CHEMICAL CONSTITUENTS AND BIOACTIVITY OF MALAYSIAN AND
INDONESIAN KAEMPFERIA ROTUNDA
YAU SUI FENG
UNIVERSITI TEKNOLOGI MALAYSIA
i
CHEMICAL CONSTITUENTS AND BIOACTIVITY OF MALAYSIAN AND
INDONESIAN KAEMPFERIA ROTUNDA
YAU SUI FENG
A thesis submitted in fulfillment of the
requirements for the award of the degree of
Master of Science (Chemistry)
Faculty of Science
Universiti Teknologi Malaysia
DECEMBER 2009
iii
Dedicated to Buddha, Dharmma, Sangha,
my beloved father, mother, sisters, brother and friends.
iv
ACKNOWLEDGEMENTS
First of all, I would like to express my deepest appreciation to my supervisor,
Prof. Dr. Hasnah Mohd. Sirat, for her guidance, motivation, and more crucially,
precious advise to all my research. I am truly grateful for her kindness, all the time
and effort she devoted to me.
I would like to extend special thanks to Assoc. Prof. Dr. Farediah Ahmad, Dr.
Shajarahtunnur Jamil and Mrs. Norazah Basar for their precious help and guidance.
I would also like to express my gratitude to Universiti Teknologi Malaysia
for granting me scholarship and to Department of Chemistry for allowing me to get
access to the GC, GC-MS, IR, UV and NMR facility.
I would like to express greatly appreciation to Ms. Mala, Mr. Emrizal and Mr.
Oh Boon Thai for their patience, support and discussions. Sincerely thanks to Mr.
Farriz, Mr. Syamil, Ms. Azlin, Mrs. Ummu and Mr. Salihin for the friendship and
help during the work.
Sincerely thanks to lab assistants especially Mrs. Mekzum, Mr. Azmi and Mr.
Hanan who helped me throughout these years.
Special thanks to my parents, my sisters, my brother and Mr. Ting Chee Ming
for their love, support and understanding.
v
PREFACE
This thesis is the result of my work carried out in the Department of Chemistry,
Universiti Teknologi Malaysia between July 2007 and June 2009 under the
supervision of Prof. Dr. Hasnah Mohd. Sirat. Parts of my work described in this
thesis have been reported in the following publications:
1. Yau Sui Feng and Hasnah Mohd. Sirat (2009). Chemical Constituents of
Kaempferia rotunda. Mal. J. Sci. 2009. 28 (special edition): 81-88.
2. Yau Sui Feng and Hasnah Mohd. Sirat (2008). Chemical Constituents of
Kaempferia rotunda. Paper presented at International Conference on Molecular
Chemistry 2008 at University of Malaya, Kuala Lumpur. 25-26 November 2008.
3. Yau Sui Feng and Hasnah Mohd. Sirat (2008). Chemical Constituents of
Kaempferia rotunda. Poster presented at the 2nd Penang International Conference
for Young Chemists 2008 at Universiti Sains Malaysia, Penang. 18-20 June
2008.
vi
ABSTRACT
The essential oils and the phytochemicals of the rhizomes of Kaempferia
rotunda cultivated in Malaysia and Indonesia have been studied. Hydrodistillation of
the fresh rhizomes of K. rotunda gave 0.09% and 0.23% oils respectively. These oils
were analyzed by GC (Kovats Indices) and GC-MS. The main constituents found in
the rhizome oil of Malaysia were bornyl acetate (9.6%), benzyl benzoate (8.4%) and
camphor (5.6%), while the rhizome oil from Indonesia was rich in benzyl benzoate
(87.7%) and n-pentadecane (4.2%). Extractions by soxhlet apparatus were carried
out on the dried samples to get the crude extracts. Fractionation and purification on
the crude extracts resulted in the isolation of three new cyclohexane oxides and ten
known compounds, comprising of cyclohexane oxides, esters, carboxylic acid,
labdane diterpene, and flavonoids.
Two new compounds identified as 2(benzoyloxymethyl)phenyl (3-O-acetyl)-β-glucopyranoside and 3-debenzoylrotepoxide A, together with seven known compounds, crotepoxide, 4benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diol
5-acetate,
1,6desoxypipoxide, curcumrinol C, 2'-hydroxy-4,4',6'-trimethoxychalcone and
naringenin 4',7-dimethyl ether were isolated from the Malaysian species, while a new
compound identified as 3-acetoxy-2-benzoyloxy-1-(benzoyloxymethyl)-cyclohexa4,6-diene with the seven known compounds namely crotepoxide, 4benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diol
5-acetate,
1,6desoxypipoxide, 6-acetylzeylenol, trans-docosyl ferulate, benzyl benzoate and
benzoic acid were isolated from the Indonesian species. The structures of all
compounds were established based on spectral studies using MS, IR, UV and NMR
spectroscopies. Naringenin 4',7-dimethyl ether, curcumrinol C, trans-docosyl
ferulate, and benzoic acid were found for the first time from K. rotunda and also the
genus of Kaempferia. Antibacterial and antioxidant screening assays using discdiffusion method and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) method,
respectively were carried out on the crude extracts and essential oils. The crude
extracts and essential oils of K. rotunda from Malaysia and Indonesia did not show
activities on antibacterial and antioxidant assay.
vii
ABSTRAK
Kajian terhadap komposisi minyak pati dan fitokimia ke atas Kaempferia
rotunda yang ditanam di Malaysia dan Indonesia telah dilakukan. Sebanyak 0.09%
dan 0.23% hasil minyak pati masing-masing diperolehi daripada penyulingan hidro
terhadap rizom segar K. rotunda. Minyak pati ini seterusnya dianalisis dengan
menggunakan KG kapilari (Indeks Kovat) dan KG-SJ. Sebatian utama dalam
minyak pati rizom dari Malaysia terdiri daripada bornil asetat (9.6%), benzil benzoat
(8.4%), dan kamfor (5.6%), manakala minyak pati rizom dari Indonesia didapati
kaya dengan benzil benzoat (87.7%) dan n-pentadekana (4.2%). Pengekstrakan
soxhlet telah dijalankan terhadap rizom kering untuk mendapatkan ekstrak mentah.
Pengasingan daripada ekstrak telah menemukan tiga sebatian baru dan sepuluh
sebatian yang telah diketahui, merangkumi sebatian sikloheksana oksida, ester, asid
karboksilik, diterpena labdana dan flavonoid. Dua sebatian baru yang diasingkan
daripada spesies Malaysia dikenali sebagai 2-(benzoiloksimetil)fenil (3-O-asetil)-βglukopiranosida dan 3-debenzoilrotepoksida A, bersama dengan tujuh sebatian yang
diketahui, iaitu krotepoksida, 4-benzoiloksimetil-3,8-dioksatrisiklo[5.1.0.02,4]oktana5,6-diol 5-asetat, 1,6-desoksipipoksida, kurkumrinol C, 2'-hidrosi-4,4',6'trimetosikalkon and naringenin 4',7-dimetil eter. Satu sebatian baru dikenali 3asetoksi-2-benzoiloksi-1-(benzoiloksimetil)sikloheksa-4,6-diena, manakala sebatiansebatian yang telah diketahui, iaitu krotepoksida, 4-benzoiloksimetil-3,8dioksatrisiklo[5.1.0.02,4]oktana-5,6-diol 5-asetat, 1,6-desoksipipoksida, 6-asetilzeilenol, trans-dokosil ferulat, benzil benzoat dan asid benzoik telah diasingkan
daripada spesies Indonesia. Struktur bagi semua sebatian telah dikenalpasti secara
spektroskopi melalui MS, IR, UV dan NMR. Naringenin 4',7-dimetil eter,
kurkumrinol C, trans-dokosil ferulat, dan benzoik asid telah ditemui pertama kali
daripada K. rotunda khususnya daripada genus Kaempferia. Ujian antibakteria dan
antioksidan melalui teknik pembauran cakera dan radikal bebas DPPH telah
dijalankan ke atas ekstrak mentah dan minyak pati. Ekstrak mentah dan minyak pati
K. rotunda dari Malaysia dan Indonesia didapati tidak menunjukkan perencatan
aktiviti terhadap bakteria dan radikal bebas DPPH.
viii
TABLE OF CONTENTS
CHAPTER
TITLE
PAGE
DECLARATION OF THE STATUS OF THESIS
SUPERVISOR’S DECLARATION
CERTIFICATION OF EXAMINATION
TITLE PAGE
i
DECLARATION OF ORIGINALITY AND
ii
EXCLUSIVENESS
1
2
DEDICATION
iii
ACKNOWLEDGEMENTS
iv
PREFACE
v
ABSTRACT
vi
ABSTRAK
vii
TABLE OF CONTENTS
viii
LIST OF TABLES
xii
LIST OF SCHEMES
xiii
LIST OF FIGURES
xiv
LIST OF ABBREVIATIONS
xvi
LIST OF APPENDICES
xviii
INTRODUCTION
1.1
General Introduction
1
1.2
Objectives
2
1.3
Scope of Study
3
LITERATURE REVIEWS
2.1
The Zingiberaceae Family
4
ix
3
2.2
Botany and Distribution of Kaempferia
5
2.3
The Usages of Kaempferia Species
6
2.4
Phytochemicals of Kaempferia Species
7
2.4.1 Chalcones
7
2.4.2 Flavones
10
2.4.3 Flavanones
11
2.4.4 Pimarane Diterpenes
12
2.4.5 Cyclohexane Oxides
14
2.4.6 Cinnamates
16
2.5.7 Phenolics
17
2.4.8 Esters
18
2.4.9 Monoterpene
19
2.5
Bioactivity Studies on Kaempferia Species
19
2.6
Essential Oil Studies on Kaempferia Species
21
2.7
Biogenetic Pathway on Cyclohexane Oxides
22
CHEMICAL COMPOSITIONS OF KAEMPFERIA ROTUNDA OILS
3.1
The Essential Oils of Kaempferia rotunda
27
3.1.1 Rhizome Oil of Malaysian Kaempferia rotunda
27
3.1.2 Rhizome Oil of Indonesian Kaempferia rotunda
31
3.1.3 Comparison of the Compositions of Malaysian
33
and Indonesian Kaempferia rotunda
4
PHYTOCHEMICAL
AND
BIOACTIVITY
STUDIES
KAEMPFERIA ROTUNDA
4.1
Phytochemical Study of Malaysian Kaempferia rotunda
36
4.1.1 Crotepoxide (54)
37
4.1.2 2'-Hydroxy-4,4',6'-trimethoxychalcone (1)
38
4.1.3 4-Benzoyloxymethyl-3,8-dioxatricyclo-
39
[5.1.0.02,4]octane-5,6-diol 5-acetate (57)
4.1.4 1,6-Desoxypipoxide (69)
41
4.1.5 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)-
42
β-glucopyranoside (131)
OF
x
4.1.6 5-Hydroxy-7,4'-dimethoxyflavanone
51
(Naringenin 4',7-dimethyl ether) (133)
4.1.7 12-Acetoxy-8α,13-dihydroxylab-14-en-7-one
52
(Curcumrinol C) (134)
4.1.8 4-Benzoyloxymethyl-3-oxabicyclo[4.1.0]heptane- 56
1,5,6,7-tetrol (3-Debenzoylrotepoxide A)(132)
4.2
Phytochemical Studies of Indonesian Kaempferia rotunda 64
4.2.1 Crotepoxide (54)
65
4.2.2 Benzyl Benzoate (82)
65
4.2.3 trans-Docosyl ferulate (137)
66
4.2.4 3-Acetoxy-2-benzoyloxy-1-(benzoyloxymethyl)-
69
cyclohexa-4,6-diene (136)
4. 3
4. 2.5 6-Acetylzeylenol (68)
78
4. 2.6 Benzoic Acid (138)
80
The Distribution of Compounds in Malaysian and
81
Indonesian Kaempferia rotunda
4.4
5
Bioactivity Studies on Kaempferia rotunda
82
4.4.1 Antibacterial Activity
83
4.4.2 Antioxidant Activity
85
EXPERIMENTAL
5.1
General Experimental Procedures
87
5.2
Chemicals
88
5.3
Plant Materials
88
5.4
Essential Oil Extraction and Analysis
89
5.5
Extraction and Isolation of Malaysian Kaempferia
90
rotunda
5.5.1 Crotepoxide (54)
90
5.5.2 2'-Hydroxy-4,4',6'-trimethoxychalcone (1)
91
5.5.3 4-Benzoyloxymethyl-3,8-dioxatricyclo-
92
2,4
[5.1.0.0 ]octane-5,6-diol 5-acetate (57)
5.5.4 1,6-Desoxypipoxide (69)
92
5.5.5 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)-
93
β-glucopyranoside (131)
xi
5.6
5.5.6 Naringenin 4',7-dimethyl ether (133)
94
5.5.7 Curcumrinol C (134)
94
5.5.8 3-Debenzoylrotepoxide A (132)
95
Extraction and Isolation of Indonesian Kaempferia
96
rotunda
5.6.1 Crotepoxide (54)
96
5.6.2 Benzyl benzoate (82)
96
5.6.3 trans-Docosyl ferulate (137)
97
5.6.4 3-Acetoxy-2-benzoyloxy-1-(benzoyloxymethyl)-
97
cyclohexa-4,6-diene (136)
5.7
5.6.5 6-Acetylzeylenol (68)
98
5.6.6 Benzoic Acid (138)
98
Bioactivity Studies
99
5.7.1 Chemicals
99
5.7.2 Microorganisms
99
5.7.3 Antimicrobial Assay
99
5.7.3.1 Microorganisms and Culture Media
99
5.7.3.2 Disc Diffusion Method
100
5.7.4 Antioxidant Screening (Free Radical
101
Scavenging Activity (DPPH))
6
CONCLUSION AND RECOMMENDATION
103
REFERENCES
105
Appendices
117
xii
LIST OF TABLES
TABLE NO.
TITLE
PAGE
2.1
Biological Properties of Several Kaempferia Species
19
2.2
Biological Properties of Compounds Isolated from
20
Kaempferia Species
3.1
Constituents of Malaysian of Kaempferia rotunda Rhizome Oil
29
3.2
Constituents of Indonesian Kaempferia rotunda Rhizome Oil
32
3.3
Comparison of the Rhizomes Oils of Malaysian and Indonesian
33
Kaempferia rotunda
4.1
1
4.2
1
H, 13C NMR and COSY Data of Compound (69)
13
H and C NMR Data of Compound (131) and 2-(Benzoyl-
42
44
oxymethyl)phenyl (3,6-di-O-acetyl)-β-glucopyranoside (135)
4.3
1
H, 13C NMR, COSY and HMBC Data of Compound (134)
55
4.4
1
H, 13C NMR, COSY and HMBC Data of Compound (132)
64
4.5
1
H, 13C NMR, COSY and HMBC Data of Compound (137)
68
4.6
1
H, 13C NMR, COSY and HMBC Data of Compound (136)
70
4.7
1
4.8
Compounds Isolated from Malaysian and Indonesian
13
H, C NMR, COSY and HMBC Data of Compound (68)
79
82
Kaempferia rotunda
5.1
The Inhibition Zones of Tested Samples
101
5.2
Percentage Inhibitions of Tested Samples
102
xiii
LIST OF SCHEMES
SCHEME NO.
4.1
TITLE
The Elimination of Water Molecule from Compound (134) and
PAGE
53
the Formation of Acylium Ion
4.2
The Suggested Mass Fragmentation Pattern of Compound (137)
68
xiv
LIST OF FIGURES
FIGURES NO.
TITLE
PAGE
2.1
Biogenetic Pathway for the Formation of Crotepoxide (54)
23
2.2
Biogenetic Pathway for the Formation of Senepoxide (95) and
24
Pipoxide (96)
2.3
Biogenetic Pathway for the Formation of (-)-Zeylenol (75),
25
Senepoxide (95), Pipoxide (63), Seneol (106) and Zeylena (107)
2.4
Biogenetic Pathway for the Formation of (+)-Zeylenol (74) and
26
(-)-Zeylenol (75)
4.1
IR Spectrum of 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)-
45
β-glucopyranoside (131)
4.2
1
H NMR Spectrum of 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)- 46
β-glucopyranoside (131)
4.3
CIMS Spectrum of 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)-
4.4
13
47
β-glucopyranoside (131)
C NMR Spectrum of 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)- 48
β-glucopyranoside (131)
4.5
13
C NMR and DEPT Spectra of 2-(Benzoyloxymethyl)phenyl
49
(3-O-acetyl)-β-glucopyranoside (131)
4.6
HMBC Spectrum of 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)-
50
β-glucopyranoside (131)
4.7
IR Spectrum of 3-Debenzoylrotepoxide A (132)
57
4.8
1
58
4.9
1
4.10
13
C NMR Spectrum of 3-Debenzoylrotepoxide A (132)
60
4.11
13
C NMR and DEPT Spectra of 3-Debenzoylrotepoxide A (132)
61
H NMR Spectrum of 3-Debenzoylrotepoxide A (132)
1
H- H COSY Spectrum of 3-Debenzoylrotepoxide A (132)
59
xv
4.12
CIMS Spectrum of 3-Debenzoylrotepoxide A (132)
63
4.13
1
72
H NMR Spectrum of 3-Acetoxy-2-benzoyloxy-1-
(benzoyloxymethyl)cyclohexa-4,6-diene (136)
4.14
1
H-1H COSY Spectrum of 3-Acetoxy-2-benzoyloxy-1-
73
(benzoyloxymethyl)cyclohexa-4,6-diene (136)
4.15
IR Spectrum of 3-Acetoxy-2-benzoyloxy-1-
4.16
13
4.17
13
74
(benzoyloxymethyl)cyclohexa-4,6-diene (136)
C NMR Spectrum of 3-Acetoxy-2-benzoyloxy-1-
75
(benzoyloxymethyl)cyclohexa-4,6-diene (136)
C NMR and DEPT Spectra of 3-Acetoxy-2-benzoyloxy-1-
76
(benzoyloxymethyl)cyclohexa-4,6-diene (136)
4.18
EIMS Spectrum of 3-Acetoxy-2-benzoyloxy-1-
77
(benzoyloxymethyl)cyclohexa-4,6-diene (136)
4.19
Graft of Percentage Scavenging Capacity of DPPH by Vitamin C, 86
5-Deoxyquercetin, Crude Extracts and Essential Oils from
Kaempferia rotunda Measured by UV Spectrometric Assay
5.1
Kaempferia rotunda cultivated in Kempas, Johor
90
5.2
Kaempferia rotunda imported from Indonesia
90
5.3
The Arrangement of the Sample Discs and Control Discs in
101
Petri Dish
xvi
LIST OF ABBREVIATIONS
br
broad
CC
Column Chromatography
COSY
Correlation Spectroscopy
13
Carbon-13
C
CDCl3
Deuterated chloroform
CD3COCD3
Deuterated acetone
CHCl3
Chloroform
CIMS
Chemical Ionization Mass Spectrometry
DPPH
2,2-Diphenyl-1-picrylhydrazyl
d
doublet
dd
doublet of doublet
ddd
doublet of doublet of doublet
DCM
Dichloromethane
DEPT
Distortionless Enhancement by Polarization Transfer
D2 O
Deuterium oxide
EtOAc
Ethyl acetate
EIMS
Electron Impact Mass Spectrometry
Et2O
Diethyl ether
GC
Gas Chromatography
GC-MS
Gas Chromatography-Mass Spectrometry
1
Proton
H
HMBC
Heteronuclear Multiple Bond Correlation
HMQC
Heteronuclear Multiple Quantum Coherence
Hz
Hertz
IR
Infrared
IC50
Inhibition Concentration at 50%
J
coupling constant
xvii
KBr
Potassium bromide
KI
Kovats Index
lit.
Literature
LWT
Lebensm.-Wiss. u.-Technol / Food Science and Technology
MIC
Minimum Inhibition Concentration
MS
Mass Spectrometry
mM
millimolar
m/z
mass to charge ion
MeOH
Methanol
m.p.
melting point
MgSO4
Magnesium sulphate
MHz
Megahertz
m
multiplet
NMR
Nuclear Magnetic Resonance
nm
nanometer
NaOH
Sodium hydroxide
NaCl
Sodium chloride
Ph
Phenyl
PE
Petroleum ether
ppm
parts per million
q
quartet
Rf
retention factor
RP-VLC
Reversed Phase Vacuum Liquid Chromatography
SD
Standard Deviation
s
singlet
t
triplet
tr
trace
TLC
Thin Layer Chromatography
UV
Ultraviolet
VLC
Vacuum Liquid Chromatography
μM
micromolar
δ
chemical shift
c
concentration
xviii
LIST OF APPENDICES
APPENDIX
1
TITLE
GC Chromatogram of Malaysian Kaempheria rotunda
PAGE
117
(Rhizome) Oil
2
GC Chromatogram of Indonesian Kaempheria rotunda
118
(Rhizome) Oil
3
CIMS Spectrum of Crotepoxide (54)
119
4
1
120
5
1
6
HMBC Spectrum of Crotepoxide (54)
122
7
IR Spectrum of Crotepoxide (54)
123
8
13
C NMR Spectrum of Crotepoxide (54)
124
9
13
C NMR and DEPT Spectra of Crotepoxide (54)
125
10
HMQC Spectrum of Crotepoxide (54)
126
11
UV Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1)
127
12
IR Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1)
128
13
1
129
14
13
C NMR Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1) 130
15
13
C NMR and DEPT Spectra of 2'-Hydroxy-4,4',6'-
H NMR Spectrum of Crotepoxide (54)
1
H- H COSY Spectrum of Crotepoxide (54)
H NMR Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1)
121
131
trimethoxychalcone (1)
16
EIMS Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1)
132
17
HMBC Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1)
133
18
CIMS Spectrum of 4-Benzoyloxymethyl-3,8-dioxatricyclo-
134
2,4
[5.1.0.0 ]octane-5,6-diol 5-acetate (57)
19
IR Spectrum of 4-Benzoyloxymethyl-3,8-dioxatricyclo2,4
[5.1.0.0 ]octane-5,6-diol 5-acetate (57)
135
xix
20
1
H NMR Spectrum of 4-Benzoyloxymethyl-3,8-dioxatricyclo-
136
2,4
[5.1.0.0 ]octane-5,6-diol 5-acetate (57)
21
13
C NMR Spectrum of 4-Benzoyloxymethyl-3,8-dioxatricyclo-
137
[5.1.0.02,4]octane-5,6-diol 5-acetate (57)
22
HMBC Spectrum of Benzoyloxymethyl-3,8-dioxatricyclo-
138
[5.1.0.02,4]octane-5,6-diol 5-acetate (57)
23
HMQC Spectrum of Benzoyloxymethyl-3,8-dioxatricyclo-
139
2,4
[5.1.0.0 ]octane-5,6-diol 5-acetate (57)
24
EIMS Spectrum of 1,6-Desoxypipoxide (69)
140
25
13
C NMR Spectrum of 1,6-Desoxypipoxide (69)
141
26
13
C NMR and DEPT Spectra of 1,6-Desoxypipoxide (69)
142
27
IR Spectrum of 1,6-Desoxypipoxide (69)
143
28
1
H NMR Spectrum of 1,6-Desoxypipoxide (69)
144
29
1
H-1H COSY Spectrum of 1,6-Desoxypipoxide (69)
145
30
IR Spectrum of Naringenin 4',7-dimethyl ether (133)
146
31
1
147
32
1
33
13
C NMR Spectrum of Naringenin 4',7-dimethyl ether (133)
149
34
13
C NMR and DEPT Spectra of Naringenin 4',7-
150
H NMR Spectrum of Naringenin 4',7-dimethyl ether (133)
1
H- H COSY Spectrum of Naringenin 4',7-dimethyl ether (133)
148
dimethyl ether (133)
35
EIMS Spectrum of Naringenin 4',7-dimethyl ether (133)
151
36
IR Spectrum of Curcumrinol C (134)
152
37
13
C NMR Spectrum of Curcumrinol C (134)
153
38
13
C NMR and DEPT Spectra of Curcumrinol C (134)
154
39
EIMS Spectrum of Curcumrinol C (134)
40
1
H NMR Spectrum of Curcumrinol C (134)
156
41
1
H NMR Spectrum of Curcumrinol C (134) (Expansion)
157
42
1
H-1H COSY Spectrum of Curcumrinol C (134)
158
43
1
H-1H COSY Spectrum of Curcumrinol C (134) (Expansion)
159
44
HMBC Spectrum of Curcumrinol C (134)
160
45
1
161
46
IR Spectrum of Benzyl Benzoate (82)
162
47
13
C NMR Spectrum of Benzyl Benzoate (82)
163
48
13
C NMR and DEPT Spectra of Benzyl Benzoate (82)
164
H NMR Spectrum of Benzyl Benzoate (82)
155
xx
49
EIMS Spectrum of Benzyl Benzoate (82)
165
50
13
C NMR Spectrum of trans-Docosyl ferulate (137)
166
51
13
C NMR and DEPT Spectra of trans-Docosyl ferulate (137)
167
52
IR Spectrum of trans-Docosyl ferulate (137)
168
53
1
H NMR Spectrum of trans-Docosyl ferulate (137)
169
54
1
H-1H COSY Spectrum of trans-Docosyl ferulate (137)
170
55
HMBC Spectrum of trans-Docosyl ferulate (137)
171
56
EIMS Spectrum of trans-Docosyl ferulate (137)
172
57
IR Spectrum of 6-Acetylzeylenol (68)
173
58
1
174
59
CIMS Spectrum of 6-Acetylzeylenol (68)
175
60
13
C NMR Spectrum of 6-Acetylzeylenol (68)
176
61
13
C NMR and DEPT Spectra of 6-Acetylzeylenol (68)
177
62
1
63
HMBC Spectrum of 6-Acetylzeylenol (68)
179
64
IR Spectrum of Benzoic Acid (138)
180
65
1
181
66
13
H NMR Spectrum of 6-Acetylzeylenol (68)
H-1H COSY Spectrum of 6-Acetylzeylenol (68)
H NMR Spectrum of Benzoic Acid (138)
C NMR Spectrum of Benzoic Acid (138)
178
182
CHAPTER 1
INTRODUCTION
1.1
General Introduction
Malaysia with its tropical forest is blessed with high biological diversity,
which enclosed over 10% of the world’s total number of species, with some of them
are unique only to Malaysia. Among more than 7,000 species of angiosperms and
600 species of ferns in Malaysia, about 12 to 18% of trees, shrubs and herbs are
reported to have medicinal properties [1]. The usage of medicinal plant products has
attracted interest since the past decade. The beneficial medicinal effects of plant
materials typically result from the secondary products in the plant [2].
Many
biologically active plant-derived compounds were discovered as a result of chemical
studies through isolation of active compounds from traditional medicine [3]. Our
Malaysian flora represents a huge, barely untapped reserve of natural resources
which is believed to contain substances with therapeutic potentials that yet to be
explored.
Research projects related to the natural products carried out in Malaysia
included: the chemistry and technology of palm oil, natural pesticides, natural
flavours and pharmalogical testing of medicinal plants [4]. Development of organic
chemistry has been closely associated with the chemistry of natural products. Many
techniques of extraction, separation, structure determination and synthesis have been
developed to understand the structural variation among the natural products.
Research in this area has certainly led to better understanding of the structural
2
requirements for a variety of physiological activities, leading to the synthesis and
modification of several lead compounds and analogues.
The study of bioactive natural products constituents is the first step in drug
discovery programs, while the eventual outcomes of blockbuster drugs may not be
that easily realised in view to the high cost and research effort [5]. Despite all these,
natural products drug discovery programs are developed all over the world, mainly
because of the high chemical diversity from natural products as compared to
synthetics.
The potential of these natural products is largely unknown and
endangered plants have added the urgency for more vigorous screening programs.
With the currently available tools of extraction, chromatographic separation and
structure identification, a fresh look at the well-studied plants may also be rewarding.
In Malaysia, local universities and research institutes are embarking on their own
programs which involve a concerted multidisciplinary approach in the discovery of
bioactive agents from plant-derived natural products. Certain novel strategies have
been carried out based on the expertise and funds available. The screening programs
implemented and administered are aimed to discover new compounds from the
Malaysian flora for use in the pharmaceutical and related industries. This in turn,
will help in the development and transfer of technology, which can be achieved
through collaborative programs.
1.2
Objectives
Phytochemical studies reported in the literature review are mostly carried out
on the Kaempferia species of Thailand. Literature search revealed only a few reports
on the studies of K. rotunda compared to other species. Therefore, this research will
focus on the chemical compositions of essential oil and chemical constituents the
rhizomes of K. rotunda cultivated in Kempas, Johor and Indonesia. In addition,
antioxidant and antimicrobe activities of the rhizomes of K. rotunda will also be
investigated.
3
The first objective is to extract the essential oils from the fresh rhizomes of
Kaempferia rotunda and to analyze the chemical compositions of the essential oils.
