elution was
carried out at a flow rate of 1.5 mL/min using 1%
acetic acid: acetonitrile 85:15 (solvent A) and
methanol (solvent B). The analysis started with 10%
B and a linear gradient was used to reach 100% B
within 30 min. From 30 to 40 min B was kept
constant at 100%. The quantification of vanillin,
Lamien-Meda et al.
vanillic acid and syringic acid (Sigma-Aldrich
Chemie, Steinheim, Germany) was conducted using
an external standard (1 – 150 µg/mL) method and
detection was at 250 nm.
GC-MS and GC-FID analysis: Samples were
derivatized as reported by Fukushima and Hatfield
[22] with some modification: 1 mL of extract was
evaporated to dryness using a rotavapor at 40°C. The
dried extract was dissolved in 40 µL of
tetrahydrofurane and trimethylsilylated by adding
100 µL of BSA and 10 µL of TMCS. The stoppered
tubes with the mixtures were put in an ultrasonicator
bath for 5 minutes and kept at 60°C for 30 min. The
tubes were cooled to room temperature before GC
analysis.
The GC/MS (HP 6890 coupled to HP 5972 mass
selective detector; Hewlett Packard, Palo Alto, USA)
was equipped with a HP-5MS column (length 30 m x
0.25 mm ID, 0.25 µm film thickness; Agilent, Palo
Alto, CA, USA), and data were analyzed on a
computer equipped with ChemStation software.
Helium (average velocity 39 cm/s) was used as
carrier gas and the temperature program consisted of
an initial temperature of 160°C (held for 5 min),
ramp at 4°C/min to 200°C, ramp at 10°C/min to
240°C (held for 5 min), followed by a ramp of
15°C/min to 300°C (held for 10 min). Samples (1 µL)
were injected at 250°C and the split ratio was 50:1.
Standards (1 mg/mL) of syringic acid, vanillic acid,
palmitic acid, linoleic acid, oleic acid, stearic acid,
and squalene were used for identification.
Squalene (0-171 µg/mL) quantification was carried
out using a GC/FID (6890N Network GC system
Agilent Technologies, Palo Alto, USA) equipped
with a flame ionization detector and a DB-5 narrow
bore column (length 10 m x 0.1 mm ID, 0.17µm film
thickness; Agilent, Palo Alto, CA, USA). Helium
(average velocity 45 cm/s) was used as carrier gas
and the oven temperature was increased from 200 to
275°C at 5°C/min, and held for 10 min. The internal
standard was 4-androstene-3,17-dione (250 µg/mL).
Samples (0.2 µL) were injected at 260°C front inlet
temperature and the split ratio was 50:1.
DPPH radical scavenging activity: The radical
scavenging activity of the sample extracts for the
radical 2,2-diphenyl-1-picrylhydrazyl (DPPH, SigmaAldrich Chemie, Steinheim, Germany) was measured
as described by Velazquez et al. [23] with some
modifications. The extracts (20 µL) were diluted to
Phytochemical analysis of wood derived pulp and fiber
Natural Product Communications Vol. 3 (12) 2008 2093
100 µL with methanol and mixed with 100 µL of
DPPH solution (0.015%). After incubation at room
temperature in the dark for 30 min, the absorbance of
the reaction mixture was measured at 490 nm using a
plate reader (BIO-RAD 450, Japan). Trolox (Fluka,
Denmark) (0 – 3.756 µg) was used as standard for the
calibration curve. A blank consisting of a high
concentration of Trolox (31.3 µg) was used to correct
all readings. The results were expressed in mg TE/ g
of dried wood or pulp weight.
Acknowledgments – This work was financially
supported by Lenzing Fiber Industry.
References
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Eucalyptus globulus. Holzforschung, 56, 143-149.
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Zule J, Moze A. (2003) GC analysis of extractive compounds in Fagus wood. Journal of Separation Science, 26, 1292-1294.
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Huber S. (2004). Charakterisierung der Extraktstoffe von Eucalyptus urophylla. Doctoral thesis, Johannes Kepler Universität Linz.
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Cruz JM, Dominguez H, Parajo JC. (2005) Anti-oxidant activity of isolates from acid hydrolysates of Eucalyptus globulus wood.
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Conde E, Cadahia E, Garcia-Vallejo MC, Tomas-Barberan F. (2007) Low molecular weight polyphenols in wood and bark of
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[6]
Gutiérrez A, del Rio JC, González-Vila FJ, Martin F. (1999) Chemical composition of lipophilic extractives from Eucalyptus
globulus Labill. wood. Holzforschung, 53, 481-486.
[7]
Rencoret J, Gutiérrez A, del Rio JC. (2007) Lipid and lignin composition of woods from different Eucalyptus species.
Holzforschung, 61, 165-174.
[8]
Esteves B, Graça J, Pereira H. (2008) Extractive composition and summative chemical analysis of thermally treated Eucalyptus
wood. Holzforschung, 62, 344-351.
[9]
Koch G, Puls J, Bauch J. (2003) Topochemical characterization of phenolics extractives in discoloured Fagus wood (Fagus
sylvatica L.). Holzforschung, 57, 339-345.
[10]
Mei W-L, Zeng Y-B, Liu J, Dai H-F. (2007) GC-MS analysis of volatile constituents from five different kinds of Chinese
eaglewood. Zhong Yao Cai, 30, 551-555.
[11]
Rana VS, Blazquez MA. (2007) Chemical constituents of Gynura cusimbua aerial parts. Journal of Essential Oil Research, 19,
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[12]
Farvin KHS, Anandan R, Kumar SHS, Shiny KS, Mathew S, Sankar TV, Nair PGV. (2006) Cardioprotective effect of squalene on
lipid profile in isoprenaline-induced myocardial infarction in rats. Journal of Medicinal Food, 9, 531-536.
[13]
Psomiadou E, Tsimidou M. (1999) On the role of squalene in olive oil stability. Journal of Agricultural and Food Chemistry, 47,
4025-4032.
[14]
Ko T-F, Weng Y-M, Chiou RY-Y. (2002) Squalene content and antioxidant activity of Terminalia catappa leaves and seeds.
Journal of Agricultural and Food Chemistry, 50, 5343-5348.
[15]
Dhandapani N, Ganesan B, Anandan R, Jeyakumar R, Rajaprabhu D, Ezhilan RA. (2007) Synergistic effects of squalene and
polyunsaturated fatty acid concentrate on lipid peroxidation and antioxidant status in isoprenaline-induced myocardial infarction in
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[16]
Kohno Y, Egawa Y, Itoh S. (1995) Kinetic study of quenching reaction of singlet oxygen and scavenging reaction of free radical by
squalene in n-butanol. Biochemica et Biophysica Acta, 1256, 52-56.
[17]
Storm MH, Oh SY, Kimler BF, Norton S. (1993) Radioprotection of mice by dietary squalene. Lipids, 28, 555-559.
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Von Gadow A, Joubert E, Hansmann CF. (1997) Comparison of the antioxidant activity of aspalathin with that of other plant
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Fernandez MA, Saenz MT, Garcia MD. (1998) Antiinflammatory activity in rats and mice of phenolic acids isolated from
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Chan P, Juei-Tang C, Chiung-Wen T, Chiang-Shan N, Chuang-Ye H. (1996) The in vitro antioxidant activity of trilinolein and
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Fukushima RS, Hatfield RD. (2001) Extraction and isolation of lignin for utilization as a standard to determine lignin concentration
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NPC
Natural Product Communications
Potential Anticancer Activity Against Human Epithelial
Cancer Cells of Peumus boldus Leaf Extract
2008
Vol. 3
No. 12
2095 - 2098
Juan Garbarinoa, Nicolas Troncosob, Giuseppina Frascac, Venera Cardile c and
Alessandra Russod*
a
Department of Chemistry, University T.F. Santa Maria, Casilla 110-V, Valparaiso, Chile
b
Lo Vicuña & Cia., www.lo vicuna.cl, Santiago, Chile
c
Department of Physiological Sciences, University of Catania, V.le A. Doria 6, 95125 Catania, Italy
d
Department of Biological Chemistry, Medical Chemistry and Molecular Biology,
University of Catania, V.le A. Doria 6, 95125 Catania, Italy
alrusso@unict.it; ales0303@libero.it
Received: July 1st, 2008; Accepted: October 20th, 2008
The potential in vitro antineoplastic effect has been studied of a methanolic extract of leaves of Peumus boldus Molina
(Monimiaceae) on two human cancer epithelial cell lines, DU-145 cells (androgen-insensitive prostate cancer cells) and KB
cells (oral squamous carcinoma cells). Our findings show that this extract exhibited comparable effects on the cancer cells
examined as judged by IC50 values (5.07±0.4 μg/mL and 5.28±0.5 μg/mL in DU-145 and KB cells, respectively). In addition,
with respect to genomic DNA damage, determined by Comet assay, the results obtained show a high fragmentation of DNA,
not correlated to lactic dehydrogenase (LDH) release, a marker of membrane breakdown, in both cell lines treated with the
extract at 5-20 μg/mL concentrations. Taken together, our experimental evidence may justify further investigation of the
chemopreventive and chemotherapeutic potential of this natural drug.
Keywords: Peumus boldus Molina, DU-145 cells, KB cells, cell growth, DNA fragmentation, LDH release.
Boldo consists of the dried leaf of Peumus boldus
Molina (Monimiaceae), an evergreen shrub or a small
tree growing from central and southern Chile. Boldo,
used traditionally in South America mainly against
liver diseases, is recognized as a herbal remedy in a
number of Pharmacopeias, and is employed in the
form of infusions, tinctures and extracts [1,2]. Boldo
leaf contains different alkaloids belonging to the
large benzylisoquinoline-derived family. Boldine
[(S)-2,9-dihydroxy-1,10-dimethoxy-aporphine], the
main aporphine alkaloid in boldo leaves and barks,
seems to be particularly important as a natural
antioxidant [1,2].
Leaves of P. boldus contain also essential oils of
complex and variable composition, tannins and
flavonoids, such as flavonol glycosides, kaempferol,
quercetin and catechin. This last compound is the
flavonoid that is most abundant and with the alkaloid
boldine is the main contributor of the antioxidant
activity of Boldo leaf extracts [3,4].
Cancer is the largest single cause of death in both
men and women. Recently, resistance to anticancer
drugs has been observed. Therefore, the research and
development of more effective and less toxic drugs
by the pharmaceutical industry has become
necessary. Many substances present in plants, and in
particular the flavonoids, are known to be effective
and versatile chemopreventive and antitumor agents
in a number of experimental models of
carcinogenesis [5-8]. In view of these considerations,
Boldo leaf could possess anticancer activity and,
therefore, could be useful in the prevention or
treatment of cancer. The present study was
undertaken to investigate the in vitro cytotoxic effect
of a methanolic extract from leaves of P. boldus on
two human epithelial cancer cell lines, DU-145 cells
(androgen-insensitive prostate cancer cells) and KB
cells (oral squamous carcinoma cells).
To evaluate the effect of the extract from P. boldus
leaves on cell growth of human cancer cells, we
2096 Natural Product Communications Vol. 3 (12) 2008
Garbarino et al.
Table 1: Cell growth inhibition, assayed using MTT test, of DU-145 and
KB cells untreated and treated with methanolic extract from P. boldus
leaves at different concentrations for 72 h.
DU-145 cells
KB cells
IC50 a (μg/mL)
P. boldus extract
5.07±0.4
5.28±0.5
Doxorubicin
9.37±1.1
1.43±0.7
KB cells
Results are expressed as IC50 values (μg/mL) ± SD. The IC50 value,
relative to untreated control, represents the concentration that inhibited
cell vitality by 50%. Doxorubicin was used as a positive control. Each
value represents the mean ± SD of three experiments, performed in
quadruplicate.
a
cultured the cells in either the absence or presence of
this natural product. After treatment for 72 h, the
MTT assay, a non-radioactive assay widely used to
quantify cell viability and proliferation, was
performed. The results, summarized in Table 1, show
that our extract was active and exhibited comparable
degrees of antigrowth effect in both cancer cells
examined as judged by IC50 values, 5.07±0.4 μg/mL
and 5.28±0.5 μg/mL in DU-145 and KB cells,
respectively.
Necrosis results in a disruption of the cytoplasmic
membrane and the necrotic cells release cytoplasmic
lactic dehydrogenase (LDH) and other cytotoxic
substances into the medium. We therefore, in a next
series of experiments, examined the membrane
permeability of treated cells by the existence of LDH
in their culture medium. No increase in LDH release
was observed in these cancer cells treated with the
methanolic extract of P. boldus leaves at 5-10-20
μg/mL concentrations. Conversely, a significant
increase (p<0.001) in LDH was observed at 40
μg/mL (Figure 1).
Nuclear DNA was analyzed using single-cell gel
electrophoresis (SCGE), known as the Comet assay,
a sensitive method for the visualization of DNA
damage measured at the level of individual cells and
a versatile tool that is highly efficacious in human
bio-monitoring of natural compounds.
The Comet assay also allows us to distinguish
apoptotic from normal and necrotic cells based on the
DNA fragmentation pattern [9]. The Comet pattern
significantly differs between apoptotic and control
cultures, as well as between apoptotic and necrotic
cultures. Quantification of the Comet data, in our
experimental conditions is reported as TDNA and
TMOM in Table 2. The results clearly display DNA
damage in cells exposed to the P. boldus leaf extract
DU-145 cells
80
% LDH release
Treatments
100
60
40
*
*
20
0
Control
5
10
20
40 μg/ml
Figure 1: Lactate dehydrogenase (LDH) release, expressed as percentage
of LDH, released into the cell medium with respect to total LDH, in DU145 and KB cells treated with methanolic extract of P. boldus leaves at
different concentrations for 72 h. Each value represents the mean ± SD of
three experiments, performed in quadruplicate. *Significant vs. control
untreated cells (p<0.001).
Table 2: Comet assay of genomic DNA of DU-145 and KB cells untreated
and treated with methanolic extract of P. boldus leaves at different
concentrations for 72 h.
Treatments
TDNA
TMOM
DU-145 cells
Control
5µg/mL
10 µg/mL
20 µg/mL
40µg/mL
15.7±3.0
99±4.0*
149±6.0*
179±4.3*
30±5.5°
88±3.7
975±15*
1035±12*
1645±43*
287±10*
KB cells
Control
5µg/mL
10 µg/mL
20 µg/mL
40µg/mL
51±4.0
205±5.0*
298±6.8*
259±3.0*
84±5.9*
175±5.4
1498±16*
2919±14*
2807±13*
337±17*
Each value represents the mean ± SD of three experiments, performed in
quadruplicate. *Significant vs. control untreated cells (p<0.001).
after 72 h, with a drastic increase in both TDNA and
TMOM at 5-10-20 μg/mL concentrations.
These findings suggest that this extract induces, in
our experimental conditions, apoptotic cell death, in
accordance with the literature data, which indicate
that only comets with high values of TMOM (tail
moments) and TD (distance between head and tail of
the comet) can be related to apoptosis [10].
Alternatively, in cells exposed to the extract at 40
μg/mL concentration for 72 h, the Comet assay did
not show typical comet–like structures that occur
during apoptosis.
The extract used in the present work could contain
boldine, catechin, tannins and other flavonoids and
Potential anticancer activity of Peumus boldus
Natural Product Communications Vol. 3 (12) 2008 2097
alkaloids, according to reported data [1].
Nevertheless, it not possible from this study to
attribute the activity to any of them. However, some
suppositions can be advanced on the base of literature
data. Flavonoids, kaempferol, quercetin and catechin
have been shown to exhibit anticancer activities in
preclinical studies [6-8]. The recent studies of Hoet et
al. [11] showed that boldine was inactive in a cell
growth inhibition assay using HeLa cells (human
epithelial cancer cell line from cervical carcinoma).
for 72 h under the same conditions. Doxorubicin was
used as a positive control. Stock solution of the tested
compound was prepared in ethanol and the final
concentration of this solvent was kept constant at
0.25%. Control cultures received ethanol alone.
Taken together, our experimental data may justify
further investigation of the chemopreventive and
chemotherapeutic potential of this natural drug.
Experimental
Chemicals: All reagents were of commercial
quality and were used as received. Boldine, catechin,
3(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium
bromide
(MTT)
and
β-nicotinamide-adenine
dinucleotide (NADH) were obtained from Sigma
Aldrich Co (St. Louis, USA). All other chemicals
were purchased from Sigma Aldrich Co (St. Louis,
USA) and GIBCO BRL Life Technologies (Grand
Island, NY, USA).
Plant material and extraction: The leaves of P.
boldo were collected at Quintay (Valparaiso) in
January 2006. A voucher specimen (voucher
specimen #12-07) was deposited in the Department
of Chemistry, Universidad Santa Maria, Valparaiso,
Chile. The leaves were exhaustively extracted with
methanol and concentrated under vacuum to give a
residue [yield 165g (14.3%)].
Cell culture and treatments: DU-145 cells
(androgen-insensitive prostate cancer cells) were
grown in Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% fetal calf serum (FCS), 100
U/mL penicillin, 100 µg/mL streptomycin, 1 mM
glutamine, and 1% non essential amino-acids. KB
cells (oral squamous carcinoma cells) were
maintained in RPMI supplemented with 10% fetal
calf serum (FCS), 100 U/mL penicillin, and 100
µg/mL streptomycin. The cells were plated at a
constant density to obtain identical experimental
conditions for the different tests, thus achieving a
high accuracy of the measurements. After 24 h
incubation at 37°C under a humidified 5% carbon
dioxide atmosphere to allow cell attachment, the cells
were treated with different concentrations of
methanolic extract of P. boldus leaves, and incubated
MTT bioassay: Cellular growth was determined
using the MTT assay on 96-well microplates, as
previously described [12]. The optical density of each
well sample was measured with a microplate
spectrophotometer reader (Digital and Analog
Systems, Rome, Italy) at 550 nm.
Lactic dehydrogenase (LDH) release: Lactic
dehydrogenase
(LDH)
activity
was
spectrophotometrically measured in the culture
medium and in the cellular lysates at 340 nm by
analyzing NADH reduction during the pyruvatelactate transformation, as previously reported [12].
The percentage of LDH released was calculated as
the percentage of the total amount, considered as the
sum of the enzymatic activity present in the cellular
lysate and that in the culture medium. A Hitachi
U-2000 spectrophotometer (Hitachi, Tokyo, Japan)
was used.
DNA analysis by Comet assay: The presence of
DNA fragmentation was examined by single cell gel
electrophoresis (Comet assay), as previously reported
[12]. At the end of the electrophoretic run, the
“minigels” were neutralized in 0.4 M Tris-HCl, pH
7.5, stained with 100 μL of ethidium bromide
(2 μg/mL) for 10 min and scored using a fluorescence
microscope (Leica, Wetzlar, Germany) interfaced
with a computer. Software (Leica-QWIN) allowed us
to analyze and quantify DNA damage by measuring:
a) tail length (TL), intensity (TI) and area (TA); b)
head length (HL), intensity (HI) and area (HA).
These parameters are employed by the software to
determine the level of DNA damage as: i) the
percentage of the fragmented DNA (TDNA), and ii)
tail moment (TMOM) expressed as the product of TD
(distance between head and tail) and TDNA.
Statistical analysis: Statistical analysis of results
was performed by using one-way ANOVA followed
by Dunnett’s post-hoc test for multiple comparisons
with control. All statistical analyses were performed
using the statistical software package SYSTAT,
version 9 (Systat Inc., Evanston IL, USA). Each
value represents the mean ± SD of three separate
experiments performed in quadruplicate.
2098 Natural Product Communications Vol. 3 (12) 2008
Garbarino et al.
References
[1]
Speisky H, Cassels BK. (1994) Boldo and boldine: an emerging case of natural drug development. Pharmacological Research, 29,
1-12.
[2]
O'Brien P, Carrasco-Pozo C, Speisky H. (2006) Boldine and its antioxidant or health-promoting properties. Chemico-Biological
Interactions, 159, 1-17.
[3]
Schmeda-Hirschmann G, Rodriguez JA, Theoduloz C, Astudillo SL, Feresin GE, Tapia A. (2003) Free-radical scavengers and
antioxidants from Peumus boldus Mol. ("Boldo"). Free Radical Research, 37, 447-452.
[4]
Quezada N, Asencio M, del Valle JM, Aguilera JM, Gómez B. (2004) Antioxidant activity of crude extract, alkaloid fraction, and
flavonoid fraction from Boldo (Peumus boldus Molina) leaves. Journal of Food Science, 69, C371-376.
[5]
Cragg GM, Newman DJ. (2005) Plants as a source of anti-cancer agents. Journal of Ethnopharmacology, 100, 72-79.
[6]
Leung HW, Lin CJ, Hour MJ, Yang WH, Wang MY, Lee HZ. (2007) Kaempferol induces apoptosis in human lung non-small
carcinoma cells accompanied by an induction of antioxidant enzymes. Food Chemical and Toxicology, 45, 2005-2013.
[7]
Hung H. (2007) Dietary quercetin inhibits proliferation of lung carcinoma cells. Forum Nutrition, 60, 146-157.
[8]
Bobe G, Weinstein SJ, Albanes D, Hirvonen T, Ashby J, Taylor PR, Virtamo J, Stolzenberg-Solomon RZ. (2008) Flavonoid intake
and risk of pancreatic cancer in male smokers (Finland). Cancer Epidemiology Biomarkers & Prevention, 17, 553-562.
[9]
Bednarek I, Sypniewski D, Klama-Baryła A, Gałka S, Machnik G. (2006) Single-cell gel electrophoresis (comet assay) as a tool for
apoptosis determination in tumor cell lines HL-60 and Jurkat cultures treated with anisomycin. Annals Academy of Medicine, 60,
278-284.
[10]
Godard T, Deslandes E, Lebailly P, Vigreux C, Sichel F, Poul JM, Gauduchon P. (1999) Early detection of staurosporine-induced
apoptosis by comet and annexin V assays. Histochemistry and Cell Biology, 112, 155-161.
[11]
Hoet S, Stévigny C, Block S, Opperdoes F, Colson P, Baldeyrou B, Lansiaux A, Bailly C, Quetin-Leclercq J. (2004) Alkaloids
from Cassytha filiformis and related aporphines: antitrypanosomal activity, cytotoxicity, and interaction with DNA and
topoisomerases. Planta Medica,70, 407-413.
[12]
Russo A, Piovano M, Lombardo L, Vanella L, Cardile V, Garbarino J. (2006) Pannarin inhibits cell growth and induces cell death
in human prostate carcinoma DU-145 cells. Anti-Cancer Drugs, 17, 1163-1169.
NPC
Natural Product Communications
Antihyperalgesic Effect of Eschscholzia californica in Rat
Models of Neuropathic Pain
2008
Vol. 3
No. 12
2099 - 2102
Elisa Vivolia*, Anna Maidecchi b, Anna Rita Biliac, Nicoletta Galeottia, Monica Norcini a and
Carla Ghelardinia
a
Dipartimento di Farmacologia, Università di Firenze, Viale G. Pieraccini 6, 50139 Firenze, Italy
b
ABOCA SpA Società Agricola, loc. ABOCA 20, 52037 Sansepolcro, (AR) Italy
c
Dipartimento di Scienze Farmaceutiche, Università di Firenze, Via U. Schiff 6,
50019, Sesto Fiorentino, (FI) Italy
elisa.vivoli@unifi.it
Received: June 11th, 2008; Accepted: October 23rd, 2008
Eschscholzia californica Cham. (Papaveraceae) is traditionally used by the Indians as a medicinal plant for its anxiolytic,
anticonflict, analgesic and sedative properties. The mechanisms of action for the sedative and anxiolytic activities have not
been clearly established and so to further investigate the pharmacological profile of E. californica in some painful conditions, a
70% v/v ethanol extract, DERnative=5:1, was tested in rat models of neuropathy induced by chronic constriction injury of the
sciatic nerve (CCI), with chemotherapeutic oxaliplatin, and osteoarthritis caused by intrarticular injection of monoiodoacetate.
In the CCI model evaluated in the rat paw-pressure test, the examined extract (100 mg kg-1 p.o.) showed an antihyperalgesic
effect. Eschscholzia extract, after single injection at a dose of 100-300 mg kg-1 p.o., produced also a statistically significant
decrease of pain perception on hyperalgesia induced by oxaliplatin and osteoarthritis, while in the same condition gabapentin
did not display any antihyperalgesic effect. Furthermore, in the range of antihyperalgesic doses, the extract was efficacious in
the hot-plate (thermal stimulus) and carrageenan tests (inflammatory model) without producing any behavioral impairment, as
evaluated by the Irwin test. The analgesic effect exhibited by Eschscholzia extract in the mouse hot-plate test was not
antagonized by naloxone, indicating that opioid neurotransmission is not involved in the effect.
The above reported results suggest that a 70% (v/v) ethanol dried extract (DERnative=5:1) of E. californica might represent a
promising product for the therapy of acute and chronic pain.
Keywords: Eschscholzia californica Cham., Papaveraceae, 70% v/v ethanol extract (DERnative=5:1), antihyperalgesic effect,
acute and chronic pain.
Eschscholzia californica Cham. (California poppy,
Papaveraceae) is an annual plant found throughout
California and traditionally used by the Indians as a
medicinal plant for its anxiolytic, anticonflict,
analgesic and sedative properties [1]. The
mechanisms of action for the sedative and anxiolytic
activities have not been clearly established, although
the involvement of several receptors have been
observed and recently it has been reported that
a 70% (v/v) ethanol extract of California poppy
was able to bind to 5-HT(1A) and 5-HT(7) receptors
at 100 μg/mL [2]. To further investigate the
pharmacological profile of E. californica in some
painful conditions, a 70% v/v ethanol extract,
DERnative=5:1, was used for the tests in rat models of
neuropathy induced by chronic constriction injury of
the sciatic nerve (CCI), repeated treatment with the
chemotherapeutic agent oxaliplatin, and osteoarthritis
caused by intrarticular injection of monoiodioacetate
and inflammation caused by carrageenan plantar
injection.
In the CCI model evaluated in the rat paw-pressure
test, the examined extract (100 mg kg-1 p.o.)
showed an antihyperalgesic activity (Figure 1).
The anti- hyperalgesic effect induced by 300 mg kg-1
did not differ from that obtained with a dose
three folds lower (data not shown). California poppy
2100 Natural Product Communications Vol. 3 (12) 2008
Vivoli et al.
70
60
*
Weight (g)
*
*
*
*
50
40
30
20
CCI
OXALIPLATIN
MIA
CARRAGEENAN
10
0
Sal .
Sal . Esch . Gab .
100
100
Sal . Esch . Gab .
100
100
Sal . Esch . Gab .
300
100
Sal . Esch . Gab .
300
100
Figure 1: Effect of Eschscholzia californica extract (ECE) in comparison with gabapentin (Gab.) in rat models of hyperalgesia induced by Chronic
Constriction injury (CCI) and by the administration of Oxaliplatin, Monoiodoacetate (MIA) and Carrageenan. Tests were performed 30 min after
administration. Doses are expressed as mg kg-1 p.o. *P<0.05 vs saline treated rats.
The examined extract, at the dose which was
effective in relieving acute and persistent pain, was
tested in order to assess its effect on mouse
behaviour. At the highest dose employed, the extract
did not induce any alteration of either mouse gross
behaviour or show any other side effect, as observed
in the Irwin test (data not shown). Moreover, mice
treated with California poppy extract were evaluated
for motor coordination by using the rota-rod test. The
endurance time, evaluated before and 15, 30 and 45
minutes after the beginning of the rota-rod test
showed the lack of any impairment in the motor
coordination of the treated mice (Figure 2).
5
4
Falls in 30 sec
extract, after single injection at a dose of 100 mg kg-1
and 300 mg kg-1 p.o., respectively, produced also
a statistically significant decrease of pain perception
on hyperalgesia induced by oxaliplatin and
osteoarthritis, while in the same condition gabapentin
did not display any antihyperalgesic effect (Figure 1).
Particularly, the extract peaked 30 min after
administration (Figure 1). Similarly, in the painful
condition caused by intraplantar injection of
carrageenan, California poppy extract reduced
hyperalgesia at the dose of 300 mg kg-1 p.o., 30 min
after administration (Figure 1). Furthermore, in the
same range of doses, the extract was efficacious in
the mouse hot-plate test in the presence of a thermal
stimulus. The analgesic effect exhibited by California
poppy extract in the mouse hot-plate test was of an
intensity comparable to that exhibited by gabapentin
and it was not antagonized by naloxone (1 mg kg-1
i.p.) indicating that opioid neurotransmission is not
involved in the effect (Table 1).
3
2
1
0
Pretest
15
30
45
60
min after treatment
Figure 2: Effect of Eschscholzia californica extract on
mouse rota-rod test: empty square is saline, filled circle is E. californica
at a dose of 1000 mg kg-1.
Table 1: Effect of Eschscholzia californica extract (ECE) in mouse hot
plate test.
Licking latency (s)
Treatment
Pre-test
SALINE
14.4±0.5
14.6±0.4
ECE
100 mg kg-1 p.o
14.4±0.7
19.6±0.9*
ECE
300 mg kg-1 p.o
14.7±0.7
18.6±0.7*
GABAPENTIN
300 mg kg-1 p.o
15.2±0.8
18.6±1.3*
NALOXONE
1 mg kg -1 i.p.
14.8±0.5
15.4±0.6
NALOXONE + ECE
100 mg kg-1 p.o.
14.3±0.6
19.7±0.8*
*P<0.05 vs saline-treated mice
30 min after treatment
Antihyperalgesic effect of Eschscholzia californica
The above reported results suggest that the freezedried extract (DERnative=5:1) of E. californica might
represent a promising material for the therapy of
acute and chronic pain.
Natural Product Communications Vol. 3 (12) 2008 2101
Table 2: Mobile phases used for HPLC analysis.
Time(min)
0.00
30.00
67
50
B%
33
50
50.00
50
50
1.500
Experimental
65.00
67
33
1.500
Plant material: Eschscholzia californica (California
poppy) was cultivated at Aboca’s Organic Farms in
2007 (Aboca, San Sepolcro (AR), Italy). After
harvest, the drying process was carried out using
special equipment, air-heated ovens, with cells in
which forced air is circulated at a temperature and
level of humidity that are carefully controlled (the
temperature is kept between 32° and 40°C). After
this, the desired plant material was separated from
any extraneous bodies present (weeds, insects, seeds)
based on specific weight, using aero-separators and
densimeters, and cut, to reduce the herb to the proper
dimensions according to its intended use (for herbal
tea, extraction, micronized powder).
HPLC-DAD system: The HPLC analyses were
performed using a HP 1100 L Palo Alto, CA, USA)
equipped with a HP 104iquid Chromatograph
(Agilent Technologies, 0 Diode Array Detector
(DAD), an automatic injector, an auto sampler, a
column oven and managed by a HP 9000 workstation
(Agilent Technologies, Palo Alto, CA, USA).
Separations were performed on a reversed phase
column
prodigy
5 μm
ODS
(3)
100 Å
(250.0 × 4.6 mm) column fitted with a 4.0 × 3.0 mm
i.d. guard column, both from Phenomenex (Torrance,
CA). The eluents were A: SDS 10 mM + DEA 0.1 M
in water adjusted to pH 2.5 by H3PO4; B: acetonitrile.
The mobile phase is reported in Table 2. The system
was operated with an oven temperature of 40oC.
Before HPLC analysis, each sample was filtered
through a cartridge-type sample filtration unit with a
polytetrafluoroethylene (PTFE) membrane [d=13
mm, porosity 0.45 µm (Lida Manufacturing Corp.)]
and immediately injected (20 μL).
Preparation of plant extract: Eschscholzia
californica freeze-dried extract was produced by
Aboca Spa (Sansepolcro, AR) and was obtained
according to the extraction procedure described
below. The dried ground tops were extracted by
percolation with a hydro ethanolic solution (70% v/v)
and herb/solvent ratio of 1:10.
After 8-10 h, the plant extract was filtered to remove
the exhausted herb and concentrated under vacuum to
remove the ethanol. The extract was freeze-dried
under vacuum, without the use of either heat or
excipients, at temperatures less than -50°C. The
Drug Extract Ratio (DER) was 5:1 and the batch
number was n. 7B1150. The content of protopin in
this batch was 0.22%, analyzed by a HPLC method.
Chemicals: Protopin was purchased from Sigma
(Sigma-Aldrich S.r.l., Milan, Italy). All the solvents
used for the extraction and HPLC analysis (EtOH,
MeOH, diethanolamine and acetonitrile) were HPLC
grade from Merck (Darmstadt, Germany). Water was
purified by a Milli-Qplus system from Millipore
(Milford, MA). The following drugs were used
in the pharmacological experiments: Naloxone
hydrochloride (Sigma, St. Louis, USA), Oxaliplatin
(Sequoia Research Products Ltd, Pangbourne, UK),
Sodium iodoacetate (Sigma-Aldrich, Germany).
Other chemicals were of the highest quality
commercially available.
Flow (mL/min)
1.500
1.500
Chromatograms were recorded between 200 and 450
nm. DAD spectra were stored for all peaks exceeding
a threshold of 0.1 mAu and detection was performed
at 280 nm.
Calibration curves: A calibration curve was obtained
from an 80% MeOH solution (containing 4% of
diluted HCl) of protopin in the range between 0.025
and 0.00625 mg/mL.
Pharmacological assays: Drugs were either
dissolved in isotonic (NaCl 0.9%) saline solution or
dispersed in 1% carboxymethylcellulose sodium salt
(CMC, Fluka Chemie GmbH, Steinheim,
Germany) immediately before use. Drug
concentrations were prepared so that the necessary
dose could be administered in a volume of 10 mL
kg-1 by i.p. and p.o. injection.
Animals: Male Sprague-Dawley albino rats (180-200
g) from Harlan (S.Piero al Natisone, Italy) and male
Swiss albino mice (24-26 g) from Morini (San Polo
d’Enza, Italy) were used. Four rats and 10 mice were
2102 Natural Product Communications Vol. 3 (12) 2008
housed per cage. For acclimatization, the cages were
placed in the experimental room 24 h before the test.
The animals were fed a standard laboratory diet and
tap water ad libitum and kept at 23±1°C with a 12-h
light/dark cycle. All experiments were carried out in
accordance with the European Communities Council
Directive of 24 November 1986 (86/609/EEC) for
experimental animal care. All efforts were made to
minimize the number of animals used and their
suffering.
Chronic constriction injury: A peripheral mono
neuropathy was produced in adult rats by placing
loosely constrictive ligatures around the common
sciatic nerve, according to the method described by
Bennet and Xie [3].
Monoiodoacetate injection: Joint damage was
induced by a single intra-articular injection of 2 mg
of sodium monoiodoacetate into the left knee joint of
anaesthetized rats in a total volume of 25 μL. The
dose of iodoacetate was chosen based on previous
literature [4] and in-house dose response data using
0.5, 1 and 2 mg.
Oxaliplatin injection: Hyperalgesia was induced
by 15 injections for 5 consecutive days every week
for 3 weeks of Oxaliplatin, 2.4 mg kg-1 (15 i.p.
injections- cumulative dose 36 mg kg-1 ) [5].
Paw-pressure test: The instrument exerts a force
which is applied at a constant rate (32 g per second)
with a cone-shaped pusher on the upper surface of the
rat hind paw. The force is continuously monitored by
a pointer moving along a linear scale. The pain
Vivoli et al.
threshold is given by the force that induces the first
struggling from the rat. Pretested rats which scored
below 40 g or over 80 g during the test before drug
administration (25%) were rejected. An arbitrary cut
off value of 250 g was adopted.
Hot plate test: Mice were placed inside a stainless
steel container, which was set thermostatically at
52.5±0.1°C in a precision waterbath from KW
Mechanical Workshop, Siena, Italy. Reaction times
(s) were measured with a stopwatch before and 15,
30, 45 and 60 min after administration of the drug.
The endpoint used was the licking of either the
forepaws or hind paws. Those mice scoring less than
12 and more than 18 s in the pretest were rejected
(30%). An arbitrary cutoff time of 45 s was adopted.
Carrageenan test: Rat paw volumes were measured
using a plethysmometer (Ugo Basile, Varese, Italy).
Rats received the investigated extract 30 min after a
0.1mL injection of 1.0% carrageenan in the right hind
paw. Four h after the injection of carrageenan, the
pain threshold of the right hind paw was measured
and compared with saline/carrageenan-treated
controls.
Statistical analysis: All experimental results are
given as the mean ± S.E.M. An analysis of variance
(ANOVA), followed by Fisher’s Protected Least
Significant Difference procedure for post-hoc
comparison, were used to verify significance between
two means. Data were analyzed with the StatView
software for the Macintosh. P values of less than 0.05
were considered significant.
References
[1]
Rolland A, Fleurentin J, Lanhers MC, Younos C, Misslin R, Mortier F, Pelt JM. (1991) Behavioural effects of the American
traditional plant Eschscholzia californica: sedative and anxiolytic properties. Planta Medica, 57, 212-216.
[2]
Gafner S, Dietz BM, McPhail KL, Scott IM, Glinski JA, Russell FE, McCollom MM, Budzinski JW, Foster BC, Bergeron C,
Rhyu M-R, Bolton JL. (2006) Alkaloids from Eschscholzia californica and their capacity to inhibit binding of [3H]8-hydroxy-2-(diN-propylamino)tetralin to 5-HT1A receptors in vitro. Journal of Natural Products, 69, 432-435.
[3]
Bennett GJ and Xie YK (1988) A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in
man. Pain, 33, 87-107.
[4]
Guingamp C, Gegout-Pottie P, Philippe L, Terlain B, Netter P, Gillet P. (1997) Mono-iodoacetato-induced experimental
osteoarthiritis: a dose-response study of loss of mobility, morphology and biochemistry. Arthritis and Rheumatism, 40, 1670-1679.
[5]
Cavaletti G, Tredici G, Petruccioli MG, Dondè E, Tredici P, Marmiroli P, Minoia C, Ronchi A, Bayssas M, Etienne GG. (2001)
Effects of different schedules of oxaliplatin treatment on the peripheral nervous system of the rat. European Journal of Cancer, 37,
2457-2463.
NPC
Natural Product Communications
Problems in Evaluating Herbal Medicinal Products
2008
Vol. 3
No. 12
2103 - 2106
Jozef Corthout
Dienst voor Geneesmiddelenonderzoek – Service de Contrôle des Médicaments, Stevinstraat 137,
B-1000 Brussels, Belgium
corthout.jozef@mail.apb.be
Received: June 24th, 2008; Accepted: October 20th, 2008
Compared to chemical drugs, the quality control of herbal medicinal products poses many problems due to the complexity of
herbal drugs, herbal drug preparations and the products themselves, and due to the use of different analytical methods
(selective and non-selective) giving different results. To illustrate this, three groups of herbal medicinal products were assayed
by different analytical methods: products containing St.John’s wort (colorimetry and HPLC), milk thistle (spectrophotometry
and HPLC) and ginkgo (HPLC). These studies show that complete and correct labeling is necessary for the evaluation of herbal
medicinal products and that for the majority of plants the knowledge of the applied analytical method is essential for a proper
verification of conformity.
Keywords: Herbal medicinal products, Hyperici herba, Silybi mariani fructus, Ginkgonis folium, analysis.
The mission of the Belgian Medicines Control
Laboratory is the post-market quality control of
medicines delivered in public pharmacies, thus also
including herbal medicinal products. Compared with
“chemical” drugs, the analysis of herbal medicinal
products poses many problems.
titrimetry) versus a selective, chromatographic
method (HPLC, GC). These problems and
differences are illustrated by the evaluation of three
groups of herbal medicinal products containing dry
extracts of Hyperici herba, Silybi mariani fructus and
Ginkgonis folium.