The second objective is to extract phytochemicals from the dried rhizomes, to purify
and identify their structures. The third goal is to evaluate the antioxidant and
antimicrobe activities of the essential oils, crude extracts and the pure isolated
compounds.
1.3
Scope of Study
This research focuses on the study of the essential oils compositions of the
fresh rhizomes and phytochemicals from the dried rhizomes of Kaempferia rotunda
from Malaysia and Indonesia.
The fresh rhizomes will be extracted by
hydrodistillation technique to obtain the essential oil. The dried rhizomes will be
extracted using soxhlet with different polarity of solvents. The crude extracts will be
fractionated by using vacuum liquid chromatography (VLC), followed by
purification of the fractions using gravity column chromatography (CC) or
chromatotron or versa-flash column chromatography and or recystallization to obtain
the pure compounds. The compositions of the essential oils will be analyzed using
GC and GC-MS, while the chemical constituents from dried samples will be
characterized using spectroscopic methods including MS, IR, 1H NMR,
2D NMR (COSY, HMQC, HMBC) and UV.
13
C NMR,
The biological activities such as
antioxidant and antibacterial will be carried out on the crude extracts, essential oils
and the pure isolated compounds.
CHAPTER 2
LITERATURE REVIEWS
2.1
The Zingiberaceae Family
Zingiberaceae is a family of ginger, comprises about 1200 species of which
about 1000 occur in tropical Asia.
The richest area is the Malesian region, a
floristically distinct region that includes Malaysia, Indonesia, Brunei, Singapore,
Philippines and Papua New Guinea, with 24 genera and about 600 species. The large
areas such as Sumatra and Borneo are still very insufficiently known and many more
new species are expected to be found in the years to come [6].
The Zingiberaceae family is divided into two subfamilies, which are
Zingiberoideae and Costoideae.
Zingiberoideae is further subdivided into three
tribes (Globeae, Hedychieae and Alpiniae), while Costoideae consists of one tribe
(Costeae) which is made up of only one genus (Costus). There is only one genus
(Globba) in the tribe of Globbeae. The tribe Hedychieae consists of eight genera
(Zingiber, Curcuma, Hedychium, Comptandra, Scaphochlamys, Boesenbergia,
Kaempferia and Haniffia), while the tribe Alpinieae consists of thirteen genera of
which the common ones are Alpinia, Phaeomeria, Achasma, Amomum and Elettaria
[7]. The highest diversity recorded in Peninsular Malaysia was Alpinieae (9 genera,
84 species) followed by Hedychineae (7 genera, 52 species). Among the genera
documented in Malaysia, only the genus Haniffia is endemic to Peninsular Malaysia
[6].
5
2.2
Botany and Distribution of Kaempferia
Kaempferia is one of the genus of the Zingiberaceae family. The name
Kaempferia commemorates Engelbert Kaempfer, a German physician and botanist in
the 17th century. Kaempferia is small herbs with short rhizomes and tuberous roots.
In the indigenous species, the flowers arise in the midst of a few leaves, while in the
introduced species, K. rotunda, the in florescence is totally enclosed in the leaf
sheaths. The genus is easily recognized as the flowers appear to consist of four lobes,
surrounded by three thin, narrow corolla lobes. In each flower, two staminodes and a
twice-as-broad labellum divided almost to the base catch the eye [6]. Kaempferia
can adapt to life in more open places and in seasonal climates, thus extended greatly
the possible range of its distribution, in Africa and Asia. There is little doubt that the
headquarters of Kaempferia is in Asia, and probably in Burma [8].
There are only four species (Kaempferia rotunda Linn., K. galanga Linn., K.
pulchra Ridl. and K. elegans Wall.) found in Malaya (now is Peninsular Malaysia
and Singapore). The genus dates from Linnaeus, included K. rotunda Linn. and K.
galanga Linn., in the first edition of his ‘Species Platarum’. Only K. rotunda and K.
galanga are widely cultivated in Malaysia and South-East Asia, and known as
‘chěkur’ (K. galanga) and ‘kecur’ (K. rotunda) [8].
Kaempferia rotunda Linn. is widely cultivated in South-East Asia.
Its
country of origin is not certainly known (possibly Indo-China). Valeton, a botanist
reported that is apparently wild in East Jawa, but he considers that it may have
escaped from cultivation. In Peninsular Malaysia, it is perhaps common in the north,
but can only keep alive in cultivation in the South [8].
Kaempferia galanga Linn. a perennial herb, is said to be native in India. It is
widely cultivated throughout South-East Asia. The Malay name ‘chěkur’ is a well
known plant. It seems to be a common village plant in all parts of Malaysia [8].
6
Kaempferia pulchra Ridl. is found on limestone in Langkawi and Southern
Thailand. It is cultivated in the Waterfall Gardens, Penang, where it maintains itself
in sandy ground in shade-rockeries, but does not flourish so well nor flower so
regularly or freely in Singapore. The flowers appear with the new leaves after rains
begin, and are produced throughout the rainy season. This species is very attractive,
floriferous under suitable conditions, and well worth cultivating for ornamental
purpose [8].
Kaempferia elegans Wall. is very closely resemble to K. pulchra. It has
larger plain green leaves on longer stalks, peduncle of inflorescences much longer
than leaf-sheath and a short broad anther-crest. It is distributing at Tenasserim
(Myanmar) and also found in Langkawi and Kedah [8].
2.3
The Usages of Kaempferia Species
Kaempferia is a popular ornamental that reputed for its beneficial medicinal
effects. It has been used for the treatment of allergy and gastrointestinal disorders,
aphrodisiac, hypertension, rheumatism and asthma [9]. For example, the rhizomes of
K. galanga have been used in a decoction or powder for indigestion, cold, pectoral
and abdominal pains, headache and toothache. Its alcoholic maceration has also
been applied as liniment for rheumatism [10]. In China, K. galanga (locally known
as ‘shan-lai’) is well known for its usage in the food spice and medicinal industry. It
is traditionally use for treating symptoms from hypertension, pectoral and abdominal
pains, headache, toothache, rheumatism, dyspepsia, coughs and inflammatory tumour.
It also has a long history of fragrance use to help restlessness, stress, anxiety and
depression. In Japan, it has been used as one of the main ingredients in a scent bag
which is recognized as improving sleep or minimizing stressful situations [11].
The rhizomes of Kaempferia rotunda have been used for flavouring agent and
medicine for stomachache, fever and for elephant medicine [12]. Among local
people in the northeast of Thailand, the rhizomes of K. parviflora have been known
7
as health-promoting herbs, and also frequently used for the treatment of colic
disorder, peptic and duodenal ulcers [13], digestive disorders and gastric ulcer [14].
Kaempferia pandurata Roxb. now known as Boesenbergia rotunda, also
known as ‘temu kunci’ in Indonesia or ‘krachai’ in Thailand, has been traditionally
used in South-East Asia as a food ingredient and a folk medicine for the treatment of
dental caries, colic disorder, fungal infection, dry cough, rheumatism and muscular
pains [15].
2.4
Phytochemicals of Kaempferia Species
Chemical investigations on Kaempferia have yielded an assortment of
flavonoids of chalcones, flavones and flavanones, pimarane diterpenes, cyclohexane
oxides, cinnamates, phenolics, esters and monoterpene.
2.4.1
Chalcones
Nineteen chalcones were isolated from the Kaempferia species including
simple chalcones (1-3), prenylated chalcones (4-6), cyclohexenyl chalcones (7-15)
and prenylated cyclohexenyl chalcones (16-19).
2'-Hydroxy-4,4',6'-trimethoxychalcone (1) was isolated from the rhizomes of
Kaempferia angustifolia and K. rotunda [16-17]. Another two simple chalcones
were isolated from the rhizomes of K. pandurata and identified as 2',6'-dihydroxy-4'methoxychalcone (2) and cardamonin (3) [18].
8
(1)
(2) R1= OH,
R2= OCH3
(3) R1= OCH3, R2= OH
The rhizomes of Kaempferia pandurata of Thailand contained two prenylated
chalcones
identified
as
(±)-(E)-1-[7'-hydroxy-5'-methoxy-2'-methyl-2'-(4''-
methylpent-3''-enyl)-2'H-l-benzopyran-8'-yl]-3-phenylprop-2-en-l-one
or
boesenbergin A (4), (±)-(E)-l-[5'-hydroxy-7'-methoxy-2'-methyl-2'-(4''-methylpent3''-enyl)-2'H-l-benzo-pyran-6'-yl]-3-phenylprop-2-en-l-one or boesenbergin B (5)
[18-19]. Study on the rhizomes of Boesenbergia pandurata (syn. K. pandurata) of
Myanmar resulted in the isolation of a new prenylated chalcone, named 2',4'dihydroxy-3'-geranyl-6'-methoxychalcone (6) [20].
H3CO
O
OH O
(4)
(5)
(6)
Cyclohexenyl chalcone was conceivably derived from a Diels-Alder reaction
of the related chalcone with an isoprenoid unit. The rhizomes of Boesenbergia
pandurata (syn. Kaempferia pandurata) of Myanmar also contained cyclohexenyl
9
chalcones identified as panduratin C (7), (1'R,2'S,6'R)-2-hydroxyisopanduratin A (8),
isopanduratin A1 (9), nicolaioidesin B (10), 6-methoxypanduratin A (11), and
isopanduratin A2 (12) [20]. Three more cyclohexenyl chalcones isolated from K.
pandurata were characterized spectroscopically as (1'RS,2'SR,6'RS)-(2,6-dihydroxy4-methoxy-phenyl) [3'-methyl-2'-(3''-methylbut-2''-enyl)-6'-phenylcyclohex-3'-enyl]
methanone or panduratin A (13), 4-hydroxypanduratin A (14) and isopanduratin A
(15) [19, 21-22]. Study on the rhizomes of K. pandurata was successfully isolated
prenylated cyclohexenyl chalcones, namely panduratins D-G (16-19) [21].
OH
CH3
O
HO
OH
O
(7)
(8) R1= OH,
R2= OH
(9) R1= OCH3, R2= OH
(10) R1= OH,
R2
R1
(11) R1= OH,
R
R3
OH
OH O
O
R2= OCH3, R3= OCH3
(12) R1= OCH3, R2= OH,
R2= OCH3
R3= OH
(13) R= OCH3
(14) R= OH
10
O
H3CO
OH
O
(15)
(16)
(17)
(18)
(19)
2.4.2
Flavones
All investigations on the rhizomes of Kaempferia parvifolia possessed
flavones. From the chemotaxanomy point of view, it is assumed that flavones from
Kaempferia are the characteristic components of this rhizome.
11
Investigation on the rhizomes of Kaempferia parvifolia has led to reports of
O-methylated flavones. These compounds were elucidated spectroscopically and
identified as 5-hydroxy-3,7-dimethoxyflavone (20), 5-hydroxy-7-methoxyflavone
(21), 5-hydroxy-3,7,4'-trimethoxyflavone (22), 5-hydroxy-7,4'-dimethoxyflavone
(23), 5-hydroxy-3,7,3',4'-tetramethoxyflavone (24), 3,5,7-trimethoxyflavone (25),
3,5,7,4'-tetramethoxyflavone
tetramethoxyflavone
(28),
(26),
5,7,4'-trimethoxy-flavone
5,7-dimethoxyflavone
(29)
(27),
and
5,7,3',4'3,5,7,3',4'-
pentamethoxyflavone (30) [23-26].
R3
H3CO
O
R2
R1
H3C
O
O
(20) R1= OCH3, R2= H,
R3 = H
(25) R1= OCH3, R2= H,
R3= H
(21) R1= H,
R3= H
(26) R1= OCH3, R2= H,
R3= OCH3
(22) R1= OCH3, R2= H,
R3= OCH3
(27) R1= H,
R2= H,
R3= OCH3
(23) R1= H,
R3= OCH3
(28) R1= H,
R2= OCH3, R3= OCH3
(24) R1= OCH3, R2= OCH3, R3= OCH3
(29) R1= H,
R2= H,
R2= H,
R2= H,
R3= H
(30) R1= OCH3, R2= OCH3, R3= OCH3
2.4.3
Flavanones
Besides chalcones and flavones, there are few reports on flavanones from
Kaempferia species. Compounds of pinostrobin (31) and pinochembrin (32) were
reported from the rhizomes of Boesenbergia pandurata (syn. K. pandurata) that
collected from Thailand [18]. A new flavanone, named (2R)-8-geranylpinostrobin
(34) together with known (-)-7,4'-dihydroxy-5-methoxyflavanone (33), (2S)-6geranylpinostrobin (35), (-)-6-geranylpinocembrin (36) and (2S)-7,8-dihydro-5hydroxy-2-methyl-2-(4''-methyl-3''-pentenyl)-8-phenyl-2H,6H-benzo-[1,2-b:5,4b']dipyran-6-one (37) were isolated from the rhizomes of B. pandurata (syn. K.
pandurata) of Myanmar [20].
12
OH
HO
O
H3C
(31) R= OCH3
O
O
(33)
(32) R= OH
O
H3CO
OH O
(34)
(35) R= OCH3
(36) R= OH
(37)
2.4.4
Pimarane Diterpenes
Pimarane diterpenes were present in Kaempferia marginata, K. pulchra and
K. sp. (locally name as chung-ngang in Thailand). The rhizomes of K. sp. (chungngang) consist of six new pimarane diterpenes. The compounds were assigned as
sandaracopimaradien-9α-ol-1-one (38), sandaracopimaradien-1α,9α-diol (39), 6βacetoxy-sandaracopimaradien-9α-ol-1-one (40), 6β-acetoxy-sandaracopimaradien-
13
6β,9α-diol-1-one
(41),
6β-acetoxy-sandaraco-pimaradien-1α,9α-diol
(42)
and
sandaracopimaradien-1α,6β,9α-triol (43) [27].
OH
O
OH
OH
R
R
(38) R= H
(39) R= H
(40) R= OAc
(42) R= OAc
(41) R= OH
(43) R= OH
Study on the Kaempferia marginata (locally name as tup mup) yielded six
new and four known sandarapimarane diterpenes.
They were identified as
(1R,2S,5S,7S,9R,10S,13R)-1,2,7-trihydroxypimara-8(14),15-diene (44), (1R,2S,5S,9S,
10S,11R,13R)-1,2,11-trihydroxypimara-8(14),15-diene (45), (1S,5S,7R,9R,10S,11R,
13R)-1,7,11-trihydroxypimara-8(14),15-diene (46), (1S,5S,9S,10S,11R,13R)-1,11dihydroxypimara-8(14),15-diene
(47),
(1R,2S,5S,7S,9R,10S,13R)-1,2-dihydroxy-
pimara-8(14),15-diene-7-one (48), (5S,6R,9S,10S,13R)-6-hydroxypimara-8(14),15di-ene-1-one (49), sandaracopimaradiene (50) and sandaracopimaradien-1α-ol (51)
[28].
The rhizomes of K. pulchra yielded pimaranes of 2α-acetoxy-sandaraco-
pimaradien-1α-ol (52) and sandaracopimaradien-1α,2α-diol (53) [29].
R3
OH
R1
H
H
R2
(44) R1= OH, R2= β-OH, R3= H
(45) R1= OH, R2= H,
R3= OH
(46) R1= H, R2= α-OH, R3= OH
(47) R1= H, R2= H,
R3= OH
(48) R1= OH, R2= O,
R3= H
14
O
H
H
OH
(49)
(50)
(51) R= H
(52) R= OAc
(53) R= OH
2.4.5
Cyclohexane Oxides
Kaempferia rotunda, K. angustifolia and K. sp. (locally name as krachaikao)
are three species that were found to contain substantial amount of cyclohexane
oxides, including cyclohexane bisepoxides (54-58), cyclohexane monoepoxides (59-
62), cyclohexene monoepoxide (63), cyclohexene oxides (64-68) and cyohexene
dienes (69-70).
Crotepoxide (54) was present in many Kaempferia species such as in the
rhizomes of K. sp. (krachaikao), K. angustifolia and K. rotunda [16-17, 30-32], while
bosenboxide (55) was isolated from the rhizomes of K. sp. (krachaikao) and K.
angustifolia [16, 30].
Three other cyclohexane bisepoxides assigned as (-)-
(1R,2R,4R,5S,6R,7R)-4-benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6diol 6-acetate (56), (+)-(1R,2R,4R,5S,6R,7R)-4-benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diol 5-acetate (57) and (-)-(1R,2R,4R,5S,6R,7R)-4-benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diol 6-benzoate (58) were isolated
from the rhizomes of K. rotunda [17, 31].
15
(54) R1 = OAc, R2 = OAc
(55) R1 = OAc, R2 = OCOPh
(56) R1 = OH, R2 = OAc
(57) R1 = OAc, R2 = OH
(58) R1 = OH, R2 = OCOPh
Reversed-phase HPLC of the MeOH extract of rhizomes of Kaempferia
rotunda yielded four new monoepoxides. They were identified as (-)-rotepoxide A
(59), (-)-rotepoxide B (60), 2-acetylrotepoxide A (61) and 2-acetylrotepoxide B (62)
[32].
(59) R1 = OH, R2 = OCOPh, R3 = OH
(60) R1 = OH, R2 = OH,
R3 = OCOPh
(61) R1 = OAc, R2 = OCOPh, R3 = OH
(62) R1 = OAc, R2 = OH,
R3 = OCOPh
A cyclohexene monoepoxide, (-)-pipoxide (63) was found in the rhizomes of
Kaempferia
sp.
(krachaikao)
[16].
hydroxymethylcyclohex-5-ene-1,2,3,4-tetrol
(+)-Zeylenol
(64),
1,4-dibenzote
(1R,2S,3R,4S)-2(66)
and
(-)-
(1R,2S,3R,4S)-2-benzoloxymethylcyclohex-5-ene-1,2,3,4-tetrol 1,4-dibenzote (67)
were isolated from the rhizomes of K. sp. (krachaikao) and K. angustifolia [16]. (-)Zeylenol (65) and (-)-6-acetylzeylenol (68) were obtained from the rhizomes of K.
rotunda [31-32].
16
HO
HO
OCOPh
OH
OCOPh
(63)
(64)
(66) R= OH
(65)
(68)
(67) R= OCOPh
Two cyclohexene dienes, (-)-1,6-desoxypipoxide (69) and rotundol (70) were
present in the rhizomes of Kaempferia rotunda [17, 32]. Rotundol (70) was deduced
from the Diels-Alder adduct by endo-addition of (-)-1,6-desoxypipoxide (69) [32].
(69)
2.4.6
(70)
Cinnamates
Cinnamate, a phenylpropanoid ester was another class of compounds
obtained especially from Kaempferia galanga. These include ethyl trans-cinnamate
(71), ethyl p-methoxy-trans-cinnamate (72), methyl p-methoxy-trans-cinammate (73)
and p-methoxy-trans-cinnamic acid (74) [33-35]. Three other cinnamates were only
isolated from the rhizomes of K. galanga cultivated in China. These compounds
were assigned as cinnamate aldehyde (75), ethyl p-methoxy-cis-cinnamate (76) and
ethyl cis-cinnamate (77) [34].
17
O
O
R
(71) R= H
(73) R= OCH3
(72) R= OCH3
(74) R= OH
O
H
(75)
(76) R= H
(77) R= OCH3
2.4.7
Phenolics
The rhizomes of Bosenbergia pandurata (syn. Kaempferia pandurata) gave a
phenolic compound, geranyl-2,4-dihydroxy-6-phenethylbenzoate (78) [20].
The
black rhizomes of K. parviflora yielded three new phenolic glycosides which
consisted of rare skeleton of 6H-benz[b]indeno[1,2-d]furan-6-one.
These
compounds were elucidated spectroscopically as rel-(5aS,10bS)-5a,10b-dihydro1,3,5a,9-tetrahydroxy-8-methoxy-6H-benz[b]indeno[1,2-d]furan-6-one
5a-O-[α-L-
rhamnopyranosyl-(1→6)-β-D-glucopyranoside] (79), rel-(5aS,10bR)-5a,10b-dihydro1,3,5a,9-tetra-hydroxy-8-methoxy-6H-benz[b]indeno[1,2-d]furan-6-one
rhamnopyranosyl-(1→6)-β-D-glucopyranoside]
(80)
and
5a-O-[α-L-
(2R,3S,4S)-3-O-[α-L-
rhamnopyranosyl-(1→6)-β-D-gluco-pyranosyl]-3'-O-methyl-ent-epicatechin(2α→O→3,4α→4)-(5aS,10bS)-5a,10b-dihydro-1,3,5a,9-tetra-hydroxy-8-methoxy6H-benz[b]indeno[1,2-d]furan-6-one 5a-O-[α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside] (81) [36].
18
HO
O OR
O
OH H
OCH3
HO
(78)
(79)
OH
HO
O
O
OR
OH
HO
O OR
O
O
RO
OH H
OCH3
H
OH
O
HO
OH
OCH3
OCH3
(80)
(81)
R= α-L-Rha-(1→6)-β-D-Glc—
2.4.8
Esters
Benzyl benzoate (82) and 2-(benzoyloxymethyl)phenyl (3,6-di-O-acetyl)-β-
glucopyranoside (83) were isolated from the rhizomes of Kaempferia rotunda [32,
37].
O
O
(82)
(83)
19
2.4.9
Monoterpene
3-Caren-5-one (84) was isolated from the rhizomes of Kaempferia galanga.
It was the only isolated monoterpene from Kaempferia species [38].
(84)
2.5
Bioactivity Studies on Kaempferia Species
Several biological and pharmacological activity studies on Kaempferia
species have been reported. The biological properties of several Kaempferia species
are listed in Table 2.1.
Table 2.1: Biological Properties of Several Kaempferia Species
Kaempferia Species
Biological Properties
K. galanga (Rhizomes)
Amebicidal activity [39]
K. galanga (Essential oil)
Antigastric cancer [40] and larvicidal activity [41]
K. pandurata (Rhizomes)
Antibacterial activity [42] and periodontal
inflammation [43]
K. parviflora (Rhizomes)
Antigastric ulcer [44], endothelial function [14],
vasorelaxation and antispasmodic effects [45]
Besides, several isolated compounds from Kaempferia species have been
characterized and evaluated for their bioactivities such as anticancer, antiinflammatory and antimicrobial activities as shown in Table 2.2.
20
Table 2.2: Biological Properties of Compounds Isolated from Kaempferia Species
Biological Properties
Active compounds
Antipancreatic cancer
Panduratin A (13) [20], and
Panduratins D-G (16-19) [20-21]
Antibreast cancer
Panduratin A (13) [46]
Anticolon cancer
Panduratin A (13) [47]
Cytotoxicity
Panduratin A (13) [48]
Anti-inflammatory
Panduratin A (13) [49],
5-Hydroxy-3,7,4'-trimethoxyflavone (22),
5-Hydroxy-7,4'-dimethoxyflavone (23),
5-Hydroxy-3,7,3',4'-tetramethoxyflavone (24) [24-25],
2α-Acetoxy-sandaracopimaradien-1α-ol (52), and
Sandaracopimaradien-1α,2α-diol (53) [50]
Antibiofilm
Panduratin A (13) [51]
Antiaging
4-Hydroxypanduratin A (14) [52]
Antibacterial
(Streptococcus mutans)
Isopanduratin A (15) [22]
Antibacterial
5,7,4'-Trimethoxyflavone (27), and
3,5,7,4'-Tetramethoxyflavone (28) [13]
Antimicrobial αglucosidase
Ethyl p-methoxy-trans-cinnamate (72) and
p-Methoxy-trans-cinnamic acid (74) [35]
Antiallergic
5-Hydroxy-7-methoxyflavone (21),
5-Hydroxy-3,7,4'-trimethoxyflavone (22) and
5-Hydroxy-7,4'-dimethoxyflavone (23) [26]
Antifungal
(Candida albicans)
5,7,4'-Trimethoxyflavone (27), and
3,5,7,4'-Tetramethoxyflavone (28) [13]
Multidrug resistance
5,7-Dimethoxyflavone (29) and
3,5,7,3',4'-Pentamethoxyflavone (30) [53-54]
Antimalarial
(Plasmodium
falciparum)
5,7,4'-Trimethoxyflavone (27) [13],
Trihydroxypimara-8(14),15-diene (46) and
1,11-Dihydroxypimara-8(14),15-diene (47) [28]
Antimalarial
(Candida albicans)
1,7,11-Trihydroxypimara-8(14),15-diene (46) and
Sandaracopimaradien-1α, 2α-diol (53) [28]
21
Biological Properties
Active compounds
Antituberculous
(Mycobacterium
tuberculosis)
Sandaracopimaradien-1α-ol (51) and
2α-Acetoxy-sandaracopimaradien-1α-ol [28]
Insecticidal activity
Crotepoxide (54) and Benzyl benzoate (82) [37]
Antiplatelet
aggregation
4-Benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-di-ol 6-acetate (56) [55]
Antifeedant activity
(-)-Zeylenol (65) [32]
Antinociceptive action
Ethyl trans-cinnamate (71) [56]
Vasorelaxant effect
Ethyl trans-cinnamate (71) [57]
Sedative activity
Ethyl trans-cinnamate (71) and
Ethyl p-methoxy-trans-cinnamate (72) [33]
Larvicidal activity
(Anisakis simple)
Ethyl trans-cinnamate (71),
Ethyl p-methoxy-cis-cinnamate (77) and
Ethyl cis- cinnamate (76) [34]
2.6
Essential Oil Studies on Kaempferia Species
Analysis of the essential oil in the fresh rhizomes of Kaempferia galanga has
led to the identification of ethyl p-methoxycinnamate (72) (31.8%), methyl
cinnamate (85) (23.2%), carvone (86) (11.1%), 1,8-cineol (87) (9.6%) and
pentadecane (88) (6.4%) as the major constituents [58].
O
OCH3
(85)
(86)
(88)
(87)
22
Investigation of the rhizome oil and the lateral part oil of Kaempferia rotunda
from Indonesia showed the presence of benzyl benzoate (82) (69.7%), and npentadecane (88) (22.9%) as the major constituents in the rhizome oil, while npentadecane (88) (53.8%), benzyl benzoate (82) (20.1%), and camphene (89) (6.2%)
as the main constituents in the lateral parts oil [59]. The rhizome oil of Malaysian K.
rotunda consisted of mainly n-pentadecane (88) (25.4 %), bornyl acetate (90)
(24.9%), benzyl benzoate (82) (15.3%) and camphor (91) (12.1%) [60].
(89)
(90)
(91)
n-Pentadecane (88) (17.8%), benzaldehyde (92) (9.1%), camphor (91) (6.2%)
and α-pinene (93) (4.3%) were identified from the rhizome oil of Kaempferia
angustifolia of Indonesia, while bornyl formate (94) (16.3%), camphene (89)
(12.4%), 1,8-cineole (87) (4.8%) and n-pentadecane (88) (5.0%) were found to be the
main constituents in the lateral part oil [59].
(92)
2.7
(93)
(94)
Biogenetic Pathway on Cyclohexane Oxides
Cyclohexane oxides are a small group of naturally occurring compounds.
The interest on the biogenesis of these compounds has recently increased.
Crotepoxide (54), senepoxide (95) and pipoxide (96) were the only representatives of
the cyclohexane oxides in 1970s.
A biogenetic pathway of crotepoxide (54),
senepoxide (95) and pipoxide (96) was proposed, as shown in Figure 2.1 and 2.2
[61].
23
(54)
(95)
(96)
The precursor of the cyclohexane oxides is believed to be (-)-(2S,3S)isochorismic acid (97). Intramolecular SN2 displacement of the enol pyruvate by the
adjacent hydroxy group of isochorismic acid (97) formed arene oxide (98a).
Enzymatic reduction to an alcohol followed by acylation to produce (99a).
Subsequent photooxygenation of diene (99a) to the endoperoxide (100), followed by
epoxide ring opening with anchimeric assistance from the neighbouring benzoate
carbonyl yielded (102) through the hemiorthoester (101).
Acetylation and
rearrangement of the endoperoxide function in (101) finally afforded crotepoxide
(54), as summarized in the Figure 2.1 [61].
OCOPh
COOH
COOH
OH
O
O
O
COOH
(97)
(99a)
(98a)
O
O
(54)
O
O
(102)
OH
OH
Ph
O
O
O
O
(100)
OCOPh
OCOPh
OAc
OAc
O
O
O
O
OH
O
Ph
OH
(101)
Figure 2.1. Biogenetic Pathway for the Formation of Crotepoxide (54)
As summarized at Figure 2.2, senepoxide (95) and pipoxide (96) which have
different absolute configuration from crotepoxide (54) would result from the
enantiomeric arene oxide (98b).
Arene oxide isomerization (98a↔98b), a
conversion that can be envisaged through the oxepin intermediate (103).
The
epoxidation at the reactive 1,6-double bond of the arene oxide (98b) afforded (104),
followed by selective epoxide ring opening and acylation of the resulting alcohol
finally led to senepoxide (95) and pipoxide (96) [61]. For this biosynthetic study, it
24
was found that the reported structure of pipoxide was incorrect. Consequently, the
structure was revised from (96) to (63) [62].