First, a plant can be used in different forms: different
parts of the plant (for example, leaf, flower, herb);
the crude drug itself or different kind of preparations:
standardized, quantified, purified or other type of
preparation. In case of standardization/quantification
this can be performed for either a single constituent
or to a group of metabolites. As a consequence of this
multiplicity of possibilities, an accurate and detailed
label is necessary otherwise it is not possible to verify
the product for its conformity. The following
information should be stated: Latin name and part of
the plant, the ratio of the herbal drug to the herbal
drug preparation (DER), the extraction solvent, the
physical state, and quantity of the extract. The
quantity may also be given as a range corresponding
to a defined quantity of constituents with known
therapeutic activity [1]. Herbal medicinal products
can be on the market as registered medicines or as
food supplements, each with their own legal
requirements. Finally, the assay of the constituent(s)
can give different results depending on the applied
method: a non-selective method (spectrophotometry,
Hyperici herba or St.John’s wort consists of the
whole or cut, dried flowering tops of Hypericum
perforatum L., harvested during flowering time,
containing not less than 0.08% of total hypericins
[2]. The most characteristic constituents are
naphthodianthrones, consisting mainly of hypericin
and pseudohypericin. Other naphthodianthrones
present, and included in the term ‘total hypericin’, are
the biosynthetic precursors protohypericin and
protopseudohypericin (which are transformed into
hypericin and pseudohypericin, respectively on
exposure to light), and a small amount of
cyclopseudohypericin. Other constituents include
phloroglucinols and flavonoids. St. John’s wort is
indicated for mild to moderate depressive episodes in
daily dosages of 450 to 1050 mg of hydroalcoholic
dry extract (with defined DER) [3].
Eight St. John’s wort preparations (tablets, coated
tablets or capsules) were analyzed by assaying total
hypericins by spectrophotometry [2] and HPLC [4].
None of the products had a comprehensive label, the
2104 Natural Product Communications Vol. 3 (12) 2008
Corthout
Table 1: Products containing St. John’s wort.
Prod
1
2
3
4
5
6
7
8
Label claim
mg TH/unit
mg extract/unit
0.3
NM
0.3
NM
0.3
NM
0.4
NM
0.4
NM
0.5
100-120
0.9
250-300
0.85
425
Spectrophotometry
mg TH/unit
% of claim
0.21
70.0%
0.20
60.6%
0.27
90.0%
0.34
85.0%
0.08
26.7%
NA
interference
0.79
87.8%
1.5
176.5%
HPLC
mg TH/unit
% of claim
0.11
36.7%
0.11
33.3%
0.05
16.7%
0.13
32.5%
0.04
13.3%
0.13
26.0%
0.38
42.2
0.72
84.7%
H, Hypericin, NM, Not Mentioned
TH, Total Hypericins, NA, Not Applicable
most common missing information being part of the
plant, type of extract, solvent, DER and even the
quantity of the extract (5 products). Five products
were quantified to a defined content of hypericin, two
to total hypericin and one to hypericinum (?). One
product gave a minimum content of hypericin.
The assay results are summarized in Table 1: the total
hypericin content varied from 26.7% to 176.5% of
their label claim for the spectrophotometric assay and
from 13.3% to 84.7% for the HPLC method. The
ratio of the spectrophotometric assay to the HPLC
assay ranged from 2.1 to 5.4, which is in accordance
with the results of the group of Prof. Vincieri and of
Dr Gaedke, who obtained for commercial dry
extracts ratios of 1.2 to 10 and 1.1 to 1.6, respectively
[5,6]. None of the products complied with its label
claim neither for the spectrophotometric result nor for
the HPLC result, taking 90.0%-110.0% of the stated
amount as requirement.
Silybi mariani fructus (Cardui mariae fructus) or milk
thistle fruit is the mature fruit, devoid of the pappus,
of Silybum marianum (L.) Gaertner, containing a
minimum of 1.5% of silymarin, expressed as
silibinin. Milk thistle refined and standardized dry
extract contains 30 to 65% of silymarin
(determination by HPLC). The content of silymarin
corresponds to 20-45% silicristin and silidianin, 4065% silibinin A and B, and 10-20% isosilibinin A
and B, with reference to total silymarin [7]. The
active constituents are flavanolignans, collectively
named silymarin, and consisting mainly of silibinin
and isosilibinin. The milk thistle dry extract, which
is the most frequently used in the pharmacological/
Table 2: Products containing milk thistle.
Prod
1
2
Label claim
mg S/caps
mg extract/caps
140
180
200
250
Colorimetry
mg S/caps
% of claim
141.6
101.1%
165.2
82.6%
3
451
NP
4
75
250
138.7
184.9%
HPLC
mg S/caps
% of claim
110.9
79.2%
115.5
57.8%
45.5
101.1%
122.3
163.1%
1
contains silibinin as silibinin phosphatidylcholine
S, silymarin; NP, Not Performed
toxicological and clinical studies, contains 74.180.9% of silymarin (determination by UV
spectroscopy; DER 36-44:1) [8]. Milk thistle
products are used in cases of toxic liver damage;
chronic inflammatory liver conditions and hepatic
cirrhosis in daily dosages equivalent to 154-324 mg
of silymarin (HPLC method) or 200-420 mg of
silymarin (colorimetric method). The silymarin
content obtained by HPLC amounts to about 77% of
the value obtained by colorimetry [8].
Four milk thistle preparations, all formulated as
capsules, were evaluated by determination of the
silymarin content by spectrophotometry [9] and
HPLC [7]. Three products contained a dry extract
standardised to different amounts of silymarin; one
product contained pure silibinin.
The results are summarized in Table 2: for the
silibinin product, 101.1% of the label claim was
found by HPLC, for the other products the silymarin
content varied from 82.6% to 184.9% of their label
claim for the colorimetric assay and from 57.8% to
163.1% for the HPLC method. The ratio of the HPLC
assay result to the colorimetric assay result ranged
from 0.7 to 0.9, which is in agreement with the
results of Ihrig [9], where the same ratio range was
obtained. Products 1 and 3 complied with their label
claim taking the content from the colorimetric
analysis and the HPLC analysis, respectively. Despite
the fact that products 2 and 4 are food supplements,
they contain silymarin in either the same or higher
amount as the medicinal product 1.
Ginkgonis folium or ginkgo leaf is the whole or
fragmented, dried leaf of Ginkgo biloba L.,
containing not less than 0.5% of flavonoids,
calculated as flavone glycosides. Ginkgo refined and
quantified dry extract contains 22.0-27.0% flavonoids
(determined by HPLC-UV), 2.6-3.2% bilobalide and
2.8-3.4% ginkgolides A, B and C (determined by
HPLC-RI) [10]. The characteristic constituents of
Problems in evaluating herbal medicinal products
Natural Product Communications Vol. 3 (12) 2008 2105
ginkgo leaf are terpenes and flavonoids. The
principal terpenes are diterpene trilactones called
ginkgolides and the sesquiterpene trilactone
bilobalide. The main flavonoids are mono-, di- and
triglycosides of the flavonols quercetin, kaempferol
and isorhamnetin. Diglycosides esterified with
p-coumaric acid are also present. Other flavonoids
include flavones, biflavones, flavan-3-ols and
proanthocyanidins. Ginkgo is indicated for mild to
moderate dementia syndromes including primary
degenerative dementia, vascular dementia and mixed
forms;
cerebral
insufficiency;
neurosensory
disturbances such as dizziness, vertigo and tinnitus;
enhancement of cognitive performance; and
peripheral arterial occlusive disease (intermittent
claudication). All these indications are supported by
clinical trials performed with preparations based on
standardized extracts, containing 22-27% flavonol
glycosides and 5-7% terpene lactones. The daily dose
for these preparations is 120-240 mg of standardized
ginkgo dry extract, corresponding to 29-58 mg
flavonoids and 7.2-14.4 mg terpene lactones [11].
between 8 mg and 44.5 mg total flavonoids per unit.
Generally the ratio of the 3 aglycones quercetin,
kaempferol and isorhamnetin in ginkgo is 5:5:1,
corresponding to a relative rutin content of 10 to
15%. Because it is known that rutin is sometimes
added to ginkgo extracts, a direct assay of rutin was
also performed [12]. Absolute and relative contents
(with reference to total flavonoids) ranged from 0.4
to 8.9 mg/unit and from 5 to 51%, respectively.
Products 4-7, 11, 13 and 14 had a relative rutin
content ranging from 24% to 51%, indicating a
possible falsification with rutin.
A total of 14 products, formulated as capsules and
tablets, were included in the study (see Table 3).
They all claim to contain ginkgo dry extract, ranging
from 30 to 200 mg per unit. For ginkgo, the problem
of different existing assay methods is not a point
because in most cases the flavonoids are determined
with HPLC-UV and the terpene lactones with
HPLC-RI [12]. The content of total flavonoids was
determined after hydrolysis to the flavonol aglycones
quercetin, kaempferol and isorhamnetin. Eleven of
the 14 products had a claim for flavonoid content
ranging from 9.6 mg up to 36 mg per unit. For these
products, between 95% and 123% of the theoretical
amount was found. For the 3 other products without
indication of flavonoid amount, the content varied
Concerning the analysis of terpene lactones, for
products 1-8 having a label claim, the assayed
amount varied from 80% (product 4) up to 194%
(product 8) of the theoretical amount. Products 9-14
(without a claim) had a content of terpene lactones
between 1.42 and 4.83 mg per unit.
Only the medicinal products 1-3 complied with all
the requirements: the content of total flavonoids and
terpene lactones were in accordance with the stated
amounts; the relative rutin content was between 10
and 15% of the total flavonoid content and their label
was complete and correct. Again, for most of the
food supplements the content and the corresponding
daily dose of flavonoids and terpene lactones was
higher when compared with the medicinal products:
product 9 had no label claim, but its daily dose for
flavonoids and terpene lactones corresponded
respectively to about 460% and 200% of a normal
daily dose. These studies show that a complete and
correct label is necessary for the evaluation of herbal
medicinal products. In addition, for the majority of
plants, the knowledge of the applied analytical
method is essential to enable a proper verification of
the conformity.
Table 3: Products containing ginkgo.
Prod
1
2
3
4
5
6
7
8
9
10
11
12
13
14
extract/unit
40 mg
60 mg
40 mg
60 mg
50 mg
80 mg
100 mg
60 mg
200 mg
150 mg
60 mg
63 mg
200 mg
30 mg
Label claim
flavonoids/
terpene
unit
lactones/unit
9.6 mg
2.4 mg
14.4 mg
3.6 mg
9.6 mg
2.4 mg
14.4 mg
3.6 mg
12.0 mg
3.0 mg
19.2 mg
4.8 mg
24.0 mg
6.0 mg
14.4 mg
3.6 mg
NM
NM
36.0 mg
NM
14.4 mg
NM
15.0 mg
NM
NM
NM
NM
NM
NM, Not Mentioned, NA, Not Applicable
Total Flavonoid Assay
A: flavonoids/unit
B: % of claim
9.14 mg
95.2%
14.53 mg
100.9%
9.23 mg
96.1%
16.97 mg
117.9%
13.45 mg
112.1%
23.63 mg
123.1%
23.02 mg
95.9%
14.70 mg
102.1%
44.50 mg
NA
35.25 mg
97.9%
16.34 mg
113.4%
15.28 mg
101.9%
13.21 mg
NA
7.98 mg
NA
Rutin Assay
A: rutin/unit
B: % of total flavonoids
1.05 mg
11%
1.57 mg
11%
1.17 mg
13%
4.19 mg
25%
6.82 mg
51%
8.89 mg
38%
5.44 mg
24%
1.54 mg
10%
4.99 mg
11%
3.41 mg
10%
5.50 mg
34%
1.70 mg
11%
3.60 mg
27%
2.89 mg
36%
Terpene lactone Assay
A: terpene lactones/unit
B: % of claim
2.39 mg
99.6%
3.71 mg
103.1%
2.31 mg
96.3%
2.88 mg
80.0%
4.27 mg
142.3%
6.10 mg
127.1%
5.25 mg
87.5%
6.98 mg
193.9%
4.83 mg
NA
4.63 mg
NA
3.93 mg
NA
4.56 mg
NA
3.64 mg
NA
1.42 mg
NA
2106 Natural Product Communications Vol. 3 (12) 2008
The analysis of ginkgo, showing a possible
falsification of ginkgo extract by adding rutin,
demonstrates the benefit of using a chromatographic
assay method. An additional remark is that most of
the examined food supplements do not comply with
either their label claim or the defined requirements
and that their content is sometimes higher than the
minimum therapeutic dose ascribed to the medicinal
products.
Experimental
Materials and apparatus: Herbal drug preparations
were purchased from a local wholesaler. The
following standards were used: hypericin CRS from
the European Pharmacopoeia, silibinin and quercetin
trihydrate from Sigma, rutin and standardized ginkgo
dry extract (1.27% ginkgolide A, 0.77% ginkgolide
B, 1.32% ginkgolide C and 2.65% bilobalide) from
Wilmar Schwabe. The UV-VIS analyses were
performed on an Agilent 8453 UV-VIS
spectrophotometer. The HPLC analyses were carried
out on either a Waters Alliance 2695 equipped with a
2996 PDA detector or a Waters 410 Differential
Refractometer.
Chromatographic conditions: Hypericin assay: The
column was a Symmetry C18 250 x 4.6 mm, 5 µm
(Waters), used at ambient temperature. The samples
were chromatographed with a mixture of 150 mL
buffer pH 2.1 (13.98 g NaH2PO4.1H2O in 1000 mL
water adjusted to pH 2.1 with H3PO4 85%), 140 mL
ethyl acetate and 760 mL methanol as mobile phase,
with detection at 590 nm, a flow rate of 1.0 mL/min
and 20 µL as injection volume.
Corthout
Silymarin assay was carried out on a Waters
Symmetry C18 150 x 4.6 mm, 5 µm column at
ambient temperature using a gradient at 1.0 mL/min.
with 2 solvents (A = H3PO4, methanol, water
(0.5:35:65); B = H3PO4, methanol, water (0.5: 50:50);
100%A/0%B to 0%A/100%B in 28 min and
continuing this for 8 min). The injection volume was
10 µL and UV detection was at 280 nm. Total
flavonoid assay utilized a Waters Symmetry C18 150
x 4.6 mm, 5 µm column held at 25°C in combination
with a gradient at 1.0 mL/min. using 2 solvents
(A = 0.3 g/L of H3PO4 in water adjusted to pH 2.0;
B = methanol; 60%A/40%B to 45%A/55%B in
20 min.). The injection volume was 10 µL and UV
detection was at 370 nm. Rutin assay was carried out
on a Varian Polaris C18 250 x 4.6 mm 5 µm column
at ambient temperature using a mixture of 3 g/L of
H3PO4 in water and acetonitrile (82:18) as mobile
phase, at a flow rate of 1 mL/min, injection volume
of 10 µL and UV detection at 357 nm. Terpene
lactone assay: the mobile phase, a mixture of water–
methanol–tetrahydrofuran (75–20–10) was pumped
at 1.0 mL/min through a Waters Symmetry C8 250 x
4.6 mm, 5 µm column at 35°C, followed by a
refractometer, also at 35°C. The injection volume
was 100 µL.
Quantitative determination: The samples were
prepared as described in the cited references. Each
determination was performed in triplicate except the
terpene lactone HPLC assay, which was undertaken
in duplicate. The contents are presented as a mean of
data obtained from the spectrophotometric and HPLC
analyses.
References
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
Guideline on quality of Herbal Medicinal Products/Traditional Herbal Medicinal Products (CPMP/QWP/2819/00 Rev1).
St.John’s wort – Hyperici herba. (2008) In: European Pharmacopoeia 6.0.
St.John’s wort – Hyperici herba. (2003) In: ESCOP Monographs, The scientific foundation for herbal medicinal products, 78-210.
Krämer W, Wiartalla R. (1992) Bestimmung von Naphtodianhronen (Gesamthypericin) in Johanniskraut (Hypericum perforatum
L.). Pharmazeutische Zeitung Wissenschaft, 5, 202-207.
Bergonzi MC, Bilia AR, Gallori S, Guerrini D, Vincieri FF. (2001) Variability in the content of the constituents of Hypericum
perforatum L. and some commercial extracts. Drug Development and Industrial Pharmacy, 27, 491-497.
Gaedke F. (1997) Johanniskraut und dessen Zubereitungen. Deutsche Apotheker Zeitung, 137, 3753-3757.
Milk thistle fruit – Silybi mariani fructus. Milk thistle dry extract, refined and standardised – Silybi mariani extractum siccum
raffinatum et normatum. In: European Pharmacopoeia 6.0. (2008)
Blaschek W, Ebel S, Hackenthal E, Holzgrabe U, Keller K, Reichling J, Schulz V. (Eds) (2007) Silybum. In: Hagers Enzyklopädie
der Arzneistoffe und Drogen 6th ed., Band 14, WVG Stuttgart, 582-605.
Ihrig M, Meiss M, Möller H. (1999) Gehaltbestimmung von Silymarin in Fertigarzneimitteln. Pharmazeutische Zeitung, 34,
2661-2671.
Ginkgo Leaf – Ginkgonis folium. Ginkgo dry extract, refined and quantified – Ginkgonis extractum siccum raffinatum et
quantificatum. In: European Pharmacopoeia 6.0.
Ginkgo Leaf – Ginkgo folium. (2003) In: ESCOP Monographs, The scientific foundation for herbal medicinal products, 178-210.
Saevels J, Corthout J. (2005) Ginkgo biloba medicines and food supplements. Journal de Pharmacie de Belgique, 4, 129-134.
NPC
Natural Product Communications
Impurities in Herbal Substances, Herbal Preparations and
Herbal Medicinal Products, IV. Heavy (toxic) Metals*
2008
Vol. 3
No. 12
2107 - 2122
SFSTP Commission, Didier Guédona, Michèle Brumb, Jean-Marc Seigneuretc, Danièle Bizetd,
Serge Bizote, Edmond Bournyf, Pierre-Albert Compagnong, Hélène Kergosienh, Luis Georges
Quintelasi, Jerôme Respaudj, Olivier Saperask, Khalil Taoubil and Pascale Urizzim
a
Laboratoires Arkopharma, ZI Carros Le Broc, BP 28, F-06511 Carros Cedex, France
48, avenue de la République, F-92500 Rueil-Malmaison, France
c
Alban Muller, 8, rue Charles-Pathé, F-94300 Vincennes, France
d
Laboratoires Boiron, 20, rue de la Libération, F-69110 Sainte-Foy-les-Lyon, France
e
Indena, 38, avenue Gustave-Eiffel, F-37095 Tours, France
f
LPPAM, avenue de la Gare, BP 47, F-26170 Buis-les-Baronnies France
g
AFSSAPS, 143-147, boulevard Anatole-France, F-93285 Saint-Denis, France
h
Euromed France, 95, route du Morgon, F-69400 Gleizé, France
i
SQUALI, 3, rue de la Pagère, F-69500 Bron, France
j
Avogadro, Parc de Genibrat, F-31470 Fontenilles, France
k
LACAPA, 3, boulevard de Clairfont, F-66350 Toulouges, France
l
Laboratoires Boiron, 20, rue de la Libération, F-69110 Sainte-Foy-les-Lyon, France
m
Institut de Recherche Pierre Fabre, 3, rue Ariane, BP 72101, F-31521 Ramonville-Saint-Agne Cedex,
France
b
didier.guedon@arkopharma.com
Received: July 29th, 2008; Accepted: October 23rd, 2008
The main source of available forms of heavy metals (toxic metals) for the plant kingdom is anthropogenic, resulting from
diverse activities such as metallurgic processing of ore, cement plants, uncontrolled discharge of sewage sludge, burning of
fossil fuels and waste incineration plants, and leaded petrol. Agricultural chemicals (e.g. phosphate fertilizers containing
cadmium) may also contribute to the contamination of cultivated plants. The main threats to human health from toxic metals
are associated with exposure to lead, cadmium, mercury (organic forms, especially methylmercury) and arsenic (mineral form
only), which have no known vital or beneficial effect on living organisms. As their toxicity often takes years to manifest and
may go unsuspected, their toxicological risk is defined on the basis of the so-called Provisional Tolerable Weekly Intake
(PTWI) values. Beside anthropogenic causes, the main factors that may lead to high levels of toxic metals in medicinal plants
are their availability in the soil with soil pH as the most important parameter for uptake by the plant. Indeed, genetic features of
certain plant species show a tendency to accumulate certain trace elements, especially cadmium (“cadmium collector”).
A very recent revision draft of the monograph “Herbal drugs” (Ph. Eur., 1433) includes acceptance criteria for lead, cadmium
and mercury. This proposal is discussed in detail, based on literature data dealing with terrestrial plants and seaweed.
Additionally, the need for inclusion of tests for inorganic impurities in quality control specification is examined, based on a
risk assessment. As the daily intake of food supplements is very similar to the one of herbal remedies, it would be advisable to
take into account the same acceptance criteria. The specific situation has also been considered of exotic herbal remedies,
particularly those of Asian origin, which have been repeatedly reported to contain toxic levels of heavy metals and/or arsenic
resulting in heavy metal poisoning.
Keywords: Herbal drugs, herbal drug preparations, herbal medicinal products, food supplements, heavy metals, toxic metals,
quality control, regulations.
The term “heavy metals” has usually been used to
________________________________________________________________________________________________________________________________________________________________________________________
* This publication pertains to an article with several parts. Parts I
(“Microbial contamination”), II (“Mycotoxins”) and III (“Pesticide
residues”) have already been published [1-3].
describe elements that should be restricted in ingested
materials because of their toxic effects. Based on
physical properties, this term includes not only lead
2108 Natural Product Communications Vol. 3 (12) 2008
and cadmium but also other elements between
chromium and bismuth on the periodic table of the
elements, having specific gravities greater than
5 g/cm3. Some of them are essential nutrients in trace
amounts (e.g. copper, iron, zinc, cobalt, manganese,
molybdenum) and others have relatively low toxicity
(e.g. nickel, chromium). Mercury, a noble metal, is
also an element that is controlled in certain foods
because of its toxic nature. Arsenic, a metalloid
(usually classified as a heavy metal), may be of
concern. So, the term “toxic metals” is presently
most often used in the literature, unlike the European
Pharmacopoeia (Ph. Eur.), which prefers so far the
wording “heavy metals”. Both of them will be used
in the present publication, along with trace element.
Heavy metals, as natural components of the earth
surface (minerals, ore), do not present any risk for
human health as they are not available for living
organisms. They are released into the environment
due to many factors including natural (e.g. erosion or
volcanic activity) and mainly anthropogenic causes,
for example, metallurgical processing of ore (mining
industry,
smelting
works),
cement
plants,
uncontrolled
discharge
of
sewage
sludge,
contaminated emissions from refineries, burning of
fossil fuels or waste incineration plants, and leaded
petrol.
Heavy metal uptake by plants from the soil is quite
variable. Root systems and soil properties, such as
pH, may determine their availability to the plant
(higher uptake in acid soils due to higher solubility of
heavy metals) [4,5]. The level of lead, cadmium and
other metallic trace elements in a raw material can
vary considerably with plant part, habitat and soil
concentration [6-8]. Industrial activities have been
identified as responsible for abnormally high
accumulations of lead and cadmium in medicinal
plants [6,9,10]. Meanwhile, heavy metals content in a
soil is not necessarily a sufficient indicator to
evaluate their possible accumulation in plants [11] as
stronger genetic features may also influence heavy
metals content [6,12-14]. Some plants have been
recommended for cleaning toxic metals from polluted
soils due to their strong absorption and concentration
from the soil [6].
There are two major reasons why it has become
necessary to consider levels of toxic metals in herbal
drugs and/or their preparations and finished products:
• contamination of the environment with toxic
metals has increased dramatically during the 20th
century, especially cadmium emissions while,
Guédon et al.
over the last decades, lead emissions in
developed countries have decreased markedly
due to the introduction of unleaded petrol [4,15].
The sources of this environmental pollution are
quite varied, ranging from industrial activities
(see above) to the use of purification mud and
agricultural treatments, such as cadmiumcontaining phosphate fertilizers, mercury
fungicides and arsenical insecticides, much used
some years ago and even used today in some
countries [8,16]. It is also generally agreed that
cadmium in fertilizers is by far the most
important source of cadmium input to soil and to
the food chain. Cadmium accumulation in
agricultural soils due to fertilizer application was
the subject of an opinion delivered by SCTEE
[17]. As an example, cadmium, lead and mercury
were significantly higher in the roots from
valerian grown on sewage sludge-amended soil,
than on unamended soil [18];
• exotic herbal remedies, particularly those of
Asian origin, have been repeatedly reported to
contain toxic levels of heavy metals and/or
arsenic [19].
1. PRESENCE OF HEAVY METALS DUE
TO ENVIRONMENTAL POLLUTION
1.1. Cadmium and lead: Most of the studies on the
presence of toxic metals in medicinal plants have
focused on lead and cadmium [5-8,12,13,15,20-30].
The contamination of herbal drugs with lead and
cadmium has been shown to be subject to broad
fluctuations depending on the plant species.
A German working group on contaminants, running a
data base on heavy metals, published a compilation
of results. A total of over 12,000 samples
corresponding to 204 herbal drugs were tested for
lead and cadmium content, comparing minimum and
maximum values, along with the 90th percentile (i.e.
level below which 90% of the findings occur) [14].
The 90th percentile of many herbal drugs was over 0.5
mg/kg of cadmium (and even over 1.0 mg/kg), while
far fewer raw materials showed a 90th percentile
exceeding 5 mg/kg of lead (see Table 1).
The Official Medicines Control Laboratories
(OMCL) network of the European Directorate for the
Quality of Medicines & Healthcare (EDQM)
conducted a market surveillance study regarding
cadmium content of different herbal drugs from the
European market [31]. The main results are
presented, with the kind permission of EDQM.
Toxic metals in herbal medicinal products
Natural Product Communications Vol. 3 (12) 2008 2109
Table 1: Cadmium and lead: 90th percentile in herbal drugs [14].
Heavy metals
Cadmium:
Herbal drugs
Speedwell (0.52 mg/kg), Celery, fruit (0.53 mg/kg), Sundew (0.53 mg/kg),
Ivy (0.53 mg/kg), Lady’s mantle (0.54 mg/kg), Dill, fruit (0.54 mg/kg),
Linseed (0.54 mg/kg), Dill, herb (0.58 mg/kg), Woodruff (0.59 mg/kg),
Kava-kava (0.63 mg/kg), Lemon balm (0.64 mg/kg),
Dandelion, herb and root (0.64 mg/kg), Birch (0.67 mg/kg),
Immortelle (0.69 mg/kg), Dandelion, herb (0.69 mg/kg), Lungwort (0.79 mg/kg), Buckwheat (0.86 mg/kg),
True golden rod (0.86 mg/kg), Spinach (0.93 mg/kg), Wild pansy (1.00 mg/kg), Bladder wrack (1.05 mg/kg),
Fumitory (1.05 mg/kg), Mallow, leaf (1.17 mg/kg), St John’s wort, herb (1.30 mg/kg),
St John’s wort, flower (1.43 mg/kg), Willow (1.80 mg/kg), Tormentil (2.13 mg/kg).
90th percentile ≥ 0,5 mg/kg
Clove (5.3 mg/kg), Sundew (6.6 mg/kg), Buckthorn (7 mg/kg),
Ginkgo (10.5 mg/kg), Mallow, leaf (12 mg/kg), Island moss (14.3 mg/kg).
Lead:
90th percentile ≥ 5,0 mg/kg
Table 2: Cadmium in herbal drugs: herbal drugs with a cadmium content higher than 0.3 mg/kg [31].
Yarrow, aerial parts
8
Number of batches
Cd > 0.3 mg/kg
7
Birch, leaf
12
11
8
---
Oak, bark
German chamomile, flower
h d
Mallow, flower
7
3
---
---
13
4
3
---
1
1
---
---
St John’s wort, flowering top
17
15
14
3
Dandelion, root
5
2
1
---
Herbal drug
Number of batches analysed
Number of batches
Cd > 0.5 mg/kg
5
Number of batches
Cd > 1.0 mg/kg
---
Water plantain, rhizome
2
1
1
---
Willow, bark
14
14
13
13
Raw materials tested included medicinal plants where
cadmium could be commonly accumulated. Results
showed that over a total of 113 different sample
batches tested, corresponding to 21 different herbal
drugs, 58 batches (51%) showed levels higher than
0.3 mg/kg, 45 batches (40%), levels over 0.5 mg/kg
and 16 batches (14%), levels higher than 1 mg/kg
(see Table 2).
It was, moreover, observed that contamination with
lead occurs rather by chance, whereas enhanced
cadmium values are restricted to some species having
a tendency to accumulate this heavy metal [13].
Some such species are St John’s wort [5,13,25,32],
yarrow [5,13], linseed [15,22,27,32,33], German
chamomile, absinth [13], valerian, passionflower,
echinacea [25] and cinchona [34].
1.2. Mercury: In the environment, several forms of
mercury occur: elemental mercury (Hg(0)),
monovalent mercury (Hg(I)) as mercurous chloride
(HgCl), divalent mercury (Hg(II)) as mercuric
chloride (HgCl2), and mercuric sulfide (HgS) or
organic mercury, where it has formed compounds
with carbon. Methylmercury (CH3Hg+), the most
common example of this form of mercury, is
particularly toxic (see II Health hazard). There are
few anthropogenic sources of methylmercury
pollution. Methylmercury is formed from divalent
inorganic mercury by action of anaerobic sulfate-
reducing bacteria that live in aquatic systems
including lakes, rivers, sediments and the ocean. It is
the form of mercury that is the most easily
bioaccumulated in organisms. It is biomagnified in
aquatic food chains from bacteria, to plankton,
through macroinvertebrates, to herbivorous fish and
to piscivorous (fish-eating) fish. The concentration of
methylmercury in the top level aquatic predators can
reach a level a million times higher than the level in
water.
When analyzed, mercury was never detected in
samples of herbal drugs [23,26,35]. From 12,000
plant samples tested, it was concluded that its content
was insignificant with regard to a threshold value of
0.1 mg/kg [14].
1.3. Other elements: Very little information is
available regarding the presence of other elements in
herbal drugs and their preparations. Chromium and
arsenic were undetectable above their limits of
detection in different formulations prepared from
ginseng [26]. In one study, where samples of herbal
drugs were tested for thallium concentration, all 80
samples tested contained less than 0.01 mg/kg [15].
1.4. Cadmium, mercury and arsenic in seaweed:
Macroalgae can survive in toxic-metal-contaminated
aquatic environments. In particular, Fucus spp. may
accumulate heavy metals and have been widely used
as biomonitors of metal pollution. So, they may be
2110 Natural Product Communications Vol. 3 (12) 2008
suitable species for use in risk assessment, for
example for coast and estuarine areas. This tolerance
to heavy metals can be explained in bladderwrack
(Fucus vesiculosus L.) by its exceptional metalbinding properties due to metallothioneins, which are
thought to act as a protective mechanism against
incoming toxic metals, such as cadmium and
mercury, [36,37].
Concentration of cadmium in bladderwrack has been
extensively studied, showing that the level in
seaweed varies with environmental levels [38-41].
Cadmium levels in bladderwrack reached values in
the range of 15-25 mg/kg [39-41]. Concentrations of
cadmium are highest in spring and lowest in autumn
in F. vesiculosus and F. serratus [38,39].
Mercury levels in Fucus species (F. vesiculosus, F.
spiralis and F. ceranoides) were found in the range
0.01-0.06 mg/kg for receptacles and 0.03-0.29 mg/kg
for holdfast and stipe [42].
Levels of arsenic are higher in the aquatic
environment than in most areas of land as it is fairly
water-soluble and may be washed out of arsenicbearing rocks. In particular, seaweed is known to
contain high concentrations of arsenic in comparison
with terrestrial plants, owing to the ability of marine
plants to concentrate the arsenic they derive from sea
water. F. vesiculosus and Laminaria digitata contain
levels of arsenic typically between 20 and 100 mg/kg
dry weight [43,44]. Arsenic compounds can be
categorized as inorganic (without an arsenic-carbon
bond) and organic compounds (with an arseniccarbon-bond). Arsenic exists in three common
oxidation states: As(0) (metalloid arsenic, 0 oxidation
state), As(III) (trivalent state, such as arsenites),
and As(V) (pentavalent state, such as arsenates).
More than 90% of arsenic present in kelp is in the
form of arsenosugars (arsenoribofuranosides), while
inorganic arsenic is less than 5% [43]. Total and
“reducible” arsenic levels (the latter level reflects the
total of inorganic arsenic and some unstable
organoarsenic species and is thus regarded as an
indicator of maximum inorganic arsenic content)
were analyzed in kelp food supplements on sale in
the UK. They yielded mean concentrations of 25
mg/kg and 0.14 mg/kg for total and reducible arsenic,
respectively. Potential daily intakes of reducible
arsenic (based on maximum manufacturer’s dose
recommendations) ranged from 0.05 to 9 µg [45].
Inorganic arsenic was found at concentrations in the
range 67-96 mg/kg in hijiki seaweed [Hizikia
fusiforme (Harv.) Okamura], a brown sea vegetable
Guédon et al.
growing wild on rocky coastlines around Japan and
China [46]. The UK Food Standards Agency issued
advice to consumers to avoid eating it [47].
2. TOXIC METALS IN ASIAN TRADITIONAL
MEDICINES
Publications about unacceptable levels of toxic
metals in non-Western folk medicines have regularly
appeared in medical and pharmaceutical literature
[48-53]. The presence of those poisonous metals is
usually not due to contamination but to deliberate
inclusion for alleged medicinal purpose or to an
accidental addition in herbal preparations during their
manufacture. In particular, Chinese and Indian
preparations have been implicated. Taking into
account that in most developed countries, Asian
herbal medicines are becoming more and more
popular [54], it seems justified to describe in this
article those preparations which might constitute a
serious health problem.
Evidence suggesting that some Asian herbal
medicines contain toxic metals have been reviewed
[19,55]. For example, traditional Chinese medicines,
which are usually complex mixtures of several (often
20 or more) herbal drugs, may contain heavy metals
such as arsenic, cadmium, mercury and lead (see
[48,50,51,56] for a review). Similarly, Indian
medicinal systems (e.g. Ayurveda and Unani) have a
long and rich history of herbal medicine, and heavy
metals have been regular constituents of traditional
Indian remedies (see [57] for a review). Surveys
carried out during 2004 in the United States have
shown that 20% of all Ayurvedic medicines in the
Boston area contained potentially harmful levels of
lead, mercury or arsenic [52]. Most common
ingredients deliberately included for a specific
curative purpose are realgar (arsenic sulfide),
cinnabar (mercuric sulfide), calomel (mercurous
chloride), hydrargyri oxydum rubrum (red mercuric
oxide) and lithargyrum (lead monoxide) [58-60]. The
presence of heavy metals may also be the result of
accidental contamination during manufacture, for
instance, from grinding weights [61] or lead-releasing
containers [62] or other manufacturing utensils.
3. HEALTH HAZARD
The main threats to human health from heavy metals
are associated with exposure to lead, cadmium,
mercury and arsenic. They have no known vital or
beneficial effect on organisms, and their toxicity
often takes years to manifest and may go unsuspected
Toxic metals in herbal medicinal products
Natural Product Communications Vol. 3 (12) 2008 2111
[63]. They have been extensively studied and their
effects on human health regularly reviewed by
international bodies, such as WHO (see [4] for a
review).
The formula, given in the section dealing with
pesticide residues [3] for calculation of theoretical
maximum tolerable levels in food commodities
(mg/kg), from ADI values, can also be used to derive
similar levels for a toxic metal [8]:
Chronic exposure to cadmium can cause
nephrotoxicity in humans, mainly due to
abnormalities of tubular re-absorption [64]. Lead and
mercury can cause adverse effects on the renal and
nervous systems and can cross the placental barrier,
with potential toxic effects on the fetus [65, 66]. The
International Agency for Research on Cancer (IARC)
classified cadmium and lead as human carcinogens:
group I (sufficient evidence in both humans and
experimental animals), and “possible human
carcinogen”: group IIA (sufficient animal data and
insufficient human data), respectively [67,68]. Longterm exposure to arsenic is mainly related to
increased risks of skin, liver, bladder and lung cancer
[69,70].
PTWI x M
MWI x 100
M = body mass in kilograms (60 kg),
MWI = mean weekly intake of raw material, in
kilograms,
100 = general safety factor.
PTWI values established by JECFA for cadmium
[71], lead [72], mercury [71, 72] and arsenic [73] are
listed in Table 3. The EU Scientific Committee on
Food (SCF) adopted an opinion endorsing the PTWI
established by JECFA (see [74]).
3.1. Mercury
Trace elements (e.g. iron, cooper, nickel) may also
have toxicity when considered in abnormally high
doses. Metals in an oxidation state abnormal to the
body may also become toxic: chromium (III) is an
essential trace element, but chromium (VI) is a
carcinogen. Radioactive metals (e.g. thorium,
uranium, polonium) have both radiation toxicity and
chemical toxicity.
FAO/WHO recommended a PTWI value of 5
µg/kg/week for mercury, while the PTWI for
methylmercury of 3.3 µg/kg/week [72] was revised
by the 61st meeting of JECFA to 1.6 µg/kg/week [71].
Ingested methylmercury is readily and completely
absorbed by the gastrointestinal tract. It is mostly
found complexed with cysteine and with peptides and
proteins containing that amino acid. The
methylmercuric-cysteinyl complex is recognized by
amino acid transporting proteins in the body, as
methionine, another essential amino acid. Because of
this mimicry, it is transferred freely throughout the
body, including across the blood-brain barrier and
across the placenta, where it is absorbed by the
developing fetus. Because of this mimicry and its
strong binding to proteins, methylmercury is not
readily eliminated.
Contrary to pesticides, where the toxicological risk is
defined on the basis of Acceptable Daily Intake
(ADI) values (see “Health hazard” under [3]), it is
preferable for toxic metals to base the risk evaluation
on the so-called Provisional Tolerable Weekly Intake
(PTWI) values, established also by the Joint
FAO/WHO Expert Committee on Food Additives
(JECFA). PTWI is an endpoint used for food
contaminants such as toxic metals with cumulative
properties. This value (µg/kg body weight/week)
represents permissible human weekly exposure to
those contaminants unavoidably associated with the
consumption of foods.
3.2. Arsenic: FAO/WHO has established a
PTWI value only for inorganic arsenic
(15 µg/kg/week) without assigning any such value
to organic arsenicals [73]. JECFA are scheduled to
Table 3: Provisional Tolerable Weekly Intake (PTWI) values for toxic metals, as established by the joint FAO/WHO Expert Committee on Food Additives
(JECFA).
Metal
Lead
Cadmium
Mercury
Arsenic (inorganic)
PTWI (µg/kg/week)
Reference
25
7
51
152
JECFA 2000 [72]
JECFA 2003 [71]
JECFA 2000 [72]
JECFA 1989 [73]
1- But not more than 1.6 µg/kg/week in the form of methylmercury [71].
2- This PTWI does not refer to organoarsenicals, as the organoarsenic compounds naturally occurring in marine products are considerably less toxic than
inorganic arsenicals.
2112 Natural Product Communications Vol. 3 (12) 2008
review the PTWI for arsenic. The relative toxicity of
an arsenical depends primarily on inorganic or
organic form, oxidation state, solubility and rates of
absorption and elimination. Inorganic arsenic is more
toxic than organic arsenic (e.g. LD50 for arsenic
trioxide in rats is 20 mg/kg, while for arsenobetaine
no signs of toxicity were observed in mice after an
oral dose of 10 g/kg) [75,76]. The toxicity of As(III)
is several times greater than that of As(V), due to a
greater cellular uptake. However, at equivalent
intracellular levels, As(III) and As(V) compounds are
equipotent. For instance, organoarsenic compounds,
naturally occurring in fish (mainly arsenobetaine), are
excreted very rapidly by man and there have been no
reports of ill effects among populations whose
consumption of large quantities of fish results in
organoarsenical intakes of about 50 µg/kg per day
[73]. Although the arsenic most commonly found in
seaweed (organosugars) is relatively non-toxic to
humans as compared with inorganic species [77],
case reports of potential arsenic toxicosis secondary
to kelp supplements intake have, surprisingly, been
described [78,79].
3.3. Other toxic metals: No FAO/WHO PTWI value
for thallium has been established. The current dietary
intake of this toxic element in the United Kingdom,
which is estimated to be 5 µg/day, is not regarded as
a cause for concern [80].
3.4. Comments:The following points deserve special
consideration:
• The safety margin between a PTWI and the
weekly exposure that produces deleterious effects
can be relatively small. For instance, the critical
organ in relation to the toxic effects of chronic
ingestion of small amounts of cadmium is the
kidney. So, intakes as low as 140-255 µg/day
(PTWI = 60 µg/day/person with 60 kg bw) have
been associated with low molecular weight
proteinuria (without specific histological
changes) in the elderly and renal dysfunction
would be expected in sensitive population groups
at cadmium exposure levels half that of the
present PTWI, i.e. 30 µg/day [64]. On the other
hand, a short-term exposure to levels exceeding
the PTWI is not necessarily a cause for concern,
provided the average intake over longer periods
does not exceed the level set [73].
• As PTWI values refer to total dietary
intakes, it is impossible to determine the
acceptability of a certain contamination level in a
herbal drug without considering the normal
Guédon et al.
dietary exposure to the metal in question. Dietary
intakes measured in seven different countries
[81] showed that the normal daily diet may
provide a substantial portion of the tolerable
daily amount that can be derived from the PTWI,
dietary load of cadmium, lead and mercury
accounting for 17-55%, 6-42% and 2-31% of
tolerable daily amounts. Meanwhile, in many
countries, lead intake from the diet can approach
or exceed the PTWI [82]. Mercury exposure for
the general population occurs mainly from
consumption of fish [82] and possibly from
dental amalgam fillings [66].
• Experts are still debating whether PTWI for
cadmium can be used in general or whether it
should be applied only to healthy adults, with the
exclusion of risk groups such as chronically ill
patients, pregnant women, and breast-feeding
mothers [8].
Three other organizations, the National Institute of
Public Health and the Environment (RIVM, the
Netherlands), the Agency for Toxic Substances and
Disease Registry (ATSDR, the United States) and the
United States Environmental Protection Agency (US
EPA) have also evaluated the noncancer oral toxicity
data and established risk values for lead [83],
cadmium [83,84], mercury (mercuric chloride,
methylmercury) [83,85-87] and arsenic [83,88,89]
(see Table 4). Critical organs or effects are:
• brain and central nervous system (lead),
• kidney damage (cadmium),
• kidney, autoimmune effects (inorganic
mercury),
• central nervous system, developmental
effects (methylmercury),
• skin (inorganic arsenic).
On the basis of sufficient evidence for an increased
risk for cancer of the urinary bladder, lung and skin,
an oral risk value has only been established for
inorganic arsenic (6.7 ng/kg/day) [89].
3.5 Toxic metals in Asian traditional medicines:
The presence of heavy metals in ethnic medicines is
considered as being a significant international
problem. Lead and, less frequently, mercury and
arsenic poisonings, associated with intentional or
accidental addition of toxic heavy metals in
unlicensed Ayurvedic medicines, have been
described in developed countries with some
regularity during the last three decades (see [57,90]
for a review). Similarly, numerous case reports and
Toxic metals in herbal medicinal products
Natural Product Communications Vol. 3 (12) 2008 2113
Table 4: Other oral risk values for toxic metals.
Metal
Lead
Cadmium
Mercury (mercuric chloride)
Methylmercury
Arsenic (inorganic)
Risk value1
RIVM [83]
3.6 µg/kg/day
25 µg/kg/week
0.5 µg/kg/day
3.5 µg/kg/week
2 µg/kg/day
14 µg/kg/week
0.1 µg/kg/day
0.7 µg/kg/week
1 µg/kg/day
7 µg/kg/week
Risk value2
ATSDR [84, 85, 88]
Risk value3
US EPA [86, 87, 89]
-----
-----
0.2 µg/kg/day
1.4 µg/kg/week
----0.3 µg/kg/day
2 µg/kg/week
0.3 µg/kg/day
2 µg/kg/week
----0.3 µg/kg/day
2 µg/kg/week
0.1 µg/kg/day
0.7 µg/kg/week
0.3 µg/kg/day
2 µg/kg/week
1- National Institute of Public Health and the Environment (RIVM): Tolerable Daily Intake (TDI). 2- Agency for Toxic Substances and Disease Registry
(ATSDR): chronic oral Minimal Risk Level (MRL). 3- United States Environmental Protection Agency (US EPA): Reference Dose (RfD).
case series of heavy metal poisoning associated with
the use of traditional Chinese medicines (TCM) have
been published in different countries (United States,
Hong-Kong, Taiwan) (see [48] for a review).
Recently, out of 247 TCM products investigated in
Australia, some preparations exceeded FAO/WHO
PTWI for arsenic (4 products), lead (1 product) and
mercury (5 products), taking into account
recommended product dose and the concentration of
element in the product. The levels were high, ranging
up to three orders of magnitude (103) higher than the
PTWI for those elements considered [91].
It has also been shown that the passage of lead and
cadmium into an extraction fluid decreased as the
polarity of the extraction fluid diminished [7]. Thus,
there can be little doubt that processing of herbal
drugs with hydroethanolic mixtures likewise results
in incomplete passage of heavy metals.
4. EFFECTS OF PROCESSING
5. ANALYTICAL METHODS
The effects of extraction with boiling water were
studied by analyzing lead and cadmium levels in 136
samples of 19 herbal drugs and in tisanes prepared
from these samples. Passage into water was above
50% in only 12% and 8% of the lead and cadmium
assays, respectively. The majority of tea samples
(67% for lead and 71% for cadmium) showed a
relatively low extraction of 25% or less. Individual
extraction values ranged from 0.1% to 87% for lead
and 1% to 68% for cadmium [21, 22]. Otherwise, the
average percentage of lead and cadmium into the
infusion from herbal teas consumed in Thailand was
6-12% and 14-24%, respectively [92] and
determination of lead content in infusions or
decoctions obtained from 11 herbal teas showed
extraction values ranged from less than 1% to 59%
[30].
Taking into account the relatively low concentrations
of investigated toxic metals and complexity of the
plant matrices, adequate physical techniques are
necessary.
Different
methods
have
been
recommended for quantitative analysis of plant
samples, in order to satisfy relevant analytical criteria
such as specificity and precision, and to provide less
time consuming and cost beneficial results:
• flame atomic absorption/emission spectrometry
(FAAS/FAES);
• graphite
furnace
atomic
absorption
spectrometry (GFAAS);
• energy disperse X-ray fluorescence (EDX-RF);
• inductively coupled plasma-atomic emission
spectrometry (ICP-AES);
• inductively coupled plasma-mass spectrometry
(ICP-MS).
When an herbal drug is contaminated at its surface
with inorganic salts, it is likely that a relatively large
amount will dissolve in the hot water. However,
when metal traces are organically bound in the plant
cell, the passage rate into a tisane will be relatively
low [21, 22]. Besides, higher amounts of heavy
metals are transferred from the raw material to water
by boiling (decoction) than by immersion in hot
water (infusion) [12].
Although FAAS/FAES and GFAAS are efficient to
determine low levels of elements, techniques such as
ICP-AES and mainly ICP-MS are superior due to
multielement capabilities, low detection limits,
isotopic capabilities and speed of analysis. The
general chapter of the Ph. Eur. “Heavy metals in
herbal drugs and fatty oils” (2.4.27) provides test
methods for the estimation of lead, cadmium,
mercury, arsenic, nickel, copper, iron and zinc by
The washing of plants while they are still fresh may
remove about 15-30% of heavy metal contamination
[8]. For example, washing valerian roots after harvest
has shown to be an effective way in reducing lead
and cadmium contamination in crops [18].
2114 Natural Product Communications Vol. 3 (12) 2008
atomic absorption spectroscopy (2.2.23). The content
of arsenic and mercury is measured by direct
calibration (Method I) using an automated
continuous-flow hydride vapour generation system,
while the content of other metals is determined by the
standard additions method (Method II) using a
graphite furnace as atomisation device.
Other physical methods recently introduced in the 6th
edition of the Ph. Eur. (6.0 January 2008) are ICPAES (2.2.57) and ICP-MS (2.2.58).
The pharmacopoeial test for heavy metals (2.4.8),
despite its limitations, is still a standard test described
in individual monographs of the Ph. Eur. dealing
mainly
with
chemical
substances.
Some
improvements to simplify the test, avoiding loss of
analytes and increasing sensitivity, have recently
been proposed [93].
Methods used for the investigation of contaminants in
the food area, i.e. standard procedures established by
the European committee for standardization (CEN:
Comité européen de normalisation), include
determination of different trace elements. General
aspects of analytical methodologies, sample
preparation, quantitative analysis, precision of the
method and results of collaborative tests are fully
presented. Published standard procedures are listed
below.
5.1. Cadmium and lead
• Determination of lead, cadmium, zinc, copper,
iron and chromium by atomic absorption
spectrometry (AAS) after dry ashing (EN 14082:
2003) [94];
• Determination of lead, cadmium, chromium and
molybdenum by graphite furnace atomic
absorption spectrometry (GFAAS) after pressure
digestion (EN 14083: 2003) [95];
• Determination of lead, cadmium, zinc, copper
and iron by atomic absorption spectrometry
(AAS) after microwave digestion (EN 14084:
2003) [96].
5.2. Mercury
• Determination of mercury by cold-vapour atomic
absorption spectrometry (CVAAS) after pressure
digestion (EN 13806: 2003) [97].
5.3. Arsenic
• Determination of total arsenic by hydride
generation atomic absorption spectrometry
Guédon et al.
(HGAAS) after dry ashing (EN 14546: 2005)
[98];
• Determination of total arsenic and selenium by
hydride
generation
atomic
absorption
spectrometry
(HGAAS)
after
pressure
digestion (EN 14627: 2005) [99];
• Determination of inorganic arsenic in
seaweed by hydride generation atomic
absorption spectrometry (HGAAS) after acid
extraction (EN 15517, 2008) [100].
5.4. Cadmium, lead, mercury and arsenic
• Determination of arsenic, cadmium, mercury
and lead in foodstuffs by inductively coupled
plasma-mass spectrometry (ICP-MS) after
pressure digestion (standard project EN 15763:
2008) [101].
6. STANDARDS/GUIDELINES
6.1.
Foodstuffs:
Commission
Regulation
1881/2006/EC from December 2006 sets maximum
levels for certain contaminants in foodstuffs,
including maximum levels for toxic metals [74].
Maximum levels in vegetables, fruits, fresh herbs and
fungi are in the range of 0.10-0.30 mg/kg and 0.050.20 mg/kg for lead and cadmium, respectively (see
Table 5). Mercury is only controlled in fishery
products, considering that levels of mercury found in
foods other than fish and seafood are of lower
concern. Moreover, it is mentioned that “the forms of
mercury present in these other foods are mainly not
methylmercury and they are therefore considered to
be of lower risk” [74]. It should be noted that
Commission Directive 61/2004/EC specifies a limit
for mercury compounds expressed as mercury
(pesticides prohibited for use in European
Community) in food commodities of plant origin at
the limit of analytical determination (0.01 mg/kg for
food of plant origin and 0.02 mg/kg for tea and hops
[102].
French regulation sets maximum levels for mineral
arsenic, iodine and certain metals in seaweeds
intended for human consumption [103]. Limits
(values apply to the dried material) are as follows:
• mineral arsenic
: ≤ 3 mg/kg;
• iodine
: ≤ 5,000 mg/kg;
• cadmium
: ≤ 0.5 mg/kg;
• mercury
: ≤ 0.1 mg/kg;
• lead
: ≤ 5 mg/kg;
• tin
: ≤ 5 mg/kg.
Toxic metals in herbal medicinal products
Natural Product Communications Vol. 3 (12) 2008 2115
Table 5: Maximum limits of metals in foodstuffs (EU regulation).
Foodstuffs
Lead (mg/kg wet weight
Cereals
Vegetables
Leaf vegetables, fresh herbs, cultivated fungi
Fruit, excluding berries and small fruit
Berries and small fruit
Regulation 1881/2006/EC [74]
Cadmium (mg/kg wet weight)
0.20
0.10
0.30
0.10
0.20
0.10
0.050
0.20
---------
Table 6: Maximum limits of metals in food supplements [104].
Heavy metals
Foodstuffs
Maximum levels (mg/kg wet weight)
3.0
Lead
Food supplements1, dried seaweed1
Cadmium
Food supplements1
1.0
Cadmium
Food supplements consisting exclusively or mainly of dried seaweed or of products derived
from seaweed, dried seaweed1
Food supplements1, dried seaweed1
3.0
Mercury
0.1
1- The maximum level applies to the finished product.
6.2. Food supplements
6.3. Herbal Medicinal Products
It has been shown that food supplements can
contribute significantly to human exposure to lead,
cadmium and mercury as high levels of these toxic
metals have been found in some food supplements
during monitoring activities in European member
states. In order to protect public health, the
Commission of the European Communities considers
that it is appropriate to set maximum levels for lead,
cadmium and mercury in food supplements via an
amendment of the Regulation 1881/2006/EC. A
revised draft Commission Regulation has recently
been released including the following comments and
proposals [104]:
• maximum levels must be safe and as low as
reasonably achievable (ALARA), based upon
good manufacturing practices. Proposals for
limits are listed in Table 6;
• due to natural accumulation of cadmium in
seaweed, food supplements consisting of either
dried seaweed or of products derived from
seaweed can contain higher levels of cadmium
than other food supplements. So, a higher
maximum level for cadmium is needed for food
supplements consisting exclusively or mainly of
seaweed (Table 6).
6.3.1. Pharmacopoeias: Up to now, there are no
binding criteria established by Ph. Eur. for toxic
metals in herbal drugs. At present, with a few
exceptions, no limit is prescribed in individual
monographs for herbal drugs. Exceptions are:
• kelp (Ph. Eur.: 1426): lead (≤ 5 mg/kg), cadmium
(≤ 4 mg/kg), mercury (≤ 0.1 mg/kg) and total
arsenic (≤ 90 mg/kg);
• linseed (Ph. Eur.: 0095): cadmium (≤ 0.5 mg/kg);
• red vine (Ph. fr.): copper (≤ 200 mg/kg) [106].
The draft Regulation favours the control at the final
product level.
Maximum levels for heavy metals in food
supplements have been set out so far only in Belgium
at the national level [105]. Limits in final products as
sold are:
• mercury
: ≤ 0.2 mg/kg;
• cadmium
: ≤ 0.5 mg/kg;
• lead
: ≤ 1 mg/kg;
•
arsenic
: ≤ 1 mg/kg.
Presently, the general monograph on “Herbal drugs”
(1433) states that a potential risk must be considered
and, unless included in an individual monograph,
limits may be required if justified.
A draft proposal for a revision of this monograph has
recently been published [107] including, amongst
changes, the introduction of limits for certain heavy
metals. These are as follows “unless otherwise stated
in an individual monograph or unless otherwise
justified and authorised:
• cadmium
: ≤ 0.5 mg/kg;
• lead
: ≤ 5 mg/kg;
• mercury
: ≤ 0.1 mg/kg.
It is also specified that “if required by the relevant
authority or by the nature or origin of the herbal drug,
suitable limits for the contents of arsenic, copper,
iron, nickel and zinc are defined”.
Additionally, the general monograph on “Extracts”
(0765) specifies “where applicable, as a result of
analysis of herbal drug or animal matter used for
production and in view of the production process,
tests for microbiological quality, heavy metals,
2116 Natural Product Communications Vol. 3 (12) 2008
aflatoxins or pesticide residues in the extract may be
required”. In relationship to the introduction of
proposed limits for certain heavy metals in the
monograph “Herbal drugs” (see above), a provision
has been introduced in a revised draft of the
monograph “Extracts” [108]. Herbal drugs used for
extraction could exceed the limits set for these ones
“provided the finished extract complies with the
requirements concerning heavy metals in the general
monograph Herbal drugs (1433)”.
6.3.2. European guidelines: The guideline on “Good
Agricultural and Collection Practice” (GACP) [109]
specifies that “medicinal plants should not be grown
in soil contaminated with sludge, heavy metals,
residues, plant protection products or other chemicals
etc.”.
According to the CPMP guideline (EMEA) on
“Quality” [110]: “as a general rule, herbal substances
must be tested, unless otherwise justified, for
microbiological quality and for residues of pesticides
and fumigation agents, toxic metals, likely
contaminants and adulterants”. For herbal
preparations, the guideline comments “if deemed
necessary by analysis of the starting material, tests on
microbiological quality, residues of pesticides,
fumigation agents, solvents and toxic metals should
be performed”. The other CPMP guideline (EMEA)
on “Specifications” [111] stresses the point that “the
need for inclusion of tests and acceptance criteria for
inorganic impurities should be studied during
development and based on knowledge of the plant
species, its cultivation and the manufacturing
process. Acceptance criteria will ultimately depend
on safety considerations”. It is also mentioned
regarding herbal preparations that “the potential for
manufacturing process to concentrate toxic residues
should be fully addressed. If the manufacturing
process will reduce the burden of toxic residues, the
tests on the herbal substance may be sufficient”.
6.3.3. National regulations: The German Ministry of
Health published in 1991 a “draft recommendation
for limits of heavy metals in medicinal products of
plant and animal origin” [112]. This draft included
the following limits for plants, parts of plants, oils,
fats and waxes of plant origin and products thereof as
well as for other products of plant origin, each with
reference to the dried matter:
• lead
: ≤ 5 mg/kg;
• cadmium
: ≤ 0.2 mg/kg;
• mercury
: ≤ 0.1 mg/kg.
Guédon et al.
The following exceptions were considered for
cadmium: 0.3 mg/kg for linseed, hawthorn, yarrow
and 0.5 mg/kg for birch, St John’s wort, willow and
maté.
The draft recommendation has never been finally
adopted. However, these maximum levels have been,
so far, widely used as acceptance criteria for the
assessment of marketing authorisation/registration
application dossiers by EU national regulatory
authorities.
6.4. WHO
WHO guidelines on “Quality control methods for
medicinal plants” [113] and “Quality of herbal
medicines with reference to contaminants and
residues” [114] recommend to take into consideration
maximum limits of heavy metals. The following
levels in dried plant material, based on ADI values,
have been proposed:
• lead
: ≤ 10 mg/kg,
• cadmium
: ≤ 0.3 mg/kg.
The following recommendation is given “the need for
the inclusion of tests for toxic metals and acceptance
criteria should be studied at the various development
stages of the plant and based on knowledge of the
medicinal plant species, its growth and/or cultivation
and the manufacturing process” [114]. Additionally,
if the heavy metals burden of the herbal material is
unknown, “it is suggested that it can be determined
qualitatively and quantitatively on several batches,
preferably collected over several years” [114].
7. LITERATURE RECOMMENDATIONS
The
German
pharmaceutical
manufacturers
association (BAH), based on results collected from
more than 20,000 samples by its contaminant
working group (see also “I.1.1. Cadmium and lead”),
showed that recommendations of the German
Ministry of Health (see above “V.3.3. National
regulations”) could not generally be applied [14,
115]. So, it made a proposal in 2002 for general
limits:
• lead
: ≤ 10 mg/kg;
• cadmium
: ≤ 1 mg/kg;
• mercury
: ≤ 0.1 mg/kg;
• arsenic
: ≤ 5 mg/kg;
• nickel
: ≤ 10 mg/kg;
• copper
: ≤ 40 mg/kg.
Based on an appropriate amount of data available,
90th percentile appears to be a practical limit in view
of the limited availability of many herbal drugs. It is
Toxic metals in herbal medicinal products
Natural Product Communications Vol. 3 (12) 2008 2117
Table 7: Proposal of cadmium maximum levels for certain herbal drugs
(BAH proposal) [116].
Cadmium : ≤ 0.3 mg/kg
Wormwood
Angelica (root)
Arnica
Hawthorn (leaf & flower)
Elecampane
Artichoke
Milk thistle
Damiana
Strawberry
Raspberry
Gentian
Kava-kava
Linseed
German chamomile
Nettle (root)
Passionflower
Buckhorn plantain
Silver linden (flower)
Valerian
Cadmium : ≤ 0.5 mg/kg
Yarrow
Cornflower
Birch
Buckthorn
St Benedic thistle
Coughgrass
Mistletoe
Mashmallow
Rupturewort
Immortelle
Island moss
Lovage
Maté
St John’s wort (herb)
Lily of the valley
Dandelion (root)
Common horsetail
Lungwort
Roselle
Wild thyme
Pot marigold
Stevia
Speedwell
Specific limits regarding cadmium :
Belladonna (leaf)
English holly
Wild pansy
Cinchona
≤ 1.0 mg/kg
≤ 3.5 mg/kg
≤ 1.5 mg/kg
≤ 1.5 mg/kg
Willow
Sanicle
Thyme
Zedoary
≤ 3.0 mg/Kg
≤ 0.6 mg/Kg
≤ 0.8 mg/Kg
≤ 1.0 mg/Kg
considered that the usual small quantities of herbal
drugs consumed do not pose a health hazard to the
population [14]. As already described (see “I.1.1.
Cadmium and lead”), many herbal drugs have a 90th
percentile over 0.5 mg/kg (and even over 1.0 mg/kg)
of cadmium [14]. So, it was observed that exceptions
should be established for specific herbal drugs with
90th percentile higher than 1 mg/kg, e.g. St John’s
wort, willow and fucus.
Previously, based on a restrictive cadmium maximum
level of 0.2 mg/kg [112], BAH made, in 1992, a
proposal for exemptions of a large number of herbal
drugs known to have a specific affinity to cadmium
[116] (see Table 7).
8. CONCLUDING REMARKS
Contamination of medicinal plants and herbal drugs
by heavy metals has been extensively documented in
published literature [5-16,18,20-35]. Based on the
present survey, different observations can be made:
• first of all, the deliberate addition or not
(accidental
contaminations
during
manufacturing) of compounds such as cinnabar
(HgS) or realgar (As4S4) to certain preparations,
allowable by Chinese and Indian traditional
medicines [19,48-62,90,91], must be clearly
differentiated from the presence of heavy metals
due
to
environmental
pollution.
Such
unacceptable levels of toxic metals in non-
Western traditional herbal remedies should not
overestimate the real risk due to the presence of
toxic metals in herbal drugs;
• mercury: no literature has been found supporting
a risk of level above a threshold value of 0.1
mg/kg in medicinal plants [14, 23, 26, 35], with
the exception of seaweed like bladderwrack
where it may accumulate [42]. Toxicity is mainly
due to methylmercury as it is readily and
completely absorbed by the gastrointestinal tract.
Mercury exposure and especially its organic
form, methylmercury, for the general population
occurs mainly from consumption of fish and
seafood [82], which explains why maximum
levels in foodstuffs apply only to fishery products
[74];
• arsenic: the ability to concentrate arsenic has
been described in marine plants such as kelp [4345]. Organic arsenic is much less toxic than
inorganic arsenic (e.g. arsenosugars) due to its
rapid excretion by man, thereby a PTWI value
has only been established for inorganic arsenic
[73].
The three main factors which may lead to high
levels of toxic metals in medicinal plants are the
following:
• anthropogenic influence: human activities lead to
the production of available (soluble) forms of
toxic metals in the soils, the main source of such
elements for terrestrial plants. Furthermore, some
anthropogenic activities may also cause heavy
metals contamination of plants by deposition of
air particles on the aboveground organs.
Contamination via air pollution may be the most
significant source of heavy metals in certain
areas (e.g. vicinity of mining industry, smelting
works, waste incineration plants).
Agricultural chemicals may also contribute to
high levels of toxic metals (e.g. phosphate
fertilizers, which contain varying quantities of
cadmium depending on the original content of
the phosphate rocks, sewage sludge-amended
soils, pesticides, such as lead arsenate or
mercury-containing products which continue to
be used today in some countries) [4,6,810,15,16,18,114];
• availability in the soil: soil characteristics such as
pH, redox-potential, content and form of organic
matter or clay minerals affect heavy metal
chemical behaviour in soils. Amongst these
factors, soil pH is probably the most important
one (availability of all elements, with the
2118 Natural Product Communications Vol. 3 (12) 2008
exception of molybdenum, increases as pH
decreases) [4,5];
• uptake by the plant: genetic features of certain
plant species show a tendency to accumulate
trace elements, the most characteristic being
cadmium [6,12,14]. Plants which are prone to
increase assimilation of that element are named
“cadmium collector” (e.g. St John’s wort, willow,
yarrow, linseed, birch, wild pansy, dandelion)
[5,13,14,22,25,27,31,32].
So, excluding species which have a natural tendency
to accumulate cadmium, cultivated plant materials
may be considered as being more at risk of
contamination than raw materials collected in wild
habitats.
9. GENERAL RECOMMENDATIONS
As already mentioned, the CPMP guideline (EMEA)
on “Specifications” [111] stresses the point that “the
need for inclusion of tests and acceptance criteria for
inorganic impurities should be studied during
development and based on knowledge of the plant
species, its cultivation and the manufacturing
process”. Taking into account previous comments,
criteria, which should be part of a risk assessment,
include:
• presence or not of anthropogenic influences in
the vicinity of the site of cultivation or wild
collection resulting in a potentially contaminated
environment;
• soil pH where the herbal drug is
harvested/collected. Medicinal plants, as all
plants, may exhibit or not different soil
preferences, e.g. preference for soils rich or poor
in lime (high and low pH values, respectively);
• cultivation techniques (e.g. use of fertilizers);
• ability of the plant species to uptake and
accumulate toxic metals (mainly cadmium).
Information justifying that testing heavy metals on a
routine basis can be waived (i.e. no contamination to
be expected following the risk assessment) should
preferably be supported by development data,
including at least three suitable representative
batches. Assurance must also be provided that the
medicinal plant was cultivated/collected in
accordance with the principles of Good Agricultural
and Collection Practice [109].
Moreover, heavy metals are most often weakly
extracted into the herbal drug preparations during
Guédon et al.
processing (e.g. infusion, alcoholic extracts).
Therefore, it is fully appropriate, as included in the
revision draft for the Ph. Eur. monograph “Extracts”
[107], to consider the possibility of using raw
materials exceeding the limits set for herbal drugs
(see proposed revision for the monograph “Herbal
drugs” [108]), provided the herbal preparation
complies with the herbal drugs requirements.
9.1. Cadmium and lead: Harmonised maximum
limits which are scheduled in the revised
pharmacopoeial monograph “Herbal drugs” (Ph. Eur.
1433) for cadmium (≤ 0.5 mg/kg) and lead
(≤ 5 mg/kg) [107] are achievable for most herbal
drugs. Meanwhile, exceptions for cadmium collectors
need to be considered, some herbal drugs containing
often cadmium levels above 0.5 mg/kg. It is presently
the case for kelp where a maximum level of 4 mg/kg
is set up (Ph. Eur.: monograph “Kelp” 1426).
Assuming a mean daily consumption of 5 g herbal
drug (average daily dose according to the monograph
“Tisanes” of the French Pharmacopoeia [117]), a
passage rate of heavy metals into hot water of 20%
[21,22] and a concentration of 5 mg/kg lead and 0.5
mg/kg cadmium, the estimated weekly intake would
represent 2% and 0.8% respectively of the PTWI
recommended by the WHO for an adult of 70 kg
(1,750 µg of lead and 490 µg of cadmium) [71,72].
So, the consumption of herbal drugs is not expected
to contribute significantly to the exposure of the
population to cadmium and lead.
9.2. Mercury: As mercury exposure through
consumption of herbal drugs of terrestrial origin does
not appear to be of health concern, as its uptake is
insignificant, inclusion of the test is not justified.
9.3. Arsenic: Arsenic is not identified as being a risk
in herbal drugs of terrestrial origin. Based on safety
considerations, it is advisable, if necessary, to include
in individual monographs testing of inorganic arsenic
rather than total arsenic, based on standard EN 15517
[100]. The official monograph “Kelp” (Ph. Eur.:
1426) should be modified accordingly as only
inorganic arsenic is to be considered a risk (presently,
Ph. Eur. sets a limit of 90 mg/kg total arsenic in
kelp).
9.4. Other trace elements: Only, if required by
the nature or origin of the herbal drug, a suitable
limit for the content of other trace elements (e.g.
copper when used as fungicide) may be defined.
Toxic metals in herbal medicinal products
Natural Product Communications Vol. 3 (12) 2008 2119
9.5. Food supplements: Current proposals for
legislation on heavy metals in food supplements
[104] are not identical to those scheduled by the Ph.
Eur. [108], i.e. for cadmium 1 mg/kg and 0.5 mg/kg,
and for lead 3 mg/kg and 5 mg/kg, respectively. An
harmonisation of maximum limits should be justified
as food supplements and herbal remedies have
similar specificities, i.e. they have a defined serving
size (e.g. 1 tablet/day) with a small amount of
product consumed daily in comparison to typical
food and a duration of use which is also limited.
Maximum levels scheduled by the Ph. Eur. [107]
represent practicable limits on the basis of the present
literature survey. Additionally, the contaminant limits
should be set for the raw materials and not for the
final products as sold, as specified in the draft
Regulation [104].
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NPC
Natural Product Communications
A Fresh Insight into the Interaction of Natural Products
with Pregnane X Receptor
2008
Vol. 3
No. 12
2123 - 2128
Salvador Máñez
Departament de Farmacologia, Universitat de València, Av. Vicent Andrés Estellés s/n,
46100 Burjassot, Spain
salvador.manez@uv.es
Received: July 29th, 2008; Accepted: October 20th, 2008
The discovery that various drugs (e.g., phenobarbital) stimulate their own metabolism through a mechanism coined as
enzymatic induction opened up a fascinating road that eventually led to the accurate biochemical characterization of the
pregnane X receptor. After numerous studies, researchers have concluded that this receptor is activated by different
endogenous steroids and a number of foreign lipophile ligands. Once activated, it induces the synthesis of oxygenases and
conjugating enzymes. The activating ligands identified to date include many synthetic drugs, along with a number of natural
products. The present review summarizes the data relating to the origin, chemistry, and pharmacological activity of the newest
natural products that have been found to interact with the pregnane X receptor.
Keywords: natural products , nuclear receptors, pregnane X, xenobiotics.
As a result of their work on the pregnane X receptor
(PXR, NR1I2), Matic et al. (2007) aptly dubbed it a
“promiscuous regulator of detoxification pathways”
[1]. Indeed, this short, clear definition of PXR, which
belongs to the much larger class of nuclear receptors,
is a perfect description. When activated by its
ligands, the receptor heterodimerizes with retinoid
receptor (RXR), binds to DNA, and induces the
expression of genes that encode the synthesis of
cytochrome
P450
(CYP)
monooxygenases.
Interestingly, both CYPs and PXR are co-localized in
many types of tissue, including the liver, intestine,
and kidney [1,2].
It is well-known that drug detoxification begins with
a hepatic metabolism carried out by CYPs, which
cause chemical modifications to xenobiotics through
the use of oxygen and a reduced flavoprotein [3].
This process also affects endogenous mediators such
as steroid hormones, bile acids, and several other
lipids, including eicosanoids. The most important
human CYP is CYP3A4, mainly because of its
abundance in human hepatocytes and the variety of
its susceptible substrates [4]. For its part, PXR
activation is responsible for the increased expression
of phase II conjugating enzymes and phase III
transporters [5].
While the term pregnane refers to the 17-ethyl-steroid
skeleton shared by some of the substrates
metabolized, the letter X means that this is an orphan
receptor, that is, without a known endogenous ligand.
Teleologically speaking, CYPs are cleansing agents
because they generally help eliminate potentially
toxic substances.
However, given that newly
generated products can sometimes be highly toxic,
activators of PXR expression may actually produce
deleterious effects. Such is the case of troglitazone, a
tocopherol-like agonist of the family of peroxisome
proliferator-activated receptors (PPARs), which was
introduced, and then later withdrawn, as an
antidiabetic drug [6].
Perhaps one of the most striking features of PXR is
the vast range of compounds that can serve as ligands
for it. In fact, there is no prerequisite chemical
structure or function for a given substance to bind
and activate the receptor. Some synthetic hormonal
steroids, such as dexamethasone, mifepristone and
pregnenolone-16-α-carbonitrile (PCN); or antibiotics,
like rifampicin, were promptly described as PXR
ligands [7].
2124 Natural Product Communications Vol. 3 (12) 2008
According to the reviews by Staudinger et al. [8] and
by Chang and Waxman [9], several natural products,
both molecular entities and herbal products (sic),
were reported to interact with PXR, mostly as
activators. Some of these compounds are wellknown drugs, such as paclitaxel, the antitumor
principle from Taxus brevifolia (Taxaceae);
artemisinin, a sesquiterpenoid from Artemisia annua
(Asteraceae); and forskolin, a diterpenoid from
Coleus forskohlii (Lamiaceae). Others are active
ingredients of pharmacologically active extracts,
including hyperforin from Hypericum perforatum
(Hypericaceae) and both E- and Z-guggulsterones
from Commiphora mukul (Burseraceae). Among the
very few PXR-antagonists, ecteinascidin-743
(Yondelis®), a complex phenolic tris-isoquinoline
from the tunicate Ecteinascidia turbinata, deserves
special mention.
2. Natural activators of PXR
2.1. Diterpenoids: Cafestol is a furanoditerpene
alcohol (Figure 1) present in Coffea seeds, and
therefore also in some drinking preparations, such as
the unfiltered coffees of Turkey and Scandinavia.
Among the biologically significant activities of this
compound, increasing blood cholesterol [10],
enhancing glutathione-based detoxification [11], and
inhibiting oxidative damage to DNA [12], are several
of the best-known. Studies on gene expression related
to lipid metabolism, for example, showed that
cafestol down-regulates cholesterol 7α-hydroxylase
(CYP7A1),
thereby
repressing
bile
acid
synthesis [13]. For our purposes, it is even more
important to note that homozygous deletion in
the human CYP7A1 gene causes pronounced
hypertriglyceridemia and LDL-hypercholesterolemia
[14]. Indeed, Ricketts et al. found that, at 20 μM,
cafestol activates PXR in HepG2 cells, with no
significant effects on other nuclear receptors such as
RXRs, glucocorticoid receptors, or the constitutive
androstane receptor (CAR) [15].
The effects of cafestol were not thoroughly extended
throughout the body; in fact, no induction of
CYP3A11 was detected in mouse livers either 3, 7, or
14 days after administration, whereas a nearly
fourfold increase was found in intestinal tissues.
However, the expression of the detoxificant enzyme
glutathione-S-transferase μ1 (GSTμ1) increased in
both the liver and the intestine. Interestingly, in
contrast with the standard PXR agonist, PCN, the
effect of cafestol on liver GSTμ1 was independent of
Máñez
Figure 1: Cafestol
PXR. As for lipid metabolism, cafestol induced ATPbinding cassette (ABC) transporter type A1
(ABCA1), a protein that is responsible for
transporting cholesterol from enterocytes into the
circulating blood [15]. This may partially explain
why this diterpene markedly raises blood cholesterol
levels.
2.2. Phenolic compounds
2.2.1. Phenylpropanoids
Coumarins: Many plant coumarins possess an
isoprene-derived pyran or furan cycle fused to the
benzo-α-pyrone basic structure, leading to the socalled pyranocoumarins or furanocoumarins. Such
compounds are quite toxic and have limited
pharmacological value, with the exception of 8methoxypsoralen (Figure 2), which is used in
combination with UV radiation for the treatment of
psoriasis and certain malignant dermatosis.
Figure 2: 8-Methoxypsoralen
This compound causes different metabolism-related
interactions with other drugs, such as nicotine [16]
and estrogens [17], and induces CYP1A1-mRNA
activation in rat hepatocytes. However, it is also
known to markedly inhibit CYP1A1 catalytic activity
[18]. The involvement of PXR in the induction not
only of CYPs, but also of carboxyl esterase 2 (HCE2)
by 8-methoxypsoralen has been assessed with the aid
of both overexpression and knockdown of PXR in
human hepatoma (Huh7) cells. In cells transfected
with the siPXR construct, CYP3A4-, HCE2-mRNA,
and PXR levels were reduced, whereas PXR
overexpression led to an increase in these
indicators [19].
Flavonoids and lignans: In a survey examining the
possible activity of several well-known phenolics,
Kluth et al. (2007) reported that 25 µM of quercetin
(3,5,7,3’,4’-pentahydroxyflavone), one of the most
widespread flavonols, activated the expression of
Interaction of natural products with pregnane X receptor
CYP3A4 in HepG2 cells transfected with the
reporter gene construct pGL3-CYP3A4-9. Its effect
was higher than that of the standard PXR activator
rifampicin [20]. However, quercetin did not increase
PXR-mediated gene expression, a finding that is in
disagreement with a previous report characterizing
the compound as a low-grade PXR activator [21]. A
moderate activation of PXR occurred with
secoisolariciresinol at 10 µM [21]. This latter
compound is a dibenzylbutane lignin present in
flaxseeds and other plant products in glycosidic form.
2.2.2. Alkylphenols
For the purpose of this review, this group includes
several non-phenylpropanoid phenols reputed to be
chemopreventive agents. As weakly suggested by
this label, these are cancer preventive substances,
generally of natural origin, which inhibit
tumorigenesis
through
various
mechanisms:
inhibition of proinflammatory transcription factors,
proliferation of malignant cells, synthesis of
antiapoptotic
proteins,
leukocyte
adhesion,
angiogenesis, etc [22].
Two of the most widely analyzed alkylphenols,
curcumin (diferuloylmethane, from turmeric,
Figure 3) and resveratrol (3,5,4’-trihydroxy-transstilbene, from red grapes), activated PXR-mediated
gene expression.
Figure 3: Curcumin.
At 25 µM, this effect was almost three times higher
for curcumin than for resveratrol. Both compounds
were also found to increase CYP3A4 promoter
activity, albeit to a lesser extent than quercetin (see
the epigraph “Flavonoids and lignans” [21]).
3. Natural inhibitors of PXR
3.1. Steroids
Plant sterols, for example, stigmasterol (Figure 4),
campesterol, and sitosterol, are widely considered to
be beneficial dietary constituents because they
allegedly lower cholesterolemia. However, in certain
cases, the effects may be surprisingly contrary.
When there is a mutation in the genes regulating the
Natural Product Communications Vol. 3 (12) 2008 2125
Figure 4: Stigmasterol
synthesis of the ABC-type transporters G5 or G8,
high sterolemia can result, causing severe
coronariopathy. Furthermore, feeding babies with
sterol-rich soy products can lead to cholestasis and
hepatic failure [23].
Given that some nuclear receptors are of key
importance in the control of bile acid toxicity, Carter
et al. investigated the possible interaction with
farnesoid X receptor (FXR) and other constructs
containing ligand binding domains of another six
nuclear receptors. In the context of the present
review, it is noteworthy that stigmasterol acetate
suppressed the activity of the PXR ligand binding
domain in HepG2 cells, with notable specificity [24].
This set of experiments reveals how sterols can be
toxic by depriving the liver of detoxification
pathways based on nuclear receptors.