Figure 2.2. Biogenetic Pathway for the Formation of Senepoxide (95) and Pipoxide
(96)
OCOPh
OH
O
OCOPh
(96)
(63)
Another alternative biogenetic pathway for the formation of pipoxide (63),
senepoxide (95) and new cyclohaxene oxides of (-)-zeylenol (75), seneol (106) and
zeylena (107) was proposed, as summarized in the Figure 2.3 [63].
HO
H3CO
OCOPh
OAc
OAc
(75)
(106)
(107)
Benzyl benzoate (82) is epoxidized to the arene oxide intermediate (99b)
which then adds to cinnamic acid, acetic acid, or benzoic acid, to give the dienes
25
(108a, 108b, or 108c) respectively. Acetylation and epoxidation of (108b) yield
senepoxide (95). The formation of pipoxide (63), (-)-zeylenol (75) and seneol (106)
can be similarly explained by the same way. On the other hand, (108a) can undergo
an intramolecular cycloaddition reaction to give zeylena (107) [63].
OCOPh
OCOPh
(82)
O
(108)
O
(108a) R= COCH=CHPh
(108b) R= COMe
(108c) R= COPh
O
OCOPh
OH
OH
OR
(109)
(99b)
Ph
OH
OH
O
OCOPh
OCOPh
OCOPh
(107)
OCOPh
HO
HO
OCOPh
(63)
OH
OCOPh
OAc
O
OCOPh
(75)
HO
H3CO
OAc
(95)
OCOPh
OAc
OAc
(106)
Figure 2.3. Biogenetic Pathway for the Formation of Pipoxide (63), (-)-Zeylenol
(75), Senepoxide (95), Seneol (106) and Zeylena (107)
The arene oxide (99b) is perceived to easily rearrange to O-hydroxybenzyl
benzoate (109), thus providing the plants with a pool of O-hydroxybenzyl groups.
This biogenetic pathway also explained the origin of compounds that contained Ohydroxybenzyl group and one cinnamic acid group, isolated from Uvaria genus [63].
Although the arene oxide (99) was postulated as the key metabolic
intermediate in both the biogenetic pathways, the former pathway speculated that the
arene oxide (99) resulted from isochorismic acid (97). The latter postulated a direct
epoxidation of benzyl benzoate (82) to give (99b), and a direct epoxide ring opening
to give the diene intermediate (108).
There was another biogenetic pathway particularly for the formation of α,βoxide at C-1 and C-6 of (+)-zeylenol (74) and (-)-zeylenol (75) isolated from
Zingiberaceae (Figure 2.4) [64]. It also explained the formation of pipoxide (63).
26
(74)
(75)
Opening of arene β-oxide (99b) at C-3 by nucleophilic attack on the arene αface leads to the deoxygenated cyclohexadiene (110). Furthermore, epoxidation of
(110) on the β- or α-face leads eventually to the various known natural products. In
particular, (-)-zeylenol (75) can be obtained from the β-epoxidation of (110)
followed by trans opening at C-6 as shown in Figure 2.4 [64].
As shown in Figure 2.4, two pathways can be considered involve in the
formation of (+)-zeylenol (74). (+)-Zeylenol (74) was proposed to be formed either
from β-oxide (99a) or (99b) through the intermediate (108c). Opening of the β-oxide
(99a) or (99b) at C-2 by α-nucleophilic attack (possibly OCOPh group) to give
(108c), followed by α-epoxidation to produce pipoxide (63), and subsequent epoxide
opening at C-6 afforded (+)-zeylenol (74) [64].
OCOPh
2
3
(99b)
OCOPh
O
2
3
(i)
(ii) (99a)
OCOPh
OCOPh
OH
(110)
OH
OCOPh
(108c)
OCOPh
O
OH
OCOPh
OCOPh
O
OH
1
OCOPh
(75)
OH
OCOPh
(63)
OCOPh
HO
HO
6
(111)
O
OCOPh
HO
HO
6
1
OCOPh
(74)
OH
OCOPh
Figure 2.4. Biogenetic Pathway for the Formation of (+)-Zeylenol (74) and (-)Zeylenol (75)
CHAPTER 3
CHEMICAL COMPOSITIONS OF KAEMPFERIA ROTUNDA OILS
3.1
The Essential Oils of Kaempferia rotunda
In this study, rhizomes oils of Kaempferia rotunda that cultivated from two
different sites were analysed and comparison were made. The first sample was
collected from Kempas, Johor while the second sample was imported from Indonesia.
The oils were obtained by using hydrodistillation technique for 8 hours and were
analyzed by GC and GC-MS. Identification of the compositions was carried out by
comparing their mass spectra obtained with Wiley Library mass spectral database,
and the calculated Kovats Indices with the values in the literature.
3.1.1
Rhizome Oil of Malaysian Kaempferia rotunda
Hydrodistillation of fresh rhizomes of the Malaysian Kaempferia rotunda
(233.7 g) for 8 hours gave a pale yellow oil in 0.09% yield. GC chromatogram
(Appendix 1) revealed the presence of sixty three compounds. Fifty compounds
which contributed 81.2% of the total compositions were identified.
Table 3.1
showed the compositions of the rhizome oil along with their retention indices, in two
different GC capillary columns, Ultra-1 (non-polar) and Carbowax (polar). The
compositions were identified by matching their mass spectra recorded with Wiley
Library mass spectral, and the calculated Kovats Indices with literature [65-66].
28
Bornyl acetate (90) (9.6%), benzyl benzoate (82) (8.4%) and camphor (91)
(5.6%) were the major constituents in the rhizome oil. The oil contained various
types of compounds including eight monoterpene hydrocarbons (2.4%), nine
oxygenated monoterpenes (20.0%), sixteen sesquiterpene hydrocarbons (17.9%),
four oxygenated sesquiterpenes (7.7%), three diterpenes (11.1%), four acids (4.5%),
two esters (8.6%), two hydrocarbons (5.1%) and two aldehydes (3.9%).
Oxgenated monoterpenes (20.0%) were the major contributor to the oil with
bornyl acetate (90) (9.6%), camphor (91) (5.6%) and borneol (112) (1.4%) as the
main constituents. Eight monoterpenes were present in the oil with β-pinene (113)
(0.6%) as the major component. Among the sixteen hydrocarbon sesquiterpenes
(17.9%), the major compounds were isocaryophyllene (114) (2.4%), β-caryophyllene
(115) (1.9%) and δ-cadinene (116) (1.8%). Four oxygenated sesquiterpenes were
present in the oil with (E,E)-farnesyl acetate (117) (5.5%) as the major component.
The ester constituted 8.6% of the oil, with benzyl benzoate (82) (8.4%) as the
major component. Two hydrocarbons and three diterpenes were identified, among
which n-pentadecane (88) and α-hydroxysandaracopimara-8(14),15-diene (118) were
the major constituents in the hydrocarbons and diterpenes, respectively. The other
classes of compounds such as acids and aldehydes in this oil were only present in
low concentration (<4.0%).
H
H
(112)
(113)
(114)
(115)
OH
H
(116)
OAc
(117)
H
H
(118)
29
Table 3.1: Constituents of Malaysian Kaempferia rotunda Rhizome Oil
Kovats Index
Carbowax
Percentage
Tricyclene
α-Pinene
Constituents
920
931
987
tr
0.4
Camphene
β-Pinene
Myrcene
1,8-Cineole
Limonene
(Z)-β-Ocimene
(E)-β-Ocimene
950
958
989
1001
1008
1025
1068
1134
1171
1159
0.4
0.6
0.4
0.7
0.3
Linalool
1086
1198
1214
1511
0.2
0.2
0.9
Camphor
Isoborneol
1105
1127
1444
5.6
0.4
Borneol
Terpine-4-ol
α-Terpineol
1139
1152
1167
1643
1597
1647
1.4
0.4
0.6
Bornyl acetate
Isobornyl acetate
1261
1538
1540
9.6
0.4
α-Copaene
β-Elemene
β-Caryophyllene
Aromadendrene
1365
1387
1417
1552
1562
0.2
0.4
1.9
0.6
Isocaryophyllene
α-Humulene
trans-β-Farnesene
ar-Curcumene
Germacrene D
1421
1432
1436
1457
δ-Cadinene
(E,E)-α-Farnesene
γ-Curcumene
Valencene
α-Selinene
Ultra-1
1628
1663
2.4
0.9
0.5
1.1
0.2
1705
1723
1.8
0.8
0.9
1.1
3.9
1458
4.0
0.9
0.2
0.8
0.3
1465
1471
1486
n-Pentadecane
Cadina-1,4-diene
n-Pentyl benzoate
(E)-Nerolidol
α-Cubebene
1496
1536
β-Caryophyllene oxide
Humulene epoxide
n-Heptadecane
1579
1593
1690
Benzyl benzoate
Tetradecanoic acid
1716
1752
8.4
0.3
(E,E)-Farnesyl acetate
Salicyldehyde
1825
1845
5.5
0.3
1849
1995
1999
1918
1.0
0.5
1.0
30
Sandaracopimara-8(14),15-diene
Benzyl salicylate
Hexadecanoic acid
Heptadecanoic acid
1931
1950
1980
2040
0.6
3.6
3.1
0.4
α-Hydroxysandaracopimara-8(14),15-diene
Octadecanoic acid
Aristolene epoxide
2103
2160
2221
9.2
0.7
1.3
Total (%)
81.2
Note: tr = trace
Previous study on the rhizome oil of Kaempferia rotunda collected from
Johor, Malaysia consisted of monoterpenes (19.0%), sesquiterpenes (10.1%), esters
(39.2%), and hydrocarbons (27.3%). n-Pentadecane (88) (25.4 %), bornyl acetate
(90) (24.9%), benzyl benzoate (82) (15.3%) and camphor (91) (12.1%) were found to
be the main constituents in the oil [60].
This study also revealed the rhizome oil of Kaempferia rotunda from Johor,
Malaysia. However, the major component of the rhizome oil in this study varies in
the quantity with the previous report [60]. In this study, n-pentadecane (88), bornyl
acetate (90), benzyl benzoate (82) and camphor (91) were only present in 4.0%, 9.6%,
8.4% and 5.6% respectively, compared to 25.4%, 24.9%, 15.3% and 12.1%
respectively in the previous report [60].
The difference quantity of the major
components was probably due to the different muturity of rhizomes.
The present result revealed a clear difference, especially α-hydroxysandaracopimara-8(14),15-diene (118) (9.2%) and four acids (4.5%) were found to
be present in the oil, were in accordance with the previous study [61]. The rhizome
oil from previous study was dominated by esters (39.2%) [61], compared to the
present study where sesquiterpenes (25.8%) and monoterpenes (22.4%) were the
main contributors to the oil. The differences in the compositions were probably due
to locality, habitat of the plants and different chemotype.
31
3.1.2
Rhizome Oil of Indonesian Kaempferia rotunda
The rhizome oil isolated from the Indonesian Kaempferia rotunda was
obtained in 0.23% yield as a pale yellow oil. A total of twenty four compounds were
identified from the GC analysis shown in the chromatogram (Appendix 2),
contributing 99.6% of the total oil. The compounds are tabulated in Table 3.2 with
their retention indices in Ultra-1 (non-polar) and Carbowax (polar) capillary GC
columns.
The compositions were identified by comparing their mass spectra
obtained with Wiley Library mass spectral database, and the calculated Kovats
Indices with literature [65-66]. A significant amount of ester (87.7%) was observed
in this oil, while monoterpenes (4.8%), sesquiterpenes (0.4%), aldehyde (2.3%) and
hydrocarbons (4.4%) constituted a minor fraction in the oil.
The oil was characterized by a high content of benzyl benzoate (82) (87.7%).
Other compounds in this oil were only present in a low concentration (<5.0%). Most
of the monoterpenes in the oil were present in low percentage (<1.0%), except for
camphene (89) (1.3%) and bornyl acetate (90) (1.1%). Only sesquiterpene
hydrocarbons (0.4%) and no oxygenated sesquiterpene were identified. Furthermore,
salicyldehyde (119) (1.4%), benzyl salicylate (108) (0.9%), n-pentadecane (88)
(4.2%) and n-heptadecane (120) (0.2%) were also detected in this oil.
(119)
(120)
Previous report on the rhizome oil of Kaempferia rotunda from Indonesia
showed the presence of benzyl benzoate (82) (69.7%), and n-pentadecane (88)
(22.9%) as the major constituents [59].
The composition of the rhizome oil
especially the major component in this study was almost identical with the previous
report [59], except varies slightly in the quantity. In this study, benzyl benzoate (82),
and n-pentadecane (88) were present in 87.7% and 4.2%, respectively compared to
69.7% and 22.9%, respectively in the previous report [59]. Esters and hydrocarbons
portions were found to be the main contributor in both oils. However, much less
32
compounds were identified in this study i.e. twenty four compounds compared to
seventy five compounds in the previous report [59].
The differences in the
compositions were probably due to different maturity, locality, habitat of the plants
and different chemotype.
Table 3.2: Constituents of Indonesian Kaempferia rotunda Rhizome Oil
Kovats Index
Ultra 1
Carbowax
Percentage
Tricyclene
α-Pinene
Camphene
Constituents
922
936
972
989
1024
0.1
0.4
1.3
Myrcene
1,8-Cineole
Limonene
(Z)-β-Ocimene
α-Terpinolene
977
1005
1009
1124
0.1
0.2
0.1
0.1
0.5
Camphor
Borneol
Bornyl acetate
Isobornyl acetate
β-Caryophyllene
1108
1448
1261
1522
1527
1558
Aromadendrene
Terpine-4-ol
α-Humulene
trans-β-Farnesene
n-Pentadecane
1424
1164
1079
1399
1433
1447
1502
1572
1597
1495
0.1
tr
0.1
tr
4.2
Linalool
δ-Selinene
n-Heptadecane
1686
tr
0.1
0.2
Benzyl benzoate
Benzyl salicylate
Salicyldehyde
1803
1851
1955
87.7
0.9
1.4
Total (%)
1500
0.7
0.2
1.1
tr
0.1
99.6
Note: tr = trace
3.1.3
Comparison of the Compositions of Malaysian and Indonesian
Kaempferia rotunda
Hydrodistillation of the rhizomes of Kaempferia rotunda from Malaysia and
Indonesia gave pale yellow oils in 0.09% and 0.23% yield respectively. This showed
33
that the Indonesian sample gave high yield of essential oil.
The chemical
compositions of the rhizomes oils of Malaysian and Indonesian K. rotunda are listed
in Table 3.3 for comparison.
Table 3.3: Comparison of the Rhizomes Oils of Malaysian and Indonesian
Kaempferia rotunda
Percentage
No.
Constituent
Malaysian
Indonesian
1
2
3
4
Tricyclene
α-Pinene
Camphene
β-Pinene
tr
0.4
0.4
0.6
0.1
0.4
1.3
-
5
6
7
8
Myrcene
1,8-Cineole
Limonene
(Z)-β-Ocimene
0.4
0.7
0.3
0.2
0.1
0.2
0.1
-
9
10
11
12
13
(E)-β-Ocimene
α-Terpinolene
Linalool
Camphor
Isoborneol
0.2
0.9
5.6
0.4
0.1
0.5
tr
0.7
-
14
15
16
17
18
Borneol
Terpine-4-ol
α-Terpineol
Bornyl acetate
Isobornyl acetate
1.4
0.4
0.6
9.6
0.4
0.2
tr
1.1
tr
19
20
21
22
23
α-Copaene
β-Elemene
β-Caryophyllene
Aromadendrene
Isocaryophyllene
0.2
0.4
1.9
0.6
2.4
0.1
0.1
-
24
25
26
27
28
α-Humulene
trans-β-Farnesene
ar-Curcumene
Germacrene D
δ-Cadinene
0.9
0.5
1.1
0.2
1.8
0.1
tr
-
29
30
31
32
(E,E)-α-Farnesene
γ-Curcumene
Valencene
δ-Selinene
0.8
0.9
1.1
-
0.1
33
34
35
α-Selinene
n-Pentadecane
Cadina-1,4-diene
3.9
4.0
0.9
4.2
-
34
36
37
38
39
40
n-Pentyl benzoate
(E)-Nerolidol
α-Cubebene
β-Caryophyllene oxide
Humulene epoxide
0.2
0.8
0.3
1.0
0.5
-
41
42
43
44
45
n-Heptadecane
Benzyl benzoate
Tetradecanoic acid
(E,E)-Farnesyl acetate
Salicylate aldehyde
1.0
8.4
0.3
5.5
4.4
0.2
87.7
1.4
46
47
48
49
Benzyl salicylate
Sandaracopimara-8(14),15-diene
Hexadecanoic acid
Heptadecanoic acid
3.6
0.6
3.1
0.4
0.9
-
9.2
0.7
1.3
81.2
99.6
2.4
20.0
17.9
7.7
5.1
2.6
2.2
0.4
4.4
8.6
3.9
4.5
11.1
0.09
87.7
2.3
0.23
50
α-Hydroxysandaracopimara-8(14),15-diene
51
Octadecanoic acid
52
Aristolene epoxide
Identified Compounds (%)
Group Components:
Monoterpene Hydrocarbons
Oxygenated Monoterpenes
Sesquiterpene Hydrocarbons
Oxygenated Sesquiterpenes
Hydrocarbons
Esters/ ester
Aldehyde
Acids
Diterpenes
Oil Yield, % (w/w)
Note: tr = trace
As can be seen from the Table 2.3, the chemical compositions of rhizomes
oils Kaempferia rotunda from Malaysia and Indonesia were very different. The
significant differences between both oils were the occurrence of their main
components. Bornyl acetate (90) (9.6%), α-hydroxysandaracopimara-8(14),15-diene
(118) (9.2%) benzyl benzoate (82) (8.4%) and camphor (91) (5.6%) were the
principal components in the oil of Malaysian species, while only benzyl benzoate (82)
(87.7%) and n-pentadecane (88) (4.2%) were the main components in the oil of
Indonesian species. The Malaysian rhizome oil contained a significant low amount
(8.4%) of benzyl benzoate (82) compared to the oil of Indonesian species (87.7%).
35
In addition, the rhizome oil of Malaysia was rich in oxygenated
monoterpenes (20.0%) and sesquiterpene hydrocarbons (17.9%) whereas the oil of
Indonesian species contained high amount of esters (88.6%).
Furthermore, the
rhizome oil of Indonesia showed a much lower concentration of monoterpenes and
sesquiterpenes (5.2%) than the Malaysian species (48.0%).
Most of the sesquiterpenes and sesquiterpenoids i.e. ar-cucumene (121),
germacrene D (122), δ-cadinene (123), humulene epoxide (124), β-caryophyllene
oxide (125), (E)-nerolidol (126) together with acids of hexadecanoic acid (127),
octadecanoic acid (128), heptadecanoic acid (129) and the diterpenes of αhydroxysandaracopimara-8(14),15-diene (118) and sandaracopimara-8(14),15-diene
(130) were absent in the rhizome oil of Indonesia.
H
(121)
(122)
(123)
(124)
O
OH
H
H
(125)
(126)
(127) n= 11
(128) n= 13
(129) n= 12
(130)
The differences in the contents and the components of the rhizomes oils from
different locations, Malaysia and Indonesia may suggest the existence of intraspecific
chemical differences among the population of Kaempferia rotunda. Generally, the
content of benzyl benzoate (82) can used for distinguish locality (Malaysia and
Indonesia) of K. rotunda.
CHAPTER 4
PHYTOCHEMICAL AND BIOACTIVITY STUDIES OF KAEMPFERIA
ROTUNDA
4.1
Phytochemical Study of Malaysian Kaempferia rotunda
Soxhlet extraction of the dried rhizomes of Kaempferia rotunda (205.0 g),
cultivated in Kempas, Johor, Malaysia (purchased from Larkin market) with nhexane, EtOAc and MeOH for 16 hours each yielded viscous dark brown semisolids
2.32 g (1.13%), dark brown oil 6.80 g (3.32%) and dark brown viscous liquid 15.27 g
(7.45%) respectively.
Preliminary TLC screening of the n-hexane, EtOAc and MeOH crude extracts
visualized under UV 254 nm and using anisaldehyde spraying reagent showed that
the crude extracts gave different profiles on TLC plate. Thus these crude extracts
were subjected to VLC for fractionation, followed by purification either by gravity
column chromatography (CC), chromatotron, versa flash column chromatography or
recrystallization techniques to afford two new compounds and seven known
compounds. Two new compounds were identified as 2-(benzoyloxymethyl)phenyl
(3-O-acetyl)-β-glucopyranoside (131) and 3-debenzoylrotepoxide A (132), while the
known compounds were characterized as crotepoxide (54), 2'-hydroxy-4,4',6'trimethoxychalcone (1), 4-benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6diol 5-acetate (57) (major compound), 1,6-desoxypipoxide (69), naringenin 4',7dimethyl ether (133), and curcumrinol C (134). Naringenin 4',7-dimethyl ether (133),
and curcumrinol C (134) were first time reported from Kaempferia species.
37
4.1.1
Crotepoxide (54)
Solids from the n-hexane crude extract was washed using Et2O to give
compound (54) (0.036 g, 1.55 %), as colourless needles with m.p. of 143-144°C (lit.
[67] 146-148°C). The CIMS spectrum (Appendix 3) displayed a molecular ion peak
at m/z 363 [M+1]+ which was consistent with the molecular formula of C18H18O8.
(54)
The 1H NMR spectrum (Appendix 4) showed two singlets at δ 2.04 and 2.14
for the two acetoxyl groups. A multiplet integrating for five protons appeared at δ
7.50-8.00 indicated the presence of phenyl protons. An AB system signal at δ 4.58
and 4.25 (J = 12.0 Hz) was attributed to the oxymethylene protons, H-7. Three
epoxide protons were observed at δ 3.67 (d, J = 2.4 Hz), 3.46 (dd, J = 4.0 and 2.4 Hz)
and 3.11 (d, J = 4.0 Hz), which attributed to H-6, H-5 and H-4 respectively. This
was confirmed by COSY spectrum (Appendix 5) which showed H-5 at δ 3.46 was
coupled with both H-4 at δ 3.11 and H-6 at δ 3.67. A set of doublets at δ 5.00 and
5.71 (J = 9.2 Hz) was assigned to H-2 and H-3 respectively. The HMBC spectrum
(Appendix 6) confirmed the position of the acetoxyl groups where correlations were
observed between acetoxyl protons at δ 2.04, 2.14 to C-3 and C-2 respectively.
The IR spectrum (Appendix 7) showed two strong carbonyl absorption bands
at 1727 cm-1, 1766 cm-1 and C-O absorption bands at 1236 cm-1 and 1211 cm-1 for
benzoate and acetoxyl esters. The 13C NMR (Appendix 8) supported the presence of
carbonyl esters at δ 170.06, 169.75 and 165.80. Analysis of the 13C NMR and DEPT
spectra (Appendices 8 and 9) revealed the presence of two acetoxyl carbons at δ
20.66 and 20.62, five methine carbons at δ 70.37 (C-3), 69.42 (C-2), 53.81 (C-6),
52.60 (C-4) and 48.06 (C-5), a methylene carbon at δ 62.46 (C-7), two quaternary
carbons at δ 59.38 (C-1) and 129.11 (C-1'), and aromatic carbons at δ 133.55 (C-4'),
38
129.80 (C-3', C-5') and 128.56 (C-2', C-6'). Full assignments of the carbons were
based on the HMQC (Appendix 10) and HMBC (Appendix 6).
Compound (54) was identified as 4-benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diyl diacetate or trivially named as crotepoxide, based on
comparison of its physical properties and spectroscopic data of compound previously
isolated from Kaempferia rotunda, K. angustifolia and K. sp. [16-17, 30-32]. The
conventional numbering system of (54) in this thesis is consistent with the literature,
while the numbering system for the IUPAC name is shown in (54*).
O
O
1'
3'
5'
O
7
O
O
2
1
3
6 5 4
O
O
O
(54)
4.1.2
(54*)
2'-Hydroxy-4,4',6'-trimethoxychalcone (1)
Purification of n-hexane extract (2.20 g) by CC gave 20 fractions.
Recystallization of fraction 13 and 14 by using n-hexane: Et2O yielded compound (1)
as yellow rhombics (6.9 mg, 0.3%), m.p. 109-110°C (lit. [64] 115°C). Compound (1)
gave yellow spot with an anisaldehyde reagent suggesting a flavonoid compound.
This was supported by the UV spectrum (Appendix 11) which showed λmax at 372.8
and 287.2 nm in MeOH were typical for a chalcone compound. The IR spectrum
(Appendix 12) showed absorption bands for hydroxyl and chelated carbonyl groups
at 3457 cm-1 and 1623 cm-1 respectively.
Two doublets (J = 16.4 Hz) at δ 7.77 and 7.85 attributed to an α,β-unsaturated
olefinic protons were observed in the 1H NMR (Appendix 13). A set of metacoupled doublets was observed at δ 6.12 and 5.98 (J = 2.2 Hz) was assigned to H-5'
and H-3' respectively. The aromatic protons in ring B appeared as a set of doublets
39
at δ 6.94 and 7.58 (J = 8.8 Hz) revealed the presence of a para-disubstituted benzene
ring. Three sharp singlets appeared at δ 3.92, 3.86 and 3.85 were attributed to the
methoxyl groups.
H3CO
6'
4'
CH3
Oα
4
OCH3
β
2'
OH O
(1)
The 13C NMR and DEPT (Appendices 14 and 15) disclosed the presence of
three methoxyl carbons at δ 55.83, 55.57 and 55.39, six CH aromatic carbons at δ
130.11 (C-3, C-5), 114.35 (C-2, C-6), 93.79 (C-5') and 91.23 (C-3'), five quaternary
carbons at δ 168.36 (C-2'), 166.01 (C-6'), 162.45 (C-4'), 161.35 (C-4) and 106.35
(C-1'), two vinyl carbons at δ 142.47 (C-8) and 125.12 (C-7), and a carbonyl carbon
at δ 192.59. This was supported by the EIMS (Appendix 16) which showed a
molecular ion peak, M+ at m/z 314 corresponding to a molecular formula C18H18O5.
The HMBC (Appendix 17) showed long range correlations from methoxyl protons
to carbons at δ 166.01 (C-6'), 162.45 (C-4') and 161.35 (C-4) suggesting the position
of the methoxyl groups at ring A and B.
Thus, compound (1) was identified as 2'-hydroxy-4,4',6'-trimethoxychalcone
based on its physical properties and comparison with spectroscopic data of
compound previously isolated from the rhizome of Boesenbergia sp. [64].
4.1.3
4-Benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diol 5-acetate
(57)
Compound (57) was obtained as colourless needles (0.145 g, 2.13 %), m.p.
141-142ºC (lit. [31] 145-148 ºC) from the EtOAc extract. It had molecular formula
of C16H16O7, in agreement with the CIMS (Appendix 18) (m/z 321, [M+1]+). Two
strong carbonyl absorption bands at 1748 cm-1 and 1723 cm-1, C-O absorption bands
40
at 1226 cm-1, and 1284 cm-1 were observed in the IR spectrum (Appendix 19). In
addition, a typical OH stretching band at 3447 cm-1 was also observed.
O
O
2'
4'
6'
7
O
O
(57)
O
2
1
3
6 5 4
OH
O
This was in agreement with the 1H NMR (Appendix 20) which showed a free
hydroxyl proton at δ 2.71, that exchangeable with D2O. The 1H NMR spectrum
(Appendix 20) was very similar to crotepoxide (54), except for the disappearance of
one methyl singlet of an acetoxyl group. In addition, H-3 of epoxide (57) was more
shielded to the upfield region at δ 3.98 (dd, J = 8.4, 4.8 Hz) confirmed that a
hydroxyl group was attached on that carbon.
Similarly, the carbon signals (Appendix 21) of epoxide (57) were almost
identical to that of crotepoxide (54) except the lack of carbonyl ester and a methyl
peak associated to an acetoxyl group and C-3 was more shielded to δ 69.17. Full
assignments of the protons and carbons were accomplished with the aid of the
HMBC (Appendix 22) and HMQC (Appendix 23) spectral analysis.
The spectroscopic data of compound (57) were identical with the reported
values of 4-benzoyloxymethyl-3,8-dioxatricyclo-[5.1.0.02,4]octane-5,6-diol 5-acetate
[17, 31]. The convention and IUPAC numbering system are shown in (57) and (57*)
respectively.
(57*)
41
4.1.4
1,6-Desoxypipoxide (69)
Compound (69) (0.03g, 0.44%) was obtained from the purification of the
fraction 3 of EtOAc extract as white solids with m.p. of 89-90°C (lit. [68] 90-91°C).
The EIMS (Appendix 24) exhibited [M]+ ion peak at m/z 350 corresponding to a
molecular formula of C21H18O5.