3.2. Phenolic compounds
Unlike the vast majority of naturally occurring
coumarins (benzo-α-pyrones), which are derived
from ortho-hydroxycinnamic acid, coumestrol
(Figure 5) and its related coumarins present a
4-phenyl substitution. These types of compounds
are also known as neoflavones because they can
be formally considered as isomers of flavones
(2-phenyl-benzo-γ-pyrones).
As is the case with many isoflavones (3-phenylbenzo-γ-pyrones), coumestrol possesses estrogenic
properties, hence the name. In addition, this agent is a
PXR antagonist, as proven by Wang et al. These
researchers found that coumestrol reduced the basal
reporter activity, as measured by the xenobiotic
responsive enhancer module (XREM)-luciferase, in
the PXR transiently transfected CV-1 cells by 20% at
a dose of 25 µM. While many other nuclear receptors
were not sensitive to the compound, ERα and ERβ
were highly activated. The activation of human PXR
by rifampicin was also inhibited by coumestrol, albeit
not by its dimethyl or diacetyl derivatives. These
results indicate that the presence of the two free
2126 Natural Product Communications Vol. 3 (12) 2008
Figure 5: Coumestrol
phenolic hydroxyls is of the utmost importance. From
the results of the dose-response studies, the IC50 value
was calculated to be 12 µM. It was also confirmed
that coumestrol competes for the ligand-binding
domain (Ki = 37 µM) with the PXR agonist SR12813
(4-[2,2-bis(diethoxyphosphoryl)-ethenyl]-2,6-ditertbutylphenol). Gene expression studies for CYP3A4
and CYP2B6 in primary cultures of hepatic cells
from two human donors helped determine that 25 µM
of coumestrol was able to abolish the induction
brought about by SR12813 or rifampicin [25].
3.3. Sulfur compounds
Sulforaphane (Figure 6) is the trivial name of
1-isothiocyanato-4-(methylsulphinyl)-butane, a linear
sulfur compound derived from the hydrolysis of
glucoraphanin, a glucosinolate found in many
cruciferous plants (e.g. the genus Brassica), specially
in broccoli sprouts.
Figure 6: Sulforaphane
It had previously been shown that sulforaphane
strongly reduces CYP3A4 mRNA in human
hepatocytes, an effect which, in addition to others
such as the inhibition of histone deacetylases or the
induction of glutathione transferases, may be
implicated in the experimental anticancer effect of
the drug [26]. Zhou et al. [27] have recently shown
how PXR inactivation correlates to the inhibition of
CYP3A4 expression. Indeed, at 1 µM, sulforaphane
inhibited the reporter activity of the PXR ligands
mifepristone and rifampicin; at 25 µM the inhibition
of mifepristone was complete. After transfection of
HepG2 cells with a GAL4-PXR vector, sulforaphane
inhibited GAL4 reporter activity with an IC50
value of 14 µM. Moreover, it was demonstrated
that the test compound binds to the ligand
binding domain of PXR with a Ki of 16 µM. The
authors also studied several related isothiocyanates
present in Brassicaceae, including iberin (1-
Máñez
isothiocyanato-3-(methylsulfinyl)propane), cheirolin
(1-isothiocyanato-3-(methylsulfonyl)propane), and
erucin
(1-isothiocyanato-4-(methylthio)butane).
Iberin and cheirolin had roughly the same potency as
sulforaphane in repressing the PXR-mediated
CYP3A4-luciferase reporter activity in HepG2 cells
[27].
4. Concluding remarks
Although the restricted scope of this review prevents
us from making too many general assumptions,
several interesting points should be emphasized.
Vegetal secondary metabolites are xenobiotics for
mammalian organisms, which is not surprising since
a long list of microbial and plant principles show
interaction with PXR; at least as activator ligands.
Indeed, the latest studies have added even more
compounds to the list of novel PXR inhibitors, which
have been dubbed, perhaps inappropriately,
“antagonists.” As there is no chemical structure that
determines the binding of PXR to date, there is
likewise no particular chemical clue which would as
yet indicate either the activation or inhibition of such
a receptor.
As noted above, it is widely thought that PXR not
only regulates the detoxification of many structurally
unrelated foreign agents, but that it also participates
in the metabolism of endogenous steroids. In this
context, it is surprising that so few plant steroids and
triterpenoids have been studied. In fact, since the
discovery of guggulsterones, no new studies have
been published in this highly interesting field.
For now, the therapeutic value of PXR activators or
inhibitors remains obscure. We refer to drugs that
owe their application to interactions with PXR, but
not to other, some times very important (paclitaxel,
rifampicin) drugs that, apart from their main action,
have been shown to be notable PXR activators. On
the other hand, the role as pharmacologically active
ingredients of foods, such as sulforaphane, deserves
careful attention.
Acknowledgment - The author is indebted with
Spanish Ministry of Science for financial support
(Project SAF2006-06726) and Laura Gatzkiewicz for
her English revision.
Interaction of natural products with pregnane X receptor
Natural Product Communications Vol. 3 (12) 2008 2127
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NPC
Natural Product Communications
Natural Products as Gastroprotective and Antiulcer Agents:
Recent Developments
2008
Vol. 3
No. 12
2129 - 2144
Rosa Tundisa,*, Monica R Loizzoa, Marco Bonesia, Federica Menichinib, FilomenaConfortia,
Giancarlo Stattia and Francesco Menichinia
a
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Nutritional and Health Sciences,
University of Calabria, I-87030 Arcavacata di Rende (CS) Italy
b
Pharmaceutical Sciences Research Division, King’s College London, 150 Stamford Street, London
SE1 9NH, UK
tundis@unical.it
Received: May 27th, 2008; Accepted: October 3rd, 2008
Peptic ulcer, one of the most common gastrointestinal diseases, is a chronic inflammatory disease characterized by ulceration
in the regions of the upper gastrointestinal tract where parietal cells are found and where they secrete hydrochloric acid and
pepsin. The anatomical sites where ulcer occurs commonly are stomach and duodenum, causing gastric and duodenal ulcer,
respectively. Physiopathology of ulcer is due to an imbalance between aggressive factors, such as acid, pepsin, Helicobacter
pylori and non-steroidal anti-inflammatory agents, and local mucosal defensive factors, such as mucus bicarbonate, blood flow
and prostaglandins. Several drugs are widely used to prevent or treat gastro-duodenal ulcers. These include H2-receptor
antagonists, proton pump inhibitors and cytoprotectives. Due to problems associated with recurrence after treatment, there is
therefore the need to seek alternative drug sources against ulcers. In recent years, a widespread search has been launched to
identify new gastroprotective drugs from natural sources. The aim of the present review is to highlight the recent advances in
current knowledge on natural products as gastroprotective and antiulcer agents and consider the future perspectives for the use
of these compounds.
Keywords: gastroprotective agents, plant extracts, terpenes, flavonoids, xanthones.
Peptic ulcer disease is a problem of the
gastrointestinal tract characterized by mucosal
damage secondary to pepsin and gastric acid
secretion. It usually occurs in the stomach and
proximal duodenum; less commonly, it occurs in the
lower esophagus, the distal duodenum or the
jejunum, as in hypersecretory states, hiatal hernias or
ectopic gastric mucosa. Helicobacter pylori infection
and the use of non-steroidal anti-inflammatory drugs
(NSAIDs) are the predominant causes of peptic ulcer
disease in the United States [1].
H. pylori infection leads to gastroduodenal
inflammation, peptic ulceration, gastric lymphoma,
and gastric cancer, which has been proven with
animal studies and human epidemiological report. H.
pylori may induce inflammatory-associated gene
expression in gastric epithelial cells, including
activation of nuclear factor kappa B (NF-κB),
enhance expression of cyclooxygenase-2 (COX-2)
and inducible nitric oxide synthase (iNOS), and
production of interleukin-8 (IL-8). H. pylori bacteria
adhere to the gastric mucosa; the presence of another
inflammatory protein and a functional cytotoxinassociated gene island in the bacterial chromosome
increases virulence and probably ulcerogenic
potential [2]. NSAIDs can cause damage to the
gastro-duodenal mucosa via several mechanisms,
including their topical irritant effect on the
epithelium, impairment of the mucosal barrier
function, suppression of gastric prostaglandin
synthesis, reduction of gastric mucosal blood flow,
and interference with the repair of superficial injury.
The presence of acid in the lumen of the stomach also
contributes to the pathogenesis of NSAIDs-induced
ulcers and bleeding by impairing the restitution
process, interfering with hemostasis and inactivating
several growth factors that are important in mucosal
2130 Natural Product Communications Vol. 3 (12) 2086
defense and repair [3]. A variety of other infections
and co-morbidities are associated with a greater risk
of peptic ulcer disease, such as Crohn’s disease,
hepatic cirrhosis, cytomegalovirus, tuberculosis,
chronic
renal
failure,
sarcoidosis
and
myeloproliferative disorder.
Most of the available gastroprotective drugs act on
the offensive factors neutralizing acid secretion, like
antacids, H2 receptor blockers, like ranitidine,
anticholinergics, like pirenzepin, proton pump
inhibitors, like omeprazole, and lansoprazole, which
interfere with acid secretion. However, the use of
these antisecretory drugs may be associated with
adverse events and ulcer relapse [4]. Thus, there is a
need for more effective, less toxic and cost-effective
anti-ulcer agents.
Herbal medicines have been used since the dawn of
civilization to maintain health and to treat diseases.
The World Health Organization estimates that about
three quarters of the world’s population currently use
herbs and other forms of traditional medicines to treat
their diseases because the use of these compounds
are considered as safe [5,6].
In recent years, a widespread search has been
launched to identify new anti-ulcer drugs from
natural sources. In traditional medicine, for example,
several plants have been used to treat gastrointestinal
disorders, including gastric ulcers [7-9].
The potential use of plants has been successfully
demonstrated in the field of gastroprotection in a
recent article that reviewed the studies on extracts
and pure compounds as gastroprotective agents
reported in the literature up to 2005 [10].
The purpose of the present review article is to
highlight the more recent data in current knowledge
on natural products as gastroprotective and antiulcer
agents. The mechanism of action and the structureactivity relationships are also discussed where it is
possible.
1. Extracts
Several studies on the gastroprotective effects of
plant extracts have been recently undertaken. In a
recent study, de Andrade et al. [11] evaluated the
effects of Maytenus robusta extract, a plant used in
folk medicine for the treatment of stomach ulcers,
using the NSAIDs-induced ulcer, ethanol-induced
ulcer and stress-induced ulcer protocols.
Tundis et al.
In the ethanol-induced ulcer model, it was observed
that the treatment with M. robusta extract (50, 250
and 500 mg/kg) and positive control omeprazole (30
mg/kg) significantly reduced the lesion index, the
total lesion area and the percentage of lesion, in
comparison with the negative control group. The
percentages of inhibition of ulcers were 75.1, 85.0,
86.6 and 75.5 for the treated groups with 50, 250 and
500 mg/kg of M. robusta and omeprazole,
respectively. Significant inhibition was also observed
in the lesion index in the indomethacin-induced ulcer
model, the decrease being 62.5, 62.5, 63.6 and 96.2
for groups treated with 50, 250 and 500 mg/kg of
Maytenus robusta and positive control (cimetidine),
respectively. Similar results were observed in the
stress-induced ulcer model, where the inhibition of
ulcer lesions was 71.3, 72.7, 76.5 and 92.3 for the
groups treated with 50, 250 and 500 mg/kg of plant
extract. Regarding the model of gastric secretion, a
reduction in the volume of gastric juice volume and
total acidity was observed, as well as an increase in
gastric pH.
At 200 mg/kg body weight (b.w.) the aqueous extract
of Decalepis hamiltonii protected swim stressinduced ulcer lesions by 77%, similar to that of
ranitidine (79%), a known antiulcer drug, at 30
mg/kg b.w. [12]. Reactive oxygen species (ROS)
have been implicated in the pathogenesis of a wide
variety of clinical disorders and gastric damage.
Preventive antioxidant enzymes such as superoxide
dismutase (SOD) and catalase (CAT) are the first
line of defence against ROS. Administration of
D. hamiltonii extract resulted in a significant increase
in the SOD, catalase and reduced glutathione (GSH)
levels, similar to those of control animals, suggesting
the efficacy of D. hamiltonii extract in preventing
free radical-induced damage during ulceration. The
extract also normalized the approximately3.1 and 2.4
folds of increased H+-K+-ATPase and gastric mucin,
respectively, in ulcerous animals, to levels similar to
those found in healthy controls.
The gastroprotective effects of an aqueous
suspension of the ethanolic extract of leaves and
flowers of Guazuma ulmifolia was assessed in a
model of acute gastric ulcer induced by diclofenac,
using the proton pump inhibitor omeprazole as a
protection reference [13]. Pretreatment with G.
ulmifolia decreased the ulcerated area by diclofenac
in a dose-dependent way. Myeloperoxidase activity,
as a marker of neutrophil infiltration, was slightly
reduced in vivo, whereas in vitro anti-inflammatory
Gastroprotective natural products
Natural Product Communications Vol. 3 (12) 2008 2131
activity was clearly inhibited in a dose-dependent
manner. The lowest doses of the extract significantly
decreased the levels of lipoperoxides, and superoxide
dismuthase activity increased to a similar extent as
with omeprazole. Examination of glutathione
metabolism reflected a significant rise in glutathione
peroxidase (GPx) activity at the highest dose of
G. ulmifolia. These results showed that the aerial
parts of G. ulmifolia had demonstrated protection of
the gastric mucosa against the injurious effect of
NSAIDs, mainly by anti-inflammatory and radical
scavenging mechanisms.
cells. The results indicate that the gastroprotective
effect of T. arjuna extract is probably related to its
ability to maintain the membrane integrity by its
antilipid peroxidative activity that protects the gastric
mucosa against oxidative damage and its ability to
strengthen the mucosal barrier, the first line of
defense against exogenous and endogenous
ulcerogenic agents.
A crude hydroalcoholic extract of Polygala
paniculata administered orally was able to protect the
gastric mucosa against lesions induced by ethanol
70% [14]. In this study the extract was given by two
routes (oral and intraperitoneal) to evaluate whether
the observed effect was related to an adherent
property of the extract on the gastric mucosa by
forming a protective barrier against the aggressive
effects of ethanol. The results showed that, when
given by the intraperitoneal route, P. paniculata
extract exhibited an important cytoprotective effect
similar to the one seen when the extract was given
orally. This suggested that the pharmacological
mechanism did not have any relationship with an
adherent property of the extract. In addition, the
extract partially protected the mucosa against
indomethacin-induced lesions. The extract did not
change the volume and acidity of gastric secretion
and exerted an antioxidant activity. The
gastroprotective effects of P. paniculata extract may
have involved prostaglandins and be related to
cytoprotective factors, such as antioxidant activity
and maintenance of mucus production.
The methanol extract of the bark of Terminalia
arjuna showed marked antiulcer and ulcer healing
activity against 80% ethanol, diclofenac sodium and
dexamethasone induced ulcer models dosedependently [15]. Pre-, post and co-administration of
extract showed 100% protection to the gastric
mucosa
against
ethanol,
diclofenac
and
dexamethasone induced ulcers. T. arjuna increased
the levels of GSH in gastric mucosa, which were
reduced upon ulcer induction with dexamethasone.
These results suggest that GSH depletion has a role to
play in the ulcerogenesis induced by dexamethasone.
The restoration of GSH levels by the extract provides
evidence for the involvement of GSH in the antiulcer
activity of T. arjuna. T. arjuna also increased
significantly the glycoprotein content of the mucosal
Some spices, namely black pepper, ginger, and
turmeric have been shown to possess significant
gastroprotective activities [16-18]. Recently, Al
Mofleh et al. [19] provided substantial evidence for
anti-ulcer and anti-secretory effects of an aqueous
suspension of anise (Pimpinella anisum). Anise
suspension significantly inhibited the ulcerative
lesions in all animals treated with necrotizing agents.
Chemical studies demonstrated that P. anisum and its
major constituents, anethol, eugenol, anisaldehyde,
methylchanicol, other terpenes and coumarins, were
free radicals or active oxygen scavengers. In addition,
the ability of anise suspension to protect gastric
mucosa against lesions induced by chemical irritants
is likely by maintaining the structural integrity of the
gastric epithelium and a balance between aggressive
factors and inherent protective mechanisms.
Previously, the same research group evaluated the
anti-ulcerogenic property of an aqueous suspension
of Mentha piperita in different ulcer models in vivo
[20]. The suspension at 250 and 500 mg/kg b.w.,
orally (i.p. in Shay rat model), had a significant effect
in pyloric ligation induced basal gastric secretion, in
indomethacin and noxious chemical (80% ethanol,
0.2 M NaOH and 25% NaCl) induced gastric
ulceration. The aqueous suspension showed
significant protection in all models used. These
findings were supported by histopathological
assessment of gastric tissue and by the determination
of non-protein sulfhydryl (NP-SH) contents of the
stomach, as these parameters showed protection of
various indices and replenishment of the depleted
NP-SH level by the suspension treatment,
respectively. The ulcer protective effect of M.
piperita may possibly be due to its anti-secretory
effect, along with antioxidative and cytoprotective
properties through a prostaglandins mediated
mechanism.
The effect of Carum carvi pretreatment on gastric
mucosal injuries caused by NaCl, NaOH, ethanol and
pylorous ligation accumulated gastric acid secretions
was investigated in rats [21]. Pretreatment at oral
doses of 250 and 500 mg/kg b.w. was found to
2132 Natural Product Communications Vol. 3 (12) 2086
provide a dose-dependent protection against the
ulcerogenic effects of different necrotizing agents,
ethanol-induced histopathological lesions, depletion
of stomach wall mucus and NP-SH groups and
pylorous ligated accumulation of gastric acid
secretions. The protective effect of C. carvi against
ethanol-induced damage of the gastric tissue appears
to be related to the free-radical scavenging property
of its constituents. The exact mechanism of action of
the gastroprotective activity is not known. However,
it might be due to flavonoid related suppression of
cytochrome P450 1A1 (CYP1A1), which is known to
convert xenobiotics and endogenous compounds to
toxic metabolites.
Another spice, Coriandrum sativum, was evaluated
for its gastroprotective activity [22]. Pretreatment at
oral doses of 250 and 500 mg/kg b.w. was found to
provide a dose-dependent protection against the
ulcerogenic effects of different necrotizing agents,
ethanol-induced histopathological lesions, pylorus
ligated accumulation of gastric acid secretions and
ethanol related decrease of NP-SH. Results obtained
from the study of gastric mucus and indomethacininduced ulcers demonstrated that the gastroprotective
activity of C. sativum might not be mediated by
gastric mucus and/or endogenous stimulation of
prostaglandins, but might be related to the freeradical scavenging property of different antioxidant
constituents (coumarins, catechins, terpenes and
polyphenolic compounds) present in C. sativum. The
inhibition of ulcers might be due to the formation of a
protective layer of either one or more than one of
these compounds by hydrophobic interactions.
An aqueous suspension of Crocus sativus was
evaluated in rats for its gastric antiulcer activity
induced by pylorus ligation, indomethacin and
various necrotizing agents, including 80% ethanol,
0.2 M NaOH and 25% NaCl [23]. Gastric wall mucus
and NP-SH contents were also estimated in rats.
Histopathological assessment of the stomach was
carried out. The C. sativus aqueous suspension at
doses of 250 and 500 mg/kg exhibited decreases in
basal gastric secretion and ulcer index in Shay rats
and indomethacin treated groups. Gastric wall mucus
elevation was observed, but no significant
histopathological changes were noted. C. sativus
exhibited significant antisecretory and antiulcer
activities without causing any deleterious effects on
acute and chronic toxicity in rodents.
Tundis et al.
In the ethanol-induced stress gastric ulcer test in rats,
it was shown that the Carlina acanthifolia essential
oil, traditionally used in the treatment of various
disorders, including stomach diseases, produced
significant dose-dependent gastroprotective activity
[24]. This was particularly noticeable when the
essential oil was given as a “pure oil” in a dose of 1.0
mL/kg. The free radical scavenging activity of the
essential oil tested might be considered as one of the
possible mechanisms of the gastroprotective effect
observed [25].
The effect of San-Huang-Xie-Xin-Tang (a traditional
oriental medicinal formula containing Coptis
chinesis, Scutellaria baicalensis and Rheum
officinale) and its main component baicalin was
recently evaluated on H. pylori-infected human
gastric epithelial AGS cells [26]. It is widely
accepted that most peptic ulcers are associated with
H. pylori infection and eradication of the organism
leads to enhanced ulcer healing and reduces the
chance of ulcer recurrence. NF-κB activation plays
an important role in H. pylori-induced inflammation
and apoptosis in gastric epithelial cells [27].
Treatment with San-Huang-Xie-Xin-Tang and
baicalin significantly inhibited IκBα degradation and
NF-κB activation in H. pylori-infected AGS cells.
Previously, H. pylori-induced inflammation has been
shown to be associated with COX-2 expression in
experimental animals and human patients [28].
Furthermore, in early gastric cancer and intestinal
metaplasia the expression of COX-2 in patients
infected by H. pylori is increased [29]. Thus, chronic
expression of COX-2 may play an important role in
H. pylori-associated gastric carcinogenesis, in
addition to propagation of gastric inflammation [30].
San-Huang-Xie-Xin-Tang and baicalin decreased
H. pylori-induced COX-2 expression in human
gastric epithelial cells. Thereby, they might inhibit
COX-2 associated gastric inflammation.
Recent studies also demonstrated that H. pylori
acts through TLR2/TLR9 to activate both the
PI-PLCγ/PKCα/c-Src/IKKα/β and
NIK/IKKα/β
pathways, resulting in the phosphorylation and
degradation of IκBα, which in turn leads to the
stimulation of NF-κB and COX-2 gene expression
[31]. Thus, it was suggested that San-Huang-XieXin-Tang and baicalin suppression of COX-2
expression might be mediated via inhibition of
degradation of IκBα.
Gastroprotective natural products
Natural Product Communications Vol. 3 (12) 2008 2133
IL-8 secreted by gastric epithelial cells is likely to be
an important host mediator inducing neutrophil
migration to the site of infection and, therefore, may
be important in the regulation of inflammatory and
immune processes in response to H. pylori [32]. SanHuang-Xie-Xin-Tang and its major component also
inhibit H. pylori-induced IL-8 production and,
therefore, increase the benefit on gastric mucosal
protection.
200 mg/kg) against aspirin-induced gastric lesions
could possibly be due to its 5-lipoxygenase inhibitory
effect. Ethanol-induced depletion of gastric wall
mucus has been significantly prevented by A. latifolia
extract. Pylorus ligation-induced ulcers are due to
auto-digestion of the gastric mucosa and breakdown
of the gastric mucosal barrier [36]. A. latifolia extract
also protected against the cold-resistant stressinduced ulcers and pylorus ligation at 200 mg/kg.
Stress-induced ulcers are probably mediated by
histamine release with enhancement in acid secretion
and a reduction in mucous production. Increase in
gastric motility, vagal overactivity, mast cell
degranulation decreased gastric mucosal blood flow,
and decreased prostaglandin synthesis is involved
in genesis of stress-induced ulcers [37-39].
Accordingly, the protective action of A. latifolia
extract against stress-induced ulceration could be due
to its histamine antagonistic, anticholinergic and
antisecretory effects.
Alchornea glandulosa (Euphorbiaceae) is a plant
used in folk medicine as an antiulcer agent. Rats
pretreated with a methanolic extract of the leaves
showed a significant, dose-dependent reduction of
gastric ulcers induced by absolute ethanol.
Pretreatment of mice with A. glandulosa extract (500,
1000 mg/kg, p.o.) showed significant, dosedependent decreases in the severity of lesions caused
by HCl/ethanol and by NSAID-induced gastric
lesions. Pretreatment with the extract also induced
antisecretory action via local and systemic routes and
a significant decrease in the total gastric acid content.
The gastroprotective effects of A. glandulosa
involved the participation of nitric oxide (NO) and
increased levels of endogenous sulfhydryl
compounds, which are defensive mechanisms of the
gastrointestinal mucosa against aggressive factors.
The results showed that single oral administrations of
A. glandulosa (250 mg/kg/once daily) potently
stimulates gastric epithelial cell proliferation that
contributes to the accelerated healing of gastric ulcers
induced by acetic acid. In addition, no sub-acute
toxicity (body weight gain, vital organs and serum
biochemical parameters) was observed during
treatment with the extract. Phytochemical
investigation led to the isolation of gallic acid, methyl
gallate, pterogynidine and different flavonoids. These
compounds may contribute to the observed
antiulcerogenic effects of A. glandulosa [33].
The gastroprotective potential of the 50% aqueous
alcoholic extract of Anogeissus latifolia (100 and 200
mg/kg b.w.) was studied on aspirin, cold-resistant
stress, pylorus ligated and ethanol-induced ulcers.
The status of the antioxidant enzymes SOD and CAT,
along with lipid peroxidation (LPO), was also studied
in cold-resistant stress-induced ulcers [34]. Synthetic
NSAIDs, like aspirin, cause mucosal damage by
interfering with prostaglandin synthesis, increasing
acid secretion and back diffusion of H+ ions, resulting
in overproduction of leukotrienes and other products
of the 5-lipoxygenase pathway [35]. Hence, the
protective action of A. latifolia extract (65.6% at
Stachytarpheta cayennensis is an herbaceous plant
popularly known as gervão-roxo, gervão-do-campo
or vassourinha-de-botão in Brazil and used in
traditional medicine for the treatment of gastritis and
ulcers [40]. Two hydroalcoholic extracts obtained
respectively with ethanol/water 70:30 and
ethanol/water 96:4 were prepared and tested. The oral
pretreatment with the second ethanolic extract
significantly inhibited the generation of gastric
lesions induced by diclofenac, whereas the first
extract induced a slight, but not statistically
significant inhibition of mucosa damage.
Mouriri pusa is another medicinal plant commonly
used in Brazil against gastric ulcer. The methanol and
dichloromethane extracts obtained by sequential
extraction from the leaves of M. pusa were evaluated
for their ability to protect the gastric mucosa against
injuries caused by necrotizing agents (0.3M HCl/60%
EtOH, absolute ethanol, NSAIDs, stress and pylorus
ligature) in mice and rats [41]. The best results were
obtained after pretreatment with methanol extract,
whereas the dichloromethane extract did not show the
same significant antiulcerogenic activity. The
mechanism involving the antiulcerogenic action of
the methanol extract seemed to be related to NO
generation and also suggested the effective
participation of endogenous sulfhydryl groups in the
gastroprotective action. Phytochemical investigation
of the methanol extract of M. pusa yielded tannins
and flavonoids. The presence of these phenolic
compounds
probably
would
explain
the
2134 Natural Product Communications Vol. 3 (12) 2086
antiulcerogenic effect of the polar extract of M. pusa
leaves.
Recently, Berenguer et al. [42] evaluated the
gastroprotective effect of Rhizophora mangle in a
model of diclofenac-induced ulcers in rats and
studied the mechanisms involved, using the proton
pump inhibitor omeprazole for comparison. The
major active principles are polyphenols [43]. These
compounds have shown cytoprotective properties
[44] and have been associated with antiulcerogenic
activity in other plants [45,46]. The topical action of
the aqueous extract of R. mangle in accelerating
wound healing has been previously explained by
several mechanisms, such as coating the wound,
forming complexes with proteins of the
microorganism cell wall, chelating free radicals and
reactive oxygen species, stimulating the contraction
of the wound and increasing the formation of new
capillaries and fibroblasts [47]. Berenguer et al. [42]
found a thick coating of R. mangle extract
macroscopically adherent to the gastric mucosa,
which suggests that in addition to antioxidant
mechanisms, the formation of a physical barrier with
similar properties as observed in topical wounds may
contribute to the gastroprotective action of the drug.
A methanolic extract, the essential oil, light
petroleum soluble and insoluble fractions of the
methanolic extract of Elettaria cardamomum were
studied in rats at doses of 100-500, 12.5-50, 12.5-150
and 450 mg/kg, respectively, for their ability to
inhibit the gastric lesions induced by aspirin, ethanol
and pylorous ligature [48]. In addition, effects on
wall mucus and gastric acid output were recorded.
All fractions significantly inhibited gastric lesions
induced by ethanol and aspirin, but not those induced
by pylorus ligation. The methanolic extract proved to
be active, reducing lesions by about 70% in the
ethanol-induced ulcer model at 500 mg/kg. The light
petroleum soluble fraction reduced the lesions by
50% at 50 and 100 mg/kg, with similar effect to that
of the insoluble fraction of the methanol extract at
450 mg/kg. In the aspirin-induced gastric ulcer, the
best gastroprotective effect was found in the light
petroleum soluble fraction, which inhibited lesions by
nearly 100% at 12.5 mg/kg.
Oral administration of Kaempferia parviflora
ethanolic extract (30-120 mg/kg) inhibited gastric
ulcer formation induced by indomethacin, HCl/EtOH
and water immersion restraint stress [49]. It was
Tundis et al.
found that pretreatment with K. parviflora at doses of
60 and 120 mg/kg significantly increased the amount
of gastric mucus content in HCl/EtOH-ulcerated rats.
The finding that K. parviflora failed to increase the
gastric pH and decrease the gastric volume and
acidity in pylorus-ligated rats suggests that the antisecretory action is unlikely to be ascribed to the
anti-gastric ulcer effect of the K. parviflora. The
gastric wall mucus is thought to play an important
role as a defensive factor against gastrointestinal
damage. The gastric wall mucus was used as an
indicator for gastric mucus secretion. The finding that
pretreatment with K. parviflora at doses of 60 and
120 mg/kg significantly increased gastric mucus
content in HCl/EtOH ulcerated rats suggests that the
gastroprotective effect of K. parviflora is mediated
only partly by preservation of gastric mucus
secretion.
Sannomiya et al. [50] evaluated the potential
antiulcerogenic effect of three different extracts
obtained from the leaves of Byrsonima crassa,
namely hydromethanolic (80% MeOH), methanolic
and chloroform extracts. The oral administration of
all the extracts reduced the formation of lesions
associated with HCl/ethanol administration in mice.
The 80% MeOH extract significantly reduced the
incidence of gastric lesions by 74, 78 and 92% at
doses of 250, 500 and 1000 mg/kg, respectively. The
methanolic extract reduced the ulceration only at
doses of 500 and 1000 mg/kg. Phytochemical
investigation of B. crassa revealed the presence of
phenolic compounds that may probably explain the
antiulcerogenic effect of the extracts of B. crassa.
In the HCl/EtOH-induced gastric ulcer model, an
hydroalcoholic extract obtained from Pradosia
huberi barks demonstrated significant inhibition of
the ulcerative lesion index by 73% (500 mg/kg) and
88% (1000 mg/kg), respectively [51]. The gastric
damage induced by absolute ethanol in rats was
effectively reduced by 84, 88 and 81% (250, 500 and
1000 mg/kg). In the NSAID-induced lesion model,
P. huberi extract also showed an antiulcerogenic
effect with decrease in gastric lesions. P. huberi
administered either orally or intraduodenally was
able to change gastric juice parameters as well as
those treated with cimetidine. The treatment with
P. huberi extract significantly increased gastric
volume, the pH values and promoted reduced acid
output. By comparison of the effects produced by the
intraduodenal and oral routes, it was observed that
P. huberi was better for local activity in gastric
Gastroprotective natural products
Natural Product Communications Vol. 3 (12) 2008 2135
mucosa than in systemic action. The hydroalcoholic
extract of P. huberi was also shown to be an inhibitor
of intestinal motility. The mechanism of action of
the.extract did not seem to be related to the NOinhibitor, but showed the participation of endogenous
sulphydryl groups in the gastroprotective action.
treated groups of animals as compared with the
control group. Histopathological examination of the
stomach of the ulcerated animals showed severe
erosion of the gastric mucosa, sub-mucosal edema
and neutrophil infiltration. All of these symptoms
were found to be normal in the treated groups. In
general, the results of this investigation revealed the
gastroprotective activity of the extract through an
antioxidant mechanism.
In order to establish the pharmacological basis for
their ethnomedicinal use in gastric disorders,
studies were made of the effects of ethanol extracts
and fractions from root tubers of Cynanchum
auriculatum, C. bungei and Cynoctonum wilfordii on
ethanol- and indomethacin-induced gastric lesions,
and histamine-induced gastric acid secretion in rats
[52]. Oral administration of the ethanol extract and
chloroform fraction of C. wilfordii at doses of 150
and 68 mg/kg, respectively, significantly inhibited
the development of ethanol- and indomethacininduced gastric lesions and also caused a significant
decrease of gastric acid secretion after histamineinduced gastric lesion. Oral administration of ethanol
extract and the chloroform fraction of C. auriculatum
at doses of 300 and 69 mg/kg, respectively,
significantly inhibited ethanol- and indomethacininduced gastric lesions.
Cissus quadrangularis is well known for the
treatment of gastric disorders owing to it being a rich
source of carotenoids, triterpenoids and ascorbic acid.
Jainu et al. [53] evaluated an ethanol extract of C.
quadrangularis against the gastric toxicity induced
by aspirin in rats with an optimum protective dose of
500 mg/kg of extract in the aspirin model. In
addition, administration of aspirin increases lipid
peroxidation status, xanthine oxidase (XO), and
myeloperoxidase, and decreases selenium-GPx
activities in the gastric mucosa, resulting in mucosal
damage at both cellular and subcellular level.
Pretreatment with C. quadrangularis ameliorated the
observed effects significantly in the gastric mucosa of
ulcerated rats. These findings suggest that the
gastroprotective activity of C. quadrangularis could
be mediated possibly through its antioxidant effect,
as well as by the attenuation of the oxidative
mechanism and neutrophil infiltration.
Administration of a 70% methanolic extract of
Punica granatum fruit rind showed inhibition in
aspirin- and ethanol-induced gastric ulceration [54].
In treated groups of animals, the SOD, CAT, GSH
and GPx levels were increased and found more or
less equal to the normal values. The tissue lipid
peroxidation level was found to be decreased in
Al-Qarawi et al. [55] evaluated, in a rat model of
ethanol-induced gastric ulceration, the beneficial
effects on gastric ulcers of a plant used in
folk medicine, Phoenix dactylifera. Aqueous and
ethanolic undialyzed and dialyzed extracts from date
fruits and pits were given orally to rats at a dose of 4
mL/kg for 14 consecutive days. On the last day of
treatment, rats were fasted for 24 h and were then
given 80% ethanol (1 mL/rat) by gastric intubation to
induce gastric ulcer. Rats were killed after 1 h of
ethanol exposure and the incidence and severity of
the ulceration were estimated, as well as the
concentrations of gastrin in plasma, and histamine
and mucus in the gastric mucosa. As a positive
control, a single group of rats that were fasted for 24
h was administered orally with lansoprazole and was
given 80% ethanol, as above, 8 h thereafter. The
results indicated that the aqueous and ethanolic
extracts of the date fruit and, to a lesser extent, date
pits, were effective in ameliorating the severity of
gastric ulceration and mitigating the ethanol-induced
increase in histamine and gastrin concentrations, and
the decrease in mucin gastric levels. The ethanolic
undialyzed extract was more effective than the other
extracts used. It is postulated that the basis of the
gastroprotective action of P. dactylifera extracts may
be multi-factorial, but may include an antioxidant
action.
The tissue lipid peroxidation level was found to be
decreased in the treated groups of animals as
compared with the control group. Histopathological
examination of the stomach of the ulcerated animals
showed severe erosion of the gastric mucosa, submucosal edema and neutrophil infiltration.
Portulaca oleracea, commonly used in Iranian folk
medicine, has been demonstrated to protect mice
from gastric aggressive factors and its administration
reduced total gastric acidity and increased pH of
gastric juice [56]. On induction of gastric ulceration
by using HCl, pretreatment with the aqueous and
ethanolic extracts showed a dose-dependent reduction
2136 Natural Product Communications Vol. 3 (12) 2086
Tundis et al.
in the severity of ulcers. The dose of 0.8 g/kg of the
aqueous extract and 1.4 g/kg of the ethanolic extract
had similar activity to sucralfate (0.1 g/kg). In lesions
induced by ethanol, the dose of 0.56 g/kg, and 0.8
g/kg of the aqueous extract, and 0.8 and 1.4 g/kg of
the ethanolic extract showed significant inhibition of
lesions. The oral and intraperitoneal doses of both
extracts inhibited the total gastric acidity in the
pylorus-ligated mice in a dose-dependent manner.
The highest dose of extracts had antisecretory
activity, which was comparable to cimetidine.
Lavandula hybrida Reverchon “Grosso” exerted
gastroprotective effects [57]. Interestingly, the
principal constituents of the oil, linalool and linalyl
acetate, were demonstrated to contribute to the
gastroprotective effect of lavender oil which, orally
administered, caused a dramatic reduction in ethanolinduced gastric injury to rats. The lack of a protective
effect against gastric mucosal damage caused by
indomethacin led to the hypothesis that
gastroprotection afforded by L. hybrida oil cannot be
attributed to interference with the arachidonic acid
metabolic cascade.
1
2
3
4
5
6
7
ROC
Of the sesquiterpenes 1-8, assessed at a single oral
dose of 50 mg/kg, the best gastroprotective effect was
observed for derivative 8, obtained as a diasteromeric
mixture by reduction of the 4,5-double bond of
cyperenoic acid (3). Compound 8 reduced the lesion
index by 86%, being the most active of the
sesquiterpenes evaluated in this work and more
active than lansoprazole at 20 mg/kg. The products
1 and 3-8 did not show significant differences
in gastroprotective activity. Cyperenol (1) and
cyperenoic acid methyl ester (4), however, were more
cytotoxic with IC50 values of 44 and 75, and 48
and 75 mM against AGS cells and fibroblasts,
respectively. The best gastroprotective effect with a
H
ROC
H
OH
13
H
R = OH
HN
CH3
14 R =
9 R = OH
10 R = OCH3
11 R =
HN
12 R =
8
R = CH2OH
R = CH2OCOCH3
R = COOH
R = COOCH3
R = CONHCH2CH3
R = CONHCH2CH2CH2CH3
R = CONHPhOCH3
H
CH3
HN
H
OCH3
ROC
H
15 R = OH
HN
16 R =
CH3
CH2OAc
CH2OAc
2. Pure compounds
2.1. Terpenes: Several plant terpenoids, including
sesquiterpenes, diterpenes and triterpenes, have been
shown to protect the gastric mucosa against the
damage caused by different ulcerogens [58]. Recently
the gastroprotective effect of the sesquiterpene
cyperenoic acid and seven semi-synthetic derivatives
was assessed in the HCl/ethanol-induced gastric ulcer
model in mice [59]. At doses of 25, 50 and 100
mg/kg, cyperenoic acid (3) showed a dose-dependent
gastroprotective effect, reducing the ulcers by 45 and
75% at 50 and 100 mg/kg, respectively, compared
with the untreated controls.
HOOC
R
HOOC
H
HOOC
17
H
18
CH2OAc
CH2OAc
ROC
H
R=
HN
D
A
C
ROC
R=
B
20
21
22
25
27
28
A=B=C=D=H
A =B = D = H, C = OCH3
A = C = OCH3, B = D = H
A = B = D = H, C = I
A = Br, B = C = D = H
A = C = D = H, B = Br
HN
H
D
A
C
B
19
23
24
26
29
30
A=B=C=D=H
A =B = D = H, C = OCH3
A = C = OCH3, B = D = H
A = B = D = H, C = I
A = Br, B = C = D = H
A = C = D = H, B = Br
lower cytotoxicity was found for compound 8,
cyperenoic acid (3) and the p-anisidyl derivative 7.