(69)
This was in agreement with the analysis of the
13
C NMR spectrum
(Appendix 25) which revealed the presence of twenty one carbons. The DEPT
(Appendix 26) exhibited a methylene at δ 64.77 (C-7), two methines at δ 75.35 (C-3)
and 70.08 (C-2), three quaternary carbons at δ 135.06 (C-1) and 129.89 (C-1', C-1'')
and ten CH aromatic carbons at δ 133.25 (C-4''), 133.18 (C-4'), 129.78 (C-2'', C-6''),
129.68 (C-2', C-6'), 128.42 (C-3'', C-5''), 128.40 (C-3', C-5'). The signals for vinyl
carbons were observed at δ 125.84 (C-6), 124.98 (C-4) and 123.01 (C-5). Two
downfield signals at δ 166.47 and 166.48 were assigned to the carbonyl groups.
Absorption bands at 1715 cm-1 (C=O) and 1269 cm-1 (C-O) in the IR
spectrum (Appendix 27) confirmed the presence of ester group. In addition, a broad
peak at 3432 cm-1 indicated the presence of hydroxyl group.
The 1H NMR (Appendix 28) supported the presence of a hydroxyl proton at
δ 2.48 which was exchangeable with D2O (d, J = 6.0 Hz), ten phenyl protons
resonated at δ 7.40-8.07, oxymethylene protons (H-7) appeared as an AB system
(13.2 Hz) at δ 5.01 and 5.13, two methine protons at δ 4.70 (t, J = 6.0 Hz, H-2) and
5.80 (dd, J = 4.0, 6.0 Hz, H-3); and three vinyl protons at δ 6.01 (dd, J = 4.0, 9.2 Hz,
H-4), 6.15 (dd, J = 5.6, 9.2 Hz, H-5) and 6.19 (d, 5.6 Hz, H-6). The 1H-1H COSY
spectrum (Appendix 29) showed that the methine proton, H-3 at δ 5.80 was adjacent
to the H-2 at δ 4.70 and H-4 at δ 6.01. The correlations between the olefinic protons
42
(H-4 to H-6) were also observed in the COSY spectrum (Appendix 29). The 1H and
13
C and COSY data of compound (69) are summarized in Table 4.1.
Based on its physical properties and comparison with literature values of
previously isolated compound from Uvaria purpurea and Kaempferia rotunda [17,
68], compound (69) was identified as 1,6-desoxypipoxide.
Table 4.1: 1H, 13C NMR and COSY Data of Compound (69)
Position
13
C NMR
δ (ppm)
1
2
3
4
5
6
7
135.06
70.08
75.35
124.98
123.01
125.84
64.77
1' , 1''
2', 6'
2'', 6''
3', 5'
3'', 5''
4'
4''
OH
C=O
C=O
129.89
129.68
129.78
128.40
128.42
133.18
133.25
166.47
166.48
4.1.5
1
H NMR
δ (ppm) (int., mult., J)
COSY
(1H-1H)
4.70 (1H, t, J = 6.0 Hz)
5.80 (1H, dd, J = 4.0 and 6.0 Hz)
6.01 (1H, dd, J = 4.0 and 9.2 Hz)
6.15 (1H, dd, J = 5.6 and 9.2 Hz)
6.19 (1H, d, J = 5.6 Hz)
5.12 (1H, d, J = 13.2 Hz)
5.01 (1H, d, J = 13.2 Hz)
8.05 (2H, m)
8.05 (2H, m)
7.43 (2H, m)
7.43 (2H, m)
7.58 (1H, t, J = 7.6 Hz)
7.58 (1H, t, J = 7.6 Hz)
2.84 (d, J = 6.0 Hz)
-
H-3, OH
H-2, H-4
H-3, H-5
H-4, H-6
H-4, H-5
H-7a
H-7b
H-3', H-5'
H-3'', H-5''
H-2', H-6', H-4'
H-2'', H-6'', H-4
H-3', H-5'
H-3'', H-5''
H-2
-
2-(Benzoyloxymethyl)phenyl (3-O-acetyl)-β-glucopyranoside (131)
Purification of fraction 4 resulted from VLC of the EtOAc extract by versa
flash CC, followed by chromatotron yielded compound (131) (8.9 mg, 0.36%), as a
pale brown gum. The IR spectrum (Figure 4.1) gave absorption bands at 1698 cm-1
(C=O), 1167 cm-1 (C-O) and 3421 cm-1 (OH). Compound (131) had an [M+1]+ ion
peak at m/z 433 in the CIMS (Figure 4.2), corresponding to a molecular formula of
C22H24O9.
43
(131)
The 1H NMR (Figure 4.3) supported the presence of hydroxyl groups which
showed three free hydroxyl protons at δ 3.32 and 3.31.
A singlet at δ 2.14
attributable to the acetoxyl group was also observed. The sugar moiety was assigned
based on the presence of the proton signals at δ 5.07 (H-1), 3.56 (H-2), 5.05 (H-3),
3.54 (H-4), 3.64 (H-5), 3.73 (H-6a) and 3.90 (H-6b) [69]. The coupling constant of
7.8 Hz for H-1 and H-2 confirmed the β-form of sugar [69]. The oxymethylene
protons, H-7' appeared as an AB system with coupling constant of 12.8 Hz at δ 5.44
and 5.56. A multiplet at δ 7.06-8.06 integrating for nine protons was assigned to the
benzoyl protons (H-2'' to H-6'') and protons in the benzene ring (H-3' to H-6') [32].
The
13
C NMR (Figure 4.4) showed the presence of twenty two carbons
which was in agreement with the molecular formula mentioned above. The DEPT
(Figure 4.5) revealed the presence of three quaternary carbons at δ 155.50 (C-1'),
130.10 (C-1'') and 125.70 (C-2'), nine CH aromatic carbons δ 132.87 (C-4''), 129.22
(C-2'', C-6''), 128.18 (C-3'', C-5''), 129.36 (C-5'), 128.98 (C-3'), 122.28 (C-4') and
115.43 (C-6'), a methylene at δ 61.79 (C-7') and a methyl carbon at δ 19.69 (OAc).
Five oxygenated methines at δ 101.30 (C-1), 77.61 (C-3), 76.59 (C-2), 71.83 (C-5)
and 68.05 (C-4) and a methylene at δ 60.79 (C-6) indicated the presence of sugar
moiety [69]. Two signals resonated at δ 171.23 and 166.68 were assigned to the
acetoxyl and benzoate carbonyls respectively.
The position of acetoxyl group at C-3 was accomplished from the HMBC
(Figure 4.6) which showed cross peak between H-3 and carbonyl of the acetoxyl
group. Long-range 1H-13C correlations were also observed between the proton at the
anomeric carbon (H-1) at δ 5.07 and C-1' at δ 155.50. The oxymethylene proton
(H-7') at δ 5.44, 5.56 was correlated with C-2 at δ 125.70 and carbonyl at 166.68.
These data confirmed the ortho-disubstituted pattern of compound (131).
complete NMR data are listed in Table 4.2.
The
44
Table
1
H and 13C NMR Data of Compound (131) and 2(Benzoyloxymethyl) phenyl (3,6-di-O-acetyl)-β-glucopyranoside (135)
4.2:
Compound (131)
Position
13
2-(Benzoyloxymethyl)phenyl (3,6-di-Oacetyl)-β-glucopyranoside (135)
1
13
1
2
3
4
5
C
NMR
101.30
76.59
77.61
68.05
71.83
6
60.79
1'
2'
3'
4'
5'
6'
7'
155.50
125.70
128.98
122.28
129.36
115.43
61.79
1''
2'', 6''
3'', 5''
4''
OAc
130.10
129.22
128.18
132.87
19.69
5.07 (1H, d, J = 7.8 Hz)
3.56 (1H, dd, J = 7.8, 9.4 Hz)
5.05 (1H, dd, J = 9.4, 9.6 Hz)
3.54 (1H, dd, J =9.0, 9.6 Hz)
3.64 (1H, ddd, J = 2.0, 4.8,
9.0 Hz)
3.73 (1H, dd, J = 12.4, 2.0
Hz)
3.90 (1H, dd, J = 12.4, 4.8
Hz)
7.43 (1H, d, J = 7.6 Hz)
7.08 (1H, t, J = 7.6 Hz)
7.35 (1H, t, J = 7.6 Hz)
7.28 (1H, d, J = 7.6 Hz)
5.44 (1H, d, J = 12.8 Hz)
5.56 (1H, d, J = 12.8 Hz)
8.06 (2H, d, J = 7.2 Hz)
7.47 (2H, dd, J = 7.2, 7.6 Hz)
7.61 (1H, t, J = 7.6 Hz)
2.14 (3H, s)
C=O (OAc)
171.23
-
C=O (OBz)
OH
OH
166.68
-
3.32 (1H, br s)
3.31 (2H, br s)
The 1H and
H NMR
13
C
NMR
102.90
73.30
78.80
69.90
75.50
64.40
156.90
127.70
130.50
124.10
130.80
117.40
63.20
131.60
130.70
129.70
134.40
21.10,
20.70
172.6,
172.70
168.20
-
1
H NMR
5.03 (1H, d, J = 7.8 Hz)
3.63 (1H, dd, J = 9.5, 7.8 Hz)
5.03 (1H, t, J = 9.4 Hz)
3.53 (1H, dd, J = 9.9, 9.3 Hz)
3.72 (1H, ddd, J = 2.4, 6.2, 9.9
Hz)
4.39 (1H, dd, J = 12.0, 2.4 Hz)
4.24 (1H, dd, J = 11.9, 6.2 Hz)
7.44 (1H, dd, J = 7.5, 1.8 Hz)
7.09 (1H, td, J = 7.5, 1.1 Hz)
7.33 (1H,ddd, J = 8.3, 7.5, 1.8 Hz)
7.22 (1H, dd, J =8.3, 1.0 Hz)
5.44 (1H, d, J = 12.8 Hz)
5.56 (1H, d, J = 12.8 Hz)
8.05 (2H, m)
7.47 (2H, m)
7.60 (1H, m)
2.13 (3H, s),
2.02 (3H, s)
-
C data (Table 4.2) of compound (131) were almost identical
with the known of 2-(benzoyloxymethyl)phenyl (3,6-di-O-acetyl)-β-glucopyranoside
(135) [32], except the lack of an acetoxyl group at C-6. The C-6 and H-6 peaks were
more shielded to δ 60.79 and 3.73, 3.90 respectively and this showed that the C-6
was hydroxylated. Therefore, compound (131) was identified as a new compound,
2-(benzoyloxymethyl) phenyl (3-O-acetyl)-β-glucopyranoside.
HO
HO
AcO
6
O
O
O
OH
O
(131)
AcO
HO
AcO
6
O
O
O
OH
O
(135)
1558.13
1'
3'
O
1''
4''
5'
64
62
3500
3000
2500
2000
1500
949.66
1113.36
1167.75
1304.64
1376.55
O
OH
1
1017.59
3
O
O
7'
1211.70
66
HO
AcO
6
1451.79
68
HO
1492.26
70
3059.45
72
2917.96
74
1602.03
76
1698.73
3023.85
78
2851.31
80
3421.34
%T
82
906.25
1270.85
84
755 10
86
832.05
88
1874.55
2728.52
92
90
1807.92
1946.13
94
2331.00
96
3847.90
98
1000
Wavenumbers (cm-1)
Figure 4.1: IR Spectrum of 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)-β-glucopyranoside (131)
45
HO
HO
AcO
6
3
O
O
OH
1
O
7'
1'
3'
O
1''
4''
5'
(M+1)+
46
Figure 4.2: CIMS Spectrum of 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)-β-glucopyranoside (131)
H-3''
H-5''
H-4''
H-2
H-3'
H-6'
H-5'
CH3 (OAc)
H-5
H-4'
H-6
H-4
HO
HO
AcO
6
3
O
O
OH
1
O
7'
1'
3'
O
1''
4''
5'
H-2''
H-6''
H-1
H-3
OH
H-7'
47
Figure 4.3: 1H NMR Spectrum of 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)-β-glucopyranoside (131)
C-2'', 6''
HO
HO
AcO
6
3
O
O
OH
1
O
7'
1'
3'
O
1''
C-3'', 5''
4''
5'
C-5'
C-1''
C-3'
C-4' C-6'
C=O
(OAc)
C=O C-1
(OBz)
C-4''
C-2'
C-1
C-2 C-5
C-7'
C-4
C-3
C-6
48
Figure 4.4: 13C NMR Spectrum of 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)-β-glucopyranoside (131)
CH3 (OAc)
All carbons
C=O
(OAc) C=O C-1'
(OBz)
C-1''
C-2'
C-5' C-2'', 6'' C-3'
DEPT 45
DEPT 90
C-4''
C-3'', 5''
C-4' C-6'
C-1
C-2
C-3
HO
C-5
C-4
6
HO
AcO
3
O
O
OH
1
O
7'
1'
3'
O
1''
4''
5'
CH3 (OAc)
DEPT 135
C-7'
C-6
Figure 4.5: 13C NMR and DEPT Spectra of 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)-β-glucopyranoside (131)
49
HO
HO
AcO
6
O
3
O
OH
1
O
7'
1'
3'
O
H-3''
H-5''
1''
4''
5'
C-6
C-7'
H-2''
H-6''
H-4''
H-1
H-3
H-5' H-4'
H-3'
H-6'
H-7'
H-2
H-4
H-5
OH
CH3 (OAc)
H-6
C-4
C-5
C-2
C-3
C-1
C-3'
C-2'', 6''
C-5'
C-1''
C-6'
C-4'
C-2'
C-4''
C-1'
C=O (OBz)
C=O (OAc)
Figure 4.6: HMBC Spectrum of 2-(Benzoyloxymethyl)phenyl (3-O-acetyl)-β-glucopyranoside (131)
50
51
4.1.6
5-Hydroxy-7,4'-dimethoxyflavanone (Naringenin 4',7-dimethyl ether)
(133)
Compound (133) (0.7 mg, 0.004%) was isolated as yellow gum, gave yellow
spot after anisaldehyde spraying which suggested a flavonoid compound. The IR
spectrum (Appendix 30) showed absorption bands at 3413 cm-1 (OH), 1639 cm-1
(chelated C=O) and 1156 cm-1 (C-O).
(133)
The 1H NMR spectrum (Appendix 31) supported the presence of hydroxyl
group which exhibited a singlet signal resonated at downfield region at δ 12.04. In
addition, two sharp methoxyl singlets were observed at δ 3.85 and 3.82. The most
characteristic peaks were the presence of an ABX type signal at δ 2.80 (dd, J = 2.8
and 17.2 Hz), 3.12 (dd, J = 13.2 and 17.2 Hz) and 5.38 (dd, J = 2.8 and 13.2 Hz) for
flavanone moiety, which were assigned to two protons H-3 and one proton H-2
respectively. This was supported by the strong 1H-1H correlation between H-3 (δ
3.12 and 2.80) and H-2 (δ 5.38) in the COSY spectrum (Appendix 32). A set of
meta-coupled doublet with coupling constant of 2.4 Hz at δ 6.06 and 6.09 was
assigned to H-6 and H-8 in ring A. A set of doublet at δ 7.40 (J = 8.8 Hz) and δ 6.79
(J = 8.8 Hz), integrating for two protons each, confirmed structure of paradisubstituted benzene ring for the ring B.
Seventeen carbons were observed in the 13C NMR spectrum (Appendix 33).
The carbons were characterized as a methylene carbon at δ 43.21 (C-3), a methine
carbon at δ 97.02 (C-2), six quaternary carbons at δ 167.97 (C-5), 164.14 (C-9),
162.90 (C-7), 160.10 (C-4'), 130.39 (C-1'), , 103.15 (C-10), six CH aromatic carbons
at δ 127.74 (C-3', C-5'), 114.24 (C-2', C-6'), 95.09, (C-6), 94.23 (C-8), two methyl
carbons at δ 55.68, 55.38 and a carbonyl carbon at δ 196.04 (C-4) by the analysis of
52
DEPT (Appendix 34). The EIMS spectrum (Appendix 35) furnished a molecular
ion peak, [M+] at m/z 300 which was consistent with a molecular formula C17H16O5.
The 1D and 2D NMR, EIMS as well as the IR of compound (133) were in
good agreement with those reported in the literature for 5-hydroxy-7,4'-dimethoxyflavanone or trivially named as naringenin 4',7-dimethyl ether (133) [70], previously
isolated from Armand pine [71]. To the best of our knowledge, this is the first report
on the isolation of naringenin 4',7-dimethyl ether (133) from the genus of
Kaempferia.
4.1.7
12-Acetoxy-8α,13-dihydroxylabd-14-en-7-one (Curcumrinol C) (134)
Compound (134) (8.3 mg, 0.54%), a yellow amorphous with the m.p. of 150-
151°C was obtained from the MeOH extract. The IR (Appendix 36) exhibited a
broad band at 3418 cm-1 for hydroxyl groups, a strong absorption band at 1722 cm-1
corresponding to carbonyl group and bands at 1250 cm-1 and 1215 for C-O stretching.
(134)
The presence of carbonyl group was supported by
13
C NMR spectrum
(Appendix 37) which displayed carbonyl ketone and carbonyl ester at δ 170.43 and
209.02 respectively. The DEPT (Appendix 38) showed six methyl at δ 15.06 (C-20),
20.66 (C-19), 21.21 (C-22), 22.26 (C-17), 26.80 (C-16), 32.62 (C-18), five
methylene at δ 38.95 (C-1), 41.57 (C-3), 35.86 (C-6), 21.06 (C-11), 18.16 (C-2),
three methine (one oxygenated) at δ 70.41 (C-12), 56.47 (C-5), 50.46 (C-9), four
53
quaternary (two oxygenated) at δ 81.64 (C-8), 76.06 (C-13), 36.30 (C-10), 33.72
(C-4) and two olefinic carbons at δ 111.46 (C-15) and 145.82 (C-14).
The EIMS spectrum (Appendix 39) displayed an ion peak at m/z 362, [M+H2O], consistent with molecular formula C22H36O5. The base peak at m/z 43 was due
to the formation of acylium ion as shown in Scheme 4.1.
Scheme 4.1: The Elimination of Water Molecule from Compound (134) and the
Formation of Acylium Ion
The presence of acetoxyl group was in agreement by 1H NMR spectrum
(Appendix 40-41) which showed a singlet at δ 2.12 for the acetoxyl group. The 1H
NMR spectrum (Appendix 40-41) also indicated the existence of methyls at δ 1.48,
1.22, 0.95, 0.86 and 0.84, corresponding to Me-17, Me-16, Me-20, Me-18 and Me-19
respectively. A doublet of doublet at δ 5.38 (J = 5.6 and 3.0 Hz) was assigned to
H-12 which was adjacent to acetoxyl group. The existence of monosubstituted
olefinic protons was suggested by signals at δ 6.05 (dd, J = 11.6 and 18.4 Hz, H-14),
5.05 (d, J = 11.6 Hz, H-15a) and 5.10 (d, J = 18.4 Hz, H-15b). Coupling between H14 and H-15 was confirmed by the 1H-1H COSY spectrum (Appendix 42). The
complete assignments of the other protons were established by 1H-1H COSY
spectrum (Appendix 43).
54
The decaline ring was characterized by 1H-13C correlations (Appendix 44)
between H-18 and H-19/C-3 and C-4, as well as H-20/C-1 and H-20/C-5, as shown
in Table 4.3. Correlations between H-17/C-7, H-6/C-7 and H-17/C-9 indicated that
the attachment of the ketone function at C-7. Furthermore, the singlet methyls at δ
1.22 (H-16) and 1.48 (H-17) were correlated with the hydroxylated carbons at δ
76.06 (C-13) and 81.64 (C-8) respectively confirmed the methyls were bonded to
carbon bearing hydroxyl groups at C-13 and C-8 respectively. The assignment of the
protons, carbons and the correlations of the protons and carbons are summarized in
Table 4.3.
Based on comparison of its physical properties and spectral data of compound
previously isolated from the root tuber of Curcuma wenyujin [72], compound (134)
was identified as 12-acetoxy-8α,13-dihydroxylabd-14-en-7-one or trivially named as
curcumrinol C. This is the first report on the isolation of labdane diterpene from the
genus Kaempferia.
Table 4.3: 1H, 13C NMR, COSY and HMBC Data of Compound (134)
Position
13
1
C NMR
δ (ppm)
38.95
2
18.16
3
41.57
4
5
6
33.72
56.47
35.86
7
8
9
10
11
209.02
81.64
50.46
36.30
21.06
12
13
14
15
70.41
76.06
145.82
111.46
16
17
18
19
20
21
22
26.80
22.26
32.62
20.66
15.06
170.43
21.21
1
H NMR
δ (ppm) (int., mult., J)
a) 1.66 (1H, dd, J = 3.2 and 14.0 Hz)
b) 0.80 (1H, dd, J = 14.0 and 4.0 Hz)
a) 1.61 (1H, m)
b) 1.52 (1H, m)
a) 1.45 (1H, m)
b) 1.17 (1H, dd, J = 12.8 and 4.0 Hz)
1.40 (1H, dd, J = 14.4 and 2.9 Hz)
a) 2.57 (1H, dd, J = 14.0 and 14.4 Hz)
b) 2.44 (1H, dd, J = 14.0 and 2.9 Hz)
1.98 (1H, m)
a) 1.98 (1H, m)
b) 1.82 (1H, dd, J = 8.8 and 3.0 Hz)
5.38 (1H, dd, J = 5.6 and 3.0 Hz)
6.05 (1H, dd, J = 11.6 and 18.4 Hz)
a) 5.05 (1H, d, J = 11.6 Hz)
b) 5.10 (1H, d, J = 18.4 Hz)
1.22 (3H, s)
1.48 (3H, s)
0.86 (3H, s)
0.84 (3H, s)
0.95 (3H, s)
2.12 (3H, s)
COSY
(1H-1H)
H-1b, H-2a, H-2b
H-1a, H-2b
H-3b, H-2b, H-1a, H-3a
H-2a, H-1b, H-1a, H-3a
H-3b, H-2a, H-2b
H-3a, H-2a
H-6a, H-6b
H-5
C-20, C-19, C-16, C-4, C-6, C-10
C-10, C-5, C-8, C-7
H-11a, H-11b
H-11b, H-9
H-11a, H-9
H-11a, H-11b
H-15a, H-15b
H-14
C-20, C-10, C-8
C-20, C-22, C-10, C-9, C-8,
C-12, C-13
C-9, C-22
C-12
C-16, C-13, C-14
-
C-12, C-13, C-14
C-8, C-9, C-7
C-19, C-4, C-3
C-4, C-3
C-10, C-1, C-9, C-5
C-21
HMBC
(1H-13C)
C-3, C-20
C-3
C-2, C-4
5
56
4.1.8
4-Benzoyloxymethyl-3-oxabicyclo[4.1.0]heptane-1,5,6,7-tetrol
(3-Debenzoylrotepoxide A) (132)
Purification of fraction 18 from RP-VLC of MeOH extract yielded compound
(132) (10.0 mg, 0.07%), as a pale brown gum. The IR (Figure 4.7) exhibited a broad
band at 3414 cm-1 for hydroxyl groups.
O
2'
4'
6'
7
O
O
OH
2
1
3
6 5 4
OH
OH
OH
(132)
The presence of hydroxyl groups was supported by the 1H NMR (Figure 4.8)
which displayed a broad singlet at δ 2.34 for four hydroxyl protons. The 1H NMR
(Figure 4.8) showed signals for a benzoate group at δ 7.47-8.05 and an AB quartet at
δ 4.63 and 4.50 (J = 12.0 Hz) was attributable to H-7. Two doublets resonated at δ
4.29 (J = 5.0 Hz) and 3.76 (J = 3.2 Hz) were assigned to H-2 and H-6, respectively.
Three doublet of doublets appeared at δ 4.01 (J = 5.0 and 1.6 Hz), 4.13 (J = 6.0 and
1.6 Hz), 4.38 (J = 6.0 and 3.2 Hz) were assigned to H-3, H-4 and H-5, respectively.
The assignment of protons in the epoxide ring was supported by 1H-1H COSY
spectrum (Figure 4.9), which showed the signal of H-5 was coupled with H-6 and H4. In addition, proton H-3 was also coupling with H-4 and H-2.
Fourteen carbon atoms were observed in the 13C NMR (Figure 4.10) which
were further classified by DEPT (Figure 4.11) as two quaternary at δ 63.14 (C-1)
and 130.06 (C-1'), five oxygenated methines at δ 72.50 (C-3), 69.33 (C-5), 69.09
(C-2), 62.90 (C-4) and 61.37 (C-6), five CH aromatic carbons at δ 129.78 (C-2', C-6'),
128.55 (C-3', C-5'), 133.51 (C-4'), a methylene at δ 64.05 (C-7) and a carbonyl at δ
166.10. The presence of fourteen carbon atoms in tetrol (132) was further supported
by the CIMS spectrum (Figure 4.12) which exhibited the [M+1]+ peak at m/z 297
corresponding to a the molecular formula of C14H16O7.
57
O
2'
25
4'
20
6'
7
O
O
2
1
3
6 5 4
876.62
OH
OH
OH
1215.40
15
929.00
1027.35
1118.20
1046.82
1273.72
OH
669.21
30
849.48
35
1071.26
40
1316.34
45
3019.18
%T
50
1477.12
3413.81
55
1451.65
60
1424.71
1602.32
1720.75
65
1631.40
2399.98
2976.20
70
3683.11
75
1524.04
80
10
5
0
4000
3500
3000
2500
2000
1500
1000
500
Wavenumbers (cm-1)
57
Figure 4.7: IR Spectrum of 3-Debenzoylrotepoxide A (132)
58
H-7
H-5
H-2
H-4
H-3
H-6
Phenyl protons
O
2'
4'
6'
7
O
O
OH
2
1
3
6 5 4
OH
OH
OH
OH
Figure 4.8: 1H NMR Spectrum of 3-Debenzoylrotepoxide A (132)
58
59
H-7
H-6
H-2
O
2'
4'
6'
7
O
O
H-5
OH
2
1
3
6 5 4
H-4 H-3
OH
OH
OH
H-6
H-3
H-4
H-2
H-5
H-7
Figure 4.9: 1H-1H COSY Spectrum of 3-Debenzoylrotepoxide A (132)
59
60
C-3', 5'
C-2', 6'
C-2
C-7
C-5
C-6
C-1 C-4
C-1'
C-4'
C-3
O
2'
C=O (OBz)
4'
6'
7
O
O
OH
2
1
3
6 5 4
OH
OH
OH
60
Figure 4.10: 13C NMR Spectrum of 3-Debenzoylrotepoxide A (132)
61
C-1
C-1'
All carbons
O
2'
4'
C=O (OBz)
6'
7
O
O
OH
2
1
3
6 5 4
OH
OH
OH
C-2', 6'
DEPT 45
C-3', 5'
C-4'
C-2
C-5
DEPT 90
C-3
C-4
C-6
DEPT 135
C-7
Figure 4.11: 13C NMR and DEPT Spectra of 3-Debenzoylrotepoxide A (132)
61
62
100
90
80
%T
70
60
50
O
2'
40
4'
6'
OH
7
O
O
2
1
3
6 5 4
30
OH
OH
OH
20
10
(M+1)+
0
Figure 4.12: CIMS Spectrum of 3-Debenzoylrotepoxide A (132)
62
63
The 1H and
13
C NMR spectra (Figures 4.8 and 4.10) were similar to those
known rotepoxide A (59) [32], except the lack of a benzoate group at position 3. The
C-3 and H-3 peaks were more shielded to δ 72.50 and 4.01 respectively and this was
in agreement with the C-3 to be a hydroxylated methine. The complete assignments
of all protons and carbons are summarized in Table 4.4.
The relative stereochemistry was proposed based on comparison with the
multiplicity and coupling constant values between H-2 and H-3, H-3 and H-4, H-4
and H-5, H-5 and H-6 in tetrol (132) and rotepoxide A (59) [32]. The J2,3 was 5.0 Hz
indicated trans orientation between H-2 and H-3. Similarly, the configuration of 1,6epoxide ring was proposed as same face, either (α,α) or (β,β) based on the value of
J5,6 (3.2 Hz) [32].
(59)
Compound (132) is a new compound, assigned as 4-benzoyloxymethyl-3oxabicyclo[4.1.0]heptane-1,5,6,7-tetrol or trivially named as 3-debenzoylrotepoxide
A based on the 1D, 2D NMR, CIMS and IR data. The convention and the IUPAC
numbering system are shown in (132) and (132*) respectively.
O
2'
4'
6'
OH
7
O
O
2
1
3
6 5 4
O
2'
OH
OH
4'
6'
OH
O
3O
OH
(132)
4 56
2
7
1
OH
(132*)
OH
OH
64
Table 4.4: 1H, 13C NMR, COSY and HMBC Data of Compound (132)
Position
13
C NMR
δ (ppm)
1
H NMR
δ (ppm) (int., mult., J)
1
2
3
4
5
6
7
63.14
69.09
72.50
62.90
69.33
61.37
64.05
1'
2', 6'
130.06
129.78
4.29 (1H, d, J = 5.0 Hz)
4.01 (1H, dd, J = 5.0 and 1.6 Hz)
4.13 (1H, dd, J = 6.0 and 1.6 Hz)
4.38 (1H, dd, J = 6.0 and 3.2 Hz)
3.76 (1H, d, J = 3.2 Hz)
a) 4.63 (1H, d, J = 12.0 Hz)
b) 4.50 (1H, d, J = 12.0 Hz)
8.05 (2H, d, J = 7.6 Hz)
3', 5'
4'
C=O
128.55
133.51
166.10
7.47 (2H, t, J = 7.6 Hz)
7.60 (1H, t, J = 7.6 Hz)
-
4.2
COSY
(1H-1H)
HMBC
(1H-13C)
H-3
H-2, H-4
H-3, H-5
H-4, H-6
H-5
H-7b
H-7a
H-3', H-5'
H-2', H-6'
H-3', H-5'
-
C-1, C-3
C-2, C-5
C-5
C-4
C-1, C-2
C-6, C-2,
C-1, C=O
C-2', C-6',
C-1', C-4'
C=O
C-2', C-6'
C-2', C-6'
-
Phytochemical Study of Indonesian Kaempferia rotunda
Soxhlet extraction of the dried rhizomes of Kaempferia rotunda (898.0 g)
cultivated in Indonesia (purchased from Larkin market) by using CHCl3 and EtOAc
for 18 hours successively afforded brown semisolids of chloroform extract (19.2 g,
2.14 %) and brown liquid of ethyl acetate extract (7.6 g, 0.85%).
The TLC profiles of these crude extracts of Indonesian sample were not
identical with the crude extracts of Malaysian sample.
However, some of the
compounds present in the Malaysian sample were also found in the chloroform
extract of Indonesian sample. Two compounds identified as 4-benzoyloxymethyl3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diol 5-acetate (57) and 1,6-desoxypipoxide
(69) were detected in the chloroform extract. The identification of these compounds
was done by comparing the TLC profiles of crude extract and the isolated
compounds.
65
Washing of the solids in chloroform extract with excessive Et2O gave
crotepoxide (54). Fractionation and purification of the chloroform extract residue by
VLC technique and repeated gravity column chromatography (CC) or chromatotron
or recystallization technique gave a new compound and three known compounds.
The structure of the new compound was elucidated spectroscopically, as 3-acetoxy2-benzoyloxy-1-(benzoyloxymethyl)cyclohexa-4,6-diene (136), while the known
compounds were identified as benzyl benzoate (82) (major compound), transdocosyl ferulate (137) and 6-acetylzeylenol (68). Fractionation on the ethyl acetate
extract by VLC technique yielded an acid, identified as benzoic acid (138). This is
the first report of the isolation and identification of trans-docosyl ferulate (137) and
benzoic acid (138) from Kaempferia species.
4.2.1 Crotepoxide (54)
The solids (1.50 g, 7.81%) obtained from chloroform extract was exactly
identical to that of crotepoxide (54) previously isolated from the Malaysian
Kamempferia rotunda by TLC, IR and 1H NMR as well as its melting point. Based
on these data, this compound was identified as crotepoxide (54).
4.2.2 Benzyl Benzoate (82)
Compound (82) was isolated from the fractionation of chloroform extract by
VLC, as colourless oil (2.25 g, 11.7%). The 1H NMR (Appendix 45) displayed the
presence of oxymethylene protons, H-7 as a singlet at δ 5.42 and two phenyl groups
of protons at δ 7.45 (m, H-2, H-3, H-4, H-5, H-6, H-3' and H-5'), 7.59 (br t, J = 7.3
Hz, H-4') and 8.14 (d, J = 7.6 Hz, H-2' and H-6') [73].
66
(82)
The IR (Appendix 46) disclosed the absorption band due to the carbonyl and
C-O groups at 1718 cm-1 and 1272 cm-1 respectively. The
13
C and DEPT spectra
(Appendices 47 and 48) showed fourteen carbon atoms consistent with a carbonyl at
δ 166.44, ten CH aromatic carbons at δ 133.06 (C-4'), 129.75 (C2', C-6'), 128.64,
128.42, 128.28, 128.20 (C-2, C-3, C-4, C-5, C-6, C-3', C-5'), two quaternary carbons
at δ 136.14 (C-1'), 130.22 (C-1) and a methylene carbon at δ 66.72 (C-7). The
EIMS spectrum (Appendix 49) supported the presence of fourteen carbons which
gave a molecular ion peak [M+] at m/z 212, corresponding to a molecular formula
C14H12O2.
Therefore, ester (82) was characterized as benzyl benzoate, based on its
spectral evidence and comparison with literature [73]. This compound has also been
isolated from Kaemphefria rotunda and Uvaria purpurea [37, 74].
4.2.3 trans-Docosyl ferulate (137)
Purification of fractions 13 to 14 resulted from the VLC chloroform extract
by CC, followed by removal of acid gave compound (137) (18.6 mg, 0.10%) as pale
yellow gum. The 13C NMR (Appendix 50) showed thirty two carbons. The DEPT
spectrum (Appendix 51) revealed the presence of three CH aromatic at δ 123.01
(C-6), 114.71 (C-5), 109.33 (C-2), three quaternary at δ 147.91 (C-3), 146.77 (C-4),
127.06 (C-1), a methoxyl at δ 55.92, two olefinic carbons at δ 144.60 (C-7), 115.70
(C-8) and a carbonyl at δ 167.36 (C-9) were assigned to the feruloyl moiety; while
the remaining carbons, twenty one methylenes at δ 64.00 (C-1'), 31.91 (C-n-2'),
29.68, 29.64, 29.58, 29.53, 29.34, 25.99 (C-4' to C-n-3'), 29.29 (C-2'), 28.77 (C-3'),
22.67 (C-n-1'), and a methyl at δ 14.08 (C-22') were assigned to the aliphatic alcohol
moiety [75].
67
(137)
The IR spectrum (Appendix 52) supported the existence of carbonyl which
showed absorption bands at 3434 cm-1, 1674 cm-1 and 1268 cm-1 that were indicative
of hydroxyl and ester groups, respectively.
The 1H NMR (Appendix 53) showed the characteristic of feruloyl moiety,
which displayed four doublets at δ 7.62 (J = 16.0 Hz, H-7) and 6.30 (J = 16.0 Hz,
H-8), 6.92 (J = 8.4 Hz, H-5), 7.08 (J = 8.4 Hz, H-6), a broad singlet at δ 7.05 (H-2)
and a methoxyl group resonated as a singlet at 3.94. The coupling constant between
H-7 and H-8, suggested the trans-geometry [75]. Long chain alcoholic residue was
observed at δ 4.20 (t, J = 6.8 Hz), 1.70 (m), 1.26 (br. s) and. 0.89 (t, J = 6.0 Hz).
Correlation between the olefinic protons (H-7 and H-8) was observed in the
1
1
H- H COSY spectrum (Appendix 54). Long range correlations in the HMBC
spectrum (Appendix 55) supported the connection between the aliphatic alcohol
moiety and feruloyl acid moiety in the ester (137). In addition, the connectivity of
the methoxyl at δ 3.94 with C-3 at δ 147.91 confirmed the position of methoxyl
group at C-3. The complete NMR data for 1H and
13
C spectra are shown in Table
4.5.
The structure was further confirmed by EIMS (Appendix 56) which gave a
molecular ion peak at m/z 502 [M]+ attributed to the molecular formula C32H54O4,
hence suggesting that compound (137) to be C24 alkyl ferulate [75]. The peak at m/z
194 was attributed to the methylcaffeic acid (139) [75] which was formed from the
intramolecular rearrangement, as shown in the Scheme 4.2, supported the existence
of feruloyl moiety.
68
Scheme 4.2: The Suggested Mass Fragmentation Pattern of Compound (137)
Based on the physical properties and comparison of its spectroscopic data
with compound previously isolated from the stem barks of Pavetta Owariensis [75],
compound (137) was identified as trans-docosyl ferulate. This is the first report on
the isolation of trans-docosyl ferulate (137) from Kaempferia rotunda and also the
genus of Kaempferia.
Table 4.5: 1H, 13C NMR, COSY and HMBC Data of Compound (137)
Position
13
C NMR
δ (ppm)
1
H NMR
δ (ppm) (int., mult., J)
COSY
(1H-1H)
1
2
3
4
5
6
7
127.06
109.33
147.91
146.77
114.71
123.01
144.60
7.05 (1H, br. s)
6.92 (1H, br. d, J = 8.4 Hz)
7.08 (1H, br. d, J = 8.4 Hz)
7.62 (1H, d, J = 16.0 Hz)
8
9
1'
2'
3'
4' to n-3'
115.70
167.36
64.00
29.29
28.77
29.68,
29.64,
29.58,
29.53,
29.34,
25.99
31.91
22.67
14.08
55.92
6.30 (1H, d, J = 16.0 Hz)
4.20 (2H, t, J = 6.8 Hz)
1.70 (2H, m)
1.70 (2H, m)
1.26 (32H, br. s)
H-7
1.26 (2H, br. s)
1.26 (2H, br. s)
0.89 (3H, t, J = 6.0 Hz)
3.94 (3H, s)
CH3
H-n-1'
-
n-2'
n-1'
CH3
OCH3
HMBC
(1H-13C)
C-3, C-4,C-6, C-7
H-6
H-5
H-8
H-2'
H-1'
-
C-1, C-3, C-4
C-2, C-3, C-4, C-7
C-1, C-2, C-6, C-8,
C-9
C-1, C-9
C-2', C-3', C-4', C-9
C-1', C-2', C-3', C-4'
C-1', C-2', C-3', C-4'
C-n-1',C-n-2',
C-n-3', C-4'
C-n-1', C-n-2'
C-3
69
4.2.4 3-Acetoxy-2-benzoyloxy-1-(benzoyloxymethyl)cyclohexa-4,6-diene (136)
Compound (136) (6.5 mg, 0.34%) was obtained as brown gum from repeated
CC of fraction 15 resulted from VLC of the chloroform extract. The 1H NMR
spectrum (Figure 4.13) indicated the presence of an acetoxyl group at δ 2.09 (s), two
benzoate groups at δ 8.06 (4H, m), 7.58 (2H, m) and 7.46 (4H, m) and oxymethylene
protons at δ 4.90 (br. s) [76]. Two broad singlet integrating for two protons at δ 5.88
was assigned to methine protons, H-2 and H-3. Three vinyl protons were observed
as a broad singlet at δ 6.00, a doublet of doublet at δ 6.10 (J = 9.6 and 2.4 Hz) and a
doublet at δ 6.19 (J = 9.6 Hz), attributed to H-4, H-5 and H-6, respectively. The
correlations between the vinyl protons were observed in the COSY spectrum (Figure
4.14), which showed that H-5 at δ 6.10 was coupled with H-4 at δ 6.00 and H-6 at δ
6.19.
(136)
The IR spectrum (Figure 4.15) displayed the carbonyl absorption band at
1720 cm-1 and C-O stretching at 1268 cm-1. The 13C NMR (Figure 4.16) supported
the presence of carbonyls at δ 165.77, 166.14 and 170.17. The remaining twenty one
carbons corresponding to an acetoxyl carbon at δ 19.39, a methylene carbon at δ
65.29 (C-7), two methine carbons at δ 74.93 (C-2) and 71.26 (C-3), three olefinic
carbons at δ 126.27 (C-5), 125.83 (C-6) and 122.07 (C-4), three quaternary carbons δ
133.27 (C-1) and 129.75 (C-1', 1'') and ten CH aromatic carbons at δ 133.27 (C-4''),
133.24 (C-4'), 129.82 (C-2', 6'), 129.72 (C-2'', 6''), 128.46,(C-3'', 5''), and 128.43
(C-3', 5') were observed in the DEPT spectrum (Figure 4.17).
The complete
assignments of the NMR data of diene (136) are tabulated in Table 4.6.
70
Table 4.6: 1H, 13C NMR, COSY and HMBC Data of Compound (136)
Position
13
C NMR
δ (ppm)
1
H NMR
δ (ppm) (int., mult., J)
COSY
(1H-1H)
1
2
3
4
5
133.27
71.93
71.26
122.07
126.27
6
125.83
5.88 (1H, br s)
5.88 (1H, br s)
6.00 (1H, br s)
6.10 (1H, dd, J = 9.6
and 2.4 Hz)
6.19 (1H, d, J = 9.6 Hz)
H-3, H-4, H-5
H-2, H-4, H-5
H-2, H-3, H-5
H-2, H-3, H-4,
H-6
H-5
7
65.29
4.90 (2H, br s)
-
1' , 1''
2', 6'
129.75
129.82
8.06 (2H, m)
H-3', H-5'
3', 5'
128.43
7.46 (2H, m)
H-2', H-6', H-4'
4'
133.24
7.58 (1H, m)
H-3', H-5'
2'', 6''
129.72
8.06 (2H, m)
H-3'', H-5''
3'', 5''
128.46
7.46 (2H, m)
4''
133.27
7.58 (1H, m)
H-2'', H-6'',
H-4''
H-3'', H-5''
C=O (OBz)
C=O (OBz)
C=O (OAc)
CH3 (OAc)
165.77
166.14
170.17
19.39
2.09 (3H, s)
-
HMBC
(1H-13C)
C-3, C-6
C-2, C-4, C-5
C-5, C-6, C-7
C-1, C-2, C-3,
C-4, C-6
C-1, C-2, C-3,
C-4, C-7
C-1, C-4, C-5,
C-6, OBz
C-2', 6';
C-3', 5'; C-4'
C-2', 6';
C-3', 5'
C-2', 6';
C-3', 5'
C-2'', 6'';
C-3'', 5'';
C-4''
C-2'', 6'';
C-3'', 5''
C-2'', 6'';
C-3'', 5''
OAc
71
Although the 1H and 13C NMR spectra (Figure 4.13 and 4.16) of diene (136)
resemble those of 1,6-desoxytingtanoxide (140) [76], the chemical shifts of H-2 was
found to be shifted to downfield (δ 5.88) while H-3 was upfiled shifted (δ 5.88).
These were suggested that H-2 was benzoylated and H-3 was acylated. The relative
stereochemistry for diene (136) was proposed to be in a trans orientation, based on
the
naturally
occurring
compounds,
1,6-desoxypipoxide
(69)
and
1,6-
desoxytingtanoxide (140) [16, 76].
O
O
O
(136)
2
O
O
3
O
(140)
Diene (136) had a molecular formula of C23H20O6 as shown in the CIMS
spectrum (Figure 4.18), with an [M]+ at m/z 392.
Based on its spectral data,
compound (136) was identified as a new compound, 3-acetoxy-2-benzoyloxy-1(benzoyloxymethyl)cyclohexa-4,6-diene.
72
H-2, 3
H-6
H-5
CH3 (OAc)
H-4
4''
2''
6''
O
1'
3'
O
7
O
3
1
O
O
O
Phenyl protons
5'
5
H-7
Figure 4.13: The 1H NMR Spectrum of 3-Acetoxy-2-benzoyloxy-1-(benzoyloxymethyl)cyclohexa-4,6-diene (136)
72
73
H-6
H-5
H-4
H-7
H-2, 3
H-7
4''
2''
6''
O
H-2, 3
H-4
H-5
H-6
1'
3'
O
7
O
3
1
O
O
O
5'
5
Figure 4.14: The COSY Spectrum of 3-Acetoxy-2-benzoyloxy-1-(benzoyloxymethyl)cyclohexa-4,6-diene (136)
73
74
4''
2''
6''
O
1'
3'
O
7
O
3
1
O
O
O
5'
5
Figure 4.15: The IR Spectrum of 3-Acetoxy-2-benzoyloxy-1-(benzoyloxymethyl)cyclohexa-4,6-diene (136)
74
75
C-3′, 5′
C-2′′, 6′′
C-3′′, 5′′
C-4′
C-5
C-6
C-4
4''
C-2′, 6′
C-4′′
C-1
2''
6''
C-1′
C-1′′
O
1'
3'
O
7
O
3
1
O
O
O
5'
5
C-2
CH3 (OAc)
C-3
C-7
C=O
(OAc)
C=O
(OBz)
Figure 4.16: The 13C NMR Spectrum of 3-Acetoxy-2-benzoyloxy-1-(benzoyloxymethyl)cyclohexa-4,6-diene (136)
75
76
4''
2''
6''
All carbons
O
3'
C=O C=O
(OAc) (OBz)
DEPT 45
C-4''
C-4'
C-3', 5' C-2', 6'
C-3'', 5'' C-2'', 6''
1'
O
7
O
3
1
O
O
O
5'
C-5
C-6
DEPT 90
5
C-4
C-2 C-3
C-4
DEPT 135
CH3 (OAc)
C-7
Figure 4.17: The 13C NMR and DEPT Spectra of 3-Acetoxy-2-benzoyloxy-1-(benzoyloxymethyl)-cyclohexa-4,6-diene (136)
76
77
100
90
80
4''
70
2''
%T
6''
60
50
O
1'
3'
O
7
O
3
1
O
O
O
40
5'
5
30
20
10
M+
0
Figure 4.18: The EIMS Spectrum of 3-Acetoxy-2-benzoyloxy-1-(benzoyloxymethyl)-cyclohexa-4,6-diene (136)
77
78
4.2.5 6-Acetylzeylenol (68)
Purification of polar fraction of the chloroform extract by chromatotron,
followed by recrystallization gave compound (68) (7.0 mg, 0.36%) as a white solids.
The IR spectrum (Appendix 57) displayed C=O stretching appeared at 1715 cm-1,
while C-O stretching was observed at 1272 cm-1. A broad absorption band at 3425
cm-1 in IR spectrum (Appendix 57) was corresponded to O-H stretching.
4'
2'
6'
HO
O
6
O
7
1
5
4
2
3
O
O
OH
4''
2''
O
O
6''
(68)
The 1H NMR spectrum (Appendix 58) showed the presence of hydroxyl
protons at δ 3.28 (d, J = 4.8 Hz), 3.42 (s) that exchangeable with D2O and an
acetoxyl group at δ 2.11. A multiplet in the downfield region interpreting for ten
protons at δ 8.05-7.42 was attributed to the benzoyl protons. Two methines, H-6 and
H-3 appeared as a doublet resonated at δ 5.55 (J = 2.4 Hz) and 5.74 (J = 6.0 Hz)
respectively, suggesting these protons were bonded on the carbon bearing the ester
groups. Another methine proton was observed as doublet of doublet at δ 4.24
(J = 4.8 and 6.0 Hz) was matched to H-2. A doublet integrating for two protons at δ
5.98 (J = 2.4 Hz) was assigned to olefinic protons, H-4 and H-5.
The CIMS spectrum (Appendix 59) exhibited a molecular ion peak [M+1]+
at m/z 427 which was in agreement with a molecular formula C23H22O8. This was
supported by the 13C NMR and DEPT spectra (Appendices 60 and 61) that showed
the presence of twenty three carbons identified as an acetoxyl carbon at δ 20.94 (CH3,
OAc), three methine carbons at δ 73.59 (C-3), 71.49 (C-2), 70.54 (C-6), two olefinic
carbons at δ 128.38 (C-4), 126.59 (C-5), three quaternary carbons at δ 129.35 (C-1''),
129.25 (C-1'), 74.72 (C-1), a methylene carbon at δ 66.56 (C-7), ten CH aromatic
79
carbons at δ 133.48 (C-4''), 133.34 (C-4'), 129.84 (C-2'', C-6''), 129.67 (C-2', C-6'),
128.45 (C-3'', C-5'', C-3', C-5'), and three carbonyl carbons at δ 170.13 (OAc),
167.07 (OBz), 167.04 (OBz).
The assignments of the protons in compound (68) were established by 1H-1H
COSY spectrum (Appendix 62). The HMBC spectrum (Appendix 63) confirmed
the positions of the benzoate and acetoxyl groups, which showed long range
connectivity from H-3 at δ 5.74 to the benzoate carbonyl and H-6 at δ 5.55 to the
acetoxyl carbonyl.
The complete 1H and
13
C NMR data together with the
correlations of the protons are shown in Table 4.7.
Therefore, compound (68) was identified as 6-acetylzeylenol based on
comparison of its physical properties and spectral data of compound previously
isolated from the rhizomes of Kaempferia rotunda of Thailand [32].
Table 4.7: 1H, 13C NMR, COSY and HMBC Data of Compound (68)
Position
13
C NMR
δ (ppm)
1
H NMR
δ (ppm) (int., mult., J)
COSY
(1H-1H)
HMBC
(1H-13C)
OH (C-2), H-3
C-1
H-2, H-4
H-6
H-6
H-4, H-5
H-7a
H-7b
H-3',5'
1
2
74.72
71.49
3
4
5
6
7
73.59
128.38
126.59
70.54
66.56
1'
2', 6'
129.25
129.67
4.24 (1H, dd, J = 4.8,
6.0 Hz)
5.74 (1H, d, J = 6.0 Hz)
5.98 (1H, d, J = 2.4 Hz)
5.98 (1H, d, J = 2.4 Hz)
5.55 (1H, d, J = 2.4 Hz)
4.59 (1H, d, J = 12.0 Hz)
4.81 (1H, d, J = 12.0 Hz)
8.00 (2H, d, J = 7.2 Hz)
3', 5'
128.45
7.42 (2H, m)
H-2',6', H-4'
4'
1''
2'', 6''
3'', 5''
4''
133.34
129.35
129.84
128.45
133.48
7.57 (1H, m)
8.05 (2H, d, J = 7.2 Hz)
7.42 (2H, m)
7.57 (1H, m)
H-3',5'
H-3'',5''
H-2'',6''
H-3'', 5'', H-4''
C-1', C-3', 5', C-2', 6'
OH (C-1)
OH (C-2)
C=O (OBz)
C=O (OBz)
C=O (OAc)
CH3 (OAc)
167.04
167.07
170.13
20.94
3.42 (1H, s)
3.28 (1H, d, J = 4.8 Hz)
2.11 (3H, s)
H-2
-
C-6
OAc
C-2, C-4, C-5, OBz
C-2, C-3, C-6
C-2, C-3, C-6
C-1, C-4, C-5, OAc
C-1, C-2, C-6, OBz
C-1', C-3', 5',
C-4' OBz
C-1', C-2', 6'
C-1'', C-4'', C-3'', 5'', OBz
C-1'', C-2'', 6''
C-1'', C-3'', 5'', C-2'', 6''
80
4.2.6 Benzoic Acid (138)
Compound (138) (0.48 g, 6.26%) was isolated as colourless crystals, m.p.
121°C (lit. [77], 122°C) was isolated from the fractionation of ethyl acetate extract
by VLC. The IR spectrum (Appendix 64) showed a broad absorption band of O-H
in the region of 3600-2800 cm-1 along with the C=O stretching band at 1693 cm-1 and
absorption for C-O (1215 cm-1) group, indicated that compound (138) was a
carboxylic acid.
(138)
1
The H NMR spectrum (Appendix 65) showed signals resonated at δ 7.488.14 integrating five protons was ascribed to the benzoate group protons. The
13
C
NMR (Appendix 66) showed six carbons at δ 133.75, 130.19, 130.02, 128.06,
127.90, 128.47 and a carbonyl acid at 171.96. On the basis of these spectral data [78]
and comparison with an authentic sample, the structure of compound (138) was
identified as benzoic acid. This compound has also been isolated from Bramley's
Seedling apple fruits [79].
81
4.3
The Distribution Compounds in Malaysian and Indonesian Kaempferia
rotunda
Phytochemicals study on the rhizomes of Kaempferia rotunda cultivated in
Kempas, Johor and Indonesia revealed the presence of cyclohexane oxides, chalcone,
flavanone, labdane diterpene, esters and acid. This study was in agreement with the
previous report that cyclohexane oxides are the characteristic components in the
rhizomes of K. rotunda, where three cyclohexane oxides, crotepoxide (54), 4benzoyloxymethyl-3,8-dioxatricyclo-[5.1.0.02,4]octane-5,6-diol 5-acetate (57) and
1,6-desoxypipoxide (69) were found in both samples. Therefore, these compounds
can be used as the chemical marker for the identification of K. rotunda species.
However, the significant differences between both samples were the
occurrence of their main components. The Indonesian rhizomes was dominated by
benzyl benzoate (82) and benzoic acid (138) were found to be absent in the rhizomes
of Malaysia. In the Malaysian sample, crotepoxide (54) was only present in 1.55 %
(0.036 g) compared to 7.8% (1.50 g) in the Indonesian sample.
A clear difference of compounds, especially chalcone (1), flavanone (133)
and labdane diterpene (134) were present in the rhizomes cultivated in Malaysia.
While,
trans-docosyl
ferulate
(137),
3-acetoxy-2-benzoyloxy-1-
(benzoyloxymethyl)cyclohexa-4,6-diene (136) and 6-acetylzeylenol (68) were only
isolated from the Indonesian rhizomes. For the comparison purpose, the compounds
isolated from both Malaysian and Indonesian Kaempferia rotunda are tabulated in
Table 4.8.
82
Table 4.8: Compounds Isolated from Malaysian and Indonesian Kaempferia
rotunda
COMPOUNDS
Crotepoxide (54)
Kaempferia rotunda
Malaysian
Indonesian
+
+
2'-Hydroxy-4,4',6'-trimethoxychalcone (1)
+
-
4-Benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]-
+
+
1,6-Desoxypipoxide (69)
+
+
2-(Benzoyloxymethyl)phenyl (3-O-acetyl)-β-
+
-
3-Debenzoylrotepoxide A (132)
+
-
Naringenin 4',7-dimethyl ether (133)
+
-
Curcumrinol C (134)
+
-
Benzyl benzoate (82)
-
+
trans-Docosyl ferulate (137)
-
+
3-Acetoxy-2-benzoyloxy-1-(benzoyloxymethyl)-
-
+
6-Acetylzeylenol (68)
-
+
Benzoic acid (138)
-
+
octane-5,6-diol 5-acetate (57)
glucopyranoside (131)
cyclohexa-4,6-diene (136)
Notes:
+ = Present
- = Absent
4.4
Bioactivity Studies on Kampferia rotunda
Ginger plants have been claimed in traditional medicine for the treatment of
some diseases. In this present study, the crude extracts and the isolated compound
from Malaysian and Indonesian Kaempferia rotunda were screened for their
antibacterial and antioxidant activities. To the best of our knowledge there have
been no antibacterial and antioxidant studies on the essential oils and the crude
extracts of K. rotunda.
83
4.4.1 Antibacterial Activity
Bacteria are single cell microorganisms that are present in all ecosystems on
earth.
Many epidemics and scores of human have been caused by pathogenic
bacteria. Most bacteria can be divided into two groups, either Gram negative or
Gram positive based on the composition of the cell wall [80].
Staphylococcus aureus and Bacillus subtilis are two common Gram positive
bacteria. S. aureus produces some components that contribute to the pathogenesis of
infection; for example enzymes that cause coagulation of the blood, tissues wound
infections, oseteomyelitis and food poisoning. In addition, B. subtilis is the most
frequent Bacillius spp. to cause invasive infection. It causes pneumonia,
meningoencephalitis, endocarditis (native and prosthetic valves), and intravascular
catheter infection [80-81].
Pseudomonas aeruginosa and Escherichia coli are two representative Gram
negative bacteria. P. aeruginosa is the most dreaded and is capable of expelling
dangerous substances into the infected host. Infections caused organism
immunocompromised, burn, wound, respiratory tract, and auditory canal, while
Escherichia coli can cause urinary tract infection and intestinal disorder [80-81].
Secondary metabolites produced by plants, the essential oils and extracts of
various species of edible and medicinal plants, herbs and spices constitute of very
potent natural sources of antibacterial agents.
Majority of antibacterial drugs
marketed today are chemically modified from natural products.
The antibacterial activity of the crude extracts, essential oils and isolated
compound of Malaysian and Indonesian Kaempferia rotunda were determined
qualitatively by disc diffusion method [82]. To the best of our knowledge there has
84
been no antibacterial study on the essential oils and the crude extracts of this species.
Therefore, this is the first study on the antibacterial studies of K. rotunda.
The bacteria used in this assay were Gram-positive bacteria, Staphylococcus
aureus and Bacillus subtilis and Gram-negative bacteria, Pseudomonas aeruginosa
and Escherichia coli. The positive control (Streptomysin sulphate) exhibited the
inhibition zones of 20.0 - 20.2 mm for all cultured bacteria. Six crude extracts, two
essential oils and benzoic acid (137) did not exhibited inhibition zone thus not active
against all the tested bacteria.