The main sesquiterpene of Fabiana imbricata, 11hydroxy-4-amorphen-15-oic acid (9), at doses of 25,
50 and 100 mg/kg showed a dose-dependent
gastroprotective effect in HCl/EtOH-induced gastric
lesions in mice, reducing the lesions by 68% at 100
mg/kg [60]. Seven derivatives of this terpene were
prepared and their gastroprotective effects were
assessed in HCl/EtOH-induced gastric lesions in
mice. Compounds 9, 10 and 12 reduced the lesion
index by 60-65%, while the mixture of compounds
13 and 15, as well as 14 and 16 presented values of
71% and 51%, respectively. The most active
compound proved to be the amide derivative 11,
Gastroprotective natural products
which reduced the lesion index by 80%. At 20 mg/kg,
lansoprazole reduced the lesion index by 70%.
Compounds 13 and 15, lacking the alcohol function
at C-11, had a better gastroprotective activity than 9.
In the case of compounds 14 and 16, where the
alcohol function at C-11 is absent and the acid
group is substituted by an amide function, the
gastroprotective activity was reduced when compared
with compound 11. The alcohol function at C-11 is
required for a better gastroprotective effect when
there is a substitution of the acid for an amide. At
doses of up to 1000 mg/kg, oral administration of 9
did not show any observable symptoms of toxicity or
mortality in mice. Therefore, the intraperitoneal LD50
for this compound in mice is higher than 1000 mg/kg
and it can be regarded as ‘not harmful’. The
cytotoxicity study revealed that compound 9, as well
as the mixtures of 13 and 15 and compounds 14 and
16 presented low toxicity towards AGS cells and
fibroblasts. In the compound series 9-12, when the
acid function at C-15 is substituted forming an amide
(11 and 12), the cytotoxicity increased significantly.
A comparison of the acids 9, 13 and 15 indicated a
comparable low cytotoxicity, thus suggesting that the
presence of the alcohol function at C-11 did not
contribute to this effect.
When the corresponding amides were prepared (i.e.
11-12 and 14-16), the presence of the hydroxy group
at C-11 determined the cytotoxicity of the products.
The labdane diterpenes 15-acetoxyimbricatolic acid
(17) and 15-acetoxylabd-8(9)-en-19-oic acid (18)
isolated from Araucaria araucana exhibited
significant gastroprotective activity at 50 and 100
mg/kg in mice, respectively.
From these compounds, some aromatic amides were
prepared and assessed for their gastroprotective effect
in the HCl/EtOH-induced gastric lesion model in
mice [61]. The analysis of the gastroprotective
activity of the benzylamides belonging to the series
8(9)- and 8(17)-ene was undertaken at doses of 12.5,
25 and 50 mg/kg in the HCI/EtOH-induced gastric
lesion model in mice.
A significant gastroprotective effect was observed for
15-acetoxylabd-8(9)-en-19-oic acid benzylamide (19)
starting at 12.5 mg/kg, reducing the gastric lesions by
50%, while 15-acetoxylabd-8(17)-en-19-oic acid
benzylamide (20) reduced lesions by 66% at
25 mg/kg. At 25 mg/kg, the highest gastroprotective
effect was observed for the benzyl- and 3-bromo
phenylamides from 17, as well as for the benzyl- and
Natural Product Communications Vol. 3 (12) 2008 2137
CH2OAc
CH2OAc
HOOC
H
HOOC
17
H
18
CH2OAc
CH2OAc
HN
ROC
H
20
21
22
25
27
28
R=
HN
D
ROC
A
A=B=C=D=H
A =B = D = H, C = OCH3
A = C = OCH3, B = D = H
A = B = D = H, C = I
A = Br, B = C = D = H
A = C = D = H, B = Br
H
C
B
R=
D
A
C
B
19
23
24
26
29
30
A=B=C=D=H
A =B = D = H, C = OCH3
A = C = OCH3, B = D = H
A = B = D = H, C = I
A = Br, B = C = D = H
A = C = D = H, B = Br
p-toluidylamides from 18, these being as active
as lansoprazole at 20 mg/kg. The presence of a
4'-methoxy or a 2',4'-dimethoxy functionality did
not result in significant differences in the
gastroprotective effects of 21 and 22, but a strong
effect was observed for 23, while the activity of 24
was lower. The effect of a halogen in the aromatic
ring on the gastroprotective activity can be assessed
by comparing 25 and 26, which bear iodine. While
the gastroprotective activity of 27 and 28 was strong
and comparable to that of 20, there was a substantial
decrease in the gastroprotective effect of 29 and 30
compared with 19. The results suggest a relevant role
of the exomethylene function in the gastroprotective
effect of the brominated derivatives, with higher
activity for 28. The effect, however, is not
statistically different from that of 27. The structural
modifications undertaken led to labdane derivatives
with an increased gastroprotective effect compared
with the parent compounds.
The gastroprotective effect of the diterpenes
jatropholone A (31), jatropholone B (32) and sixteen
semisynthetic derivatives was assessed in the
HCl/ethanol-induced gastric lesion model in mice
and the cytotoxicity was determined towards
fibroblasts and AGS cells [62]. In a dose-response
study, 32 reduced gastric lesions by 65% at 6 mg/kg
and 31 by 54% at 100 mg/kg. The jatropholone B
derivatives 33-38 and the compounds 39-42 were
compared at a single oral dose of 25 mg/kg, while the
jatropholone A derivatives 43-48 were assessed at
100 mg/kg. A decrease in gastroprotective activity
was observed for the ether as well as for the ester
derivatives of 32. The methyl and propyl ethers of 31
were more gastroprotective than the natural product
The placement of an additional methyl group at C-2
in the jatropholone B derivatives led to a loss of
selectivity; the methyl and propyl ethers lack a
2138 Natural Product Communications Vol. 3 (12) 2086
gastroprotective effect. At the dose of 25 mg/kg
compound 32 reduced the lesions by 83%, while
compound 31 inhibited them by only 36%. At 100
mg/kg, all the derivatives of 31 were active.
Compounds 45-47 showed a similar activity to that of
their parent 31, while derivatives 43, 44 and 48 were
the most active. Considering the derivatives of 32, at
25 mg/kg, compounds 34, 41 and 42 showed the best
gastroprotective effect, while compounds 38 and 39
were the less active.
The gastroprotective mechanism of the natural
diterpene ferruginol (49) was assessed in vivo. The
involvement of gastric prostaglandins PGE(2),
reduced GSH, NO or capsaicin receptors was
evaluated in mice either treated or untreated with
indomethacin, N-ethylmaleimide (NEM), N-nitro-Larginine methyl ester (L-NAME) or ruthenium red,
respectively, and then orally treated with 49 or
vehicle. Gastric lesions were induced by oral
administration of ethanol. The effects of ferruginol
(49) on the parameters of gastric secretion were
assessed in pylorus-ligated rats. Gastric PGE(2)
content was determined in rats treated with 49 and/or
indomethacin.
Tundis et al.
OR1
OR
H
H
H
R
O
O
H
H
31
43
44
45
46
47
48
HO
HO
HO
R=H
R = CH3
R = C3H7
R = Ac
R = COCH=CH2
R = COC6H4NO2p
R = COC6H4Clp
O
OH
O
O HO
RO
OH
OH
H
OH
32
33
34
35
36
37
38
39
40
41
42
OH
O
COOH
O
O
O
OH
49
Glc - O
OH
Glc - Glc - O
H
OH O
O
OH
R = R1 = H
R = H, R1 = CH3
R = H, R1 = C3H7
R = H, R1 = Ac
R = H, R1 = COCH=CH2
R = H, R1 = COC6H4NO2p
R = H, R1 = COC6H4Clp
R = R1 = CH3
R = CH3, R1 = C3H7
R = C3H7, R1 = CH3,
R = R1 = C3H7
52
50 R = H
51 R = Ac
H
OH
stronger than those of the reference compounds,
omeprazole and cimetidine [64].
The oligoglycoside fraction from the flower buds of
Panax ginseng was found to show protective effects
on ethanol-induced gastric mucosal lesions in rats.
From this fraction, ginsenoside Rd (protopanaxadiol
3,20-O-bisdesmoside) (52) was isolated, together
with new dammarane-type triterpene tetraglycosides.
Ginsenoside Rd (52) exhibited inhibitory effects on
ethanol- and indomethacin-induced gastric mucosal
lesions in rats. The effect of 52 on ethanol-induced
gastric lesions was equipollent to that of a reference
compound, cetraxate hydrochloride [65].
The reduction of gastric GSH content was determined
in rats treated with ethanol after oral administration
of ferruginol (49), lansoprazole or vehicle. Finally,
the acute oral toxicity was assessed in mice.
Indomethacin reversed the gastroprotective effect of
ferruginol (49) (25 mg/kg), but not NEM, ruthenium
red or L-NAME. The diterpene (25 mg/kg) increased
the gastric juice volume and its pH value, and
reduced the titrable acidity, but was devoid of effect
on the gastric mucus content. Ferruginol (49)
increased gastric PGE(2) content in a dose-dependent
manner and prevented the reduction in GSH observed
due to ethanol-induced gastric lesions in rats. Single
oral doses up to 3 g/kg 49 did not elicit mortality or
acute toxic effects in mice. The results showed that
ferruginol (49) acted as a gastroprotective agent
stimulating gastric PGE(2) synthesis, reducing gastric
acid output and improving the antioxidant capacity of
the gastric mucosa by maintaining GSH levels [63].
The triterpene oleanolic acid (53) and its
semisynthetic derivatives 54-59 were studied for
gastroprotective and ulcer-healing effect using AGS
cells and human lung fibroblasts (MRC-5) [66]. The
assessment of the effect of the oleanolic acid
derivatives on the PGE(2) content showed a
significant increase of this prostaglandin when the
AGS cell cultures were treated with compounds 53,
54, 56 and 58.
The principal 28-noroleanane-type triterpene
oligoglycosides camelliosides A (50) and B (51),
isolated from the flowers buds of Camellia japonica,
showed protective effects on both ethanol- and
indomethacin-induced gastric lesions and their
gastroprotective effects were either equivalent or
The gastroprotective effect of oleanolic acid
derivatives was assessed also in the HCl/EtOHinduced gastric lesions in mice. All the assayed
compounds exhibited gastroprotective activity at the
dose of 50 mg/kg, reducing the gastric lesions to
different degrees ranging from 38% for compound 54
Gastroprotective natural products
Natural Product Communications Vol. 3 (12) 2008 2139
R1
R1
H
R = H, β OH; R1 = H2; R2 = H
R = H, β Ac; R1 = H2; R2 = H
R = H, β OH; R1 = H2; R2 = CH3
R = H, β Ac; R1 = H2; R2 = CH3
R = R1 = O; R2 = H
O
OH
COOH
O
O
O
H
OH O
O
HO
O
RO
O
OH
OH
OH H
H
OH
OH
HO
O
O
OH
O
O
67
OH
HO
O
O
HO
OH
OR1
CH2OR2
OH
R3
60
61
62
63
64
OH
65 R = OH
66 R = H
H
58 R = R1 = O
59 R = H, β OH; R1 = H, α OH
R
HO
HO
O
H
R
H
53
54
55
56
57
CO
H
OR2
H
R
O
R
O
R = OAng, R1 = H, R2 = H, R3 = CH2OH
R = OAng, R1 = H, R2 = Ac, R3 = CH2OH
R = OAng, R1 = H, R2 = Ac, R3 = COOCH3
R = H, R1 = Ang, R2 = H, R3 = CHO
R = OAng, R1 = Ac, R2 = H, R3 = CH2OH
and up to 76% for compound 57. The most active
products were compounds 57 and 59. In the
compound group 53-56, differing in the free or
esterified hydroxyl group at C-3 and the free or
methylated carboxylic acid function at C-28,
acetylation of the hydroxyl group at C-3 with a free
COOH at C-28 reduced the gastroprotective activity,
as can be observed for compound 54. Methylation of
the COOH at C-28 in compound 57 significantly
lowered the gastroprotective effect. Therefore, the
effect should be related to the presence of a free
carboxylic acid at C-28 when there is an oxo group at
C-3 and C-11. In compounds 58 and 59, the free
hydroxyl groups at C-3 and C-12 increased the
gastroprotective effect, the activity of compound 58
being in the same range as that of oleanolic acid (53).
The triterpene saponins theasaponins A1 (60), A2
(61), F3 (62), assamsaponin A (63) and assamsaponin
D (64), isolated from the seeds of Camellia sinensis,
were tested for their gastroprotective effects.
Theasaponin A2 (61) showed an inhibitory effect on
ethanol-induced gastric mucosal lesions in rats at a
dose of 5.0 mg/kg, p.o. and its activity was more
potent than that of omeprazole. Structure-activity
relationships for theasaponins on ethanol-induced
gastroprotective activities may suggest that (a) the
28-acetyl moiety enhances activity and (b)
theasaponins having a 23-aldehyde group exhibit
more potent activities than those with either a 23hydroxymethyl group or a 23-methoxycarbonyl
group [67].
R
O
HO
O
R1
OCH 3
H 2 CO
O
O
OCH 3
OCH 3
. H 2O
OH
OH
O
71
68 R = OH,
R1 = H
69 R = OH,
R 1 = OH
70 R = OCH 3 , R 1 = OH
2.2. Flavonoids: The flavonoids minimiflorin (65)
and mundulin (66) and the chalcone lonchocarpin
(67), isolated from Lonchocarpus oaxacensis and L.
guatemalensis, respectively, were tested on H+,K+ATPase isolated from dog stomach [68].
The flavanone minimiflorin (65) was the most potent
inhibitor, while mundulin (66) was 7.3-fold less
potent than 65. Hydroxylation at C-2' accounts for
this difference in potency. Thus, hydroxylation plays
an important role in conferring inhibitory activity of
the gastric H+,K+-ATPase to the flavanones.
Lonchocarpin (67), which has only one hydroxyl
group in its molecule showed only moderate
inhibition of ATPase (about 18-fold less potent than
65). A comparison of the relative potencies of these
active compounds with omeprazole shows that many
of these isolated compounds have higher inhibitory
activity of H+,K+-ATPase than the reference
compound, from 2 to 44 times higher for the most
potent inhibitor of H+,K+-ATPase tested here,
mundulin (66).
Kolaviron is a mixture of three compounds, Garcinia
biflavonoid GB1 (68), GB2 (69) and kolaflavanone
(70) and has been extensively studied for its antiinflammatory property in various experimental
models [69-72]. The antioxidant and scavenging
properties of kolaviron have also been demonstrated
[73]. Recently, it was demonstrated also that
treatments with kolaviron significantly inhibited
gastric lesions produced by indomethacin and
acidified ethanol [74]. The effects of kolaviron on
both indomethacin and ethanol-induced hemorrhagic
erosion may be associated with an increase in gastric
mucosal blood flow and gastric mucus secretion.
2140 Natural Product Communications Vol. 3 (12) 2086
OCH3O
O
O
O
CH2R
N
72 R = H
73 R = OCH3
O
N
O
75 R =
N
NCH3
OH O
R1
O
R
79 R =
80 R =
O
O
Some furoflavones (74-88), synthesized from the
naturally occurring chromones visnagin (72) and
khellin (73), exhibited gastroprotective activity in the
ethanol damage model [77].
81 R = H, R1 = C6H5
82 R = H, R1 = p-ClC6H4
R1 = C6H5
R1 = p-ClC6H4
83 R =
84 R =
OCH3O
N
O
R1 = C6H5
R1 = p-ClC6H4
OCH3O
CH2R1
O
R1
R
76 R = H, R1 = C6H5
77 R = H, R1 = p-ClC6H4
78 R = H, R1 = p-CH3C6H4
H2CN
N
74 R =
OCH3O
O
tablets to float in gastric fluid and release the drug
continuously. The release of DA-6034 (71) from
tablets in acidic media was significantly improved by
using EFMS, which is attributed to the effect of the
solubilizers and the alkalizing agent, such as sodium
bicarbonate used as a gas generating agent. DA-6034
EFMS tablets showed enhanced gastroprotective
effects in gastric ulcer-induced beagle dogs,
indicating the therapeutic potential of EFMS tablets
for the treatment of gastritis [76].
OCH3O
O
CH2R
CH2 N
O
O
R
N
O
87 R = OCH3 , R1 =
NCH3
N
O
O
88
85 R = H, R1 = N(CH3)2
86 R = OCH3, R1 =
N
Tundis et al.
The gastroprotective activity of DA-6034 (71), a new
flavonoid derivative, against various ulcerogens
including ethanol, aspirin, indomethacin, stress, and
acetic acid was evaluated [75]. The basic
mechanisms of DA-6034 (71) as a defensive factor,
such as mucus secretion and endogenous PGE(2)
synthesis were determined. Rats with gastric lesions
induced by ethanol-HCl, aspirin, indomethacin, and
stress that had been pretreated with 71 orally showed
either a statistically significant decrease or decreasing
tendency of the gastric lesion. In acetic acid-induced
gastric lesions, repeated oral administration of 71
exhibited a U-shape activity in ulcer healing, with the
maximum and minimum inhibition being observed at
30 and 10 mg/kg/day, respectively. DA-6034 (71)
also increased the mucus content in the gel layer, as
well as endogenous PGE(2) synthesis. These results
suggest that 71 prevents gastric mucosal injury, and
these gastroprotective activities appear to be due to
the increase in the gastric defensive systems. The
therapeutic limitations of 71 caused by its low
solubility in acidic conditions were overcome by
using the effervescent floating matrix system
(EFMS), which was recently designed to cause
In the benzopyrone portion of the furoflavone
system, the type of aromatic substitution at the 7position affected the gastroprotective effect. The pmethoxyphenyl derivative 78 was more active than
the p-chlorophenyl 77, which was more active than
the phenyl derivative 76.
The presence of a 9-alkylaminomethyl substituent in
these compounds decreased the activity of 79 and 80,
while the presence of a 6-alkylaminomethyl
substituent increased the activity (compound 86
showed more activity than 76). When the aromatic
group in position 7 was pyridinyl, the activity was
slightly decreased (compounds 74 and 85), except in
the case of the 9-N-methylpiperazinomethyl
derivative
75,
which
showed
promising
gastroprotective activity. It was found that the
presence of a methoxy group showed great effect on
the activity.
Substitution at the 4-position with a methoxy group
(compounds 76-80) enhanced the gastroprotective
activity, in contrast to 4-hydroxy derivatives 81-83,
which showed a marked decrease in activity.
Substitution with another methoxy group (87)
produced a potent level of gastroprotection.
In
summary,
furoflavones
exhibited
good
gastroprotective activity in the ethanol damage model
when there was a methoxy group (either in the 4, 9 or
7-position as methoxyphenyl) and an appropriate
substitution
in
the
6-position
with
an
alkylaminomethyl group.
2.3. Xanthones: Four xanthones, 6-desoxyjacareubin
(89), jacareubin (90), 1,3,5,6-tetra-hydroxy-2-(3hydroxy-3-methylbutyl)-xanthone (91) and 1-
Gastroprotective natural products
O
R
OH
O
OH
Natural Product Communications Vol. 3 (12) 2008 2141
O
O
HO
O
OH
91
OH
O
O
OAc
HO
OH
O
OH
OAc
92
content in mice, suggesting an antioxidant action.
These findings provide evidence that mangiferin (93)
affords gastroprotection against gastric injury
induced by ethanol and indomethacin, most possibly
through
the
antisecretory
and
antioxidant
mechanisms of action [78].
β-D-glucopyranosyl
HO
AcO
OH
OH
89 R = H
90 R = OH
O
OH
93
hydroxy-3,5,6-tri-O-acetyl-2(3,3-dimethylallyl)
xanthone (92), isolated from Calophyllum
brasilienses were tested on H+,K+-ATPase isolated
from dog stomach [68].
The compounds showed IC50values ranging from 47
μM to 1.6 mM. Steric hindrance by the substituents
at C-6 and C-3 appears to influence the potency of
inhibition of H+,K+-ATPase activity of these
compounds. The presence of a hydroxyl group at C-6
seems to play a prime role in the activity of
xanthones on gastric ATPase. In accord with this,
groups at C-6 in xanthone 89 reduced the potency of
H+,K+-ATPase inhibition by 34-fold. In addition,
acetylation at this position (xanthone 91) also
reduced the activity of the enzyme by a similar
amount. Also, the presence of a bulky substituent at
C-3 significantly reduced the potency of inhibition of
gastric H+,K+-ATPase activity by xanthone 91.
In search of novel gastroprotective agents, mangiferin
(93), a naturally occurring glucosylxanthone from
Mangifera indica, was evaluated in mice suffering
gastric injury induced by ethanol and indomethacin.
The effects of 93 on gastric mucosal damage were
assessed by determination of changes in either mean
gastric lesion area or ulcer score in mice and on
gastric secretory volume and total acidity in 4 hour
pylorus-ligated rats. Mangiferin (93) (3, 10 and 30
mg/kg p.o.) significantly attenuated the gastric
damage induced by ethanol and indomethacin. NAcetylcysteine (750 mg/kg, i.p.) and lansoprazole (30
mg/kg, p.o.), used as positive controls in these
ulcerogenic models, resulted in 50% and 76%
suppression of gastric injury, respectively. In 4 hourpylorus-ligated rats, intraduodenally applied 93 (30
mg/kg) caused significant diminutions in gastric
secretory volume and total acidity. In addition, like
N-acetylcysteine, a donor of sulfhydryls, mangiferin
(93) effectively prevented the ethanol-associated
depletion of gastric mucosal non-protein sulfhydryl
Conclusions: The development of safe and effective
drugs capable of preventing stomach damage induced
by NSAIDs or other gastric-damaging substances
represents an important goal of medicinal research
considering the large use of these drugs and the
increased healthcare costs when peptic ulcer disease
becomes a chronic condition. It is well established
that natural products are an excellent source of
chemical structures with a wide variety of biological
activity, including gastroprotective properties. The
large number of compounds derived from natural
sources that are currently undergoing evaluation in
clinical trials is another positive indicator that natural
product discovery provides good value for human
medicine.
This paper gives an up-to-date review of plant
extracts, natural compounds and their derivatives as
gastroprotective agents. This knowledge should
encourage further in vitro and in vivo
pharmacological studies and help to provide leads to
the
ultimate
goal
of
developing
novel
gastroprotective drugs.
List of abbreviations
AGS
CAT
COX-2
CYP1A1
EFMS
GPx
GSH
IC50
IL-8
iNOS
LD50
L-NAME
LPO
MRC-5
NEM
NF-κB
NO
NP-SH
NSAIDs
PGE
ROS
SOD
XO
= Human gastric epithelial cells
= Catalase
= Cyclooxygenase-2
= Cytochrome P450 1A1
= Effervescent floating matrix system
= Glutathione peroxidase
= Glutathione
= Inhibitory concentration 50%
= Interleukin-8
= Inducible nitric oxide synthase
= Lethal dose 50%
= N-nitro-L-arginine methyl ester
= Lipid peroxidation
= Human lung fibroblasts
= N-ethylmaleimide
= Nuclear factor kappa B
= Nitric oxide
= Non-protein sulfhydryl
= Nonsteroidal anti-inflammatory drugs
= Prostaglandin
= Reactive oxygen species
= Superoxide dismutase
= Xanthine oxidase
2142 Natural Product Communications Vol. 3 (12) 2086
Tundis et al.
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by kolaviron, a natural biflavonoid of Garcinia kola seed. Phytotherapy Research, 20, 14-20.
Choi SM, Shin JH, Kang KK, Ahn BO, Yoo M. (2007) Gastroprotective effects of DA-6034, a new flavonoid derivative, in various
gastric mucosal damage models. Digestive Diseases Sciences, 52, 3075-3080.
Jang SW, Lee JW, Park SH, Kim JH, Yoo M, Na DH, Lee KC. (2008) Gastroretentive drug delivery system of DA-6034, a new
flavonoid derivative, for the treatment of gastritis. International Journal of Pharmaceutics, 356, 88-94.
Ragab FA, Hassan GS, Yossef HA, Hashem HA. (2007) Synthesis of 6- and 9-alkylaminomethyl furoflavones as gastroprotective
agents. European Journal of Medicinal Chemistry, 42, 1117-1127.
Carvalho AC, Guedes MM, de Souza AL, Trevisan MT, Lima AF, Santos FA, Rao VS. (2007) Gastroprotective effect of
mangiferin, a xanthonoid from Mangifera indica, against gastric injury induced by ethanol and indomethacin in rodents. Planta
Medica, 73, 1372-1376.
NPC
Natural Product Communications
Phytochemistry and Pharmacology of Boronia pinnata Sm.
2008
Vol. 3
No. 12
2145 - 2150
MassimoCurinia*, Salvatore Genovesea, Luigi Menghinib, Maria Carla Marcotullioa and
Francesco Epifanob
a
Dipartimento di Chimica e Tecnologia del Farmaco, Sezione di Chimica Organica,
Università degli Studi di Perugia, Via del Liceo, Perugia, Italy 06123
b
Dipartimento di Scienze del Farmaco, Università “G. D’Annunzio”, Via dei Vestini 31,
66013, Chieti Scalo (CH), Italy 66013
curmax@unipg.it
Received: July 1st, 2008; Accepted: October 21st, 2008
Boronia pinnata Sm. (Rutaceae) is a plant that is widespread in New South Wales (Australia). Although there are no reports
about the use of this species in the local ethnomedical traditions, recent investigations led to the characterization of several
secondary metabolites, most belonging to the class of prenyloxyphenylpropanoids. Some of the compounds extracted from
B. pinnata showed valuable biological properties, such as anti-inflammatory activity and in vitro inhibition of growth of
Helicobacter pylori. The aim of this review is to cover what has been reported so far in the literature on the title plant from a
phytochemical and pharmacological point of view.
Keywords: Anti-inflammatory activity, Boronia pinnata, Helicobacter pylori, prenyloxyphenylpropanoids.
Prenylation is a chemical or enzymatic addition of an
hydrophobic side chain to an accepting molecule
(another terpenoid molecule, an aromatic compound,
a protein, etc.). In particular, prenylation of aromatic
secondary metabolites plays a critical role in the
biosynthesis of a wide range of molecules exerting
valuable
pharmacological
effects
across
phylogenetically different classes of living
organisms, from bacteria to mammals and plants.
Frequently, the addition of an isoprenoid chain
renders the molecule more effective than the parent
compound from a pharmacological point of view.
These ‘‘hybrid’’ natural products represent nowadays
a new frontier for the development of novel drugs, in
particular as antimicrobial, anti-oxidant, antiinflammatory and anti-cancer agents. Oxyprenylated
natural products are compounds of mixed
biosynthetic origin for which the final step of the
biosynthetic process is the prenylation of either an
alkaloid or a phenylpropanoid core using prenyl
diphosphate as alkylating agent [1], the latter coming
in turn from either the mevalonate [2] or 1-DOXP
pathways [3]. Oxyprenylated secondary metabolites
have been considered for decades merely
as biosynthetic intermediates of C-prenylated
compounds and only in the last ten years have been
characterized as phytochemicals exerting interesting
and valuable biological activities [4]. Considering the
length of the carbon chain, three types of prenyloxy
skeletons can be identified: C5 (isopentenyl), C10
(geranyl) and C15 (farnesyl). Isopentenyloxy and
geranyloxy chains are quite abundant in nature, while
farnesyloxy ones are less common. The skeleton may
consist only of carbon and hydrogen or may contain
oxygen atoms, usually in the form of alcohols, ethers
or ketones. Several species have been identified up to
now as producing prenyloxyphenylpropanoids.
Among these Boronia pinnata Sm. (Rutaceae) has
been characterized as one of the species to
biosynthesize a wide variety of the above cited
secondary metabolites.
While several plants of the genus Boronia have been
reported to be used in local ethnomedical traditions
[5-10], B. pinnata has never been cited in the
literature in this regard. However, recent
investigations led to a detailed phytochemical profile
of the main secondary metabolites of this plant and
have revealed that some of these compounds exert
valuable anti-cancer and anti-inflammatory effects,
2146 Natural Product Communications Vol. 3 (12) 2008
mainly targeting the lipoxygenase system, and an
inhibitory activity in vitro against Helicobacter
pylori. The aim of this review is to cover what has
been reported so far in the literature on the title plant
from a phytochemical and pharmacological point of
view.
Curini et al.
OH
CH3O
HO
O
OH
CH3O
OGlcRhm
OCH3
OH
1
O
2
R1
Botany of Boronia pinnata
B. pinnata is a species belonging to the family
Rutaceae that typically grows in a very restricted area
of Australia, namely New South Wales. The name of
the genus comes from Francesco Borone, an Italian
botanist (1769-1794), while “pinnata” alludes to the
paired leafets that are an anatomical feature of this
plant [10]. B. pinnata grows in sandstone country up
to lower mountain levels in dry sclerophyll forests
and in well-drained sandy heaths, mainly along
coasts. It is a beautiful waxy-flowered shrub, 0.5-1.5
m high. Branchlets are glabrous and slightly angled
to prominently 4-angled. The foliage is ornamental
and ferny, light to mid-green. Leaves are up to 25
mm long, opposite and with several pairs of widely
spaced leaflets. Flowers are grouped in inflorescences
that are subterminal to axillary, collected in
corymbose cymes, each comprising from 3 to 8
flowers. Petals are in group of 4, imbricate, 5-10 mm
long, colored bright to purplish pink. The flowering
stage occurs from June to February [11].
R2
R4
OR3
3
R1
CHO
4
CHO
R2
R3
H
R4
H
H
H
O
5
CHO
OCH3
H
6
CHO
H
H
7
8
9
CHO
CHO
CH2OH
OCH3
OCH3
OCH3
CH3
CH3
H
OCH3
H
10
11
CH2OCH3
CH2OH
OCH3
OCH3
CH3
OCH3
OCH3
12
13
14
15
CH2OH
CH2OH
COOH
COOH
OCH3
OCH3
H
OCH3
CH3
CH3
H
H
OCH3
H
H
Phytochemistry of B. pinnata
The first studies describing the phytochemical
composition of B. pinnata refer to the analysis of the
essential oil obtained from the aerial parts of the
plant, which were reported in two manuscripts
published in 1919 and 1922 [12,13]. The essential oil
was obtained with a yield of 0.1% and showed a very
high quantity of elemicin (1) (> 75%) that was also
claimed as a distinctive feature of B. pinnata in
comparison with other Boronia species. At the same
time, Morrison reported the isolation of rutin (2) from
the aqueous extract of the leaves [14].
R1
R3
OR2
R1
H
R2
16
R3
OCH3
17
OCH3
CH3
H
OCH3
O
O
H
OH
H
OH
O
Studies on the title plant were then abandoned for
about 80 years. In 1999 and 2000 Itoigawa et al
reported the isolation and structural characterization
of 23 novel and already known secondary
metabolitesfrom the roots of B. pinnata [15,16]. In
particular, they isolated six cinnamic aldehyde
derivatives, boropinals A (3), B (4) and C (5),
together with 3’,4’-dimethoxycinnamic aldehyde
(7) and 3’,4’,5’-trimethoxycinnamic aldehyde (8),
five cinnamoyl alcohol derivatives, boropinol A (9),
OH
N
OCH3
O
CH3
18
19
OCH3
R1
R2
N
O
R3
20
21
R1
H
H
R2
H
OCH3
R3
H
H
Phytochemical and pharmacological aspects of Boronia pinnata
Natural Product Communications Vol. 3 (12) 2008 2147
OCH3
1
N
OCH3 H
R
a
O
HO
O
25
R1
OCH3
R2
23
H
H
15
CH3O
O
OCH3
OCH3
22
O
CHO
CHO
R2
methoxyboropinol B (10), boropinol C (11), 3’,4’dimethoxycinnamyl alcohol (12) and 3’,4’,5’trimethoxycinnamyl alcohol (13), two cinnamic
acids, p-hydroxycinnamic (14) and boropinic acid
(15), three phenylpropenes, 3-(3’-methoxy-4’prenyloxy)phenyl-1-propene (16), methyleugenol
(17) and elemicin (1), one lignan, boropinan
(18),
five alkaloids, pinolinone (19), dictamnine
(20), evolitrine (21), preskimmianine (22) and
folimine (23), and finally one coumarin, braylin (24).
Some of the novel secondary metabolites extracted
from the roots of B. pinnata have been also obtained
by chemical synthesis.
The first synthesis in this regard was reported by Hou
and coworkers in 2003 [9]. They synthesized
boropinol A (9), boropinal C (5) and boropinic acid
(15) using commercially available vanillin (25) as
starting material (Scheme 1).
Condensation of the latter with α-carbethoxy-methylphosphorane in DME furnished the corresponding
ester, followed by alkaline hydrolysis to give
boropinic acid (15) in 91% yield. Reduction of 15
with LiAlH4 in Et2O yielded boropinol A (9),
although not in a sufficiently pure form. So the
mixture was oxidized with K2Cr2O7 in DMSO to give
boropinal C (5) in 61% yield. Finally, reduction of 5
with NaBH4 in MeOH yielded pure boropinol A (9)
(70%) [17].
The reaction of 25 with isopentenyl bromide in DMF
gave the O-prenylated adduct 26 in 84% yield.
Three years later Curini and coworkers reported a
short and high-yielding synthesis of boropinic acid
(15) starting from readily commercially available
ferulic acid (7) (Scheme 2) [18].
d,e
5
26
f
9
Scheme 1: Reagents and conditions: (a) isopentenyl bromide, DMF; (b)
Ph3P=CH-CO2Et, DME; (c) KOH (aq); (d) LAH, dry Et2O; (e) K2Cr2O7,
DMSO; (f) NaBH4, MeOH.
CO2H
O
24
b,c
CO2CH3
a
HO
HO
OCH3
OCH3
7
b,c
28
15
Scheme 2: Reagents and conditions: (a) MeOH, conc. H2SO4, reflux; (b)
isopentenyl bromide, K2CO3, acetone, reflux; (c) NaOH 70°C.
Ferulic acid (7) was first converted to the
corresponding methyl esters 28 in 99% yield by
reaction in refluxing MeOH under the catalysis of
conc. H2SO4; compound 28 was submitted to
prenylation using isopentenyl bromide as alkylating
agent and dry K2CO3 as base in refluxing acetone,
followed by alkaline hydrolysis in the same reaction
vessel to obtain boropinic acid (16) in 96% yield.
So, compound 16 has been easily synthesized from
widely available and non-toxic starting materials by a
high-yielding, environment friendly, and cheap
synthetic route.
Pharmacological properties of active principles
from B. pinnata
The fact that B. pinnata was not part of local
ethnomedical traditions, mostly due to its restricted
habitat, has not attracted, for many decades, the
attention of researchers to carry out pharmacological
studies of extracts or single secondary metabolites of
this plant. The first study, aimed at investigating the
anticancer properties of selected compounds
extracted from roots of B. pinnata, was reported by
Itoigawa and coworkers in 1999 [15]. Twelve
phenylpropanoids, namely compounds 1, 3, 5, 7-12,
16 and 17 were tested in vitro as inhibitors of
Epstein-Barr virus early antigen (EBV-EA) activation
induced by 12-O-tetradecanoylphorbol-13-acetate
(TPA) in Raji cells (EBV genome-carrying human
lymphoblastoid cells; EBV non –producer type).
One of the extracted compounds, 3-(3’-methoxy-4’isopentenyloxy)phenyl-1-propene (16), was also
tested in vivo as an inhibitor of effects on skin tumor
2148 Natural Product Communications Vol. 3 (12) 2008
Curini et al.
Table 1: Inhibitory effects of selected phenylpropanoids from roots of B.
pinnata on TPA-induced EBV-EA activation.
Table 2: Inhibitory effects of compounds 3 and 14 on TPA-induced
EBV-EA activation.
Compd.
Compd.
1
3
5
7
8
9
10
11
12
13
16
17
EBV-EA positive cells (% vialibility)
Compd. concentration (mol ratio/32 pmol TPA)
500
100
10
29.2 ± 1.6
71.2 ± 2.2
90.1 ± 1.3
26.7 ± 1.3
69.7 ± 2.1
86.2 ± 2.2
23.2 ± 1.1
67.7 ± 2.0
84.5 ± 2.1
33.5 ± 1.3
75.5 ± 2.1
91.3 ± 1.7
30.6 ± 1.1
71.4 ± 2.2
89.3 ± 1.8
29.8 ± 1.1
69.4 ± 2.0
87.6 ± 2.1
37.2 ± 1.4
77.3 ± 2.3
92.4 ± 1.5
26.4 ± 1.4
69.5 ± 1.9
87.3 ± 1.3
36.2 ± 1.4
77.0 ± 2.3
92.3 ± 1.8
32.5 ± 1.3
75.8 ± 2.1
90.6 ± 1.2
26.2 ± 1.3
69.5 ± 1.9
87.3 ± 1.1
31.5 ± 1.5
74.2 ± 2.3
92.4 ± 1.6
promotion by means of a two-stage carcinogenesis
test
of
mouse
skin
papilloma
using
dimethylbenz[a]anthracene (DMBA) as initiator and
TPA as promoter. Results of the test on Raji cells are
reported in Table 1.
Although not reported in Table 1, all compounds
showed a 100% inhibitory effect at the concentration
value of 1000, expressed as mol ratio/32 pmol TPA.
Among the tested aldehydes, boropinal C (5)
exhibited the most significant inhibitory activity on
EBV-EA activation. Among the cinnamyl alcohols,
boropinol A (9), boropinol C (11) and compound (16)
showed similar activities to that of boropinal C (5).
It’s interesting to note that secondary metabolites
lacking a prenyloxy side chain like compounds 7, 8,
10, 12 and 13 are less effective as inhibitors of EBVEA activation. This suggests that the presence of a
prenyloxy side chain in position 4’ on 3-phenyl-2propenal and –propenol cores enhance the inhibitory
effects. The same observation was made for the
activity of several other prenyloxyphenylpropanoids
[4].
Results of the in vivo two-stage carcinogenesis test of
mouse skin papillomas induced by DMBA and
promoted by TPA using compound 15 in comparison
with a positive control that developed papillomas
after only 10 weeks of promotion, showed that when
applied before TPA treatment, 15 delayed the
formation of papillomas. In each group of animals
treated with compound 16 only about 20% of mice
bore papillomas at 10 weeks after promotion and
even after 20 weeks of promotion 80% of the mice
bore tumors. Moreover, 16 reduced the incidence of
papillomas (average number of tumors per mouse):
less than 5 papillomas were formed per mouse after
11 weeks of promotion and only about 3.8 papillomas
6
15
EBV-EA positive cells (% vialibility)
Compd. concentration (mol ratio/32 pmol TPA)
500
100
10
27.2 ± 1.8
65.5 ± 1.0
88.5 ± 0.4
23.1 ± 1.1
62.2 ± 1.5
84.0 ± 0.3
Table 3: Inhibition of 5-LOX mediated PUFAs peroxidation by boropinic
acid (15).
Compds.
15
Ascorbic acid
BHT
Trolox
a
IC50 (μmol/mL)a
2.89 x 10-5 ± 2.62 x 10-6
0.105 ± 0.0072
0.023 ± 0.0052
0.047 ± 0.0048
p<0.05 at Student’s t test
per mouse even after 20 weeks of promotion. The
same in vitro test on EBV-EA activation was later
carried out on 4’-hydroxy-3’-prenylcinnamaldehyde
(6) and boropinic acid (15), which had been isolated
in a second step of the ongoing studies of Itoigawa
and coworkers [16]. Results are reported in Table 2.
Both compounds showed a 100% inhibitory activity
at a concentration value of 1000, expressed as mol
ratio/32 pmol TPA, as had the previous compounds,
and a good inhibitory activity on TPA-induced EBVEA activation, also at lower doses. All the other
secondary metabolites that had been extracted from
the roots of B. pinnata were not active in the same
test.
In the frame of an ongoing study devoted to the
synthesis and characterization of pharmacological
properties of prenyloxyphenylpropanoids, Curini and
coworkers first studied the in vitro anti-inflammatory
and anti-bacterial properties of boropinic acid (15).