4.4.2 Antioxidant Activity
Free radicals in the form of reactive oxygen and nitrogen species are an
integral part of human physiology. An over-production of free radical will due to
oxidative stress, the imbalance on the antioxidant defense system and free radical
formation. These reactive species can react with biomolecules cause cellular injury
and death. This may lead to the development of chronic diseases such as cancers and
those that involve the cardio- and cerebrovascular systems [83].
The harmful action of the free radicals can be blocked by antioxidant
substances which scavenge the free radicals and detoxify the organism [84].
Antioxidants are the chemical substances that reduce or prevent oxidation. It has
been claimed have ability to counteract the damaging effects of free radicals in
tissues [85]. Current research on free radicals has confirmed antioxidants play an
essential role in the prevention against cancer [86], arteriosclerosis, heart disease [85]
and neurodegenerative diseases, including Parkinson’s and Alzheimer’s diseases [86]
as well as inflammation [88] and problems caused by cell and degenerative aging
[89].
Plants are a source of compounds with antioxidant activity such as phenolic
acids, flavonoids, anthocyanins, tannins and carotenoids that may be used as
85
pharmacologically active products. These naturally occurring antioxidants can be
formulated to give neutraceuticals that can help to prevent oxidative damage from
occurring in the body [83].
2,2-Diphenyl-1-picrylhydrazyl radical (DPPH•) assay is a well known
method for the evaluation of free radical scavenging activity. This method is rapid,
simple and reproducible [90]. The DPPH• radical was observed as a purple colour
and gave a strong absorption at 517 nm. The colour will turn from purple to yellow
when the odd electron of DPPH• radical becomes paired with hydrogen from
antioxidant compound [91].
In this study, the radical scavenging activities on the six crude extracts, two
essential oils were determined by the free radical scavenging method (DPPH).
Vitamin C and 5-deoxyquercetin were used as the references antioxidant. Figure
4.19 showed the percentage of scavenging (S) by Vitamin C, 5-deoxyquercetin,
crude extracts and essential oils. The scavenging concentration (SC) to obtain 50%
of maximum scavenging capacity of DPPH by Vitamin C and 5-deoxyquercetin were
30.30 μg/mL and 11.65 μg/mL respectively. All crude extracts and the essential oils
showed no ability to act as free radical scavengers thus did not possessed antioxidant
activity.
86
100
90
80
70
60
50
40
30
20
10
0
-10
-20
-30
-40
-50
S (%)
Vitamin C
5-Deoxyquercetin
KRH
KREA
KRM
KRC2
KREA2
KRM2
EO1
EO2
0
100 200 300 400 500 600 700 800 900 1000 1100
Concentration (µg/mL)
Note:
KRH = n-Hexane extract of Malaysian K. rotunda
KREA = Ethyl acetate extract of Malaysian K. rotunda
KRM = Methanol extract of Malaysian K. rotunda
KRC2 = Chloroform extract of Indonesian K. rotunda
KREA = Ethyl acetate extract of Indonesian K. rotunda
KRM2 = Methanol extract of Indonesian K. rotunda
EO1 = Essential oil of Malaysian K. rotunda
EO2 = Essential oil of Indonesian K. rotunda
Figure 4.19: Graft Of Percentage Scavenging Capacity of DPPH by Vitamin C, 5Deoxyquercetin, Crude Extracts and Essential Oils from Kaempferia
Rotunda Measured By UV Spectrometric Assay
CHAPTER 5
EXPERIMENTAL
5.1
General Experimental Procedures
Two extraction techniques were applied in this study. Soxhlet extraction
technique was used to the dried samples, while hydrodistillation technique was
applied to the fresh samples.
Vacuum liquid chromatography (VLC) and gravity column chromatography
(CC) were carried out by using Merck silica gel (230-400 mesh) or LiChroprep RP18 (40-63μm) and Merck silica gel (70-230 mesh) respectively. Versa flash column
chromatography technique was carried out by using 4.0 × 7.5 cm of silica catridge.
Chromatotron (Model 7924T) with 1 mm plate thickness was also used in the
purification of samples. The plate was coated with silica gel 60 PF254 containing
gypsum.
Thin layer chromatography (TLC) were performed on 0.20 mm precoated
silica gel aluminium sheets (Merck Kieselgel 60 F254). The spots on TLC were
detected by ultraviolet, 254 nm and 365 nm illumination before sprayed with
anisaldehyde reagent that prepared with p-anisaldehyde (0.5 mL), MeOH (85 mL),
sulphuric acid (10 mL) and ethanolic glacial acid (0.5 mL).
The 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on
Bruker Avance 400 spectrometer using deuterated solvents CDCl3 or otherwise
stated. Residual solvent was used as an internal standard. Infrared spectra (IR) were
88
recorded on Perkin-Elmer series 1600 spectrophotometer as KBr disc or thin film
(NaCl windows). Gas chromatography (GC) analyses were carried out on Hewlett
Packard HP6890. Ultraviolet (UV) spectra were recorded on Shimadzu UV 1601PC
spectrophotometer. Mass spectral data were obtained from Kent Mass Spectrometry
Service, UK.
Melting points were measured by using melting point apparatus
equipped with microscope, Leica Gallen III and were uncorrected.
The essential oils were analyzed by using GC and GC-MS instruments. The
samples were injected by splitter by using helium gas as a carrier gas. The column
ovens were programmed from 40°C (after 5 min) to 300°C at 4°C/min and the final
temperature was held for 3 min. GC-MS analysis were equipped with Wiley Library
Software. Capillary GC-MS conditions were performed as above except for the oven
temperature program. The oven temperature programmed were 50°C (5 min) with
4°C/min to 300°C (5 min).
5.2
Chemicals
Petroleum ether (60-80°C) was redistilled before used. Distilled petroleum
ether, n-hexane, diethyl ether (Et2O), ethyl acetate (EtOAc), acetone and methanol
were used as solvent system in chromatographic method. Anhydrous MgSO4 was
used to dry the essential oil. A series of standard hydrocarbons (C6-C20) was used in
Kovats Indices analysis. Deuterated solvents used in NMR analysis were purchased
from Merck.
5.3
Plant Materials
Two samples of rhizomes of Kaempferia rotunda were purchased from
Larkin Market, Johor Bharu on the 27th July 2007 and 9th July 2008 respectively.
The first sample cultivated in Kempas, Johor while the second sample was imported
from Indonesia, as shown in the Figure 5.1-5.2.
89
Figure 5.1. Kaempferia rotunda cultivated in Kempas, Johor
Figure 5.2. Kaempferia rotunda imported from Indonesia
5.4
Essential Oil Extraction and Analysis
The fresh rhizomes of Malaysian sample (704.0 g) and Indonesian sample
(800.0 g) were chopped into small size and loaded in the round bottom flask (5 L).
Then, distilled water was added until the water covered the entire sample. The flask
was equipped with a Dean-Stark apparatus together with a water condenser. The
samples were hydrodistilled for 12 hours. The oils were extracted with Et2O (3 x 10
mL) and dried over anhydrous MgSO4. The MgSO4 was filtered and the ether layer
was evaporated at room temperature to yield pale yellow oils of 0.20 g (0.03%) and
1.85 g (0.23 %) respectively.
The essential oils were analysed by using GC (Appendices 1-2) and GC-MS.
The constituents of the essential oils were identified by the mass spectra from the
Wiley Library in the GC-MS and Kovats Indices. The Kovats Index, KI relates the
90
retention of the sample component to the retention of saturated hydrocarbons eluted
before and after the sample. The Kovats Index can be calculated using equation:
⎡ (log tsample ) − (log tx ) ⎤
KI = 100 ⎢
⎥ + 100 x
⎣ (log tx + 1) − (log tx ) ⎦
Where,
tsample = the retention time of the sample component,
tx = the retention time of the saturated hydrocarbons elute before the sample
component,
tx+1 = the retention time of the saturated hydrocarbon which contains x+1 carbon
and that just elutes after the sample component
x = number of carbon atoms in saturated hydrocarbons
5.5
Extraction and Isolation of the Malaysian Kaempheria rotunda
The dried rhizomes of Kaempferia rotunda (205.0 g) were ground into
powder and extracted successively with n-hexane (4.5 L), EtOAc (4.5 L) and MeOH
(4.5 L) by soxhlet extraction for 16 hours. Then, the extracts were concentrated by
using rotary evaporator to give dark brown semisolids of n-hexane crude extract
(2.32 g, 1.13 %), and dark brown oil of EtOAc crude extract (6.80 g, 3.32%) and
dark brown viscous liquid of MeOH crude extract (15.27 g, 7.45%).
5.5.1
Crotepoxide (54)
The solids in the n-hexane extract was filtered and washed with Et2O to yield
crotepoxide (54) (0.036 g, 1.55 %). Colourless needles; Rf = 0.51 (n-hexane: EA =
1: 1); m.p. 143-144°C (lit. [67] 146-148°C); IR υmax cm-1: 2925, 2848, 1766, 1727,
1631, 1450, 1373, 1283, 1236, 1211; 1H NMR (CDCl3): δ 8.03 (2H, d, J = 8.0 Hz,
H-2', H-6'), 7.61 (1H, t, J = 7.6 Hz, H-4'), 7.48 (2H, dd, J = 8.0, 7.6 Hz, H-3', H-5'),
5.71 (1H, d, J = 9.2 Hz, H-2), 5.00 (1H, d, J = 9.2 Hz, H-3), 4.58 (1H, d, J = 12.0 Hz,
H-7a), 4.25 (1H, d, J = 12.0 Hz, H-7b), 3.67 (1H, d, J = 2.4 Hz, H-6), 3.46 (1H, dd, J
91
= 4.0 Hz, 2.4 Hz, H-5), 3.11 (1H, d, J = 4.0 Hz, H-4), 2.14 (3H, s, OAc), 2.04 (3H, s,
OAc); 13C NMR (CDCl3): δ 170.06 (C=O, OAc), 169.75 (C=O, OAc), 165.80 (C=O,
OBz), 133.55 (CH, C-4'), 129.80 (2CH, C-3', C-5'), 129.11 (C, C-1'), 128.56 (2CH,
C-2', C-6'), 70.37 (CH, C-3), 69.42 (CH, C-2), 62.46 (CH2, C-7), 59.38 (C, C-1),
53.81 (CH, C-6), 52.60 (CH, C-4), 48.06 (CH, C-5), 20.66 (CH3, OAc); CIMS m/z
363 [(M+1)+, C18H18O8].
5.5.2
2'-Hydroxy-4,4',6'-trimethoxychalcone (1)
The n-hexane extract residue (2.20 g) was purified by CC (93.0 g, column
size: 4.5 × 45.0 cm) with n-hexane: Et2O (99: 1, 98: 2 and 97: 3) as eluents, to give
20 fractions. Fractions 13 (0.08 g) and 14 (0.02 g) were combined and recrystallized
by using n-hexane: Et2O to get 2'-hydroxy-4,4',6'-trimethoxychalcone (1) (6.9 mg,
0.3 %). Yellow rhombics; Rf = 0.39 (PE: EtO2 = 1: 1); m.p. 109-110°C (lit. [64]
115°C); IR υmax cm-1: 3457, 2924, 2841, 1623, 1582, 1514, 1440, 1347, 1291, 1257,
1221, 11179, 1158, 1116, 825; UV (MeOH) λmax nm: 372.8, 287.2; 1H NMR
(CDCl3): δ 14.43 (1H, s, OH), 7.85 (1H, d, H-β, J = 16.4 Hz), 7.77 (1H, d, H-α, J =
16.4 Hz), 7.58 (2H, d, J = 8.8 Hz, H-3 and H-5), 6.94 (2H, d, J = 8.8 Hz, H-2, H-6),
6.12 (1H, d, J = 2.2 Hz, H-5'), 5.98 (1H, d, J = 2.2 Hz, H-3'), 3.92 (3H, s, OMe), 3.86
(3H, s, OMe), 3.85 (3H, s, OMe);
13
C NMR (CDCl3): δ 193.59 (C=O), 168.36 (C,
C-2'), 166.01 (C, C-6'), 162.49 (C, C-4'), 161.35 (C, C-4), 142.47 (CH, C-8), 130.10
(2CH, C-3, C-5), 128.30 (C, C-1), 125.12 (CH, C-7), 114.35 (2CH, C-2, C-6), 106.35
(C, C-1'), 93.79 (C, C-5'), 91.23 (CH, C-3'), 55.83 (CH3, 4'-OMe), 55.57 (CH3,
6'-OMe), 55.39 (CH3, 4-OMe); EIMS m/z (rel. int.): 314 (100) [M+, C18H18O5], 301
(40), 288 (28), 207 (42), 180 (47), 134 (67), 115 (37), 103 (50), 87 (44), 75 (47).
92
5.5.3
4-Benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diol 5-acetate
(57)
The EtOAc extract (6.80 g) was fractionated by VLC (260 g, column size:
10.0 × 12.0 cm) using PE, Et2O, EtOAc with increasing polarity to afford 4 major
fractions, KRREA 1 (0.02g), KRREA 2 (0.15g), KRREA 3 (2.70g) and KRREA 4
(2.50 g).
Solids from fraction KRREA 3 was washed by Et2O gave 4-
benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diol 5-acetate (57) (0.145
g, 2.13 %). Colourless needles; Rf = 0.24 (n-hexane: EtOAc = 1: 1); m.p. 141-142ºC
(lit. [31] 145-148ºC); IR υmax cm-1: 3447, 2929, 1748, 1723, 1635, 1450, 1374, 1284,
1226, 1129, 1047, 852, 711; 1H NMR (CDCl3): δ 8.03 (2H, d, J = 7.8 Hz, H-2', H-6'),
7.61 (1H, t, J = 7.6 Hz, H-4'), 7.48 (2H, dd, J = 7.8, 7.6 Hz, H-3', H-5'), 5.47 (1H, d,
J = 8.6 Hz, H-2), 4.60 (1H, d, J = 12.0 Hz, H-7a), 4.25 (1H, d, J = 12.0 Hz, H-7b),
3.98 (1H, dd, J = 8.6 Hz and 4.8 Hz, H-3), 3.64 (1H, d, J = 2.8 Hz, H-6), 3.44 (1H,
dd, J = 3.6 Hz and 2.8 Hz, H-5), 3.19 (1H, d, J = 3.6 Hz, H-4), 2.71 (1H, d, J = 4.8
Hz, OH), 2.20 (3H, s, OAc); 13C NMR (CDCl3): δ 171.61 (C=O, OAc), 165.90 (C=O,
OBz), 133.58 (CH, C-4'), 129.77 (2CH, C-3', C-5'), 129.09 (C, C-1'), 128.58 (2CH,
C-2', C-6'), 73.39 (CH, C-2), 69.17 (CH, C-3), 62.62 (CH2, C-7), 59.83 (C, C-1),
54.72 (CH, C-4), 53.76 (CH, C-6), 48.13 (CH, C-5), 20.84 (CH3, OAc); CIMS m/z
321 [(M+1)+, C16H16O7].
5.5.4
1,6-Desoxypipoxide (69)
The residue of KRREA 3 (2.60g) was further purified by using CC (81 g,
column size: 4.3 × 14.5 cm) with solvent system PE: E 95: 5 to 80: 20 yielded 1240
fractions. Fractions 789-842 was combined to give 1,6-desoxypipoxide (69) (0.03g,
0.44%). White solids; Rf = 0.48 (DCM: MeOH = 49: 1); m.p. 89-90ºC (lit. [68] 9091ºC); IR υmax cm-1: 3432, 2974, 2896, 2413, 1715, 1647, 1474, 1451, 1316, 1269,
1112, 1027; 1H NMR (CDCl3): 8.05 (4H, m, H-2', H-6', H-2'', H-6''), 7.58 (2H, t,
J = 7.6 Hz, H-4', H-4''), 7.43 (4H, m, H-3', H-5', H-3'', H-5''), 6.19 (1H, dd, J = 1.2
and 5.6 Hz, H-5), 6.15 (1H, dd, J = 5.6 and 9.2 Hz, H-6), 6.01 (1H, dd, J = 4.0 and
9.2 Hz, H-4), 5.80 (1H, dd, J = 4.0 and 6.0 Hz, H-3), 5.13 (1H, d, J = 13.2 Hz, H-7a),
93
5.01 (1H, d, J = 13.2 Hz, H-7b), 4.70 (1H, dd, J = 6.0 and 6.0 Hz, H-2), 2.84 (1H, d,
J = 6.0 Hz, OH);
13
C NMR (CDCl3): δ 166.47 (C=O, OBz), 166.48 (C=O, OBz),
135.06 (C, C-1), 133.25 (CH, C-4''), 133.18 (CH, C-4), 129.89 (2C, C-1', C-1''),
129.78 (2CH, C-2'', C-6''), 129.68 (2CH, C-2', C-6'), 128.42 (2CH, C-3'', C-5''),
128.40 (2CH, C-3', C-5'), 125.84 (CH, C-6), 123.01 (CH, C-5), 124.98 (CH, C-4),
75.35 (CH, C-3), 70.08 (CH, C-2), 64.77 (CH2, C-7); EIMS m/z (rel. int.): 350 (100)
[M+, C21H18O5], 250 (100), 122 (55), 105 (100), 77 (65).
5.5.5
2-(Benzoyloxymethyl)phenyl (3-O-acetyl)-β-glucopyranoside (131)
Fraction KRREA 4 (2.50 g) was subjected to a versa flash CC (silica catridge
size: 4.0 × 7.5 cm) using n-hexane: EtOAc (60: 40, 58: 42 and 55: 45) to give 25
fractions. Fractions 16 (0.08 g) and 17 (0.06 g) were combined and further purified
by chromatotron (thickness of plate = 1.0 cm) using DCM: acetone (100: 0, 97: 3,
95: 5 and 94: 6) as eluents to yield 2-(benzoyloxymethyl)phenyl (3-O-acetyl)-βglucopyranoside (131) (8.9 mg, 0.36%). Pale brown gum; Rf = 0.37 (DCM: Acetone
= 2: 1); IR υmax cm-1: 3431, 3025, 2917, 1698, 1602, 1492, 1451, 1167, 1017, 533;
1
H NMR (CD3OD): δ 8.06 (2H, d, J = 7.2 Hz, H-2'', H-6''), 7.61 (1H, t, J = 7.6 Hz,
H-4''), 7.47 (2H, dd, J = 7.2 and 7.6 Hz, H-3'', H-5''), 7.43 (1H, d, J = 7.6 Hz, H-3'),
7.35 (1H, t, J = 7.6 Hz, H-5'), 7.28 (1H, d, J = 7.6 Hz, H-6'), 7.08 (1H, t, J = 7.6 Hz,
H-4'), 5.56 (1H, d, J = 12.8 Hz, H-7'a), 5.44 (1H, d, J = 12.8 Hz, H-7'b), 5.07 (1H, d,
J = 7.8 Hz, H-1), 5.05 (1H, dd, J = 9.4 and 9.6 Hz, H-3), 3.90 (1H, dd, J = 12.4 and
4.8 Hz, H-6a), 3.73 (1H, dd, J = 12.4 and 2.0 Hz, H-6b), 3.64 (1H, dd, J = 8.0 and
9.0 Hz, H-5), 3.56 (1H, dd, J = 7.8 and 9.4 Hz, H-2), 3.54 (1H, dd, J = 9.0 and 9.6
Hz, H-4), 3.32 (2H, s, OH), 3.21 (1H, s, OH), 2.14 (3H, s, OAc); 13C NMR (CD3OD):
δ 171.23 (C=O, OAc), 166.68 (C=O, OBz), 155.50 (C, C-1'), 132.87 (CH, C-4''),
130.10 (C, C-1''), 129.36 (CH, C-5'), 129.22 (2CH, C-2'', C-6''), 128.98 (CH, C-3'),
128.18 (2CH, C-3'', C-5''), 125.70 (C, C-2'), 122.28 (CH, C-4'), 115.43 (CH, C-6'),
101.30 (CH, C-1), 77.61 (CH, C-3), 76.59 (CH, C-2), 71.83 (CH, C-5), 68.05 (CH,
C-4), 61.79 (CH2, C-7'), 60.79 (CH2, C-6), 19.69 (CH3, OAc); CIMS m/z 433
[(M+1)+, C22H24O9].
94
5.5.6
Naringenin 4',7-dimethyl ether (133)
The MeOH extract (15.27 g) was fractionated by RP-VLC (250 g, column
size: 10.0 × 12.0 cm) and eluted with H2O: MeOH (70: 20, 60: 40, 50: 50, 40: 60,
20: 80, 15: 85, 10: 90, 0: 100) followed by MeOH: DCM (90: 10, 75: 25, 50: 50,
0: 100) to afford 23 fractions, labeled as KRRM 1 to KRRM 23. Fractions KRRM 9
to KRRM 12 (0.12 g) were combined and subjected to a short CC (3.5 g, column size:
1.0 x 21.0 cm) using n-hexane: DCM (1: 4) to afford more 1,6-desoxypipoxide (69)
(15.0 mg, 0.92 %).
Fraction KRRM 14 (0.09 g) was purified by a short CC (3.5 g, column size:
1.0 x 21.0 cm) with DCM: MeOH (100: 0, 98: 2) to give naringenin 4',7-dimethyl
ether (133) (0.7 mg, 0.004%). Yellow gum; Rf = 0.51 (n-hexane: DCM = 1: 4);
IR υmax cm-1: 3413, 1639, 1517, 1425, 1156, 1056, 1043; 1H NMR (CDCl3): δ 12.04
(1H, s, OH), 7.40 (2H, d, J = 8.8 Hz, H-3', H-5'), 6.79 (2H, d, J = 8.8 Hz, H-2', H-6'),
6.09 (1H, d, J = 2.4 Hz, H-6), 6.06 (1H, d, J = 2.4 Hz, H-8), 5.38 (1H, dd, J = 2.8
and 13.2 Hz, H-2), 3.85 (1H, s, OMe), 3.82 (1H, s, OMe), 3.12 (1H, dd, J = 13.2 and
17.2 Hz, H-3a), 2.80 (1H, dd, J = 2.8 and 17.2 Hz, H-3b);
13
C NMR (CDCl3):
δ 196.04 (C=O, C-4), 167.97 (C, C-5), 164.14 (C, C-9), 162.90 (C, C-7), 160.10 (C,
C-4'), 130.39 (C, C-1'), 127.74 (2CH, C-3', C-5'), 114.24 (2CH, C-2', C-6'), 103.15
(C, C-10), 97.02 (CH, C-2), 95.09, (CH, C-6), 94.23 (CH, C-8), 55.68 (CH3, OMe),
55.38 (CH3, OMe), 43.21 (CH2, C-3); EIMS m/z (rel. int.): 300 (71) [M+, C17H16O5],
299 (38), 242 (26), 206 (71), 161 (96), 149 (70), 134 (60), 121 (42), 105 (59), 86
(67), 84 (100).
5.5.7
Curcumrinol C (134)
Fractions 13, 15-17 (0.10 g) were combined and further purified by using CC
(3.0 g, column size: 1.0 × 21.0 cm) using n-hexane: DCM (1: 4, 0: 100) yielded
curcumrinols C (134) as a yellow gum (8.3 mg, 0.54%); Rf = 0.51 (n-hexane: DCM
= 1: 4); IR υmax cm-1: 3418, 3001, 2929, 1722, 1631, 1460, 1378, 1250, 1095, 1067,
1023, 992, 920, 613; 1H NMR (CDCl3): δ 6.05 (1H, dd, J = 11.6 and 18.4 Hz, H-14),
95
5.38 (1H, dd, J = 5.6 and 3.0 Hz, H-12), 5.05 (1H, d, J = 11.6 Hz, H-15a), 5.10 (1H,
d, J = 18.4 Hz, H-15b), 2.57 (1H, dd, J = 14.0 and 14.4 Hz, H-6a), 2.44 (1H, dd, J =
14.0 and 2.9 Hz, H-6b), 2.12 (3H, s, H-22), 1.98 (2H, m, H-9, H-11a), 1.82 (1H, dd,
J = 8.8 and 3.0 Hz, H-11b), 1.66 (1H, dd, J = 3.2 and 14.0 Hz , H-1a), 1.61 (1H, m,
H-2a), 1.52 (1H, m, H-2b), 1.45 (1H, m, H-3a), 1.40 (1H, dd, J = 14.4 and 2.9 Hz,
H-5), 1.17 (1H, dd, J = 12.8 and 4.0 Hz, H-3b), 0.80 (1H, dd, J = 14.0 and 4.0 Hz,
H-1b); 13C NMR (CDCl3): δ 209.02 (C=O, C-7), 170.43 (C=O, C-21), 111.46 (CH2,
C-15), 145.82 (CH, C-14), 81.64 (C, C-8), 76.06(C, C-13), 70.41 (CH, C-12), 56.47
(CH, C-5), 50.46 (CH, C-9), 41.57 (CH2, C-3), 38.95 (CH2, C-1), 36.30 (C, C-10),
35.86 (CH2, C-6), 33.72(C, C-4), 32.62 (CH3, C-18), 26.80 (CH3, C-16), 22.26 (CH3,
C-17), 21.21 (CH3, C-22), 21.06 (CH2, C-11), 20.66 (CH3, C-19), 18.16 (CH2, C-2),
15.06 (CH3, C-20); EIMS m/z (rel. int.): 362 (71) [(M-H2O)+, C22H36O5], 250 (55),
123 (47), 109 (43), 84 (44), 81 (43), 69 (57), 55 (60), 43 (100).
5.5.8
3-Debenzoylretopoxide A (132)
Fraction 18 (0.20 g) was subjected to chromatotron (thickness of plate = 1.0
cm) with DCM: MeOH (49: 1, 48: 2) as the eluent to get 100 fractions. Fractions 80
to 90 were combined to give 3-debenzoylrotepoxide A (132), pale brown gum (10.0
mg, 0.07%); Rf = 0.51 (n-hexane: DCM = 1: 4); IR υmax cm-1: 3414, 2976, 1720,
1631, 1602, 1273, 1118, 1071, 929; 1H NMR: δ 8.05 (2H, d, J = 7.6 Hz, H-2', H-6'),
7.60 (1H, t, J = 7.6 Hz, H-4'), 7.47 (2H, t, J = 7.6 Hz, H-3', H-5'), 4.63 (1H, d, J =
12.0 Hz, H-7a), 4.50 (1H, d, J = 12.0 Hz, H-7b), 4.38 (1H, dd, J = 6.0 and 3.2 Hz,
H-5), 4.29 (1H, d, J = 5.0 Hz, H-2), 4.13 (1H, dd, J = 6.0 and 1.6 Hz, H-4), 4.01 (1H,
dd, J = 5.0 and 1.6 Hz, H-3), 3.76 (1H, d, J = 3.2 Hz, H-6);
13
C NMR: δ 166.10
(C=O, OBz), 133.51 (CH, C-4'), 130.06 (C, C-1'), 129.78 (2CH, C-2', C-6'), 128.55
(2CH, C-3', C-5'), 72.50 (CH, C-3), 69.33 (CH, C-5), 69.09 (CH, C-2), 64.05 (CH2,
C-7), 63.14 (C, C-1), 62.90 (CH, C-4), 61.37 (CH, C-6); CIMS m/z 297 [(M+1)+,
C14H16O7].
96
5.6
Extraction and Isolation of Indonesian Kaempferia rotunda
The dried rhizomes of Kaempferia rotunda (898.0 g) were ground into
powder and extracted with chloroform (4.5 L) and EtOAc (4.5 L) by soxhlet
extraction for 16 hours.
The extract was then concentrated by using rotary
evaporator to give dark brown semi-solid of chloroform extract (19.20 g, 2.14 %)
and dark brown liquid of EtOAc crude extract (7.60 g, 0.84%).
5.6.1
Crotepoxide (54)
Solids from chloroform extract (19.2 g) was filtered with buchner funnel and
washed several times with Et2O to yield crotepoxide (54) (1.50 g, 7.8%). The m.p.,
IR, 1H NMR,
13
C NMR and EIMS data of this compound (54) were identical with
previously isolated compound.
5.6.2
Benzyl Benzoate (82)
The residue extract (13.50 g) was fractionated by using VLC (10.5cm I.D,
260g silica gel) and eluted with PE, DCM and acetone with increasing polarity to
give 29 fractions. Fractions 5 to 9 were combined to yield benzyl benzoate (82)
(2.25 g, 11.7%) as a colourless oil; Rf = 0.36 (PE: DCM = 3: 2); IR υmax cm-1: 1718,
1451, 1376, 1314, 1272, 1112, 755, 712; 1H NMR (CDCl3): δ 8.14 (2H, d, J = 7.6 Hz,
H-2', H-6' ), 7.59 (1H, br t, J = 7.3 Hz, H-4'), 7.45 (7H, m, H-2, H-3, H-4, H-5, H-6,
H-3', H-5'), 5.42 (2H, s, H-7); 13C NMR (CDCl3): δ 166.44 (C=O), 136.14 (C, C-1'),
133.06 (CH, C-4'), 130.22 (C, C-1), 129.75 (2CH, C-2', C-6'), 128.64, 128.42, 128.28,
128.20 (7CH, C-2, C-3, C-4, C-5, C-6, C-3', C-5'), 66.72 (CH2, C-7); EIMS m/z (rel.
int.): 39 (5), 51 (18), 65 (10), 77 (30), 90 (40), 105 (95), 167 (5), 194 (10), 212 (20)
[M+, C14H12O2].