These authors found first that compound 15 did not
exhibit significant antioxidant properties when
submitted to the DPPH radical scavenging assay. To
enforce this finding they also performed the assay for
inhibition of polyunsaturated fatty acids (PUFAs)
peroxidation mediated by soybean 5-lipoxygenase (5LOX), using ascorbic acid, butyl hydroxytoluene
(BHT) and Trolox as positive controls [18]. Results
of the latter test are reported in Table 3.
Contrasting results obtained by means of chemical
and enzymatic assays suggested that boropinic
acid (15) acted as an effective 5-LOX inhibitor. It is
noteworthy that other prenyloxyphenylpropanoids,
like cinnamic acid bearing longer O-chains
or coumarins, were not active in both tests.
Phytochemical and pharmacological aspects of Boronia pinnata
Table 4: MIC values for inhibition of growth against H. pylori by
boropinic acid (15).
Compd
15
Metronidazole
Amoxicillin
Tetracycline
Clarithromycin
a
MIC (μg/mL)a
1.62
> 200
0.781
4.00
1.25
Values are means of three experiments.
To rationalize tentatively the inhibitory mechanism
observed for boropinic acid and the lack of efficacy
of some other prenyloxyphenylpropanoids, Curini
and coworkers inferred a possible 5-LOX/ligand
docking by comparative modelling. As a result of this
analysis, a peculiar feature of the modelled 5
LOX/boropinic acid complex is the possibility for the
hydrophobic side chain represented by the
isopentenyloxy moiety to be oriented and to enter in
van der Waal’s contact with a cluster of hydrophobic
amino acids. This interaction is enforced by polar
interactions at the same site of the carboxylic group
with Ile 857 and the amide side chain Gln 514 of the
enzyme. Since these additional interactions might
contribute to the enhancement of the complex
stability, it may be hypothesized that the loss of
activity of 5-LOX in the presence of 15 could be the
result of enzyme inhibition as a consequence of
stable ligand docking in the active site.
The same research group studied the anti-bacterial
properties of boropinic acid (15). After having
screened several Gram positive and Gram negative
bacterial strains, they found that 15 is an effective
inhibitor in vitro of the growth of Helicobacter pylori
[19]. Results of the test, performed by the agar
dilution method with metronidazole, amoxicillin,
tetracycline and clarithromycin as reference drugs,
are reported in Table 4, expressed as minimum
inhibitory concentration (MIC).
Although from the data reported in Table 4 it is
evident that the strain of H. pylori (namely DSMZ
4867 obtained from human gastric samples) used to
perform the test is clearly resistant to metronidazole,
Natural Product Communications Vol. 3 (12) 2008 2149
the activity of boropinic acid (15) as an inhibitory
agent of the growth of H. pylori is comparable to that
of most common antibiotics currently used in therapy
to eradicate bacterial infections. Based on these
preliminary results, boropinic acid (15) could be
viewed as a potential lead compound for a novel class
of H. pylori inhibitors. However, studies aimed to
clearly depict the mechanism of action of this
secondary metabolite, and in vivo tests using a
suitable animal model, and in vitro and in vivo tests
to evaluate the activity of 15 against strains of H.
pylori isolated from clinical patients have to be
carried out in the near future.
Conclusions and future perspectives
In this review we have reported what is known so far
in the literature about the Australian shrub B.
pinnata. Twenty-four secondary metabolites have
been isolated in low concentrations and structurally
characterized from different parts of this plant and
the major part of these natural compounds belong to
the class of prenyloxyphenylpropanoids. With the
aim of obtaining these compounds in sufficient
quantities to determine a detailed pharmacological
profile, bypassing difficulties linked to the low
quantities obtainable from natural sources, a few of
the compounds have been obtained by chemical
synthesis by means of environmentally sound,
friendly and high yielding methodologies. In
particular, boropinic acid has been obtained in nearly
quantitative yield. Preliminary pharmacological
studies on simple and oxyprenylated phenylpropanoids from B. pinnata have shown that
compounds like boropinal C and boropinic acid show
valuable biological properties, such as anti-cancer,
anti-inflammatory and anti-ulcer activities. In the
search for novel therapeutic remedies from nature,
the data reported in this review will certainly prompt
further studies on this plant to better define the
profile of its secondary metabolites and their
pharmacological properties, in particular by means of
in vivo studies employing suitable animal models.
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Smith HG. (1919) Essential oil of Boronia pinnata Sm. and the presence of elemicin. Proceeding Royal Society of Victoria, 32,
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Ito C, Itoigawa M, Furukawa H, Ichiishi E, Mukainaka T, Okuda M, Ogata M, Tokuda H, Nishino H. (1999) Anti-tumor-promoting
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NPC
Natural Product Communications
Therapeutic Potential of Kalanchoe Species: Flavonoids and
other Secondary Metabolites
2008
Vol. 3
No. 12
2151 - 2164
Sônia S. Costaa,*, Michelle F. Muzitanoa,b, Luiza M. M. Camargoa and Marcela A. S. Coutinhoa
a
Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, RJ, Brazil
b
Laboratório de Biologia do Reconhecer, Centro de Biociências e Biotecnologia,
Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, RJ, Brazil
sscosta@nppn.ufrj.br
Received: July 22nd, 2008; Accepted: November 5th, 2008
The Kalanchoe genus (syn. Bryophyllum), family Crassulaceae, comprises 125 species, most of them native to Madagascar.
The great importance of several of these species for the traditional medicine in several regions of the World, esspecially India,
Africa, China and Brazil, stimulated research programs into these plants from both a pharmacological and chemical point of
view. The present review focuses on the main results obtained during the last decade on the secondary metabolites isolated
from these species – endowed or not with a specific biological profile – with emphasis on flavonoids. The distribution of these
molecules in the genus will be summarized and special attention will be given to K. brasiliensis and K. pinnata, two species
well-known for healing inflammatory and infectious processes. Ornamental Kalanchoe species are also discussed as a potential
source of bioactive compounds. This review covers the period 1970-2008.
Keywords: Kalanchoe, Crassulaceae, flavonoids, biological activity, Kalanchoe pinnata, Kalanchoe brasiliensis.
1. Introduction
The genera Kalanchoe, Rhodiola and Sedum are three
medicinally important genera in the family
Crassulaceae. The genus Kalanchoe (syn.
Bryophyllum), first established by Adanson (1763),
comprises 125 species, most of them native to
Madagascar [1]. The great importance of some
Kalanchoe species for traditional medicinal use in
many regions of the World, especially India, Africa,
China and Brazil, stimulated several research groups
to investigate the pharmacological properties of the
active plant extracts as well as their chemical
composition [2-17].
Several Kalanchoe species occur in Brazil, some of
which, such as K. pinnata and K. brasiliensis, are
used for the treatment of rheumatism, inflammation,
burns, wounds and abscesses [2]. Other species such
as K. fedtschenkoi and K. blossfeldiana are cultivated
for ornamental purposes [18]. Indeed, a significant
number of Kalanchoe species – regardless of their
medicinal interest – have a great ornamental value as
they exhibit extremely beautiful flowers with a broad
spectrum of colors varying from white, light yellow
to orange, pink, red and purple.
Although Kalanchoe plants have important medicinal
applications, some of them are known to be toxic to
cattle [19] and chickens [20]. Flowering plants were
found to be responsible for the most important forms
of poisonings in Australia [19]. As far as we know,
Kalanchoe flowers are not used for medicinal
purposes; only leaves or non-flowering aerial parts
are usually used.
Succulent plants, especially those from the genus
Kalanchoe, have been thoroughly studied with
respect to their Crassulacean-acid metabolism
(CAM) ranging from ecological, physiological
and cell biological aspects, transport and
compartmentalization to molecular biology [21-24].
The early studies on the secondary metabolites from
Kalanchoe species mainly focused on their flavonoid
content. Gaind & Gupta, at the beginning of the
1970s, accomplished the pioneer studies on the
2152 Natural Product Communications Vol. 3 (12) 2008
isolation and identification of glycosyl flavonoids
from K. pinnata [25,26].
Flavonoids can occur naturally as aglycones or
glycosides [27]. Plants containing flavonoids have
been used for thousands of years in traditional
medicine [28].
Bufadienolides - perhydrophenanthrene derivatives
substituted at C-17 with a pentadienolide – are
another metabolite class present in some
Kalanchoe species. These compounds originate
from
mevalonate-isopentenyl
pyrophosphate–
pregnenolone metabolism [29] and are involved in
poisoning of pets and cattle after ingestion of the
flowers of some Kalanchoe species [20,30].
A brief review of the different classes of secondary
metabolites isolated from Kalanchoe species, such as
bufadienolides, terpenoids and flavonoids, as well as
some biological aspects, has already been published
by one of us [31]. The increasing interest in
Kalanchoe species for the discovery of new bioactive
substances, led us to review the state of the art in the
study of their bioactive chemical composition. The
present review will particularly focus on flavonoid
metabolites, the main bioactive chemical constituents
of plants of this genus.
2. Chemical, ethnomedicinal and potential
therapeutic aspects of the genus Kalanchoe: state
of the art.
Among the most widespread Kalanchoe species, two
of them - K. pinnata and K. brasiliensis - are widely
employed in Brazil to treat different infectious,
injurious and inflammatory processes [2]. These
medicinal herbs are locally known under the same
common names, "saiao" or "coirama", in the
different Brazilian regions. In most cases, one of
these species is employed in substitution for the
other, generally in an undifferentiated manner.
K. brasiliensis Camb. is the only native Kalanchoe
species in Brazil and was the first one to be studied
in depth by our group. Although the leaves are
largely used in Brazil, their chemical composition
and pharmacological effects were yet unknown when
we started our studies.
First results from our group demonstrated that K.
brasiliensis (Kb) juice, aqueous and alcoholic
extracts exerted a direct in vitro inhibitory effect on
human lymphocyte proliferation [32]. Bioguided
purification of the juice of the combined aerial parts,
Costa et al.
based on its in vitro lymphocyte anti-proliferative
activity, led to the isolation and identification of
seven patuletin rhamnosides [5]. Among them,
patuletin 3-O-(4"-O-acetyl-α-L-rhamnopyranosyl)-7O-(2"'-O-acetyl-α-L-rhamnopyranoside),
patuletin
3-O-α-L-rhamnopyranosyl-7-O-(2"'-O-acetyl-α-Lrhamnopyranoside) and patuletin 3-O-(4"-O-acetylα- L -rhamnopyranosyl)-7-O-rhamnopyranoside
named kalambroside A, B and C, respectively, were
described for the first time.
The four other flavonoids were the known patuletin
rhamnosides initially isolated from K. gracilis, a
medicinal plant used in Taiwan for the treatment of
tissue inflammation [33]. One characteristic of
the main patuletin rhamnosides isolated from
K. brasiliensis is the presence of either a mono or a
di-acetylated sugar residue, such as those found in
kalambroside A, B and C (Figure 1). The known
flavonoids quercitrin and isoquercitrin were also
isolated from the same specimens [34].
OH
3'
OH
4'
O
7
O 2
1'
Me
O 1'''
HO
3 O
2''' MeO 6
HO
OR 2
OH O
Me
O 1"
R1 O
R 1 R2
4"
HO OH
Kalambroside A Ac Ac
Kalambroside B
H
Ac
Kalambroside C Ac
H
Figure 1 - Kalambrosides A-C from K. brasiliensis
From a biological point of view, among the seven
flavonoids isolated, kalambroside A, B and C were
shown to be potent inhibitors of lymphocyte
proliferation. Diacetylated flavonoids with two
rhamnosyl units, such as kalambroside A, were more
active than their monoacetylated congeners
kalambrosides B and C. It was generally observed
that the pure components showed higher activity then
either the crude flavonoid fraction or extract. Their
lymphoproliferative inhibitory activities seem to be
dependent on the presence of at least one acetyl
group and on its position on the rhamnosyl unit of
these flavonoids. Non-acetylated derivatives, such as
patuletin 3-O-rhamnoside and patuletin 3,7-di-Orhamnoside, did not show any activity [5]. An
hydroalcoholic extract of leaves of K. brasiliensis
showed acetylcholine esterase inhibitory effects.
This activity was attributed to a flavonoid
mixture, containing 8-methoxyquercetin-3,7-di-Orhamnopyranoside and 8-methoxykaempferol-3,7-diO-rhamnopyranoside [35]. The authors also reported
a larvicidal effect for the plant extract.
Secondary metabolites from Kalanchoe species
Natural Product Communications Vol. 3 (12) 2008 2153
Antimicrobial activity was described for K.
brasiliensis extracts [36]. These findings support, at
least partially, the popular use of the plant for healing
infected wounds.
any inhibitory activity on human lymphocyte
proliferation [40]. The observed inhibitory activity of
lymphocyte proliferation was attributed to a fraction
composed of palmitic and stearic acids [11]. This
activity can be correlated with an anti-inflammatory
effect and could partially explain the use of the plant
against inflammatory conditions. In continuation of
our phytochemical investigation of the bioactive
flavonoid fraction from K. pinnata leaf extract, we
isolated five flavonoids, including the already
reported quercitrin and afzelin. The three others were
the minor methoxylated flavones 5,7,4’-trihydroxy8,3’-dimethoxyflavone, galangustin and jaceosidin,
which had not previously been reported for the
Kalanchoe genus [40]. These flavones are common
in the Asteraceae family [41], but methoxylation at
the C-8 position is rare when compared with
methoxylation at C-6 or C-7 of the A ring. Despite
the unusual methoxylation at C-8, there is one
example described for the Crassulaceae family. This
compound, a limocitrin glycoside, was isolated from
Sedum acre [42]. Both of the flavonols isolated,
quercitrin and afzelin, significantly inhibit human
lymphocyte proliferation (IC50 = 1.0 – 10.0 μg/mL)
in vitro.
Leaves of K. brasiliensis are widely used in Brazil
against the inflammation and allergic reactions
provoked by insect bites. The anti-histaminic
property of the plant extract could be observed [37].
Additionally, the antiophidic and thyroid peroxidase
inhibitory activities were demonstrated [38,39]. The
compounds responsible for these pharmacological
activities are not yet identified [36-39].
K. pinnata (Lamarck) Persoon (=Bryophyllum
pinnatum, B. calycinum, K. calycina), one of the
oldest Kalanchoe species introduced to European
botanical gardens, is considered native to Madagascar
and has become naturalized in the tropics throughout
the World. This species is certainly the most
widespread member of the Kalanchoe genus,
particularly in the tropical regions, and is reputed for
its medicinal properties [18]. For these reasons it has
been the object of many pharmacological and
chemical studies. The first of these led to the
characterization of some organic acids, alkanes,
terpenes, sterols and waxes [31]. The occurrence in
K. pinnata of the flavonoids quercetin 3-O-diarabinoside and rutin was first reported by Gaind and
Gupta [25].
Although employed for the same medicinal purposes
as K. brasiliensis, K. pinnata shows a very different
chemical profile concerning its phenolic metabolites,
as well as different biological activities. In Brazil,
this plant is known, among other names, as "saiao",
"folha-da-fortuna" and "folha-do-pirarucu" [18].
The main common biological activity shared both by
K. pinnata and K. brasiliensis is their antiinflammatory effect and the correlation of this with
the immune system. The immunomodulatory activity
observed for the different types of K. pinnata leaf
extracts (juice, aqueous and ethanolic extracts)
led us to carry out the first biomonitored
chemical investigation of this plant. The study
of a hydrosoluble fraction of a K. pinnata
ethanolic extract led to the isolation of its major
flavonoid component, the known quercetin 3-O-α-Larabinopyranosyl
(1→2)-α-L-rhamnopyranoside
[40]. Antiallergic activity was reported for this
quercetin diglycoside that had been previously
isolated in 1986 [4]. This flavonoid, despite its
high concentration in the extract, did not show
The immunomodulatory ability demonstrated by
K. pinnata extracts, besides its ethnomedicinal use
for healing wounds and skin diseases, led us to
investigate the effect of this plant on Leishmania
amazonensis-infected macrophages. L. amazonensis
is one of several Leishmania protozoa that cause
serious endemic diseases, leishmaniases, in tropical
and subtropical regions of the World. Leishmaniases
may range from single cutaneous to fatal kala-azar,
affecting more than 12 million people in 88 countries
[43]. There is no vaccine ready for use in humans and
chemotherapy still relies on toxic i.m. injections with
pentavalent antimonials, Pentostam and Glucantime,
or Amphotericin B [44].
Oral treatment of L. amazonensis-infected mice with
aqueous leaf extract of K. pinnata (Kp) significantly
reduced the lesion size and parasite load to levels
comparable to i.p. injection with Glucantime [6,9].
Furthermore, an important remission of human
cutaneous leishmaniasis upon oral Kp treatment,
without toxic effects, has been reported in one case
study [45].
A biomonitored fractionation of Kp extract has
demonstrated quercitrin to be a potent antileishmanial
compound, with a low toxicity profile [46]. Along
2154 Natural Product Communications Vol. 3 (12) 2008
with quercitrin, other flavonoids were isolated, such
as the new kaempferol 3-O-α-L-arabinopyranosyl
(1→2)-α-L-rhamnopyranoside
(kapinnatoside),
quercetin 3-O-α-L-arabinopyranosyl (1→2)-α-Lrhamnopyranoside
and
4’,5-dihydroxy-3’,8dimethoxyflavone 7-O-β-D-glucopyranoside, all of
which showed antileishmanial activity to different
extents [47]. All these results show an interesting
medicinal potential for the use of Kp extract and its
flavonoids against leishmania.
Recently, the protective effect of K. pinnata aqueous
extract against fatal anaphylactic shock in mice was
reported [48]. Oral protection was accompanied by a
reduced production of specific IgE antibodies,
reduced eosinophilia, and impaired production of the
IL-5, IL-10 and TNF-α cytokines. Kp extract
prevented antigen-induced mast cell degranulation
and histamine release in vitro. This activity was
mainly attributed to the flavonoid quercitrin
previously reported from Kp extract [48].
Indeed, the phytotherapeutic potential of this species
is high, as can be deduced from the wide spectrum of
biological activities so far reported for it (Table 1)
[6,9,11,15,16,46-59]. It is remarkable that despite the
large number of reported pharmacological properties,
to date, only few bioactive molecules have been
identified from this species. In the perspective of a
commercial use of Kp as a standardized qualitycontrolled phytotherapeutic drug there is a
requirement for the definition of a specific
chemical marker for this species. Quercetin 3-O-α-Larabinopyranosyl
(1→2)-α-L-rhamnopyranoside,
which shows a restricted occurrence in nature, could
be considered as a convenient chemical marker for
K. pinnata. This flavonoid is an uncommon molecule
not reported to date in other plant species, except for
Alphitonia philippinensis (Rhamnaceae) [60] and
K. blossfeldiana flowers, as a minor metabolite [61].
Although K. pinnata is largely used as a medicinal
plant and is therefore considered as safe, some toxic
bufadienolides were isolated from its leaves and also
from the whole plant [62-64]. A bufadienolide from
K. pinnata – bryophyllin A – was shown to be a
potent anti-tumor promoter inhibitor when compared
with other bufadienolides isolated from K. pinnata
and K. daigremontiana x tubiflora [12]. However,
these substances were not detected in the K. pinnata
specimens employed in our studies. Other less
studied Kalanchoe species will be discussed below
under chemical and pharmacological aspects.
Costa et al.
Table 1: Pharmacological activities and bioactive compounds reported
for extracts from Kalanchoe pinnata.
Pharmacological
Activity
Antibacterial
Antidiabetic
Bioactive compounds
-
References
[49]
[50]
Antihypertensive
-
[51]
Anti-inflammatory
-
[52-54]
Antimalarial
Quercitrin, quercetin 3-O-αL-arabinopyranosyl (1→ 2)α-L-rhamnopyranoside,
kaempferol 3-O-α-Larabinopyranosyl (1→ 2)-αL-rhamnopyranoside and
4’,5-dihydroxy-3’, 8dimethoxyflavone 7-O- β-Dglucopyranoside
-
Antinociceptive
-
[50]
Antiulcerogenic
-
[53]
CNS depressant
-
[56]
Hepatoprotective
-
[16]
Immunosuppression
Fatty acids
[11]
Muscle relaxing
-
[57]
Neurosedative
-
[57]
Tocolytic
-
[58,59]
Anti-anaphylactic
Quercitrin
Antileishmanial
[6,9,15,46,47]
[55]
[48]
Kalanchoe gastonis-bonnieri R. Hamet & H. Perrier
(= Bryophyllum gastonis-bonnieri), known popularly
as “life-plant”, “donkeys-ear” and “mala-madre”,
possesses large leaves that can measure up to 50 cm
in length [18]. Its leaf juice is employed vaginally as
a contraceptive in Mexican traditional medicine [14].
The plant is also employed against genito-urinary
troubles and vaginal infections [65]. In Madagascar it
is employed to treat wounds caused by insect bites
[18]. The chemical composition of the juice obtained
from K. gastonis-bonnieri leaves is under
investigation in our laboratories.
Kalanchoe gracilis Hance is used in Taiwan for the
treatment of tissue inflammation [33]. Nineteen
flavonoids were isolated from the aerial parts of this
species, most of them exhibiting a patuletin skeleton
bearing a mono or a di-acetylated sugar residue [66].
Four eupafolin rhamnosides, some of them bearing
an acetyl substitution in the sugar moiety, were also
reported. Luteolin and four other flavonoids
(eupafolin, kaempferol, quercetin and quercitrin)
were also identified [33]. Table 2 summarizes the
flavonoids reported for the Kalanchoe species.
Recently, new cytotoxic bufadienolides were
described from K. gracilis (kalanchosides A-C)
(Figure 2) [67] and K. hybrida (kalanhybrins A-C)
(Figure 3) [68].
Secondary metabolites from Kalanchoe species
Natural Product Communications Vol. 3 (12) 2008 2155
Table 2: Flavonoids isolated from Kalanchoe species organized by their classes and frequency in the genus.
Flavonoids
Glycosides
Flavonols
Patuletin 3,7-di-O-α-L-rhamnopyranoside
Patuletin 3-O-α-L-rhamnopyranoside
Kaempferol 3-O-α-L-rhamnopyranoside (afzelin)
Kaempferol 3-glucoside (astragalin)
Kaempferol 3-O-β-D-xylopyranosyl (1→2)-α-L-rhamnopyranoside-7-O-α-L-rhamnopyranoside (sagittatin A)
Kaempferol 3-O-β-D-glucopyranoside-7-O-α-L-rhamnopyranoside
Kaempferol 3-O-β-D-xylopyranosyl (1→2)-O-α-L-rhamnopyranoside
Kaempferol 3-O-α-L-arabinopyranosyl (1→2)-α-L-rhamnopyranoside (kapinnatoside)
Kaempferol arabinosyl-coumaroyl (bryophylloside)
Quercetin 3-O-di-arabinoside
Quercetin 3-O- β -D-glucopyranoside (isoquercitrin)
Quercetin 3-O-α-L-arabinopyranosyl (1→2)-α-L-rhamnopyranoside
Quercetin 3-O-glucoside-7-O-rhamnoside
Quercetin 3-O-α-L-rhamnopyranoside (quercitrin)
Quercetin 3-O-α-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside (rutin)
Quercetin 3-O-β-D-glucopyranosyl (1→2)-β-D-xylopyranoside
Flavones
Eupafolin 4’-O-rhamnoside
Eupafolin 3,7-di-O-rhamnoside
4’,5-dihydroxy-3’,8-dimethoxyflavone-7-O-β-D-glucopyranoside
Anthocyans
Cyanidin 3-O-β-D-glucoside
Cyanidin 3,5-O-β-D-diglucoside
Pelargonidin 3,5-O-β-D-diglucoside
Peonidin 3,5-O-β-D-diglucoside
Delfinidin 3,5-O-β-D-diglucoside
Petunidin 3,5-O-β-D-diglucoside
Malvidin 3,5-O-β-D-diglucoside
Acyl-glycosides
Flavonols
Patuletin 3-O-α-L-rhamnopyranosyl-7-O-(3"'-O-acetyl-α-L-rhamnopyranoside)
Patuletin 3-O-α-L-rhamnopyranosyl-7-O-(4"'-O-acetyl-α-L-rhamnopyranoside)
Patuletin 3-O-α-L-rhamnopyranosyl-7-O-(3"', 4"'-O-diacetyl-α-L-rhamnopyranoside
Patuletin 3-O-(4"-O-acetyl-α-L-rhamnopyranosyl)- 7-O-(3"'-O-acetyl-α-L-rhamnopyranoside)
Patuletin 3-O-(3"-O-acetyl-α-L-rhamnopyranosyl)- 7-O-(3"'-O-acetyl-α-L-rhamnopyranoside)
Patuletin 3-O-(4"-O-acetyl-α-L-rhamnopyranosyl)-7-O-(2"'-O-acetyl-α-L-rhamnopyranoside) (kalambroside A)
Patuletin 3-O-α-L-rhamnopyranosyl-7-O-(2"'-O-acetyl-α-L-rhamnopyranoside) (kalambroside B)
Patuletin 3-O-(4"-O-acetyl-α-L-rhamnopyranosyl)-7-O-rhamnopyranoside (kalambroside C)
Patuletin 3-O-(4"-O-acetyl-α-L-rhamnopyranosyl)-7-O-(2"', 4"'-O-diacetyl-α-L-rhamnopyranoside)
Kaempferol 3-O-β-D-xylopyranosyl (1→2)-α-L-rhamnopyranoside-7-O-4""-O-acetyl-α-L-rhamnopyranoside (4””acetylsagittatin A)
Flavones
Eupafolin 3-O-rhamnopyranosyl-7-O-(4"'-O-acetyl-α-L-rhamnopyranoside)
Eupafolin 3-O-(3"-O-acetyl-α-L-rhamnopyranosyl)-7-O-(3"'-O-acetyl-α-L-rhamnopyranoside)
Aglycones
Flavonols
Patuletin
Species
Reference
K. brasiliensis
K. gracilis
K. spathulata
K. brasiliensis
K. gracilis
K. gracilis
K. pinnata
K. spathulata
K. pinnata
K. fedtschenkoi
K. streptantha
K. blossfeldiana
K. fedtschenkoi
K. blossfeldiana
K. fedtschenkoi
K. blossfeldiana
K. pinnata
K. daigremontiana
K. pinnata
K. blossfeldiana
K. brasiliensis
K. blossfeldiana
K. pinnata
K. blossfeldiana
K. spathulata
K. brasiliensis
K. gracilis
K. pinnata
K. pinnata
K. prolifera
[5]
[33]
[69]
[5]
[33]
[66]
[7]
[69]
[25]
[70]
[71]
[61]
[70]
[61]
[70]
[61]
[47]
[26]
[25]
[61]
[5]
[61]
[4]
[61]
[69]
[34]
[66]
[46]
[25]
[72]
K. gracilis
K. gracilis
K. pinnata
[66]
[66]
[47]
K. blossfeldiana
K. blossfeldiana
K. blossfeldiana
K. blossfeldiana
K. blossfeldiana
K. blossfeldiana
K. blossfeldiana
[26]
[26], [61]
[26], [61]
[61]
[61]
[61]
[61]
K. brasiliensis
K. gracilis
K. gracilis
K. gracilis
K. brasiliensis
K. gracilis
K. gracilis
K. brasiliensis
K. brasiliensis
K. brasiliensis
K. gracilis
K. streptantha
[5]
[33]
[33]
[33]
[5]
[33]
[33]
[5]
[5]
[5]
[66]
[71]
K. gracilis
K. gracilis
[33]
[33]
K. gracilis
K. spathulata
[33]
[69]
2156 Natural Product Communications Vol. 3 (12) 2008
Kaempferol
K. gracilis
K. pinnata
K. spathulata
K. crenata
K. gracilis
K. pinnata
K. spathulata
K. crenata
Quercetin
Flavones
Eupafolin
Luteolin
5,7-dihydroxy-8,4’-dimethoxy-flavone (Galangustin)
5,7,4’-trihydroxy-6,3’-dimethoxy-flavone (Jaceosidin)
5,7,4’-trihydroxy-8,3’-dimethoxy-flavone
Others
Flavan-3-ol
Leucocyanidin
R2
OHC
R1
O
Bufadienolides
O
Kalanchoside A Me
Me
Kalanchoside B Me
OH
OH
OH
Kalanchoside C Me
H2
OH
O
HO
OH
H2
OH
O
OH
O
OH
Figure 2: Bufadienolides from K. gracilis.
OHC
MeO
Me
R1O
R3
H
H
R2O
OH
O
OH
Table 2 (Contd.)
[66]
[26]
[69]
[26]
[66]
[26]
[69]
[26]
K. gracilis
K. gracilis
K. pinnata
K. pinnata
K. pinnata
[66]
[66]
[40]
[40]
[40]
K. pinnata
K. blossfeldiana
[73]
[26]
R2
O
HO
H
H
R 1O
Costa et al.
Bufadienolides
R1
R2
R3
Kalanhybrin A
H
Ac
CHO
Kalanhybrin B
Ac
H
CHO
Kalanhybrin C
H
Ac
Me
Figure 3: Bufadienolides from K. hybrida.
The study of a methanolic extract of the leaves
of Kalanchoe streptantha Baker, one of the
fifty nine Kalanchoe species reported in Madagascar,
led to the new kaempferol 3-O-β-D-xylopyranosyl
(1→2)-α-L-rhamnopyranoside-7-O-4""-O-acetyl-αL-rhamnopyranoside (4""-acetylsagittatin A) and
sagittatin A [71].
The flavonoids 4""-acetylsagittatin A and sagittatin
A, at 25 μg/mL, inhibited 50% of human lymphocyte
proliferation stimulated by phytohemaglutinin A
[71]. These two flavonoids showed weaker
antiproliferative activity than patuletin 3-O-(4"-Oacetyl-α-L-rhamnopyranosyl)-7-O-(3"'-O-acetyl-αL-rhamnopyranoside),
kalambroside
A
and
kalambroside B, previously isolated from K.
brasiliensis, for which the IC50 values ranged from
0.25 to 1.0 μg/mL [5].
In Nepal, leaves from Kalanchoe spathulata DC are
used to treat burns and skin diseases [74]. Leaves and
flowers from this species were reported to exhibit the
same flavonoid content - patuletin 3,7-di-Orhamnoside, together with patuletin, quercetin,
quercetin
3-O-glucoside-7-O-rhamnoside,
kaempferol and afzelin - as reported by Gaind et al.
[69].
Kalanchoe prolifera (Bowie) R. Hamet, similarly to
other Kalanchoe species in Brazil, is used against
rheumatism in Madagascar [18]. Razanamahefa et al.
[72] isolated, from this widespread medicinal
plant, quercetin-3-O-β-D-glucopyranosyl (1→2)-β-Dxylopyranoside, which was later synthesized [75].
Kalanchoe tubiflora Raym.-Hamet (= Bryophyllum
tubiflorum) originates from Madagascar and has an
ornamental value due to its very beautiful flowers
[18]. Previous studies with K. tubiflora revealed that
its leaves and flowers, when consumed by cows,
caused intoxication symptoms like diarrhea [30].
The same kind of toxic event was observed for
K. lanceolata (Forsskäl) Persoon in Zimbabwe
[76]. Two bufadienolides were described from K.
lanceolata, lanceotoxin A and B [77].
Kalanchoe crenata (Baker) R. Hamet (= K. laxiflora)
is a medicinal species, whose infusions are known for
their vermifugous properties [18]. In African
traditional medicine it is also used as a remedy
against otitis, headache, inflammations, convulsions
and general debility [78]. Nguelefack et al. [17,78]
suggest peripheral and central analgesic activities, as
well as an anticonvulsant effect for leaf extracts of K.
crenata. Aditionally, in Cameroon, this species is
widely used in the treatment of diabetes mellitus.
Recently, the effect of the water-ethanol extract of
Secondary metabolites from Kalanchoe species
Natural Product Communications Vol. 3 (12) 2008 2157
this plant on blood glucose levels was investigated in
fasting normal and diet-induced diabetic rats. This
study reported a significant improvement in glucose
clearance and/or uptake and resistance to bodyweight gain and insulin sensitivity of K. crenata
extract [79]. The bioactive compounds remain to be
identified. The presence of quercetin and kaempferol
in this species was early described [26].
Kalanchoe thyrsiflora Harv. has a high ornamental
value and is commercialized in many parts of the
world under the names of “flap-jack” or “dog-tongueplant”. Recently, in a random screening for
anticancer activity of South African plant extracts, a
methanolic extract from roots and leaves of K.
thyrsiflora was tested against three human cell lines
(breast MCF7, renal TK10 and melanoma UACC62).
This extract did not show general cytotoxicity against
all three cell lines, but displayed potent activity
against only one of the cell lines (renal TK10). The
growth inhibitory activity against the other two-cell
lines was considered moderate by the authors [83].
The anticancer activity of K. thyrsiflora is supposed
to be related to the bufadienolides generally
occurring in Kalanchoe species. Root decoctions are
traditionally used in Africa as anthelmintic enemas
and administered to pregnant women who do not feel
well [83]. The phenolic composition of this plant is
under investigation in our laboratories, as well as four
other Kalanchoe species with ornamental value.
Our group is currently developing projects for the
search of new molecules with pharmacological
properties from flowers and leaves of some
ornamental Kalanchoe species, with emphasis on
flavonoids. Preliminary results are mentioned further.
Kalanchoe blossfeldiana Poelln. is a dark green
succulent with large umbels of flower clusters held
above the foliage. This plant has a great commercial
value as an ornamental. Nielsen et al. [61], in their
study on the flavonoid composition of flowers from
K. blossfeldiana varieties, reported the isolation of
pelargonidin, cyanidin, peonidin, delphinidin,
petunidin and malvidin 3,5-O-β-D-diglucosides. Pink,
red and magenta varieties contain relatively high
amounts of quercetin derivatives. Recently we
identified
the
known
quercetin
3,7-di-Orhamnopyranoside from the juice of K. blossfeldiana
leaves, in a study searching for antiviral compounds
[80]. This flavonoid was a new report for the
Kalanchoe genus.
Kalanchoe tomentosa Baker, a beautiful ornamental
species, known as “panda plant”, is a perennial
succulent covered in white felt punctuated with
delightful brown patches along the leaf edges [18].
Few studies have been devoted to the chemical
profile of this species [20, 81].
Another species native to Madagascar, K.
daigremontiana R. Hamet & H. Perrier, is
known as “mother-of-thousands” or “devil’sbackbone”. Two toxic bufadienolides with an
unusual substitution pattern were reported for this
species. Daigremontianin and bersaldegenin-1,3,5orthoacetate, which was also found in K. tubiflora,
were shown to have pronounced sedative, positive
inotropic and CNS-activities [82]. From the leaves of
K. daigremontiana×tubiflora were isolated an
insecticidal bufadienolide, methyl daigremonate,
along with four known bufadienolides [12]. Their
insecticidal activities were assessed against larvae of
silkworm (Bombyx mori).
Kalanchoe fedtschenkoi R. Hamet & H. Perrier is an
ornamental plant widespread in Brazil and reputed to
be toxic to chickens [20]. This species was
exhaustively studied for its CAM metabolism [84].
The flavonoid composition of leaves infusion
from K. fedtschenkoi has been studied in our
laboratories. Two main fractions (F2-C and
F2-E) were purified and fractionated to give
three flavonoids: kaempferol-3-O-β-D-xylopyranosyl
(1→2)-O-α-L-rhamnopyranoside, kaempferol-3-O-βD-glucopyranoside-7-O-α-L-rhamnopyranoside
and
sagittatin A. This last compound was previously
isolated from K. streptantha [71]. Crude extract as
well as fractions F2-C and F2-E exhibited strong
cytotoxic activities, especially against resistant
leukemia cells. Fraction F2-C reduced cellular
viability of both parental tumor line and the resistant
tumor line, besides presenting low toxicity in renal
cells [70].
3. Flavonoids and their distribution in the genus
Kalanchoe
Although there has been an increasing interest in the
biological activities of Kalanchoe extracts in the last
20 years, little progress was made towards the
chemical identification of the bioactive components.
It is unquestionable that flavonoids are the most
important chemical class in Kalanchoe species,
2158 Natural Product Communications Vol. 3 (12) 2008
Costa et al.
Table 3: Bioactive flavonoids from Kalanchoe species. Entries were organized in the same sequence used for Table 2.
Flavonoids
Glycosides
Some biological activities
Reference
Flavonols
Patuletin 3-O-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranoside
Lymphocyte growth inhibitor
[5]
Patuletin 3-O-α-L-rhamnopyranoside
Kaempferol 3-O-rhamnoside (afzelin)
Lymphocyte growth inhibitor
Lymphocyte growth inhibitor, antimalarial, antioxidant,
anti-complement activity
Antiallergic
Antileishmanial
[5]
Kaempferol 3-glucoside (astragalin)
Kaempferol 3-O-α-L-arabinopyranosyl (1→2)-α-L-rhamnopyranoside
Quercetin 3-O-β-D-glucopyranoside (isoquercitrin)
Antioxidant, antiprotozoal
Quercetin 3-O-α-L-arabinopyranosyl (1→2)-α-L-rhamnopyranoside
Antiallergic, antileishmanial
Quercetin 3-O-α-L-rhamnopyranoside (quercitrin)
Antiviral, antileishmanial, anti-anaphylactic, anti-complement,
antioxidant
Antiviral, antiallergic, antioxidant, anti-inflammatory, antiulcer,
gastro-protective
Quercetin 3-O-β-L-rhamnopyranosyl (1→6)-β-D-glucopyranoside (rutin)
[7, 85-87]
[88]
[47]
[89, 90]
[4, 47]
[28, 46, 48, 87,
91]
[28, 92, 93]
Flavones
4’,5-dihydroxy-3’,8-dimethoxyflavone-7-O-β-D-glucopyranoside
Anthocyans
Antileishmanial
Cyanidin 3-O-glucoside
Skin photoprotective agent, antioxidant
[47]
[94, 95]
Acyl-glycosides
Flavonols
Patuletin 3-O-(4"-O-acetyl-α-L-rhamnopyranosyl)- 7-O-(3"'-O-acetyl-α-Lrhamnopyranoside)
Patuletin 3-O-(4"-O-acetyl-α-L-rhamnopyranosyl)- 7-O-(2"'-O-acetyl-α-Lrhamnopyranoside)
Patuletin 3-O-α-L-rhamnopyranosyl-7-O-(2"'-O-acetyl-α-Lrhamnopyranoside) (kalambroside B)
Patuletin 3-O-(4"-O-acetyl-α-L-rhamnopyranosyl)-7-O-rhamnopyranoside
(kalambroside C)
Kaempferol 3-O-β-D-xylopyranosyl (1→2)-α-L-rhamnopyranoside-7-O- 4""O-acetyl-α-L-rhamnopyranoside
Aglycones
Lymphocyte growth inhibitor
[5]
Lymphocyte growth inhibitor
[5]
Lymphocyte growth inhibitor
[5]
Lymphocyte growth inhibitor
[5]
Lymphocyte growth inhibitor
[71]
Flavonols
Patuletin
Kaempferol
Quercetin
Antiplatelet activity, antioxidant, aldose reductase inhibitor,
lipoxygenase and cyclooxygenase inhibitor, antimicrobial
Antiulcer, antiinflammatory, antiallergic, antinociceptive,
neuroprotective, antiatherogenic,
inhibition of HL-60 cell growth, induces apoptosis in human lung
non-small carcinoma cells, antioxidant
Antioxidant, spasmolytic effect (smooth muscle relaxation),
antiviral, anti-inflammatory, antiulcer, antiallergic,
hepatoprotector
[96-100]
[28, 93, 101-106]
[28, 93, 107]
Flavones
Eupafolin
Luteolin
5,7,4’-trihydroxy-6,3’-dimethoxy-flavone (Jaceosidin)
Hepatoprotector,
antiproliferative activity against HeLa cell, antioxidant,
cardiopreventive
Anti-inflammatory, hepatoprotector, antiallergic,
vascular relaxation, antioxidant
Anti-inflammatory, citotoxicity to MCF10A-ras cells,
oncogene inhibitor, antiallergic
[108-110]
[106, 111]
[112-114]
Others
Flavan-3-ol
regarding the number of reports and biological
activities. The presence of flavonols, flavones,
anthocyanins, leucoanthocyanins and flavan-3-ols has
been reported by several authors. Flavonols are the
most frequent flavonoids in Kalanchoe species,
representing aproximately 60% (Figure 4). Patuletin,
kaempferol and quercetin are the most reported
aglycones (Figures 5 and 6).