97
5.6.3
trans-Docosyl ferulate (137)
Fractions 13 and 14 (0.4 g) were combined and chromatographed on a silica
gel column (1.0 cm I.D, 20.0 g silica gel) with PE: Et2O (94: 6) mixtures giving 300
fractions. Fractions 221-240 were combined to get 0.03 g mixtures that containing
acid. The acid was removed from the mixture by adding 0.06 g of K2CO3 anhydrous
and stirred in EtO2 for 6 hours, thus yielded trans-docosyl ferulate (137) (18.6 mg,
0.10%) as a pale yellow gum; Rf = 0.52 (DCM = 100 %); IR υmax cm-1: 3434, 2925,
2853, 1674, 1462, 1377, 1268, 1186, 1113, 971, 864; 1H NMR (CDCl3): δ 7.62 (1H,
d, J = 16.0 Hz, H-7), 7.08 (1H, br. d, J = 8.4 Hz, H-6), 7.05 (1H, br. s, H-2), 6.92
(1H, br. d, J = 8.4 Hz, H-5), 6.30 (1H, d, J = 16.0 Hz, H-8), 4.20 (2H, t, J = 6.8 Hz,
H-1'), 3.94 (3H, s, OCH3), 1.70 (4H, m, H-2', H-3'), 1.26 (36H, br. s, H-4' to H-n-3',
H-n-2', H-n-1'), 0.89 (3H, t, J = 6.0 Hz, CH3);
13
C NMR (CDCl3): δ 167.36 (C=O,
C-9), 147.91 (C, C-3), 146.77 (C, C-4), 144.60 (CH, C-7), 127.06 (C, C-1), 123.01
(CH, C-6), 115.70 (CH, C-8), 114.71 (CH, C-5), 109.33 (CH, C-2), 64.00 (CH2,
C-1'), 55.92 (CH3, OMe), 31.91 (CH2, C-n-2'), 29.68, 29.64, 29.58, 29.53, 29.34,
25.99 (16CH2, C-4' to C-n-3'), 29.29 (CH2, C-2'), 28.77 (CH2, C-3'), 22.67 (CH2,
C-n-1'), 14.08 (CH3, C-22'); EIMS m/z (rel. int.): 502 (100) [M+, C32H54O4], 194
(70), 177 (45), 150 (18), 137 (20), 43 (45).
5.6.4
3-Acetoxy-2-benzoyloxy-1-(benzoyloxymethyl)cyclohexa-4,6-diene (136)
Fraction 15 (0.31g) was purified by short CC (1.0 cm I.D, 12.0g silica gel)
with a PE: Et2O gradient (80: 20; 70: 30; 60: 40) to yield 480 fractions. Fractions
340-409 (0.03g) were combined and further purified with CC (20mL pipet, 1.0 g
silica gel), n-hexane: DCM (80: 20 to 60: 40) as eluents to get 3-acetoxy-2benzoyloxy-1-(benzoyloxymethyl)cyclohexa-4,6-diene (136) (6.5 mg, 0.34%)as a
brown gum; Rf = 0.55 (DCM = 100 %); IR υmax cm-1: 3020, 2926, 2854, 1720, 1647,
1452, 1268, 1216, 1110, 1070, 1026, 686; 1H NMR (CDCl3): δ 8.06 (4H, m, H-2',
H-6', H-2'', H-6''), 7.58 (2H, m, H-4', H-4''), 7.46 (4H, m, H-3', H-5', H-3'', H-5''),
6.19 (1H, d, J = 9.6 Hz, H-6), 6.10 (1H, dd, J = 9.6 and 2.4 Hz, H-5), 6.00 (1H, br s,
H-4), 5.88 (2H, br s, H-2, H-3), 4.90 (2H, br s, H-7); 13C NMR (CDCl3): δ 170.17
98
(C=O, OAc), 166.14 (C=O, OBz), 165.77 (C=O, OBz), 133.27 (C, C-1), 133.27 (CH,
C-4''), 133.24 (CH, C-4'), 129.82 (2CH, C-2', C-6'), 129.75 (2C, C-1', C-1''), 129.72
(2CH, C-2'', C-6''), 128.46 (2CH, C-3'', C-5''), 128.43 (2CH, C-3', C-5'), 126.27 (CH,
C-5), 125.83 (CH, C-6), 122.07 (CH, C-4), 71.93 (CH, C-2), 71.26 (CH, C-3), 65.29
(CH2, C-7), 19.39 (CH3, OAc); CIMS m/z 392 [(M)+, C23H20O6].
5.6.5
6-Acetylzeylenol (68)
Fraction 21 (0.16 g) was subjected to chromatotron (thickness of plate = 1.0
cm) with PE: Et2O (70: 30) gave 377 fractions. Fractions 170-210 were combined
and recrystallized with DCM and n-hexane to obtain 6-acetylzeylenol (68) (7.0 mg,
0.36%) as a white solids, Rf = 0.51 (PE: E = 1: 9); IR υmax cm-1: 3435, 3018, 2926,
2851, 1715, 1602, 1452, 1372, 1317, 1272, 1178, 1115, 1070, 1027, 973, 930, 711;
1
H NMR (CDCl3): δ 8.05 (2H, d, J = 7.2 Hz, H-2'', H-6''), 8.00 (2H, d, J = 7.2 Hz,
H-2', H-6'), 7.57 (2H, m, H-4', H-4''), 7.42 (4H, m, H-3'', H-5'', H-3', H-5'), 5.98 (2H,
d, J = 2.4 Hz, H-4, H-5), 5.74 (1H, d, J = 6.0 Hz, H-3), 5.55 (1H, d, J = 2.4 Hz, H-6),
4.81 (1H, d, J = 12.0 Hz, H-7a), 4.59 (1H, d, J = 12.0 Hz, H-7b), 4.24 (1H, dd, J =
4.8 and 6.0 Hz, H-2), 3.42 (1H, s, OH), 3.28 (1H, d, J = 4.8 Hz, OH), 2.11 (3H, s,
OAc) ; 13C NMR (CDCl3): δ 170.13 (C=O, OAc), 167.07 (C=O, OBz), 167.04 (C=O,
OBz), 133.48 (CH, C-4''), 133.34 (CH, C-4'), 129.84 (2CH, C-2'', C-6''), 129.67
(2CH, C-2', C-6'), 129.35 (C, C-1''), 129.25 (C, C-1'), 128.45 (4CH, C-3'', C-5'', C-3',
C-5'), 128.38 (CH, C-4), 126.59 (CH, C-5), 74.72 (C, C-1), 73.59 (CH, C-3), 71.49
(CH, C-2), 70.54 (CH, C-6), 66.56 (CH2, C-7), 20.94 (CH3, OAc); CIMS m/z 442
[M+, C23H22O8].
5.6.6
Benzoic Acid (138)
The ethyl acetate extract (7.60 g) was fractionated by using VLC (10.5cm I.D,
250.0g silica gel) and eluted with PE, DCM and MeOH with increasing polarity to
give 5 major fractions (KRR2EA 1-5). KRR2EA 2 was washed with Et2O to yield
benzoic acid (138) (0.48 g, 6.26%) as a colourless crystal; Rf = 0.32 (n-hexane:
99
EtOAc = 1: 1); m.p. 118°C (lit. [75] 122°C); IR υmax cm-1: 3417, 3020, 2928, 1693,
1645, 1215; 1H NMR: δ 8.14-7.18 (5H, m, H-2, H-3, H-4, H-5, H-6);
13
C NMR: δ
171.96 (C=O), 133.75 (CH, C-4), 130.19 (CH, C-2), 130.02 (CH, C-6), 128.47 (C,
C-1), 128.06 (CH, C-3), 127.90 (CH, C-5).
5.7
Bioactivity Studies
5.7.1
Chemicals
2,2-Diphenyl-1-picrylhydrazyl (DPPH) and dimethylsulfoxide (DMSO) were
purchased from Fluka, while Vitamin C was purchased from Merck. Nutrient agar
(NA), nutrient broth (NB) and standard of streptomysin sulphate antimicrobial disc
(10 μg/disc) were obtained from Oxoid.
5.7.2
Microorganisms
All bacterium i.e. Staphylococcus aureus, Bacillus subtilis (Gram-positive
bacteria), Pseudomonas aeruginosa and Escherichia coli (Gram-negative bacteria)
were obtained from Department of Biology, Faculty of Science, Universiti Teknologi
Malaysia.
5.7.3
Antimicrobial Assay
5.7.3.1 Microorganisms and Culture Media
The bacterium were cultured in an appropriate nutrient broth (NB) at 37°C
overnight. The concentrations of the cultures were adjusted by comparison with the
McFarland solution. The McFarland solution was prepared by mixing the H2SO4
solution (1% in broth) and BaCl2 solution (1% in broth). The H2SO4 solution was
100
prepared by dissolving H2SO4 (1.02 mL) in broth (100 mL), while BaCl2 solution
was prepared by dissolving BaCl2 (1 g) in broth (100 mL). The mixture of H2SO4
solution (9.95 mL) and BaCl2 solution (0.05 mL) were equivalent with 150 x 106
coloni/unit.
Nutrient agars (NA) (20 g/L) and nutrient broths (NB) (8 g/L) were dissolved
in distilled water. All solutions were sterilized by autoclave for 15 minutes at 121°C.
5.7.3.2 Disc Diffusion Method
The antibacterial assay was conducted by using disc diffusion method as
described by Mbaveng et al. [82]. The NA solution (17 mL) was placed into petri
dishes. Then, the agar plates were kept in refrigerator for overnight. Whatman filter
paper discs of 6 mm diameter were impregnated with 10 μL of the solution of crude
extract or essential oil (at 4 mg/mL) or isolated compound (at 1 mg/mL) prepared
using DMSO. Standard disc of streptomycin sulphate (10 μg/disc) was used as
positive control, while DMSO was used as a negative control. The bacteria stock
(400 μL) was spread onto the surface of agar in the petri dish by using a glass rod.
Then, the sample discs and the control discs were placed on the agar surface. Figure
5.1 showed the arrangement of the discs. The petri dishes were inverted and
incubated for 24 hours at 37°C. Clear inhibition zones around the discs indicated the
presence of antimicrobial activity. All tests and analysed were carried out in
triplicates. The inhibition zones of all tested samples are tabulated in Table 5.1.
1
D
Notes:
2
S
2
Disc 2 = Sample B
D
1
Disc 1 = Sample A
Disc D = DMSO
Disc S = Streptomycin sulphate
Figure 5.1: The Arrangement of the Sample Discs and Control Discs in Petri Dish
101
Table 5.1: The Inhibition Zones of Tested Samples
Inhibition zones diameter (mm) *
S.a
B.s
P.a
E.c
Samples
Crude Extracts
n-Hexane extract of Malaysian K. rotunda
Ethyl acetate extract of Malaysian K. rotunda
Methanol extract of Malaysian K. rotunda
Chloroform extract of Indonesian K. rotunda
Ethyl acetate extract of Indonesian K. rotunda
Methanol extract of Indonesian K. rotunda
-
-
-
-
Essential Oils
Essential oil of Malaysian K. rotunda
Essential oil of Indonesian K. rotunda
-
-
-
-
Pure Compounds
Benzoic acid (137)
-
-
-
-
20.0 ± 0.0
20.0 ± 0.5
20.2 ± 0.3
20.0 ± 1.0
Streptomysin sulphate
Experiments were done in triplicate and results are mean values ± SD; inhibition zone including the diameter of
the paper disc (6 mm); S.a = Staphylococcus aureus (Gram positive) E.c = Escherichia coli (Gram positive); B.s
= Bacillus subtilis (Gram negative); P.a = Pseudomonas aeruginosa (Gram negative); -: not active.
5.7.4
Antioxidant Screening (Free Radical Scavenging Activity (DPPH))
The method was carried out as described by Tagashira and Ohtake [92] with
some minor modifications. Each sample stock solution (1.0 mg/mL) was diluted to
final concentrations of 500, 250, 125, 62.5, 31.3, 15.63 and 7.81 μg/mL in methanol.
A total of 3.8 mL of 50 μM DPPH methanolic solution (1 mg/50 mL) was added to
0.2 mL of sample solution of different concentrations and allowed to react at room
temperature for 30 min. After 30 min, the absorbance of the reaction mixture was
measured at 517 nm. The DPPH solution and methanol was used as control while
vitamin C and 5-deoxyquercetin were used as standard antioxidant. The absorbance
of control was measured immediately at 0 min. The percentage inhibition was
calculated using the following formula:
Percentage Inhibition(%) =
Abs (DPPH) - Abs (DPPH + sample)
× 100
Abs (DPPH)
102
The IC50 value was determined as the concentration of each sample required
to give 50% of the absorbance shown by the control. All tests and analyses were
carried out in triplicates and averaged. The percentage inhibitions of the tested
samples are displayed in Table 5.2.
Table 5.2: Percentage Inhibitions of Tested Samples
IC50
(μg/mL)
Percent inhibition
at 7.81 μg/mL
Crude Extracts
n-Hexane extract of Malaysian K. rotunda
Ethyl acetate extract of Malaysian K. rotunda
Methanol extract of Malaysian K. rotunda
Chloroform extract of Indonesian K. rotunda
Ethyl acetate extract of Indonesian K. rotunda
Methanol extract of Indonesian K. rotunda
-
-
Essential Oils
Essential oil of Malaysian K. rotunda
Essential oil of Indonesian K. rotunda
-
-
30.30
11.65
17.46 ± 0.005
37.90 ± 0.01
Samples
References
Vitamin C
5-Deoxyquercetin
Data represent mean ± SD of three independent experiments performed in triplicate; -: not active.
CHAPTER 6
CONCLUSION AND RECOMMENDATION
Study on the essential oils of the rhizomes of Malaysian and Indonesian
Kaempferia rotunda gave pale yellow oils in 0.09% and 0.23% respectively. Bornyl
acetate (90) (9.6%), α-hydroxysandaracopimara-8(14),15-diene (118) (9.2%), benzyl
benzoate (82) (8.4%), camphor (91) (5.6%) and n-pentadecane (88) (4.0%) have
been identified as the major components from the rhizome oil of Malaysian sample.
Analysis of the essential oil of Indonesian fresh rhizome has led to the identification
of benzyl benzoate (82) (87.7%) and n-pentadecane (88) (4.2%) as the main
component together with camphene (89) (1.3%) and bornyl acetate (90) (1.1%),
salicyaldehyde (119) (1.4%), benzyl salicylate (108) (0.9%) and n-heptadecane (120)
(0.2%).
Three new compounds and ten known compounds, comprising of
cyclohexane oxides, esters, carboxylic acid, labdane diterpene, and flavonoids have
been isolated from the rhizomes Kaempferia rotunda. Two new compounds, 2(benzoyloxymethyl)phenyl (3-O-acetyl)-β-glucopyranoside (131), 3-debenzoylrotepoxide A (132), together with six known compounds, crotepoxide (54), 2'hydroxy-4,4',6'-trimethoxychalcone
(1),
4-benzoyloxymethyl-3,8-dioxatricyclo-
2,4
[5.1.0.0 ]octane-5,6-diol 5-acetate (57), 1,6-desoxypipoxide (69), naringenin 4',7dimethyl ether (133) and curcumrinol C (134) have been successfully isolated from
the Malaysian rhizomes.
The Indonesian rhizomes gave a new compound
characterized as 3-acetoxy-2-benzoyloxy-1-(benzoyloxymethyl)cyclohexa-4,6-diene
(136) and seven known compounds, identified as crotepoxide (54), 4benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diol 5-acetate (57), 1,6-
104
desoxypipoxide (69), benzyl benzoate (82), trans-docosyl ferulate (137), 6acetylzeylenol (68) and benzoic acid (138). To the best of our knowledge, this is the
first report on the new cyclohexane oxides (131, 132 and 136), while naringenin 4',7dimethyl ether (133), curcumrinol C (134), trans-docosyl ferulate (137), and benzoic
acid (138) were found for the first time in Kaempferia species.
This study shows that cyclohexane oxides are the characteristic components
of rhizomes of Kaempferia rotunda.
Study on the rhizomes also successfully
isolated labdane diterpene (134), flavonoids (1, 133), esters (82, 131, 137) and acid
(138). The isolation of benzyl benzoate (82) and dienes (69, 136) in the rhizomes of
K. rotunda provides strong evidence in supporting the proposed biogenetic pathway
[63] to cyclohexane oxides via the common benzyl benzoate (82) precursor and the
key intermediates (69, 136). The Indonesian species expressed significant amount of
benzyl benzoate (82) which was present in minute quantity in the Malaysian species.
The small amount of benzyl benzoate (82) in the Malaysian species is probably due
to high transformation of ester (82) to the high amount of other cyclohexane oxides.
The crude extracts were screened for antimicrobial and antioxidant properties
using disc-diffusion method and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH)
method respectively. The crude extracts and essential oils from Malaysian and
Indonesian Kaempferia rotunda did not show any activities on the antibacterial and
antioxidant assay.
Several recommendations are suggested to be done in the future study such as
study on the other parts of Kaempferia rotunda which include the aerial parts. Some
more potential bioassay studies such as antiplatelet aggregation, insecticidal activity,
antifeedant and anticancer are proposed to be carried out. Besides that, several
phytochemicals can be modified by using simple reactions such as hydrolysis,
acetylation or epoxidation, for example modification on benzyl benzoate (82) to get
its derivatives for antihypertension activity [93].
105
REFERENCES
1.
Kumara, K., Dan, Y. M. and Tuan Marina, T. I. Economic Significance of
Medicinal Plants in Peninsular Malaysia. Kuala Lumpur: Forestry
Department, Peninsular Malaysia. 1998.
2.
Donald, P. B. Medicinal Plants and Phytomedicines. Linking Plant
Biochemistry and Physiology to Human Health. Plant Physiol. 2000. 124:
507-514.
3.
Itokawa, H., Morris, N. S. L., Akiyama T. and Lee K. H. Plant-Derived
Natural Product Research Aimed at New Drug Discovery. J. Nat. Med. 2008.
62: 263-280.
4.
Feng, M. C, Sam, T. W. and Khoo, L. E. Chemistry and Structural
Elucidation of Natural Products: Lectures from UNESCO Regional
Workshop. Penang: Universiti Sains Malaysia. 1984.
5.
Rahmani, M., Mahmood, M., Sukari, M. A. H., Ee, C. L., Yap, Y. H. and Ali,
D. A. I. Natural Products Research: Prospects and Retrospect. In: Goh, S. H.,
Xu, Y. J., Wu, J., Ho, S. H. and Imiyabir, Z. Interdisciplinary Approach in
Natural Products Research. Malaysia: Universiti Putra Malaysia. 1-4. 2000.
6.
Larsen, K., Ibrahim, H., Khaw, S. H. and Saw, L. G. Gingers of Penisular
Malaysia and Singapore. Kota Kinabalu, Malaysia: Natural Product
Publications (Borneo) Sdn. Bhd. 1999.
7.
Zakaria, M. and Ibrahim, H. Phytochemical Screening of Some Malaysia
Species of Zingiberaceae. Mal. J. Sci. 1986. 8: 125-128.
106
8.
Holttum, R. E. The Zingiberaceae of the Malay Peninsular. Singapore: S. N.
Publisher. 1950.
9.
Zakaria, M. and Mustafa, A. M. Traditional Malay Medicinal Plants. Kuala
Lumpur: Fajar Bakti. 1994.
10.
Kanjanapothi, D., Panthong, A., Lertprasertsuke, N., Taesotikul, T.,
Rujjanawate, C., Kaewpinit D., Sudthayakorn, R., Choochote, W., Chaithong,
U., Jitpakdi, A. and Pitasawat, B. Toxicity of Crude Rhizome Extract of
Kaempferia galanga L. (Proh Hom). J. Ethnopharmacol. 2004. 90: 359-365.
11.
Huang, L. F., Yagura, T. and Chen, S. L. Sedative Activity of Hexane Extract
of Keampferia galanga L. and Its Active Compounds. J. Ethnopharmacol.
2008. 120: 123-125.
12.
Burkill, I. H. Dictionary of the Economic Products of the Malay Peninsula. 2.
Kuala Lumpur: Ministry of Agriculture and Cooperatievs. 1966.
13.
Yenjai, C., Prasanphen, K., Daodee, S., Wongpanich, V. and Kittakoop, P.
Bioactive Flavonoids from Kaempferia parviflora. Fitoterapia. 2004. 75: 8992.
14.
Wattanapitayakul, S. K., Suwatronnakorn, M., Hularojmontri, L., Herunsalee,
A., Niumsakul, S. Charuchongkolwongse, S., Chansuvanich, N. Kaempferia
parviflora Ethanolic Extract Promoted Nitric Oxide Production in Human
Umbilical Vein Endothelial Cells. J. Ethnopharmacol. 2007. 110: 559-562.
15.
Trakoontivakorn, G., Nakahara, K., Shinmoto, H., Takenaka, M., OnishiKameyama, M., Ono, H., Yoshida, M., Nagata, T. and Tsushida, T. Structural
Analysis of a Novel Antimutagenic Compound, 4-Hydroxypanduratin A, and
Antimutagenic Activity of Flavonoids in a Thai Spice, Fingerroot
(Boesenbergia pandurata Schult.) against Mutagenic Heterocyclic Amines. J.
Agri. Food Chem. 2001. 49: 3046-3050.
107
16.
Pancharoen, O., Tuntiwachwuthkul, P. and Taylor, W. C. Cyclohexane
Oxides
from
Kaempferia
angustifolia
and
Kaempferia
Species.
Phytochemistry. 1989. 28 (4): 1143-1148.
17.
Sirat, H. M., Jamil, S., and Siew, L. W. Constituents of Kaempferia rotunda.
ACGC Chem. Res. Comm. 2001. 13: 48-54.
18.
Jaipetch T, Kanghae S, Pancheroen O. Constituents of Boesenbergia
pandurata (syn. Kaempferia pandurata): Isolation, Crystal Structure and
Synthesis of (±)-Boesenbergin A. Aust. J. Chem. 1982. 35: 351-361.
19.
Mahidol, C., Tuntiwachwuttikul, P., Reutraakul, V. and Taylor, W. C. (1984).
Constituents of Boesenbergia pandurata (syn. Kaempfevia pandurata). III*
Isolation and Synthesis of (±)-Boesenbergin B. Aust. J. Chem. 1984. 37:
1739-1745.
20.
Win, N. N., Awale, S., Esumi, H., Tezuka, Y., and Kadota, S. Bioactive
Secondary Metabolites from Boesenbergia pandurata of Myanmar and Their
Preferential Cytotoxicity against Human Pancreatic Cancer PANC-1 Cell
Line in Nutrient-Deprived Medium. J. Nat. Prod. 2007. 70 (10): 1582-1587.
21.
Win, N. N., Awale, S., Esumi, H., Tezuka, Y., and Kadota, S. Panduratins DI, Novel Secondary Metabolites from Rhizomes of Boesenbergia pandurata.
Chem. Pharm. Bull. 2008. 56 (4): 491-496.
22.
Hwang, J. K., Chung, J. Y., Baek, N. I., Park, J. H. Isopanduratin A from
Kaempferia pandurata as an Active Antibacterial Agent Against Cariogenic
Streptococcus mutans. Int. J. Antimicrob. Agents. 2004. 23: 377-381.
23.
Sutthanut, K., Sripanidkulchai, B., Yenjai, C., Jayd, M. Simultaneous
Identification and Quantitation of 11 Flavonoid Constituents in Kaempferia
parviflora by Gas Chromatography. J. Chromatogr. A. 2007. 1143: 227-233.
108
24.
Tewtrakul, S., Subhadhirasakul, S., Karalai C., Ponglimanont, C. and
Cheenpracha, S. Anti-inflammatory Effects of Compounds from Kaempferia
parviflora and Boesenbergia pandurata. Food Chem. 2009. 115: 534-538.
25.
Tewtrakul, S. and Subhadhirasakul, S. Effects of Compounds from
Kaempferia parviflora on Nitric Oxide, Prostaglandin E2 and Tumor Necrosis
Factor-Alpha
Productions
in
RAW264.7
Macrophage
Cells.
J.
Ethnopharmacol. 2008. 120: 81-84.
26.
Tewtrakul, S., Subhadhirasakul, S. and Kummee, S. Anti-allergic Activity of
Compounds from Kaempferia parviflora. J. Ethnopharmacol. 2008. 116:
191-193.
27.
Prawat, U., Tuntiwachwuttikul, P., Taylor, W. C., Engelhardt, L. M., Skelton,
B. W. and White, A. H. Diterpenes from a Kaempferia species.
Phytochemistry. 1993. 32 (4): 991-997.
28.
Thongnest, S., Mahidol, C., Sutthivaiyakit, S., Ruchirawat, S. Oxygenated
Pimarane Diterpenes from Kaempferia marginata. J. Nat. Prod. 2005. 68:
1632-1636.
29.
Tuchinda, P., Udchachon, J., Reutrakul, V., Santisuk, T., Skeltona, B. W.,
White, A. H. and Taylor, W. C. Pimarane Diterpenes from Kaempferia
pulchra. Phytochemistry. 1994. 36 (3): 731-734.
30.
Pancharoen, O., Patrick, V. A. and Reutrakul, V. Constituents of
Boesenbergia sp. Isolation and Cystal Structure of Crotepoxide ([1R(1α,2α,4α,5β,6α,7α)]-4-[(Benzoyloxy)methyl]-3,8-dioxatricyclo[5,1,0,02,4]octane-5,6-diyl Diacetate. Aust. J. Chem. 1984. 37: 221-225.
31.
Pancharoen, O., Tuntiwachwuthkul, P., and Taylor, W. C. Cyclohexane
Diepoxides from Kaempferia rotunda. Phytochemistry. 1996. 43 (1): 305308.
109
32.
Stevenson, P.C., Veitch, N. C., and Simmonds, M. S. J. Polyoxygenated
Cyclohexane Derivatives and Other Constituents from Kaempferia rotunda L.
Phytochemistry. 2007. 68: 1579-1586.
33.
Huang, L. F., Yagura, T. and Chena, S. Sedative Activity of Hexane Extract
of Kaempferia galanga L. and Its Active Compounds. J. Ethnopharmacol.
2008. 120: 123-125.
34.
Suzuki, J., Yasuda, I., Murata, I., Murata, R., and Nishitani, K. Studies on
Larvicidal Compounds Isolated from the Rhizomes of Kaempferia galanga
Effective on Anisakis simplex. Ann. Rep. Tokyo Metr. Res. Lab. P. H. 2002.
53: 35-39.
35.
Adisakwattana, S. Sookkongwaree, K., Roengsumran, S., Petsom, A.,
Ngamrojnavanich, N., Chavasiri, W., Deesamer, S., and Yibchokanun, S.
Structure-Activity Relationship of Trans-Cinnamic Acid Derivatives on αGlucosidase Inhibition. Bioorg. Med. Chem. Lett. 2004. 14: 2893-2896.
36.
Azuma, T., Tanaka, Y. and Kikuzaki, H. Phenolic Glycosides from
Kaempferia parviflora. Phytochemistry. 2008. 69: 2743-2748.
37.
Nugroho, W. B., Schwarz, B., Wray, V., Proksch, P. Insecticidal constituents
from Rhizomes Of Zingiber cassumunar and Kaempferia rotunda.
Phytochemistry. 1996. 41 (1): 129-132.
38.
Kuichi, F., Nakamura, N. and Tsuda, Y. 3-Caren-5-one from Kaempferia
galanga. Phytochemistry. 1987. 26 (12): 3350-3351.
39.
Chu, D. M., Miles, H., Toney, D., Chi, N. Y. and Marciano-Cabral, F.
Amebicidal Activity of Plant Extracts from Southeast Asia on Acanthamoeba
spp. Parasitol. Res. 1998. 84: 746-752.
110
40.
Xiao, Y., Wei, P. K., Li, J., Shi, J., Yu, Z. H. and Lin H. M. Effects of
Rhizoma Kaempferiae Volatile Oil on Tumor Growth and Cell Cycle of
MKN-45 Human Gastric Cancer Cells Orthotopically Transplanted in Nude
Mice. J. Chin. Integr. Med. 2006. 4 (4): 384-388.
41.
Yang, Y. C., Park, II-K., Kim, E. H., Lee, H. S. and Ahn, Y. J. Larvicidal
Activity of Medicinal Plant Extracts Against Aedes aegypti, Ochlerotatus
togoi, and Culex pipiens pallens (Diptera: Culicidae) J. Asia-Pacific Entomol.
2004. 7 (2): 227-232.
42.