Antioxidant, anticarcinogen, cardiopreventive, antimicrobial,
antiviral, neuroprotective, anti-amyloidogenic effect
[115, 116]
The distribution of Kalanchoe flavonoids following
their respective parent aglycone is shown in Table 4.
Table 2 presents all flavonoids reported for
Kalanchoe species, while Table 3 shows only those
for which specific bioactivities have been reported.
Several authors reported biological activities for
patuletin aglycone, among them, antiplatelet activity,
Secondary metabolites from Kalanchoe species
70
Flavan-3-ol
(01)
Leucocyanidin
(01)
Malvidin
(01)
Petunidin
(01)
40
Delfinidin
(01)
30
Peonidin
(01)
Pelargonidin
(01)
(29)
60
Occurrence (%)
Natural Product Communications Vol. 3 (12) 2008 2159
50
(10)
20
(07)
Cyanidin
10
(01)
Galangustin
(01)
Luteolin
(01)
5.7.4'-tri-OH-8.3'-di-OMe-flavone
(01)
Fl
av
on
es
A
nt
ho
cy
an
Le
s
uc
oc
ya
ni
di
n
Fl
av
an
-3
-o
ls
Fl
av
on
ol
s
0
Eupafolin
OH
Patuletin
Quercetin
Kaempferol
Eupafolin
R3
R2
OH
O
R1 R 2 R 3
OH OMe OH
OH H OH
H
H OH
OH OMe H
OH
OH
HO
O
OH
OH
(07)
Kaempferol
(08)
Patuletin
(03)
0
10
20
30
Occurrence (%)
Figure 6: The different aglycones reported for Kalanchoe flavonoids.
The number of Kalanchoe species corresponding to each aglycone
reported is shown in brackets.
R1
O
(01)
Quercetin
Figure 4: The distribution of flavonoid classes in the Kalanchoe genus.
The number of flavonoids corresponding to each class is shown in
brackets.
HO
(01)
(01)
Jaceosidin
(01)
Cyanidin
Figure 5: The most common flavonoid aglycones reported for Kalanchoe
species
oxidative stress protection, aldose reductase
inhibition, lipoxygenase and cyclooxygenase
inhibition [96-99]. Excluding the first one, all the
activities described are involved with antiinflamatory
processes. Patuletin glycosides and acetyl-glycosides
reported for K. brasiliensis and K. gracilis were
less explored for their biological activities.
Lymphocyte antiproliferative activity was reported
for K. brasiliensis patuletin acetyl-glycosides. This
inhibitory activity on the immune system could be
useful in situations where an intensive immune
response is undesirable, as in chronic inflammation,
organ transplantation and auto-immune diseases.
The second more reported aglycone in Kalanchoe
species is kaempferol. Several studies focusing on
antiulcer, antiinflammatory, bactericidal, antiallergic,
antinociceptive, neuro-protective, antiatherogenic,
HL-60 cell growth inhibitory, human lung non-small
carcinoma cells apoptosis induction and antioxidant
activities have been reported for this flavonoid
Table 4: Occurrence of Kalanchoe flavonoids based on their respective
parent aglycones.
Parent
aglycone
Patuletin
Kaempferol
Quercetin
Eupafolin
Luteolin
Galangustin
Jaceosidin
5,7,4’-tri-OH8,3’-diOMeflavone
Cyanidin
Pelargonidin
Peonidin
Delfinidin
Petunidin
Malvidin
Leucocyanidin
Flavan-3-ol
K.
blo
3
3
-
K.
bra
7
2
-
K.
cre
1
1
-
K.
dai
1
-
K.
fed
3
-
K.
gra
9
2
2
5
1
-
K.
pin
4
5
1
1
2
K.
pro
1
-
K.
spa
2
2
2
-
K.
str
2
-
2
1
1
1
1
1
1
-
-
-
-
-
-
1
-
-
-
⇒ - = no reports; K. blo= K. blossfeldiana, K. bra= K. brasiliensis, K. cre= K.
crenata (=K. laxiflora), K. dai= K. daigremontiana, K. fed= K. fedtschenkoi, K.
gra= K. gracilis, K.pin= K. pinnata, K.pro= K. prolifera, K. spa= K. spathulata, K.
str= K. strepthanta.
(Table 3). Quercetin and its glycosides exert antiinflammatory, antiulcer, antiallergic, hepatoprotective, antiviral and antileishmanial effects, as
can be seen in Table 3. The efficacy of quercetin as
an anti-inflammatory and immunosuppressor agent
has been extensively demonstrated [117,118].
Mookerjee et al. (1986) [119] reported the reversible
lymphoproliferative inhibition effect of flavonoids,
such as quercetin, in response to phytomitogens,
soluble antigens, and phorbol esters by blocking
events that follow the exposure to the stimulus. This
activity profile was also observed for K. pinnata
quercetin glycosides, as mentioned before. K. pinnata
is the richest species with respect to the number of
flavonoids possessing a quercetin skeleton.
2160 Natural Product Communications Vol. 3 (12) 2008
Our group has been contributing to a better
knowledge of the therapeutic potential of Kalanchoe
species in an interdisciplinary approach that
correlates chemical and pharmacological studies.
These works afford interesting molecules from the
point of view of pharmaceutical application, as for
example, the antileishmanial flavonoids from K.
pinnata. Flavonoids possess several biological
activities, including antimicrobial, antiviral, antiinflammatory
and
immunomodulatory
[28].
Regarding this bioactive metabolite class, the genus
Kalanchoe is particularly fruitful, leading to many
interesting preliminary results that remain to be
explored.
In conclusion, the present review summarizes the
chemical and biological data reported for Kalanchoe
species during these last decades, with special
attention to their flavonoid profile. The so far
accumulated data show that Kalanchoe is a promising
Costa et al.
and rich genus, not only in terms of its outstanding
ornamental value, but also in term of its numerous
pharmaceutical promising perspectives. A few
species from this genus have been studied in
interdisciplinary approaches in view of finding new
bioactive compounds. Much remains to be done for a
better understanding of the ethnopharmacological use
of various Kalanchoe species and to elucidate the
chemical composition of the substances responsible
for their pharmacological activities. We are pursuing
our efforts in studying Kalanchoe species not yet
explored with these perspectives in mind.
Acknowledgments - We are grateful to all students
and research partners for their contribution to the
studies reported here. We thank Dr J-P Férézou
(Ecole Polytechnique, France) for helpful suggestions
and advice. Thanks to CNPq, CAPES, FAPERJ and
SUS (Ministério da Saúde) for grants and
fellowships.
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Natural Product Communications Vol. 3 (12) 2008 2165
Natural Product Communications
Manuscripts in Press
Volume 3, Number 12 (2008)
Characteristic Flavonoids from Acacia burkittii and A.
acuminata Heartwoods and their Differential Cytotoxicity to
Normal and Leukemia Cells
Mary H. Grace, George R. Wilson, Fayez E. Kandil, Eugene
Dimitriadis and Robert M. Coates
Bioassay-Guided Isolation of Antiproliferative Compounds
from Grape (Vitis vinifera) Stems
Vincenzo Amico, Vincenza Barresi, Rosa Chillemi, Daniele
Filippo Condorelli, Sebastiano Sciuto, Carmela Spatafora and
Corrado Tringali
Content of Total Carotenoids in Calendula officinalis L. from
Different Countries Cultivated in Estonia
Ain Raal, Kadri Kirsipuu, Reelika Must and Silvi Tenno
Plant Growth Regulating Activity of Three Polyacetylenes
from Helianthus annuus L.
Si Won Hong, Koji Hasegawa, and Hideyuki Shigemori
Bioactive Complex Triterpenoid Saponins
Leguminosae Family
José P. Parente and Bernadete P. da Silva
from
the
Effects of the Essential Oil from Leaves of Alpinia zerumbet on
Behavioral Alterations in Mice
Shio Murakami, Mariko Matsuura, Tadaaki Satou, Shinichiro
Hayashi and Kazuo Koike
Discovering Protein Kinase C Active Plants Growing in
Finland Utilizing Automated Bioassay Combined to LC/MS
Anna Galkin, Jouni Jokela, Matti Wahlsten, Päivi Tammela,
Kaarina Sivonen and Pia Vuorela
Chemical Composition and Antimicrobial Activity of the
Essential Oil from Chaerophyllum aureum L. (Apiaceae)
Branislava Lakušić, Violeta Slavkovska, Milica Pavlović, Marina
Milenković, Jelena Antić Stankovićc and Maria Couladis
Isoprenylated Flavanones and Dihydrochalcones from
Macaranga trichocarpa
Yana M. Syah, Euis H. Hakim, Sjamsul A. Achmad, Muhamad
Hanafi and Emilio L. Ghisalberti
Abietane Diterpeniods from Hyptis verticillata
Roy B. R. Porter, Duanne A. C. Biggs and William F. Reynolds
Flavone and Flavonol Glycosides from Astragalus eremophilus
and Astragalus vogelii
Angela Perrone, Milena Masullo, Alberto Plaza, Arafa Hamed
and Sonia Piacente
Synthesis of Daldinol and Nodulisporin A by Oxidative
Dimerization of 8-Methoxynaphthalen-1-ol
Karsten Krohn and Abdulselam Aslan
Diterpenoid Alkaloids from Aconitum longzhoushanense
Ping He, Xi-Xian Jian, Dong-lin Chen and Feng-Peng Wang
Alkaloids from Lindera aggregata
Li-She Gan, Wei Yao, and Chang-Xin Zhou
Antioxidants from the Leaf Extract of Byrsonima bucidaefolia
G. Margarita Castillo-Avila, Karlina García-Sosa and Luis M.
Peña-Rodríguez
Salt Stress Induces Production of Melanin Related Metabolites
in the Phytopathogenic Fungus Leptosphaeria maculans
M. Soledade C. Pedras and Yang Yu
Occurrence of Sesquiterpene Derivatives in Scleria striatonux
De Wild (Cyperaceae)
Kennedy Nyongbela, Karine Ioset Ndjoko, Reto Brun, Sergio
Wittlin, James Mbah, Felix Makolo, Marc Akam, Claire
Wirmum, Simon Efange and Kurt Hostettmann
Cytotoxic Activity of Cycloartane Triterpenoids from
Sphaerophysa salsula
Dan Wang and Zhongjun Ma
GC-MS Analysis and Antimicrobial Activity of Essential Oil
of Stachys cretica subsp. smyrnaea
Mehmet Öztürk, Mehmet Emin Duru, Fatma Aydoğmuş-Öztürk,
Mansur Harmandar, Melda Mahlıçlı, Ufuk Kolak and Ayhan
Ulubelen
Antifungal and Insecticidal Activity of two Juniperus
Essential Oils
David E. Wedge, Nurhayat Tabanca, Blair J. Sampson,
Christopher Werle, Betul Demirci, K. Husnu Can Baser, Peng
Nan, Jian Duan and Zhijun Liu
Minor Chemical Constituents of Vitex pinnata
Athar Ata, Nathan Mbong, Chad D. Iverson and Radhika
Samarasekera
Essential Oil Compositions of Three Lantana Species from
Monteverde, Costa Rica
Ashley B. Walden, William A. Haber and William N. Setzer
Design and Optimization of Ultrasound Assisted Extraction
of Curcumin as an Effective Alternative for Conventional
Solid Liquid Extraction of Natural Products
Vivekananda Mandal, Saikat Dewanjee, Ranabir Sahu and
Subhash C. Mandal
Radical Scavenging Activity and Total Phenolic Content of
Extracts of the Root Bark of Osyris lanceolata
Elizabeth M. O. Yeboah and Runner R. T. Majinda
Chemical Transformations of Parthenin, a Natural Bioactive
Sesquiterpenoid
Biswanath Das, G. Satyalakshmi, Nisith Bhunia, K. Ravider
Reddy, V. Saidi Reddy and G. Mahender
Cytotoxicity, Antimicrobial Activity and Composition of
Essential Oil from Tanacetum balsamita L.subsp. balsamita
Morteza Yousefzadi, Samad Nejad Ebrahimi, Ali Sonboli, Farah
Miraghasi, Shahla Ghiasi, Mitra Arman and Nariman Mosaffa
Betaines in Four Additional Phyla of Green Plants
Gerald Blunden, Asmita Patel, Maricela Adrian Romero and
Michael D. Guiry
Natural Product Communications
2008
Volume 3
Natural Product Communications 3 (1-12) 1-2166 (2008)
ISSN 1934-578X (print)
ISSN 1555-9475 (online)
NPC
Natural Product Communications
EDITOR-IN-CHIEF
DR. PAWAN K AGRAWAL
Natural Product Inc.
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EDITORS
PROFESSOR GERALD BLUNDEN
The School of Pharmacy & Biomedical Sciences,
University of Portsmouth,
Portsmouth, PO1 2DT U.K.
axuf64@dsl.pipex.com
PROFESSOR ALESSANDRA BRACA
Dipartimento di Chimica Bioorganicae Biofarmacia,
Universita di Pisa,
via Bonanno 33, 56126 Pisa, Italy
braca@farm.unipi.it
PROFESSOR DEAN GUO
State Key Laboratory of Natural and Biomimetic Drugs,
School of Pharmaceutical Sciences,
Peking University,
Beijing 100083, China
gda5958@163.com
PROFESSOR J. ALBERTO MARCO
Departamento de Quimica Organica,
Universidade de Valencia,
E-46100 Burjassot, Valencia, Spain
alberto.marco@uv.es
PROFESSOR YOSHIHIRO MIMAKI
School of Pharmacy,
Tokyo University of Pharmacy and Life Sciences,
Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan
mimakiy@ps.toyaku.ac.jp
PROFESSOR STEPHEN G. PYNE
Department of Chemistry
University of Wollongong
Wollongong, New South Wales, 2522, Australia
spyne@uow.edu.au
PROFESSOR MANFRED G. REINECKE
Department of Chemistry,
Texas Christian University,
Forts Worth, TX 76129, USA
m.reinecke@tcu.edu
PROFESSOR WILLIAM N. SETZER
Department of Chemistry
The University of Alabama in Huntsville
Huntsville, AL 35809, USA
wsetzer@chemistry.uah.edu
PROFESSOR YASUHIRO TEZUKA
Institute of Natural Medicine
Institute of Natural Medicine, University of Toyama,
2630-Sugitani, Toyama 930-0194, Japan
tezuka@inm.u-toyama.ac.jp
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Karachi, Pakistan
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Bergen, Norway
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Novara, Italy
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Tokushima, Japan
Prof. Maurizio Bruno
Palermo, Italy
Prof. Carlos Cerda-Garcia-Rojas
Mexico city, Mexico
Prof. Josep Coll
Barcelona, Spain
Prof. Geoffrey Cordell
Chicago, IL, USA
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New York, NY, USA
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Hyderabad, India
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Tucson, AZ, USA
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Montreal, Canada
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London, UK
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Lausanne, Switzerland
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Mexico, D. F, Mexico
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Rosario, Argentina
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Gottingen, Germany
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Dijon, France
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Szeged, Hungary
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Johannesburg, South Africa
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Delhi, India
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Manhattan, KS, USA
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Düsseldorf, Germany
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Coleraine, UK
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Natural Product Communications
Contents of Volume 3
2008
Number 1
A Novel Sesquiterpene from Pulicaria crispa (Forssk.) Oliv.
Michael Stavri, Koyippally T. Mathew and Simon Gibbons
1
Cassane diterpenoids from Lonchocarpus laxiflorus
John O. Igoli, Samuel O. Onyiriuka, Matthias C. Letzel, Martin N. Nwaji and Alexander I. Gray
5
COX-2 Inhibitory Activity of Cafestol and Analogs from Coffee Beans
Ilias Muhammad, Satoshi Takamatsu, Jamal Mustafa, Shabana I. Khan, Ikhlas A. Khan, Volodymyr Samoylenko,
Jaber S. Mossa, Farouk S. El-Feraly and D. Chuck Dunbar
11
Antibacterial Diterpenes from the Roots of Ceriops tagal
Musa Chacha, Renameditswe Mapitse, Anthony J. Afolayan and Runner R. T. Majinda
17
Boswellic Acids with Acetylcholinesterase Inhibitory Properties from Frankincense
Masahiro Ota and Peter J. Houghton
21
Synthesis of Pregnenolone and Methyl Lithocholate Oxalate Derivatives
Lutfun Nahar, Satyajit D. Sarker and Alan B. Turner
27
Annona muricata (Graviola): Toxic or Therapeutic
Sambeet Mohanty, Jackie Hollinshead, Laurence Jones, Paul Wyn Jones, David Thomas, Alison A. Watson,
David G. Watson, Alexander I. Gray, Russell J. Molyneux and Robert J. Nash
31
Two New Alkylated Piperidine Alkaloids from Western Honey Mesquite: Prosopis glandulosa Torr. var. torreyana
Volodymyr Samoylenko, D. Chuck Dunbar, Melissa R. Jacob, Vaishali C. Joshi, Mohammad K. Ashfaq and Ilias Muhammad
35
Selective Metabolism of Glycosidase Inhibitors by a Specialized Moth Feeding on Hyacinthoides non-scripta Flowers
Alison A. Watson, Ana L. Winters, Sarah A. Corbet, Catherine Tiley and Robert J. Nash
41
Antimicrobial Activities of Alkaloids and Lignans from Zanthoxylum budrunga
M. Mukhlesur Rahman, Alexander I. Gray, Proma Khondkar and M. Anwarul Islam
45
A Pyranochalcone and Prenylflavanones from Tephrosia pulcherrima (Baker) Drumm
Seru Ganapaty, GuttulaV.K. Srilakshmi, Steve T. Pannakal and Hartmut Laatsch
49
Phenolic Glycosides from Phlomis lanceolata (Lamiaceae)
Hossein Nazemiyeh, Abbas Delazar, Mohammed-Ali Ghahramani, Amir-Hossein Talebpour, Lutfun Nahar and
Satyajit D. Sarker
53
Bisresorcinols and Arbutin Derivatives from Grevillea banksii R. Br.
Hao Wang, David Leach, Michael C. Thomas, Stephen J. Blanksby, Paul I. Forster and Peter G. Waterman
57
Antioxidant and Membrane Stabilizing Properties of the Flowering Tops of Anthocephalus cadamba
M. Ashraful Alam, Abdul Ghani, Nusrat Subhan, M. Mostafizur Rahman, M. Shamsul Haque, Muntasir M. Majumder,
M. Ehsanul H. Majumder, Raushan A. Akter, Lutfun Nahar and Satyajit D. Sarker
65
A Method of Selecting Plants with Anti-inflammatory Potential for Pharmacological Study
G. David Lin, Rachel W. Li, Stephen P. Myers and David N. Leach
71
Recent Advances of Biologically Active Substances from the Marchantiophyta
Yoshinori Asakawa
77
Non-Protein Amino Acids: A Review of the Biosynthesis and Taxonomic Significance
E. Arthur Bell (the late), Alison A. Watson and Robert J. Nash
93
Number 2
New cis-Chrysanthenyl Esters from Eryngium planum L.
Emilia Korbel, Ange Bighelli, Anna Kurowska, Danuta Kalemba and Joseph Casanova
113
ii Contents of Volume 3 (1-12) 2008
Secondary Metabolites from Eremostachys laciniata
İhsan Çalış, Ayşegül Güvenç, Metin Armağan, Mehmet Koyuncu, Charlotte H. Gotfredsen and Søren R. Jensen
117
A Novel Iridoid from Plumeria obtusa
Firdous Imran Ali, Imran Ali Hashmi and Bina Shaheen Siddiqui
125
Terpenoids from Neolitsea dealbata
Xiujun Wu, Bernhard Vogler, Betsy R. Jackes and William N. Setzer
129
Volatile Components from Selected Mexican, Ecuadorian, Greek, German and Japanese Liverworts
Agnieszka Ludwiczuk, Fumihiro Nagashima, Rob S. Gradstein and Yoshinori Asakawa
133
New ent–Kaurane type Diterpene Glycoside, Pulicaroside-B, from Pulicaria undulata L.
Nasir Rasool, Viqar Uddin Ahmad, Naseem Shahzad, Muhammad A. Rashid, Aman Ullah, Zahid Hassan,
Muhammad Zubair and Rasool Bakhsh Tareen
141
Anti-babesial Quassinoids from the Fruits of Brucea javanica
Ahmed Elkhateeb, Masahiro Yamasaki, Yoshimitsu Maede, Ken Katakura, Kensuke Nabeta and Hideyuki Matsuura
145
Triterpenoids and Alkaloids from the Roots of Peganum nigellastrum
Zhongze Ma, Yoshio Hano, Feng Qiu, Gang Shao, Yingjie Chen and Taro Nomura
149
Saikosaponins from Bupleurum chinense and Inhibition of HBV DNA Replication Activity
Feng Yin, Ruixiang Pan, Rongmin Chen and Lihong Hu
155
Brauhenoside A and B: Saponins from Stocksia brauhica Benth.
Viqar Uddin Ahmad, Sadia Bader, Saima Arshad, Faryal Vali Mohammad, Amir Ahmed, Shazia Iqbal,
Saleha Suleman Khan and Rasool Bakhsh Tareen
159
Saponins from Fresh Fruits of Randia siamensis (Lour) Roem. & Schult. (Rubiaceae)
Rapheeporn Khwanchuea, Emerson Ferreira Queiroz, Andrew Marston, Chaweewan Jansakul and Kurt Hostettmann
163
New Alkaloid from Aspidosperma polyneuron Roots
Tatiane Alves dos Santos, Dalva Trevisan Ferreira, Jurandir Pereira Pinto, Milton Faccione and Raimundo Braz-Filho
171
Acanthomine A, a new Pyrimidine-β-Carboline Alkaloid from the Sponge Acanthostrongylophora ingens
Sabrin R. M. Ibrahim, RuAngelie Ebel, Rainer Ebel and Peter Proksch
175
Phytochemical and Microscopic Characterization of the Caribbean Aphrodisiac Bois Bandé: Two New Norneolignans
Ingrid Werner, Pavel Mucaji, Armin Presser, Christa Kletter and Sabine Glasl
179
3-Acetoxy-7-methoxyflavone, a Novel Flavonoid from the Anxiolytic Extract of Salvia elegans (Lamiaceae)
Silvia Marquina, Yolanda García, Laura Alvarez and Jaime Tortoriello
185
Struthiolanone: A Flavanone-Resveratrol Adduct from Struthiola argentea
Sloan Ayers, Deborah L. Zink, Robert Brand, Seef Pretorius, Dennis Stevenson and Sheo B. Singh
189
New Acylated Flavonol Diglycosides of Cynanchum acutum
Mona A. Mohamed, Wafaa S. Ahamed, Mortada M. El-Said and Heiko Hayen
193
Phenolic Constituents of Platanus orientalis L. Leaves
Taha S. El-Alfy, Hamida M.A. El-Gohary, Nadia M. Sokkar, Amani A. Sleem and Dalia A. Al-Mahdy
199
Strepsiamide A-C, New Ceramides from the Marine Sponge Strepsichordaia lendenfeldi
Sabrin R. M. Ibrahim, Gamal A. Mohamed, Ehab S. Elkhayat, Yaser G. Gouda and Peter Proksch
205
Free Radical Scavenging and Cytoprotective Activity of Salacia euphlebia Merr.
Sanan Subhadhirasakul, Niwat Keawpradub, Charuporn Promwong and Supreeya Yuenyongsawad
211
Antialactone: A New γ-Lactone from Antiaris africana, and its Absolute Configuration Determined
Vouffo Bertin, Hidayat Hussain, Simeon F. Kouam, Etienne Dongo, Gennaro Pescitelli, Piero Salvadori,
Tibor Kurtán and Karsten Krohn
215
Subereaphenol A, a new Cytotoxic and Antimicrobial Dibrominated Phenol from the Red Sea Sponge Suberea mollis
Lamiaa A. Shaala, Sherief I. Khalifa, Mostafa K. Mesbah, Rob W. M. van Soest and Diaa T. A. Youssef
219
A New Ferulic Ester and Related Compounds from Bombax malabaricum DC.
Pahup Singh, Durga K. Mewara and Mahesh C. Sharma
223
Role of Turmeric in Ultraviolet Induced Genotoxicity in a Bacterial System
Arijit Pal, Mita Ghosh and Arun Kumar Pal
227
Excited-State pKa Values of Curcumin
Qian Zhao, De-Xin Kong and Hong-Yu Zhang
229
Antibacterial and Antifungal Activities of Some Phenolic Metabolites Isolated from the Lichenized Ascomycete
Ramalina lacera
Lumír O Hanuš, Marina Temina and Valery M Dembitsky
233
Contents of Volume 3 (1-12) 2008 iii
Phenolic Constituents of Hypericum Flowers
Carolina Nör, Ana Paula Machado Bernardi, Juliana Schulte Haas, Jan Schripsema, Sandra Beatriz Rech and
Gilsane Lino von Poser
237
Seasonal Variation of Hypericin and Pseudohypericin Contents in Hypericum scabrum L. Growing Wild in Turkey
Ali Kemal Ayan, Cüneyt Çırak and Kerim Güney
241
Molluscicidal Polyphenols from Species of Fucaceae
Asmita V. Patel, David C. Wright, Maricela Adrian Romero, Gerald Blunden and Michael D. Guiry
245
Anti-diabetic Activity of Triphala Fruit Extracts, Individually and in Combination, in a Rat Model of Insulin
Resistance
Venkateshan S. Prativadibhayankaram, Samir Malhotra, Promila Pandhi and Amritpal Singh
251
Biotransformation of Mefenamic Acid by Cell Suspension Cultures of Solanum mammosum
Suzana Surodjo, Angela A. Salim, Suciati, Achmad Syahrani, Gunawan Indrayanto and Mary J. Garson
257
Natural Variability in Enantiomeric Composition of Bioactive Chiral Terpenoids in the Essential Oil of
Solidago canadensis L. from Uttarakhand, India
Chandan S. Chanotiya and Anju Yadav
263
Germacrone Dominates the Leaf Oil of Siparuna grandiflora from Monteverde, Costa Rica
William N. Setzer, Brittany R. Agius, Tameka M. Walker, Debra M. Moriarity and William A. Haber
267
Leaf Oil Composition of Piper aduncum subsp. ossanum (C. CD.) Saralegui from Cuba
Orlando Abreu and Jorge A. Pino
271
Volatile Constituents from the Leaves of Phyllanthus salviaefolius H. B. K. from the Venezuelan Andes
Silvana Villarreal, Luis B. Rojas, Alfredo Usubillaga, Irama Ramírez and Mariana Solórzano
275
Synergistic Antifungal Activities of Thymol Analogues with Propolis
Chi-Pien Chen and Ai-Yu Shen
279
Argan oil, Functional Food, and the Sustainable Development of the Argan Forest
Zoubida Charrouf and Dominique Guillaume
283
Chemical Constituents of Selected Japanese and New Zealand Liverworts
Yoshinori Asakawa, Masao Toyota, Fumihiro Nagashima and Toshihiro Hashimoto
289
Number 3
Synthesis and Antibacterial Activity of Highly Oxygenated 1,8-Cineole Derivatives
Margarita B. Villecco, Julieta V. Catalán, Marta I. Vega, Francisco M. Garibotto, Ricardo D. Enriz and César A. N. Catalán
303
Structure – Activity Relationships of Modified Eremophilanes and Anti-inflammatory Activity using the
TPA Mouse Edema Ear Test
Noemi Acevedo-Quiroz, Valeri Domínguez-Villegas and María Luisa Garduño-Ramírez,
313
New 3,4-Seco ent-Kaurenes from Croton caracasana Flowers
Alírica I. Suárez, Katiuska Chavez, Franco Delle Monache, Luis Vasquez, Daniela M. Delannoy, Giovannina Orsini
and Reinaldo S. Compagnone
319
Myrsicorianol, A New Prenylated Benzoic Acid Derivative from Myrsine coriacea
Juan Manuel Amaro-Luis, Sonia Koteich-Khatib, Freddy Carrillo-Rodríguez and Alí Bahsas
323
Diastereoselective Synthesis of β-Hydroxyketones
Teresa Mancilla Percino, Marisol López Martínez and José Luis León
329
Revised Structure by Computational Methods for a Coumarin Isolated from Zanthoxylum rhoifolium (Rutaceae)
Augusto Rivera and Jaime Rios-Motta
333
1
13
Enantiodifferentiation by H and C NMR Spectroscopy (Dirhodium Method) – Selectivity of Oxygen Functionalities
Edison Díaz Gómez, Sándor Antus, Renáta Ferenczi, Barbara Rys, Anna Stankiewicz and Helmut Duddeck
339
Antioxidant Activity of Fruits toward Iron under Gastrointestinal Conditions
Helena Morais
345
Substrate Specificity of a Cationic Peptidase from Bromelia hemisphaerica L.
Cortés-Vázquez Ma. Isabel, Muñoz-Sánchez José Luis and Briones-Martínez Roberto
351
New Sources of Oilseeds from Latin American Native Fruits
Lilia Masson, Conrado Camilo, Katherine Gonzalez, Andrea Caceres, Neuza Jorge and Esperanza M. Torija
357
Experimental Design to Determine the Factors Affecting the Preparation of Extracts for Antibacterial Use
Alejandro Pérez-López, Marcela Orozco-Hayek, Verónica Rivas-Galindo and Noemí Waksman de Torres
363
iv Contents of Volume 3 (1-12) 2008
Chemical Composition and Antibacterial Activity of the Essential Oil of Baccharis trinervis (Lam.) Pers.
(Asteraceae) Collected in Venezuela
Janne Rojas, Judith Velasco, Antonio Morales, Luis Rojas, Tulia Díaz, Maria Rondón and Juan Carmona
369
Supercritical Extraction of Essential Oil from Ilex paraguariensis Leaves
Eduardo Cassel, Rubem M. F. Vargas and Gerti W. Brun
373
Cultivars of Lavandula lanata Boiss., a Good Source of Lavandulol
Alejandro F. Barrero, M. Mar Herrador, Pilar Arteaga, Jesús F. Arteaga and Jesús Burillo
379
Essential Oils from Bolivia. X. Asteraceae: Gnaphalium viravira Molina
Javier B Lopez Arze, Guy Collin, François-Xavier Garneau, France-Ida Jean and Hélène Gagnon
383
Synthesis of Sesquiterpenes via Silicon-Guided Rearrangement of Epoxydecalins
Gonzalo Blay, Ana M. Collado, Begoña García and José R. Pedro
385
13
Structural Elucidation of Pimarane and Isopimarane Diterpenoids: The C NMR Contribution
Ana M. L. Seca, Diana C. G. A. Pinto and Artur M. S. Silva
399
Anti-inflammatory and Cytotoxic Cycloartanes from Guayule (Parthenium argentatum)
Graciela Flores-Rosete and Mariano Martínez-Vázquez
413
Rubus - A Review of its Phytochemical and Pharmacological Profile
Guillermo Omar Rocabado, Luis Miguel Bedoya, María José Abad and Paulina Bermejo
423
Therapeutic Potential and Chemical Composition of Plants from the Genus Rubus: A Mini Review of the
Last 10 Years
Rivaldo Niero and Valdir Cechinel Filho
437
n
Very Long-Range Correlations ( JC,H n > 3) in HMBC Spectra
Ramiro Araya-Maturana, Hernán Pessoa-Mahana and Boris Weiss-López
445
Vibrational Circular Dichroism: A New Tool for the Solution-State Determination of the Structure and
Absolute Configuration of Chiral Natural Product Molecules
Laurence A. Nafie
451
Number 4
Screening Study of Potential Lead Compounds for Natural Product Based Fungicides from Juniperus lucayana
Yarelis Ortíz Nuñez, Iraida Spengler Salabarria, Isidro G. Collado and Rosario Hernández-Galán
469
Inhibitory Activity of α,β-Unsaturated Lactones on Histamine Release from Rat Peritoneal Mast Cells
Alicia B. Penissi, María I. Rudolph, Mariano E. Vera, María L. Mariani, Juan P. Ceñal, Carlos E. Tonn,
Oscar S. Giordano and Ramón S. Piéis
475
Reactivity of Several Reactive Oxygen Species (ROS) with the Sesquiterpene Cacalol
Manuel Jiménez-Estrada, Ricardo Reyes-Chilpa, Arturo Navarro-Ocaña and Daniel Arrieta-Báez
479
Ring Contraction of Gummiferolic Acid, a Diterpene Isolated from Margotia gummifera, Leading to Atisagibberellins
Josefa Anaya, Juan José Fernández, Manuel Grande, Justo Martiáñez and Pascual Torres
483
A Convenient Synthesis of the Central Core of Helioporins, seco-Pseudopterosins and Pseudopterosins via
BCA-Annulation Sequence
Gema Esteban, Rocío Rincón, Aurelio G. Csákÿ and Joaquín Plumet
495
Role of Prostaglandins, Nitric oxide, Sulfhydryls and Capsaicin-sensitive Neurons in Gastroprotection of
Stigmasterol and β-Sitosterol
María Elena Sánchez-Mendoza, Jesús Arrieta and Andrés Navarrete
505
New Guanidine Alkaloids from the Leaves of Verbesina peraffinis
Reinaldo S. Compagnone, Jhorman Bermudez, Glorymar Ibáñez, Beth Díaz, María R. Garrido, Anita Israel and
Alírica I. Suárez
511
Aporphine Alkaloids from Guatteria stenopetala (Annonaceae)
María Rodríguez, Elsy Bastidas, Mildred Rodríguez, Edgar Lucena, Anibal Castillo and Masahisa Hasegawa
515
Effects of Simple and Angular Chromones on Tumor Cell Respiration
Ramiro Araya-Maturana, Jorge Heredia-Moya, Oscar Donoso-Tauda, Mario Vera, Jorge Toledo Hernández,
Mario Pavani, Hernán Pessoa-Mahana, Boris Weiss-López and Jorge Ferreira
519
Effect on Hantavirus Replication of Resins from Heliotropium species and Other Selected Compounds
René Torres Gaona, Héctor Galeno Araya and Brenda Modak Canobra
525
Total Synthesis of 5-(5–Ethenyl-2-Methoxyphenyl)–3-Furancarboxaldehyde and Related Compounds
Leticia León–Galeana and Luis Ángel Maldonado G.
529
Contents of Volume 3 (1-12) 2008 v
Seasonal Phytochemical Variation and Antifungal Evaluation of Different Parts of Epidendrum mosenii
Patrícia Walter Rosa, Marina da Silva Machado, Rivaldo Niero, Susana Zacchino, Maria de los Ángeles Gette,
Franco Delle Monache and Valdir Cechinel Filho
535
Acyl Sucroses from Salpichroa origanifolia
Carmelo Dutra, María Verónica Cesio, Patrick Moyna and Horacio Heinzen
539
Influence of N-Deacetylation Conditions on Chitosan Production from α-Chitin
Gemma Galed, Erika Diaz, Francisco M. Goycoolea and Angeles Heras
543
Searching for Natural Bioactive Compounds in Four Baccharis species from Bolivia
Marcelo Dávila, Ingrid Loayza, Daniel Lorenzo and Eduardo Dellacassa
551
In vitro Antiprotozoal Activity and Chemical Composition of Ambrosia tenuifolia and A. scabra Essential Oils
Valeria P. Sülsen, Silvia I. Cazorla, Fernanda M. Frank, Paola M. R. Di Leo Lira, Claudia A. Anesini, David Gutierrez
Yapu, Alberto GiménezTurba, Arnaldo L. Bandoni, Emilio L. Malchiodi, Liliana V. Muschietti and Virginia S. Martino
557
Composition and Antioxidant activity of Essential Oils of Lippia origanoides H.B.K. grown in Colombia
Elena Stashenko, Carlos Ruiz, Amner Muñoz, Martha Castañeda and Jairo Martínez
563
Germacrone: Occurrence, Synthesis, Chemical Transformations and Biological Properties
Alejandro F. Barrero, M. Mar Herrador, Pilar Arteaga and Julieta V. Catalán
567
Terpenoids in Grapes and Wines: Origin and Micrometabolism during the Vinification Process
Francisco M. Carrau, Eduardo Boido and Eduardo Dellacassa
577
Toxic Chemical Compounds of the Solanaceae
Alicia B. Pomilio, Elvira M. Falzoni and Arturo A. Vitale
593
Synthesis of Marine Indole Alkaloids from Flustra foliacea
Martha S. Morales-Ríos and Oscar R. Suárez-Castillo
629
Monoaminergic, Ion Channel and Enzyme Inhibitory Activities of Natural Aporphines, their Analogues and
Derivatives
Bruce K. Cassels and Marcelo Asencio
643
Number 5
Simultaneous Determination of Nine Bioactive Constituents of Caulis Lonicerae Japonicae by High-Performance
Liquid Chromatography Coupled with Mass Spectrometry
Hui-Jun Li, Zheng-Ming Qian, Ping Li , Mei-Ting Ren, Jun Chen and Gui-Zhong Xin
655
Comparative Analysis of Microbial and Rat Metabolism of the Total Saponins from Panax notoginseng by
HPLC-ESI-MS/MS
Guang-tong Chen, Min Yang, Si-jia Tao, Zhi-qiang Lu, Jin-qiang Zhang, Hui-lian Huang, Li-jun Wu and De-an Guo
661
Analysis on the Stability of Total Phenolic Acids and Salvianolic Acid B from Salvia miltiorrhiza by HPLC and
HPLC-MSn
Man Xu, Jian Han, Hui-feng Li, Li Fan, Ai-hua Liu and De-an Guo
669
Simultaneous Determination of Vitexin Rhamnoside and Vitexin Glucoside in Rats by Liquid Chromatography
Coupled with Mass Spectrometry
MingJin Liang, WeiDong Zhang, Chuan Zhang, YunHeng Shen, XiaoLin Wang and Xiangwei Wang
677
Identification and Quantification of three Tubeimosides in Rhizoma Bolbostematis by High Performance Liquid
Chromatography with Evaporative Light Scattering Detection and Electrospray Mass Spectrometric Detection
Hao Huang, MingJin Liang, Wen Xu, Chuan Zhang and WeiDong Zhang
683
Identification of the Major Constituents in Shi-Quan-Da-Bu Decoction by HPLC-ESI-MS/MS
Sijia Tao, Guangtong Chen, Min Yang, Shaosheng Deng, Jinqiang Zhang and De-an Guo
689
Analysis of Major Chemical Constituents in Luan-Pao-Prescription Using Liquid Chromatography Coupled
with Electrospray Ionization Mass Spectrometry
Jin-qiang Zhang, Min Yang, Bao-hong Jiang, Hui-lian Huang, Guang-tong Chen, Zhi-qiang Lu, Xing-nuo Li,
Kai-shun Bi and De-an Guo
697
Quantitative Determination of Ecdysteroids in Sida rhombifolia L. and various other Sida Species Using LC-UV,
and their Anatomical Characterization
Bharathi Avula, Vaishali Joshi, Yan-Hong Wang, Atul N. Jadhav and Ikhlas A. Khan
705
Antibacterial Effects of Guava Tannins and Related Polyphenols on Vibrio and Aeromonas Species
Fumi Yamanaka, Tsutomu Hatano, Hideyuki Ito, Shoko Taniguchi, Eizo Takahashi and Keinosuke Okamoto
711
vi Contents of Volume 3 (1-12) 2008
Phytochemical Analysis of Nunavik Rhodiola rosea L.
Vicky J. Filion, Ammar Saleem, Guy Rochefort, Marc Allard, Alain Cuerrier and John T. Arnason
721
1
Assessment of H NMR Spectroscopy for Specific Metabolite Fingerprinting of Angelica sinensis
Dongmin Su, Jinglan Han, Shishan Yu and Hailin Qin
727
Steroidal Glycosides from the Seeds of Hyoscyamus niger L.
Irina Lunga, Carla Bassarello, Pavel Kintia, Stepan Shvets, Sonia Piacente and Cosimo Pizza
731
A New Type of Modified Brassinosteroids for Enzyme-linked Immunosorbent Assay
Vladimir Khripach, Vladimir Zhabinskii, Alexey Antonchick, Raissa Litvinovskaya, Svetlana Drach, Oleg Sviridov,
Andrey Pryadko, Tatyana Novik, Vitaliy Matveentsev and Bernd Schneider
735
Puguflavanones A and B; Prenylated Flavanones from Baphia puguensis
Modest C. Kapingu, Joseph J. Magadula, Zakaria H. Mbwambo and Dulcie A. Mulholland
749
New Flavonoids from Baeckea frutescens and their Antioxidant Activity
Tran Hong Quang, Nguyen Xuan Cuong, Chau Van Minh, and Phan Van Kiem
755
Sideroxylin from Miconia ioneura: Monohydrate Crystal Structure from High Resolution X-Ray Powder Diffraction
Silvina Pagola, María I. Tracanna, Sara M. Amani, Ana M. González, Ana B. Raschi, Elida Romano, Alba M. Benavente
and Peter W. Stephens
759
Colletinin A and 2,2´´-Diepicolletinin A: Two New Bisflavan-3-ols from Rhododendron collettianum
Viqar Uddin Ahmad, Farman Ullah, Hidayat Hussain, Gilles Dujardin, Arnaud Martel and Ivan Robert Green
765
Isoquinoline Alkaloids and Homoisoflavonoids from Drimiopsis barteri Bak and D. burkei Bak
Dieudonne Ngamga, Josephine Bipa, Pearl Lebatha, Christabel Hiza, Joan Mutanyatta, Merha-Tibeb Bezabih,
Pierre Tane and Berhanu M. Abegaz
769
Flavonoid Constituents of Carduncellus mareoticus (Del.) Hanelt and their Biological Activities
Marwan M. Shabana, Moshera M. El-Sherei, Mohamed Y. Moussa, Amany A. Sleem and Hosam M. Abdallah
779
Isolation of Pyranocoumarins from Angelica gigas
V.L. Niranjan Reddy, Atul N. Jadhav, Bharathi Avula and Ikhlas A. Khan
785
Constituents of Zanthoxylum flavum and their Antioxidant and Antimalarial Activities
Samir A. Ross, Kesanapallis Krishnaven, Mohamed M. Radwan, Satoshi Takamatsu and Charles L. Burandt
791
Two New Xanthone Glycosides from Ventilago leiocarpa Benth.
Li-Li Wang, Jian-Ping Zuo, Lei Ma, Xia-Chang Wang and Li-Hong Hu
795
New Derivatives of Chromene and Acetoxyeudesmane Obtained by Microbial Transformation
Mamdouh S. A. Haridy and Abou El-Hamd H. Mohamed
799
Dihydrostilbenes from Indigofera pulchra
Aliyu Musa, A. K. Haruna, M. Ilyas, Augustine Ahmadu, Simon Gibbons and M. Mukhlesur Rahman
805
Membrane Activity-Guided Isolation of Antiproliferative and Antiplatelet Constituent from Evodiopanax innovans
Hironori Tsuchiya, Toshiyuki Tanaka, Motohiko Nagayama, Masayoshi Oyama and Munekazu Iinuma
809
O-Methylheptaphylline from Clausena suffruticosa
Rokeya Begum, Mohammad S. Rahman, A. M. Sarwaruddin Chowdhury , M. Mukhlesur Rahman and Mohammad A. Rashid
815
The Anti-tumor Effect of the Light Petroleum Extract from Pulsatilla chinensis (Bunge) Regel
Min Zhang, Zhihang Song, Dan Wang, Lin Cheng, Wenrong Jin, Peng Zhang, Yang Huo and Zhongjun Ma
819
Thymol and its Derivatives as Antimicrobial Agents
Ajai Kumar, Suriya P. Singh and Sudarshan S. Chhokar
823
Seasonal Variation in the Leaf Essential Oil Composition of Sassafras albidum
Kristi M. Kaler and William N. Setzer
829
Essential Oil Composition of Two Subspecies of Nepeta glomerulosa Boiss. from Iran
Katayoun Javidnia, Ramin Miri, Shaghayegh Rasteh Rezazadeh, Mohammad Soltani and Ahmad Reza Khosravi
833
Antioxidant Activities of Eleven Australian Essential Oils
Qian Zhao, Esther Joy Bowles and Hong-Yu Zhang
837
5α, 8α-Epidioxysterol from the marine sponge Biemna triraphis Topsent.
Julia Bensemhoun, Isabelle Bombarda, Maurice Aknin, Jean Vacelet and Emile M. Gaydou
843
Number 6
Iridoid Glucosides from Viburnum macrocephalum
Lamberto Tomassini, Sebastiano Foddai, Antonio Ventrone and Marcello Nicoletti
845
Contents of Volume 3 (1-12) 2008 vii
Chemotaxonomy of Linaria Genus by Nor-Iridoids Distribution
Mauro Serafini, Antonella Piccin, Alessandra Stanzione, Valentina Petitto and Marcello Nicoletti
847
A Guaiane Diol from Actinolema eryngioides
Hasan Çetin Özen, Federica Pollastro and Giovanni Appendino
849
Anti-inflammatory Activity of New Guaiane Acid Derivatives from Achillea Coarctata
Mohamed-Elamir F. Hegazy, Ahmed Abdel-Lateff, Amira M. Gamal-Eldeen, Fatma Turky, Toshifumi Hirata,
Paul W. Paré, Joe Karchesy, Mohamed S. Kamel and Ahmed A. Ahmed
851
Chemical Constituents of Ximenia americana
Mônica Regina Silva de Araújo, João Carlos da Costa Assunção, Ivana Nogueira Fernandes Dantas,
Letícia Veras Costa-Lotufo and Francisco José Queiroz Monte
857
Isolation and Cytotoxic Activity of Compounds from the Root Tuber of Curcuma wenyujin
Dan Wang, Wei Huang, Qiang Shi, Chengtao Hong, Yiyu Cheng, Zhongjun Ma and Haibin Qu
861
Cruzain Inhibition by Terpenoids: A Molecular Docking Analysis
Ifedayo V. Ogungbe and William N. Setzer
865
A new 14,15-dinor-labdane Glucoside from Crassocephalum mannii
Mohamed-Elamir F. Hegazy, Ashraf A. Aly, Ahmed A. Ahmed, Djemgou C. Pierre, Pierre Tane and Mehawed M. Ahmed
869
Furanolabdane Diterpene Glycosides from Eremostachys laciniata
Abbas Delazar, Masoud Modarresi, Hossein Nazemiyeh, Fatemeh Fathi-Azad, Lutfun Nahar and Satyajit D. Sarker
873
Secondary Metabolites from the Aerial Parts of Salvia aethiopis L.
Anna Malafronte, Fabrizio Dal Piaz, Giuseppina Cioffi , Alessandra Braca, Antonella Leone, and Nunziatina De Tommasi
877
Platelet Antiaggregating Activity and Chemical Constituents of Salvia x jamensis J. Compton
Angela Bisio, Giovanni Romussi, Eleonora Russo, Nunziatina De Tommasi, Nicola Mascolo, Alessio Alfieri,
Maria Carmela Bonito and Carla Cicala
881
Triterpenoids from Anthocleista grandiflora (Gentianaceae)
Joseph J. Magadula, Dulcie A. Mulholland and Neil R. Crouch
885
An Immunomodulator from Terminalia arjuna and Biological Evaluation of its Derivatives
Mohit Saxena, Sachidanand Yadav, Dhyaneshwar U. Bawankule, Santosh K. Srivastava, Anirban Pal, Rupal Mishra,
Madan M. Gupta, Mahendra P. Darokar, Priyanka and Suman P. S. Khanuja
891
New Antiinflammatory Cycloart-23-ene-3β-ol from Senefelderopsis chibiriquetensis
Dilsia J. Canelón, Alírica I. Suárez, Juan De Sanctis, Michael Mijares and Reinaldo S. Compagnone
895
New Cycloartane Glycosides from Camptosorus sibiricus Rupr.
Peng Zhang, Yiyu Cheng and Zhongjun Ma
899
Triterpene Bisdesmosides from the Stems of Akebia quinata
Yoshihiro Mimaki and Saya Doi
903
Chemotaxonomic relationships in Celastraceae inferred from Principal Component Analysis (PCA) and Partial
Least Squares (PLS)
Ana V. Mello Cruz, Marcelo J. P. Ferreira, Marcus T. Scotti, Maria A. C. Kaplan and Vicente P. Emerenciano
911
Chemical Composition of the Essential Oils of Centaurea sicana and C. giardinae Growing Wild in Sicily
Carmen Formisano, Daniela Rigano, Felice Senatore, Maurizio Bruno, Sergio Rosselli, Francesco Maria Raimondo
and Vivienne Spadaro
919
Chemical Composition of the Essential Oil of the Flowering Aerial Parts of Craniotome furcata
Rajesh K. Joshi and Chitra Pande
923
Chemical Composition of the Essential Oils from Flower, Leaf, and Stem of Senecio trapezuntinus Boiss.
Grown in Turkey
Osman Üçüncü, Nuran Yaylı, Ahmet Yaşar, Salih Terzioğlu and Nurettin Yaylı
925
GC/MS Analysis of the Essential Oil from the Oleoresin of Pistacia atlantica Desf. subsp. Atlantica from Algeria
Hachemi Benhassaini, Fatima Z. Bendeddouche, Zoheir Mehdadi and Abderrahmane Romane
929
Antibacterial Activity and Composition of the Essential Oil of Peperomia galioides HBK (Piperaceae) from Peru
Vincenzo De Feo, Alberto Juarez Belaunde, Joe Guerrero Sandoval Felice Senatore and Carmen Formisano
933
Chemical Composition and in vitro Antibacterial Activity of the Essential Oil of Calycolpus moritzianus (O. Berg)
Burret from Mérida, Venezuela
Teresa Díaz, Flor D. Mora, Judith Velasco, Tulia Díaz, Luis B. Rojas, Alfredo Usubillaga and Juan Carmona A
937
Essential Oil Analysis and Antimicrobial Activity of Paeonia mascula from Turkey
Nurettin Yaylı, Ahmet Yaşar, Nuran Yaylı, Mesut Albay and Kamil Coşkunçelebi
941
viii Contents of Volume 3 (1-12) 2008
Antimicrobial Activity of Nepeta Isolates
Chandra S. Mathela and Neeta Joshi
945
Sesquiterpenes of Lactarius and Russula (Mushrooms): An Update
Marco Clericuzio, Gianluca Gilardoni, Omar Malagòn, Giovanni Vidari and Paola Vita Finzi
951
Virescenols: Sources, Structures and Chemistry
Maria Carla Marcotullio, Ornelio Rosati and Massimo Curini
975
Rearranged Clerodane and Abietane Derived Diterpenoids from American Salvia Species
Baldomero Esquivel
989
Diterpenes from Euphorbia as Potential Leads for Drug Design
Elisa Barile, Gabriella Corea and Virginia Lanzotti
1003
neo-Clerodane Diterpenoids from Verbenaceae: Structural Elucidation and Biological Activity
Amaya Castro and Josep Coll
1021
Number 7
Chemical Composition, Olfactory Evaluation and Antioxidant Effects of the Essential Oil of Satureja montana L.
Ivanka Stoilova, Stefanie Bail, Gerhard Buchbauer, Albert Krastanov, Albena Stoyanova, Erich Schmidt and Leopold Jirovetz
1035
Chemical Composition, Olfactory Evaluation and Antioxidant Effects of an Essential Oil of Origanum vulgare L.
from Bosnia
Ivanka Stoilova, Stefanie Bail, Gerhard Buchbauer, Albert Krastanov, Albena Stoyanova, Erich Schmidt and Leopold Jirovetz
1043
Chemical Composition, Olfactory Evaluation and Antioxidant Effects of an Essential Oil of Thymus vulgaris L. from
Germany
Ivanka Stoilova, Stefanie Bail, Gerhard Buchbauer, Albert Krastanov, Albena Stoyanova, Erich Schmidt and Leopold Jirovetz
1047
Chemical Composition, Olfactory Evaluation and Antioxidant Effects of the Essential oil of Origanum majorana L.
from Albania
Erich Schmidt, Stefanie Bail, Gerhard Buchbauer, Ivanka Stoilova, Albert Krastanov, Albena Stoyanova and Leopold Jirovetz
1051
GC-MS-Analysis, Antimicrobial Activities and Olfactory Evaluation of Essential Davana
(Artemisia pallens Wall. ex DC) Oil from India
Stefanie Bail, Gerhard Buchbauer, Erich Schmidt, Juergen Wanner, Alexander Slavchev, Albena Stoyanova,
Zapriana Denkova, Margit Geissler and Leopold Jirovetz
1057
Comparative Evaluation of Antimicrobial Activity andComposition of Rose Oils From Various Geographic Origins,
in Particular Bulgarian Rose Oil
Velizar Gochev, Katrin Wlcek, Gerhard Buchbauer, Albena Stoyanova, Anna Dobreva, Erich Schmidt and Leopold Jirovetz
1063
Composition and Antimicrobial Analysis of the Essential Oil of Litsea laevigata Nees. (Lauraceae)
Muhammed Arif M, Subbu Raj M, Leopold Jirovetz and Mohamed Shafi P
1069
Chemical Composition and Antifungal Activity of Angelica sinensis Essential Oil against three Colletotrichum species
Nurhayat Tabanca, David E. Wedge, Xiaoning Wang, Betul Demirci, Kemal Husnu Can Baser, Ligang Zhou and
Stephen J. Cutler
1073
Development of a Miniaturized 24-well Strawberry Leaf Disk Bioassay for Evaluating Natural Fungicides
Xiaoning Wang, David E. Wedge, Nurhayat Tabanca, Robert D. Johnson, Stephen J. Cutler, Patrick F. Pace,
Barbara J. Smith and Ligang Zhou
1079
Investigation of Anticancer and Antiviral Properties of Selected Aroma Samples
Boris Ryabchenko, Elena Tulupova, Erich Schmidt, Katrin Wlcek, Gerhard Buchbauer and Leopold Jirovetz
1085
Chemical and Biological Investigations of Essential Oils from Stem Barks of Enantia chlorantha Oliv. and
Polyalthia suaveolens Engler. & Diels. from Cameroon
Maximilienne Nyegue, Paul-Henri Amvam-Zollo, François-Xavier Etoa, Huguette Agnaniet and Chantal Menut
1089
Evaluation of the Activities of Five Essential Oils against the Stored Maize Weevil
Oluwakemi O. Odeyemi, Patrick Masika and Anthony J. Afolayan
1097
Physiological and Behavioral Effects of 1,8-Cineol and (±)-Linalool: A Comparison of Inhalation and Massage
Aromatherapy
Eva Heuberger, Josef Ilmberger, Engelbert Hartter and Gerhard Buchbauer
1103
Synergistic and Antagonistic Interactions of Essential Oils on the Biological Activities of the Solvent Extracts from
Three Salvia species
Guy P. P. Kamatou, Robyn L. van Zyl, Hajierah Davids, Sandy F. van Vuuren and Alvaro M. Viljoen
1111
Contents of Volume 3 (1-12) 2008 ix
Essential Oil Analysis of the Follicles of Four North American Magnolia Species
Wolfgang Schühly, Samir A. Ross, Zlatko Mehmedic and Nikolaus H. Fischer
1117
Essential Oil Composition of Eryngium campestre L. Growing in Different Soil Types. A Preliminary Study
Jesús Palá-Paúl, Jaime Usano-Alemany, A. Cristina Soria, M. José Pérez-Alonso and Joseph J. Brophy
1121
Essential Oil Compounds of Origanum vulgare L. (Lamiaceae) from Corsica
Brigitte Lukas, Corinna Schmiderer, Ulrike Mitteregger, Chlodwig Franz and Johannes Novak
1127
Comparative Study of the Chemical Profiles of the Essential Oils of Ripe and Rotten Fruits of
Citrus aurantifolia Swingle
Anthony J. Afolayan and Olayinka T. Asekun
1133
Chemical Variation in the Essential Oil Composition of Hyptis suaveolens (L.) Poit. (Lamiaceae)
Paolo Grassi, Marvin José Nuñez, Tomás Sigfrido Urías Reyes and Chlodwig Franz
1137
Variability of the Volatile Oil Composition in a Population of Silaum silaus from Eastern Austria
Remigius Chizzola
1141
The Bark Essential Oil Composition and Chemotaxonomical Appraisal of Cedrelopsis grevei H. Baillon
from Madagascar
Miarantsoa Rakotobe, Chantal Menut, Hanitriniaina Sahondra Andrianoelisoa, Voninavoko Rahajanirina,
Philippe Collas de Chatelperron, Edmond Roger and Pascal Danthu
1145
Essential Oil Polymorphism of Hungarian Common Thyme (Thymus glabrescens Willd.) Populations
Zsuzsanna Pluhár, Szilvia Sárosi, Ildikó Novák and Gabriella Kutta
1151
Diversity of Essential Oil Glands of Spanish Sage (Salvia lavandulifolia Vahl, Lamiaceae)
Corinna Schmiderer, Paolo Grassi, Johannes Novak and Chlodwig Franz
1155
Micro-bore Column Fast Gas Chromatography-Mass Spectrometry in Essential Oil Analysis
Peter Quinto Tranchida, Rosaria Costa, Paola Dugo, Giovanni Dugo and Luigi Mondello
1161
Carbon Isotope Ratio Analysis of Authentic and Commercial Essential Oils of Lemon Balm
Susanne Wagner, Polona Vreca, Albrecht Leis and Herbert Boechzelt
1165
Essential Oil Compounds for Thrips Control – A Review
Elisabeth H. Koschier
1171
Linalool – A Review of a Biologically Active Compound of Commercial Importance
Guy P. P. Kamatou and Alvaro M. Viljoen
1183
Limonene - A Review: Biosynthetic, Ecological and Pharmacological Relevance
Paul Erasto and Alvaro M.Viljoen
1193
Number 8
Plant Secondary Metabolism: Diversity, Function and its Evolution
Michael Wink
1205
Major Constituents of the Predominant Endophytic Fungi from the Nigerian Plants Bryophyllum pinnatum,
Morinda lucida and Jathropha gossypiifolia
Abimbola A. Sowemimo, RuAngelie Edrada-Ebel, Rainer Ebel, Peter Proksch, Olanrewaju R. Omobuwajo and
Saburi A. Adesanya
1217
Integration of Plasma Membrane and Nuclear Signaling in Elicitor Regulation of Plant Secondary Metabolism
Gastón Stockman and Ricardo Boland
1223
Selective Elicitation of the Phytoalexin Rutalexin in Rutabaga and Turnip Roots by a Biotrophic Plant Pathogen
M. Soledade C. Pedras, Ravi S. Gadagi, Qing-An Zheng and S. Roger Rimmer
1239
Transient Induction of Antimicrobial 3-Deoxyflavonoids does not affect Pharmacological Compounds in Hawthorn
Karin Schlangen, Heidi Halbwirth, Silke Peterek, Christian Gosch, Alexandra Ringl, Thilo C. Fischer, Dieter Treutter,
Gert Forkmann, Brigitte Kopp and Karl Stich
1245
Trafficking and Sequestration of Anthocyanins
Erich Grotewold and Kevin Davies
1251
Unraveling the Biochemical Base of Dahlia Flower Coloration
Heidi Halbwirth, Gerlinde Muster and Karl Stich
1259
Anthocyanins Reduce Fungal Growth in Fruits
H. Martin Schaefer, Michael Rentzsch and Michael Breuer
1267
Phenolic Compounds Produced by Secretory Structures in Plants: a Brief Review
Marilia de M. Castro and Diego Demarco
1273
x Contents of Volume 3 (1-12) 2008
Surface Flavonoids in Catalpa ovata, Greyia sutherlandii and Paulownia tomentosa
Eckhard Wollenweber, Rüdiger Wehde, Matthias Christ and Marion Dörr
1285
Chemodiversity of Artemisia dracunculus L. from Kyrgyzstan: Isocoumarins, Coumarins, and Flavonoids
from Aerial Parts
Tshering D. Bhutia and Karin M. Valant-Vetschera
1289
Occurrence and Distribution of the Flavone Tricetin and its Methyl Derivatives as Free Aglycones
Eckhard Wollenweber and Marion Dörr
1293
Variation in Flavonoids in Leaves, Stems and Flowers of White Clover Cultivars
Sandra C.K. Carlsen, Anne G. Mortensen, Wieslaw Oleszek, Sonia Piacente, Anna Stochmal and Inge S. Fomsgaard
1299
Plants as a Still Unexploited Source of New Drugs
Kurt Hostettmann and Andrew Marston
1307
Activity-guided Isolation and Identification of Free Radical-scavenging Components from Ethanolic Extract
of Boneset (Leaves of Eupatorium perfoliatum)
Solomon Habtemariam
1317
Activity-guided Isolation and Identification of Antioxidant Components from Ethanolic Extract of
Peltiphyllum peltatum (Torr.) Engl.
Solomon Habtemariam
1321
Two New Kaempferol Glycosides from Matthiola longipetala (subsp. livida) (Delile) Maire and
Carcinogenic Evaluation of its Extract
Mona M. Marzouk, Salwa A. Kawashty, Lamyaa F. Ibrahium, Nabiel A. M. Saleh and Abdel-Ssalam M. Al-Nowaihi
1325
Newer Constituents of Derris indica stem
Thangaraj Shankar, Shanmugam Muthusubramanian and Rathinasamy Gandhidasan
1329
Structural Characterization of Genuine (–)-Pipermethystine, (–)-Epoxypipermethystine, (+)-Dihydromethysticin
and Yangonin from the Kava Plant (Piper methysticum)
Panče Naumov, Klaus Dragull, Masahiro Yoshioka, Chung-Shih Tang and Seik Weng Ng
1333
Naturally Occurring Flavanones: An Overview
Goutam Brahmachari
1337
Bioisosteric Replacement of Molecular Scaffolds: From Natural Products to Synthetic Compounds
Kristina Grabowski, Ewgenij Proschak, Karl-Heinz Baringhaus, Oliver Rau, Manfred Schubert-Zsilavecz and
Gisbert Schneider
1355
Composition of Essential Oil from Lavandula angustifolia and L. intermedia VarietiesGrown in British Columbia,
Canada
W. Alexander Lane and Soheil S. Mahmoud
1361
Chemical Compositions and Biological Activities of Leaf Essential Oils of Twelve Species of Piper from Monteverde,
Costa Rica
William N. Setzer, Grace Park, Brittany R. Agius, Sean L. Stokes, Tameka M. Walker and William A. Haber
1367
Number 9
Anti-tuberculosis Compounds from two Bolivian Medicinal Plants, Senecio mathewsii and Usnea florida
Qi Hong, David E. Minter, Scott G. Franzblau and Manfred G. Reinecke
1377
Natural Products from Polygonum perfoliatum and their Diverse Biological Activities
Chi-I Chang, Fei-Jane Tsai and Chang-Hung Chou
1385
Phytochemical Characterization of the Australian (Aboriginal) Medicinal Plant Dolichandrone heterophylla and
Influence of Selected Isolated Compounds on Human Keratinocytes
Thomas Dzeha, Kristian Wende, Manuela Harms, Ju Ju (Burriwee) Wilson, Jim Kohen, Subra Vemulpad, Joanne Jamie
and Ulrike Lindequist
1387
Analysis of Saponin Mixtures from Alfalfa (Medicago sativa L.) Roots using Mass Spectrometry with
MALDI Techniques
H. Ewa Witkowska, Zbigniew Bialy, Marian Jurzysta and George R. Waller
1395
Control of Allantoin Accumulation in Comfrey
Paulo Mazzafera, Kátia Viviane Gonçalves and Milton Massao Shimizu
1411
Trigonelline (N-methylnicotinic acid) Biosynthesis and its Biological Role in Plants
Hiroshi Ashihara
1423
Contents of Volume 3 (1-12) 2008 xi
Biosynthesis and Catabolism of Purine Alkaloids in Camellia Plants
Misako Kato and Hiroshi Ashihara
1429
A View on the Active Site of Firefly Luciferase
Franklin R. Leach
Study on the Chemical Constituents of Premna integrifolia L.
Nguyen Thi Bich Hang, Pham Thanh Ky, Chau Van Minh, Nguyen Xuan Cuong, Nguyen Phuong Thao and Phan Van Kiem
1449
Τ-Cadinol Nerolidol Ether from Schisandra chinensis
Asmita V. Patel, Gerald Blunden, Peter D. Cary, Lubomír Opletal, Markéta Beránkova, Kersti Karu, David E. Thurston
and Milan Pour
1453
Bioactive Semisynthetic Derivatives of (S)-(+)-Curcuphenol
Helena Gaspar, Cristina Moiteiro, João Sardinha and Azucena González-Coloma
1457
Eco-contribution to the Chemistry of Perezone, a Comparative Study, Using Different Modes of
Activation and Solventless Conditions
Joel Martínez, Benjamín Velasco-Bejarano, Francisco Delgado, Rocío Pozas, Héctor M. Torres Domínguez,
José G. Trujillo Ferrara, Gabriel A. Arroyo and René Miranda
1465
Chemical Composition of Diterpenes from the Brown Alga Canistrocarpus cervicornis (Dictyotaceae, Phaeophyceae)
Aline Santos de Oliveira, Diana Negrão Cavalcanti, Éverson Miguel Bianco, Joel Campos de Paula, Renato Crespo Pereira,
Yocie Yoneshigue-Valentin and Valéria Laneuville Teixeira
1469
Cembranoid Diterpenes from the Soft Corals Sarcophyton sp. and Sarcophyton glaucum
Daniela Grote, Kamel H. Shaker, Hesham S. M. Soliman, Muhammmad M. Hegazi and Karlheinz Seifert
1473
Dolabellane Diterpenes from Cleome droserifolia
Hoda M. Fathy, Mohamed I. Aboushoer, Fathallah M. Harraz, Abdallah A. Omar, Gilles Goetz and Rafael Tabacchi
1479
Constituents of ‘Caincin’, a Bioactive Saponin Fraction from the Root-bark of Chiococca alba (L.) Hitch.
Jnanabrata Bhattacharyya, S. K. Srivastava, M. F. Agra and George Majetich
1483
A New Biflavanone from Ochna lanceolata
Shaik I. Khalivulla, Nimmanapalli P. Reddy, Bandi A.K. Reddy, Ramireddy V.N. Reddy, Duvvuru Gunasekar,
Alain Blond and Bernard Bodo
1487
Isoflavonoids from an Egyptian Collection of Colutea istria
Mohamed M. Radwan
1491
A Flavonoid Glycoside from the Leaves of Morinda tinctoria
Atish K. Sahoo, Nisha Narayanan, S. Rajan and Pulok K. Mukherjee
1495
New Anthocyanins from Stem Bark of Castor, Ricinus communis
Robert Byamukama, Monica Jordheim, Bernard Kiremire and Øyvind M. Andersen
1497
Prenylated Xanthones and a Benzophenone from Baphia kirkii
Modest C. Kapingu and Joseph J. Magadula
1501
Phytochemical and Antimicrobial Investigation of Latex from Euphorbia abyssinica Gmel.
Fathy EL- Fiky, Kaleab Asres, Simon Gibbons, Hala Hammoda, Jihan Badr and Shemsu Umer
1505
Fenugreek Extract Rich in 4-Hydroxyisoleucine and Trigonelline Activates PPARα and Inhibits LDL Oxidation:
Key Mechanisms in Controlling the Metabolic Syndrome
Alvin Ibarra, Kan He, Naisheng Bai, Antoine Bily, Marc Roller, Aurélie Coussaert, Nicolas Provost and Christophe Ripoll
1509
Comparison of Different Techniques for Extraction of Biologically Active Compounds from Achillea Millefolium Proa
Antoaneta Trendafilova and Milka Todorova
1515
Extraction Methods Play a Critical Role in Chemical Profile and Biological Activities of Black Cohosh
Bei Jiang, Kurt A. Reynertson, Amy C. Keller, Linda S. Einbond, Debra L. Bemis, I. Bernard Weinstein, Fredi Kronenberg
and Edward J. Kennelly
1519
Antimicrobial Activity and Chemical Composition of Callistemon pinifolius and C. salignus Leaf Essential Oils
from the Northern Plains of India
Mohit Saxena, Kunal Shrivastava, Santosh K. Srivastava, Suaib Luqman, Ajai Kumar, Mahendra. P. Darokar,
Kodakandla V. Syamsundar, Tota Ram and Suman P. S. Khanuja
1533
Analysis of Chemical Constituents of Tithonia rotundifolia Leaf Essential Oil Found in Nigeria
Adebayo A. Gbolade, Vânia Tira-Picos and J.M.F. Nogueira
1537
Sedative Effect of Eucalyptus urophylla and E. brassiana in mice
Gisele F.D. Teixeira, Roberto C.P. Lima Júnior, Edilberto R. Silveira, Marinalva O. Freitas and Adriana R. Campos
1539
1437
xii Contents of Volume 3 (1-12) 2008
In Vitro Antifungal Activity of Polysulfides-Rich Essential Oil of Ferula latisecta Fruits against Human
Pathogenic Dermatophytes
Mehrdad Iranshahi, Abdolmajid Fata, Bahareh Emami, Bibi Mohadeseh Jalalzadeh Shahri and Bibi Sedigheh Fazly Bazzaz
1543
Biological Activity and Composition of the Essential Oil of Dracocephalum moldavica L. Grown in Iran
Ali Sonboli, Mehran Mojarrad, Abbas Gholipour, Samad Nejad Ebrahimi and Mitra Arman
1547
Antioxidant Activity and Chemical Composition of Essential Oils from Schinus fasciculata (Griseb.)
I.M. Johnst and S. praecox (Griseb.) Speg.
Ana P. Murray, Silvana A. Rodriguez and María G. Murray
1551
Essential oil Composition of Dendropanax gonatopodus from Monteverde, Costa Rica. An ab initio
Examination of Aromadendrane Sesquiterpenoids
William N. Setzer
1557
Number 10
Oxidative Transformations of Lappaconitine and 19-Oxolappaconine, Structural Revision of an obtained
8,9-Seco Product
Elza U. Shafikova, Elena M. Tsyrlina, Leonid V. Spirikhin, Alsu A. Balandina, Shamil K. Latypov, Marat S. Yunusov
and Oleg G. Sinyashin
1565
Oxidative Decarboxylation of Triterpene C-28 Acids of Lupane Series
Nataliya G. Кomissarova, Nataliya G. Belenkova, Olga V. Shitikova, Leonid V. Spirikhin and Marat S. Yunusov
1569
Proapoptotic and Anticarcinogenic Activities of Leviusculoside G from the Starfish Henricia leviuscula and
Probable Molecular Mechanism
Sergey N. Fedorov, Larisa K. Shubina, Alla A. Kicha, Natalia V. Ivanchina, Jong Y. Kwak, Jun O. Jin, Ann M. Bode,
Zigang Dong and Valentin A. Stonik
1575
Two New Steroid Oligoglycosides from the Caribbean Sponge Mycale laxissima
Shamil Sh. Afiyatullov, Alexandr S. Antonov, Anatoly I. Kalinovsky and Pavel S. Dmitrenok
1581
New Polar Steroids from Starfish
Valentin A. Stonik, Natalia V. Ivanchina and Alla A. Kicha
1587
New Angucyclinones from the Marine Mollusk Associated Actinomycete Saccharothrix espanaensis An 113
Nataliya I. Kalinovskaya, Anatoly I. Kalinovsky, Lyudmila A. Romanenko, Mikhail A. Pushilin, Pavel S. Dmitrenok and
Tatyana A. Kuznetsova
1611
6-Bromo-5-hydroxyindolyl-3-glyoxylate from the Far Eastern Ascidian Syncarpa oviformis
Elena A. Santalova, Vladimir A. Denisenko, Dmitry V. Berdyshev, Dmitry L. Aminin and Karen E. Sanamyan
1617
Analgesic Properties of New Pyrrolidinomorphinane Derivatives: Revealing Potential Pathways
Ekaterina A. Morozova, Tatiana G. Tolstikova, Alexey V. Bolkunov, Margarita P. Dolgikh and Elvira E. Shul’ts
1621
2,4-Dihydroxypentanoic Acids: New Non-sugar Components of Bacterial Polysaccharides
Alexander S. Shashkov, Nina A. Kocharova, Filip V. Toukach, Vadim V. Kachala and Yuriy A. Knirel
1625
Structural Analysis of Antibiotic INA 9301 from Amycolatopsis orientalis
Alexander S. Shashkov, Dmitry E. Tsvetkov, Alexey A. Grachev, Olda A. Lapchinskaia, Maia F. Lavrova-Balashova,
Valerii I. Ponomarenko, Genrikh S. Katrukha and Nikolay E. Nifantiev
1631
Structural Analysis of Fucoidans
Maria I. Bilan and Anatolii I. Usov
1639
Guaiane Sesquiterpenoids from Jatropha curcas
Xia-Chang Wang, Shi-Ping Ma, Jing-Han Liu and Li-Hong Hu
1649
Sesquiterpenes of the Brazilian Marine Red Alga Laurencia filiformis (Rhodophyta, Ceramiales)
Bruno Lopes Antunes, Beatriz Grosso Fleury, MutueToyota Fujii and Valéria Laneuville Teixeira
1653
Inhibition of Mushroom Tyrosinase and Melanogenesis B16 Mouse Melanoma Cells by Components Isolated from
Curcuma longa
Jeong Ah Kim, Jong Keun Son, Hyun Wook Chang, Yurngdong Jahng, Youngsoo Kim, MinKyun Na and Seung Ho Lee
1655
Reaction of Furanoeremophilans with Pyridoxal
Atsushi Torihata and Chiaki Kuroda
1659
A Bioactive Diterpene from Smallanthus sonchifolius
Consolacion Y. Ragasa, Agnes B. Alimboyoguen, Sylvia Urban and Dennis D. Raga
1663
Antiproliferative Oleanane Saponins from Polyscias guilfoylei
Giuseppina Cioffi, Antonio Vassallo, Laura Lepore, Fabio Venturella, Fabrizio Dal Piaz and Nunziatina De Tommasi
`1667
Contents of Volume 3 (1-12) 2008 xiii
Steroidal Saponins from the Rhizomes of Ruscus hypophyllum
Yoshihiro Mimaki, Tsukasa Aoki, Maki Jitsuno, Akihito Yokosuka, Ceyda Sibel Kiliç and Maksut Coşkun
1671
A New Carbazole Alkaloid from Murraya koenigii Spreng (Rutaceae)
Suvra Mandal, Anupam Nayak, Samir K. Banerjee, Julie Banerji and Avijit Banerji
1679
Secondary Metabolites from Teclea amanuensis (Rutaceae) from Tanzania
Joseph J. Magadula, Modest C Kapingu, Zakaria H. Mbwambo and Dulcie A. Mulholland
1683
Chemical Constituents and Insecticidal Activity of Rollinia leptopetala (Annonaceae)
Ângela M.C. Arriaga, Edinilza M.A. Feitosa, Telma L.G. Lemos, Gilvandete M.P. Santiago, Jefferson Q. Lima, Maria C.F.
de Oliveira, Jackson N. e Vasconcelos, Francisco E. A. Rodrigues, Tathilene B. M. Gomes and Raimundo Braz-Filho
1687
New Pyrenocines from an Endophytic Fungus
Karsten Krohn, Md. Hossain Sohrab, Siegfried Draeger and Barbara Schulz
1689
Chemoenzymatic Synthesis and Some Biological Properties of O-phosphoryl Derivatives of (E)-resveratrol
Danilo Aleo, Venera Cardile, Rosa Chillemi, Giuseppe Granata and Sebastiano Sciuto
1693
Comparative Photobleaching Behavior of Hypocrellin A and Elsinochrome C
Qian Zhao and Hong-Yu Zhang
1701
In vitro Cytotoxic Activity of Sesamin Isolated from Vismia baccifera var. dealbata Triana & Planch (Guttiferae)
Collected from Venezuela
Fabiola Salas, Janne Rojas, Antonio Morales, Maria E. Ramos-Nino and Nelida G. Colmenares
1705
A New Flavonoid Glycoside from the fern Dryopteris villarii
Filippo Imperato
1709
Variation of Bioactive Secondary Metabolites in Hypericum triquetrifolium Turra from Wild Populations of Turkey
Necdet Çamaş, Jolita Radušienė, Ali Kemal Ayan, Cüneyt Çırak, Valdimaras Janulis and Liudas Ivanauskas
1713
Anti-inflammatory Activity of Piper magnibaccum (Piperaceae)
Emrizal, Farediah Ahmad, Hasnah M. Sirat, Fadzureena Jamaludin, Nik Musa’adah Mustapha, Rasadah M. Ali and
Dayar Arbain
1719
Self-Organizing Maps as a New Tool for Classification of Plants at Lower Hierarchical Levels
Mauro V. Correia, Marcus T. Scotti, Marcelo J. P. Ferreira, Sandra A. V. Alvarenga, Gilberto V. Rodrigues
and Vicente P. Emerenciano
1723
Fatty Acid Composition of Heliotropium Species (Boraginaceae): A First Chemical Report on the New Species
H. thermophilum
Ahmet C. Gören, Gülendam Tümen, Ali Çelik and Simay Çıkrıkçı
1731
Comparative Analysis of the Essential Oils from two Asteraceous Plants Found in Nigeria, Acanthospermum hispidum
and Tithonia diversifolia
Adebayo A. Gbolade, Daniela M. Biondi and Giuseppe Ruberto
1735
Chemical Composition of the Essential Oil from Carramboa pittieri (Cuatrec.) Cuatrec. (Asteraceae)
Luis B. Rojas, Rebeca Gutiérrez, Yndra Cordero de Rojas and Alfredo Usubillaga
1739
Antimicrobial Activity and Chemical Composition of Melaleuca genistifolia Leaf Essential Oil from the
Northern Plains of India
Ajai Kumar, Santosh K. Srivastava, Gaurav R. Dwivedi, Merajuddin Khan, Mahendra P. Darokar, Mohit Saxena,
Kodakandla V. Syamsundar, Suaib Luqman and Suman P. S. Khanuja
1741
Number 11
New Drimane Sesquiterpenoids from Tidestromia oblongifolia
Sandeep Chaudhary, Vladimir Thomas, Louis Todaro, Onica LeGendre, Stevan Pecic and Wayne W. Harding
1747
Three New Cassane Diterpenes from Caesalpinia pulcherrima
Jun Cheng, Joy S. Roach, Stewart McLean, William F. Reynolds and Winston F. Tinto
1751
21,28-Epoxy-18β,21β-dihydroxbaccharan-3-one and Other Terpenoids from the Liverwort
Lepidozia chordulifera T. Taylor
Hildegard Zapp, Kerstin Orth, Josef Zapp, Joseph D. Connolly and Hans Becker
1755
A New Hyptadienic Acid Derivative from Hyptis verticillata (Jacq.)