Hwang, J. K., Shim, J. S. and Chung, J. Y. Anticariogenic Activity of Some
Tropical Medicinal Plants against Streptococcus mutans. Fitoterapia. 2004.
75: 596-598.
43.
Yanti, Anggakusuma, Gwon, S. H. and Hwang, J. K. Kaempferia pandurata
Roxb. Inhibits Porphyromonas gingivalis Supernatant-Induced Matrix
Metalloproteinase-9 Expression via Signal Transduction in Human Oral
Epidermoid Cells. J. Ethnopharmacol. 2009. 123 (2): 315-324.
44.
Rujjanawate, C., Kanjanapothi, D., Amornlerdpison, D. and Pojanagaroon, S.
Anti-Gastric Ulcer Effect of Kaempferia parviflora. J. Ethnopharmacol.
2005. 102: 120-122.
45.
Wattanapitayakul,
S.
K.,
Chularojmontri,
L.,
Herunsalee,
A.,
Charuchongkolwongse, S. and Chansuvanich, N. Vasorelaxation and
Antispasmodic Effects of Kaempferia parviflora Ethanolic Extract in Isolated
Rat Organ Studies. Fitoterapia. 2008. 79: 214-216.
46.
Yun, J. M., Kweon, M. H., Kwon, H. J., Hwang, J. K., and Mukhtar, H.
Induction of Apoptosis and Cell Cycle Arrest by a Chalcone Panduratin A
Isolated from Kaempferia pandurata in Androgen-independent Human
Prostate Cancer Cells PC3 and DU145. Carcinog. 2006. 27 (7): 1454-1464.
111
47.
Yun, M. J., Kwon, H. J., Mukhtar, H. and Hwang, J. K. Induction of
Apoptosis by Panduratin A Isolated from Kaempferia pandurata in Human
Colon Cancer HT-29 Cells. Planta Med. 2005. 71: 501-507.
48.
Sohn, J. H., Han, K. L., Lee, S. H., and Hwang, J. K. Protective Effects of
Panduratin A against Oxidative Damage of tert-Butylhydroperoxide in
Human HepG2 Cells. Bio. Pharm. Bull. 2005. 28 (6): 1083-1086.
49.
Yun, J. M., Kwon, H. J. and Hwang, J. K. In vitro Anti-Inflammatory of
Panduratin A Isolated from Kaempferia pandurata in RAW264.7 Cells.
Planta Med. 2003. 69: 1101-1108.
50.
Sematong, T., Reutrakul, V., Tuchinda, P., Claeson, P., Pongprayoon, U. and
Nahar, N. Topical Antiinflammatory Activity of Two Pimarane Diterpenes
from Kaempferia pulchra. Phytother. Res. 1996. 10 (6): 534-535.
51.
Yanti, Rukayadi, Y., Lee, K. H. and Hwang, J. K. Activity of Panduratin A
Isolated from Kaempferia pandurata Roxb. against Multi-species Oral
Biofilms In vitro. J. Oral Sci. 2009. 51 (1): 87-95.
52.
Shim, J. S., Han, Y. S. and Hwang, J. K. The Effect of 4-Hydroxypanduratin
A on the Mitogen-Activated Protein Kinase-Dependent Activation of Matrix
Metalloproteinase-1 Expression in Human Skin Fibroblasts. J. Dermatol. Sci.
2009. 53: 129-134.
53.
Patanasethanont, D., Nagai, J., Yumoto, R., Murakami, T., Sutthanut, K.,
Sripanidkulchai, B. O., Yenjai, C. and Takano, M. Effects of Kaempferia
Parviflora Extracts and Their Flavone Constituents on P-glycoprotein
Function. J. Pharm. Sci. 2007. 96 (1): 223-233.
54.
Patanasethanont, D., Nagai, J., Matsuura, C., Fukui, K., Sutthanut, K.,
Sripanidkulchai, B. O., Yumoto, R. and Takano, M. Modulation of Function
of Multidrug Resistance Associated-Proteins by Kaempferia parviflora
Extracts and Their Components. Euro. J. Pharmacol. 2007. 566: 67-74.
112
55.
Jantan, I., Raweh, S. M., Sirat, H. M., Jamil, S., Yasin, Y. H. M., Jalil, J. and
Jamala J. A. Inhibitory Effect of Compounds from Zingiberaceae Species on
Human Platelet Aggregation. Phytomedicine. 2008. 15: 306-309.
56.
Ridtitid, W., Wong, C. S., Reanmongkol, W. and Wongnawa, M.
Antinociceptive Activity of the Methanolic Extract of Kaempferia galanga
Linn. in Experimental Animals. J. Ethnopharmacol. 2008. 118: 225-230.
57.
Othman, R., Ibrahim, H., Mohd., M. A., Mustafa, M. R. and Awang K.
Bioassay-Guided Isolation of a Vasorelaxant Active Compound from
Kaempferia galanga L. Phytomedicine. 2006. 13: 61-66.
58.
Tewtrakul, S., Yuenyongsawad, S., Kummee, S., and Atsawajaruwan, L.
Chemical Components and Biological Activities of Volatile Oil of
Kaempferia galanga Linn. Songklanakarin J. Sci. Technol. 2005. 27 (2): 503507.
59.
Woerdenbag, H. J., Windono, T., Bos R., Riswan, S., and Quax, W. J.
Composition of the Essential Oils of Kaempferia rotunda L. and Kaempferia
angustifolia Roscoe Rhizomes from Indonesia. Flav. Fragr. J. 2004. 19: 145148.
60.
Sirat, H. M., Jamil, S., Siew, L. W. The Rhizome Oil of Kaempferia rotunda
Val. J. Essent. Oil Res. 2005. 17 (3): 306-307.
61.
Ganem, B. and Holbert, G. W. Arene Oxides in Biosynthesis. On The Origin
of Crotepoxide, Senepoxide, and Pipoxide. Bioorg. Chem. 1977. 6: 393-396.
62.
Holbert, G. W., Ganem, B., Engen, D. V., Clardy, J.,
Borsub, L.,
Chantrapromma, K., Sadavongvivad, C. and Thebtaranonth, Y. ShikimateDerived Metabolites Revised Structure and Total Synthesis of Pipoxide.
Tetrahedron Lett. 1979. 20 (8): 715-718.
113
63.
Jolad, S. D., Hoffmann, J. J., Schram, K. H., Cole, J. R., Tempesta, M. S.
and Bates, R. B. Structures of Zeylenol and Zeylena, Constituents of Uvaria
zeylanica (Annonaceae). J. Org. Chem. 1981. 46 (21): 4267-4272.
64.
Tuntiwachwuttikul, P., Pancharoen, O. and Bubb, W. A., Hambley, T. W.,
Taylor, W. C. and Reutrakul. Constituents of Zingiberaceae. XI* Structure of
(+)-[(1R,2R,4R,5R,6R,7R)-4-Benzoyloxy)methyl]-3,8-dioxatricyclo[5,1,0,02,4] octane-5,6-diol 5-Acetate 6-Benzoate. Aust. J. Chem. 1987. 40:
2049-2061.
65.
Verzera, A., Trozzi, A., Zappala, M., Condurso, C., and Cotroneo, A.
Essential Oil Composition of Citrus meyerii Y. Tan. and Citrus medica L. cv.
Diamante and Their Lemon Hybrids. J. Agric. Food Chem. 2005. 53: 48904894.
66.
Bicchi, C., Rubiolo, P., Marschall, H., Weyerstahl, P. and Laurent, R.
Constituents of Artemisia roxburghiana Besser Essential Oil. Flavour Fragr.
J. 1998. 13: 40-46.
67.
Shing, T. K. M. First Enantiospecific Syntheses of Crotepoxide and isoCrotepoxide from (-)-Quinic Acid. Tetrahedron: Asymm. 1998. 7 (2): 353-35.
68.
Schulte, G. R. Constitution of a New Key Metabolite Intermediate.
Tetrahedron Lett. 1982. 23 (42): 4303-4304.
69.
Roslund, M. U., Tähtinen, P., Niemitz, M., Sjöholm, R. Complete
Assignments of the 1H and 13C Chemical shifts and JH,H Coupling Constant in
NMR
Spectra
of
D-glucopyranose
and
All
D-Glucopyranosyl-D-
glucopyranosides. Carbohydr. Res. 2008. 343: 102-112.
70.
Oyama, K. and Kondo T. Total Synthesis of Flavocommelin, a Component of
the Blue Supramolecular Pigment from Commelina communis, on the Basis
of Direct 6-C-Glycosylation of Flavan. J. Org. Chem. 2004. 69 (16): 52405246.
114
71.
Fang, J. M, Lang, C. I., Chen, W. I. and Cheng, Y. S. Diterpenoid Acids from
the Leaves of Armand Pine. Phytochemistry. 1991. 30 (8): 2793-2795.
72.
Huang, W., Zhang, P., Jin, Y. C., Shi, Q., Cheng, Y. Y., Qu, H. B. and Ma, Z.
J. Cytotoxic Diterpene from the Root Tuber of Curcuma wenyujin. Helv.
Chim. Acta. 2008. 91 (5): 944-950.
73.
Kim, S., and Yi, K. Y. Synthetic Applications of Di-2-pyridyl
Thionocarbonate
as
a
Dehydration,
a
Dehydrosulfuriation,
and
a
Thiocarbonyl Transfer Reagent. Bull. Korean Chem. Soc. 1987. 8 (6): 466470.
74.
Kodpinid, M., Sadavongvivad, C., Thebtaranonth C., Thebtaranonth, T.
Benzyl Benzoate from the Root of Uvaria purpurea. Phytochemistry. 1984.
23 (1): 199-200.
75.
Baldé, A. M., Claeys, M., Pieters, L. A., Wray, V., Vlietinck, A. J. Ferulic
Acid Esters from Stem Bark of Pavetta Owariensis. Phythochemistry. 1991.
30 (3): 1024-1026.
76.
Kopinid, M., Sadavogvivad, C., Thebtaranonth., C. and Thebtaranonth, Y.
Structure of β-Senepoxide, Tingtangnoxide and Their Diene Precursors.
Constituents of Uvaria Ferruginea. Tetrahedron Lett. 1983. 24 (19): 20192022.
77
Christensen, H. N. The Isolation of Valylvaline from Gramicidin
Hydrolysates. J. Biol. Chem. 1943. 151: 319-324.
78.
Moore, B. S., Poralla, K. and Floss, H. G. Biosynthesis of the
Cycloxanecarboxylic Acid Started Unit of ω-Cyclohexyl Fatty Acids in
Alicyclobacillus acidocaldarius. J. Am. Chem. Soc. 1993. 115: 5267-5274.
115
79.
Brown, A. E. and Swinburne, T. R. Benzoic Acid. An antifungal Compound
Formed in Bramley's Seedling Apple Fruits Following Infection by Nectria
galligena Bres. Physiol. Plant Pathol. 1971. 1 (4): 469-475.
80.
Dax, S. L. Antibacterial Chemotherapeutic Agent. U. K.: Blanckie Academic
and Professional. 1997.
81.
Singleton, P. Bacteria in Biology, Biotechnology and Medicine. (6th ed.). U.
K.: John Wiley and Sons, Ltd. 2004.
82.
Mbaveng, A. T., Ngameni, B., Kuete, V., Simo, I. K., Ambassa, P., Roy, R.,
Bezabih, M., Etoa, F. X., Ngadjui, B. T., Abegaz, B. M., Meyer, J. J. M.,
Lall, N. and Beng, V. P. Antimicrobial Activity of the Crude Extracts and
Five Flavonoids from the Twigs of Dorstenia barteri (Moraceae). J.
Ethnopharmacol. 2008. 116: 483-489.
83.
Wong, S. P., Leong, L.P. and Koh, W. J. H. Antioxidant Activities of
Aqueous Extracts of Selected Plants. Food Chem. 2006. 99 (4): 775-783.
84.
Dasgupta, N. and De, B. Antioxidant Activity of Piper betle L. Leaf Extract
In Vitro. Food Chem. 2004. 88: 219-224.
85.
Bandyopadhyay, M., Chakraborty, R. and Raychaudhuri, U. A Process for
Preparing a Natural Antioxidant Enriched Dairy Product (Sandesh). LWT.
2007. 40: 842.
86.
Serafini, M., Bellocco, R., Wolk, A. and Ekström, A. M. Total Antioxidant
Potential of Fruit and Vegetables and Risk of Gastric Cancer. Gastroenterol.
2002. 123 (4): 985-991.
87.
Matteo, D. V. and Esposito, E. Biochemical and Therapeutic Effects of
Antioxidants in the Treatment of Alzheimer’s Disease, Parkinson’s Disease,
and Amyotrophic Lateral Sclerosis. Curr. Drug Target CNS Neurol. Disord.
2003. (2): 95-107.
116
88.
Maçao, L. B., Filho, D. W., Pedrosa, R. C., Pereira A., Backes, P., Torres, M.
A. and Fröde, T. S. Antioxidant Therapy Attenuates Oxidative Stress in
Chronic Cardiopathy Associated with Chagas’ Disease. Int. J. Cardiol. 2007.
123 (1): 43-49.
89.
Hu, H. L., Forsey, R. J., Blades, T. J., Barratt, M. E. J., Parmar, P. and
Powell, J. R. Antioxidants May Contribute in the Fight Against Ageing: An
In Vitro Model. Mech. Ageing Dev. 2001. 121 (1-3): 217-230.
90.
Brand-Williams, W., Cuvelier, M. E. and Berset, C. Use of a Free Radical
Method to Evaluate Antioxidant Activity. LWT. 1995. 28: 25-30.
91.
Heim, K. E., Tagliaferro, A. R. and Bobilya, D. J. Flavonoid Antioxidants:
Chemistry, Metabolism and Structure-Activity Relationship. J. Nutr.
Biochem. 2002. 13: 572-584.
92.
Tagashira, M. and Ohtake, Y. A. New Antioxidative 1,2-Benzodioxole from
Melissa officianalis. Planta Med. 1998. 64: 555-558.
93.
Ohno, O., Ye, M., Koyama, T., Yazawa, K., Mura, E., Matsumoto, H.,
Ichino, T, Yamada, K., Nakamura, K., Ohno, T., Yamaguchi, K., Ishida, J.,
Fukamizu, A. and Uemura, D. Inhibitory Effects of Benzyl Benzoate and Its
Derivatives on Angiotensin II-Induced Hypertension. Bioorg. Med. Chem.
2008. 16 (16): 7843-7852.
117
Appendix 1: GC Chromatogram of Malaysian Kaempferiaq rotunda (Rhizome) Oil
118
Appendix 2: GC Chromatogram of Indonesian Kaempferia rotunda (Rhizome) Oil
(M+1)+
O
O
7
1'
3'
5'
O
O
O
2
1
3
6 5 4
O
O
O
119
Appendix 3: CIMS Spectrum of Crotepoxide (54)
OAc
H-6
H-5
H-4
H-7
O
O
7
1'
3'
5'
Phenyl protons
O
O
O
2
1
3
6 5 4
O
O
O
H-4
H-2
H-7
H-6 H-5 H-4
H-3
120
Appendix 4: 1H NMR Spectrum of Crotepoxide (54)
O
O
7
1'
3'
5'
O
O
O
2
1
3
6 5 4
O
O
O
121
Appendix 5: 1H-1H COSY Spectrum of Crotepoxide (54)
O
O
7
1'
3'
5'
O
O
O
2
1
3
6 5 4
O
O
O
122
Appendix 6: HMBC Spectrum of Crotepoxide (54)
O
O
7
1'
3'
5'
O
O
O
2
1
3
6 5 4
O
O
O
123
Appendix 7: IR Spectrum of Crotepoxide (54)
C-3', 5' C-2', 6'
O
O
7
1'
3'
O
O
5'
O
2
1
3
6 5 4
O
O
O
C-3
C-2
2x CH3 (OAc)
C-6 C-4
C-1
C-5
C-7
C-4'
C=O (OAc)
C-1'
C=O (OBz)
124
Appendix 8: 13C NMR Spectrum of Crotepoxide (54)
O
O
All carbons
7
1'
3'
(OAc)
C-1'
C=O (OBz)
5'
O
O
O
2
1
3
6 5 4
O
C-1
O
O
C-3', 5'
C-2', 6'
DEPT 45
C-4'
DEPT 90
C-3
C-4
C-2 C-6
C-5
CH3 (OAc)
DEPT 135
125
Appendix 9: 13C NMR and DEPT Spectra of Crotepoxide (54)
O
O
7
1'
3'
5'
O
O
O
2
1
3
6 5 4
O
O
O
126
Appendix 10: HMQC Spectrum of Crotepoxide (54)
Da te: 11/10/2009
T im e: 4:10:53 PM
1 .0 0
0 .9
0 .8
CH3
0 .7
H3CO
219.37
6'
4'
O
4
OCH3
364.68
8
0 .6
7
2'
A
OH O
0 .5
0 .4
0 .3
0 .2
0 .1
0 .0 0
2 00 .0
2 20
Desc rip tio n:
2 40
2 60
2 80
3 00
nm
3 20
3 40
3 60
3 80
4 00 .0
Appendix 11: UV Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1)
Sc an Sp eed: 240.00 nm/min
Date Created : Mo n May 18 10:50:05 2009
Slit Wid th: 1.0000 nm
Ins trument Model: Lambda 25
Smoo th Bandwid th: 2.00 nm
Data Interval: 1.0000 nm
127
Sp ec trum Name: C:\UVWINLAB\DATA\MEOH.SP
CH3
H3CO
6'
4'
O
4
OCH3
8
7
2'
OH O
128
Appendix 12: IR Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1)
OCH3
H-7, 8
H-3, 5
H-2 , 6
H-5'
H-3'
CH3
H3CO
6'
4'
O
4
OCH3
8
7
2'
H-7, 8 H-3, 5
OH
OH O
H-2 ,6
H-5' H-3'
129
Appendix 13: 1H NMR Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1)
C-3, 5
C-2, 6
3 x OCH3
C-8
C-5' C-3'
C-7
CH3
H3CO
C=O
6'
4'
O
C-1
C-6'
C-2'
C-4'
C-4
4
OCH3
8
7
2'
OH O
C-1'
130
Appendix 14: 13C NMR Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1)
CH3
H3CO
All
C-2' C-6' C-4' C-4
C-1
C=O
6'
4'
O
4
OCH3
8
7
C-1'
2'
OH O
DEPT 45
C-8
C-3, 5
C-7
C-2, 6
C-5' C-3'
DEPT 90
DEPT 135
3 x OCH3
Appendix 15: 13C NMR and DEPT Spectra of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1)
131
M+
CH3
H3CO
6'
4'
O
4
OCH3
8
7
2'
OH O
132
Appendix 16: EIMS Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1)
CH3
H3CO
6'
4'
O
4
OCH3
8
7
2'
OH O
133
Appendix 17: HMBC Spectrum of 2'-Hydroxy-4,4',6'-trimethoxychalcone (1)
O
O
2'
4'
6'
7
O
O
O
2
1
3
6 5 4
OH
O
(M+1)+
134
Appendix 18: CIMS Spectrum of 4-Benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diol 5-acetate (57)
O
O
2'
4'
6'
7
O
O
O
2
1
3
6 5 4
OH
O
135
Appendix 19: IR Spectrum of 4-Benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diol 5-acetate (57)
H-6
CH3 (OAc)
H-5
OH
H
O
H-7
O
2'
4'
6'
7
O
O
O
2
1
3
6 5 4
OH
O
Phenyl protons
H-2
H-6
H-7
H-5 H-4
H-3
OH
136
Appendix 20: 1H NMR Spectrum of 4-Benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diol 5-acetate (57)
C-3', 5'
C-2', 6'
O
O
2'
4'
6'
O
7
O
O
2
1
3
6 5 4
OH
O
C-6 C-4
CH3 (OAc)
C-3
C-4'
C-5
C-2
C-1
C=O (OAc)
C-1'
C=O (OBz)
Appendix 21: 13C NMR Spectrum of 4-Benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diol 5-acetate (57)
137
O
O
2'
4'
6'
7
O
O
O
2
1
3
6 5 4
OH
O
138
Appendix 22: HMBC Spectrum of Benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diol 5-acetate (57)
O
O
2'
4'
6'
7
O
O
O
2
1
3
6 5 4
OH
O
139
Appendix 23: HMQC Spectrum of Benzoyloxymethyl-3,8-dioxatricyclo[5.1.0.02,4]octane-5,6-diol 5-acetate (57)
350
M+
O
7
O
4'
6'
4''
2''
OH
2'
O
1
2
6''
3
4
6
O
5
Appendix 24: EIMS Spectrum of 1,6-Desoxypipoxide (69)
140
C-2'', 6'' C-2', 6'
C-3'', 5''
C-3', 5'
C-1
C-4'' C-4'
C-1',1''
O
7
O
4'
C-6
C-5
2x C=O (OBz)
C-4
6'
4''
2''
OH
2'
O
1
2
6''
3
4
6
O
5
C-3
C-2
C-7
141
Appendix 25: 13C NMR Spectrum of 1,6-Desoxypipoxide (69)
All carbons
O
C-1',1''
C=O
4'
C-1
DEPT 45
C-4''
C-4'
DEPT 90
C-2'',2'
C-6'',6'
7
O
6'
4''
2''
OH
2'
O
1
2
6''
3
4
6
O
5
C-6
C-5
C-4
C-3
C-2
C-3'',3'
C-5'',5'
DEPT 135
C-7
Appendix 26: 13C NMR and DEPT Spectra of 1,6-Desoxypipoxide (69)
142
O
7
O
4'
6'
4''
2''
OH
2'
O
1
2
6''
3
4
6
O
5
143
Appendix 27: IR Spectrum of 1,6-Desoxypipoxide (69)
O
7
O
4'
6'
4''
2''
OH
2'
O
1
2
4
6
H-7
6''
3
O
5
H-6, H-5
H-2', 6'
H-2'', 6''
H-4
H-3
H-2
H-4'
H-4''
H-6 H-5
H-3', 5'
H-3'', 5''
H-7
H-2
H-4
OH
H-3
144
Appendix 28: 1H NMR Spectrum of 1,6-Desoxypipoxide (69)
H-5, 6
H-4
H-7
H-3
H-2
OH
OH
H-2
H-7
O
H-3
H-4
H-5, 6
7
O
4'
6'
4''
2''
OH
2'
O
1
2
6''
3
4
6
O
5
Appendix 29: 1H-1H COSY Spectrum of 1,6-Desoxypipoxide (69)
145
1043.91
70
1156.78
1517.37
75
2086.95
2399.87
80
4'
7
9
10
3
OH O
1215.54
45
1'
2
5
50
O
OCH3
669.09
H3CO
55
3413.67
40
759.59
%T
60
1638.99
3019.42
65
35
4000
3500
3000
2500
2000
1500
1000
500
Wavenumbers (cm-1)
146
Appendix 30: IR Spectrum of Naringenin 4',7-dimethyl ether (133)
2x OCH3
4'
H3CO
7
9
O
OCH3
H-2
1'
H-3
2
5
10
3
OH O
OH
H-2' ,6'
H-3', 5'
H-6
H-8
H-2
147
Appendix 31: 1H NMR Spectrum of Naringenin 4',7-dimethyl ether (133)
H-3
H-3', 5'
H-2' ,6'
H-3
H-2
H-3
H-2
4'
H3CO
H-3', 5'
H-2' ,6'
7
9
O
OCH3
1'
2
5
10
3
OH O
148
Appendix 32: 1H-1H COSY Spectrum of Naringenin 4',7-dimethyl ether (133)
4'
H3CO
C-3', 5'
7
C-2', 6'
9
O
OCH3
1'
2
5
10
3
OH O
2 x OCH3
C-8
C-6
C-2
C=O
C-5 C-9
C-10
C-7
C-4'
C-3
C-1'
149
Appendix 33: 13C NMR Spectrum of Naringenin 4',7-dimethyl ether (133)
4'
All carbons
C=O
H3CO
C-5
C-9
C-7
C-4'
C-1'
DEPT 45
C-3', 5'
C-10
9
O
1'
2
5
10
3
OH O
C-2', 6'
DEPT 90
C-2 C-6
DEPT 135
7
OCH3
C-8
2 x OCH3
C-3
C-3
150
Appendix 34: 13C NMR and DEPT Spectra of Naringenin 4',7-dimethyl ether (133)
krrm14c #3 RT: 0.50 AV: 1 NL: 7.61E6
T: + c EI Full ms [ 19.50-800.50]
84
100
161
95
90
85
80
M+
75
206
149
70
300
86
Relative Abundance
65
4'
134
105
60
H3CO
55
7
9
O
1'
2
50
3
10
5
45
OCH3
OH O
121
299
40
35
51
30
91
20
242
137
77
25
133
57
167
69
15
97
10
109
178
301
119
193
150
122
67
147
151
5
177
180
192
207
271
205
195
279
243
228
223
241
257
253 259
302
283
298
314
0
60
80
100
120
140
160
180
200
m/z
220
240
260
280
300
320
340
Appendix 35: EIMS Spectrum of Naringenin 4',7-dimethyl ether (133)
151
78
76
74
O
56
52
50
11
O 20
1
3001.65
54
3
18
10
5
8
6
1378.25
OH
O
19
920.91
1152.38
1458.34
17
992.03
15
1023.51
13
58
1095.05
OH
1067.96
16
1215.91
22
1250.72
60
3418.42
46
44
665.87
48
755.51
%T
62
1425.65
64
1631.61
2929.68
66
1722.52
68
2868.10
70
2847.67
72
42
4000
3500
3000
2500
2000
1500
1000
500
Wavenumbers (cm-1)
152
Appendix 36: IR Spectrum of Curcumrinol C (134)
15.081
18.163
22.258
21.209
21.063
20.656
26.799
32.619
33.724
36.303
35.861
38.949
41.570
22
OH
16
13
O
C-4
C-10
C-6
C-3 C-1
C-18
3
18
C-13
C-5
43 42 41 40 39 38 37 36 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 ppm
C-15
200
190
C-14
17
8
6
OH
O
19
C-9
C-12
C-8
C=O (C-21)
C=O (C-7)
210
10
5
C-20
C-2
C-11
C-16
11
O 20
1
C-22
C-19
C-1 7
15
180
170
160
150
140
130
120
110
100
90
80
70
50
40
30
20
10
ppm
153
Appendix 37: 13C NMR Spectrum of Curcumrinol C (134)
60
22
16
OH
C-10
13
O
All carbons
C=O
3
C=O
18
DEPT 45
11
O 20
1
15
10
5
8
6
C-13
17
C-4
C-8
OH
O
19
C-12
C-15
C-5 C-9
DEPT 90
DEPT 135
C-18
C-16
C-17 C-22 C-19
C-20
C-14
C-3
C-6
C-1
154
Appendix 38: 13C NMR and DEPT Spectra of Curcumrinol C (134)
C-2
C-11
105%
01231208: Scan 4 (0.93 min)
Base: 43.00 Int: 6.42018e+006 Sample: VG70-SE Electron Impact
90%
80%
70%
55
60%
250
69
22
50%
16
OH
13
123
84
O
109
40%
3
30%
95
20%
18
207
163
10%
10
5
362
100
150
200
m/z
6
OH
O
[M+-H2O]
334
0%
40
8
17
19
292
232
11
O 20
1
15
250
350
400
155
Appendix 39: EIMS Spectrum of Curcumrinol C (134)
300
H-15
2.455
2.447
2.420
2.413
2.535
2.571
2.606
5.036
5.075
5.065
5.121
5.383
5.379
5.373
6.090
6.061
6.044
6.016
H-14
H-6
22
OH
16
13
O
H-12
1
6.1
6.0
ppm
ppm 5.15
5.10
5.05 ppm
2.6
ppm
2.5
11
O 20
3
18
15
10
5
17
8
6
OH
O
19
H-15
H-6
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.012
9.0
1.000
9.5
2.5
2.0
1.5
1.0
0.5
ppm
156
Appendix 40: 1H NMR Spectrum of Curcumrinol C (134)
3.0
1.903
2.885
1.993
1.063
1.012
3.467
1.048
3.165
0.685
0.574
0.551
0.368
3.207
0.892
0.193
0.120
2.897
0.297
5.512
0.947
0.369
H-14 H-12
H-22
22
OH
16
H-17
13
O
11
O 20
1
15
10
3
5
8
6
H-16
17
OH
O
H-1a H-2
H-3b
H-1b
ppm
0.947
5.512
0.297
0.9
2.897
0.120
1.0
157
Appendix 41: 1H NMR Spectrum of Curcumrinol C (134) (Expansion)
0.193
1.1
0.892
1.2
3.207
1.3
0.368
1.4
0.551
1.5
0.574
1.6
H-3a H-5
0.685
1.7
3.467
1.8
1.012
1.9
1.063
1.993
2.885
2.0
H-2b
3.165
H-11b
1.048
H-9, H-11a
2.1
H-18 H-19
19
18
2.2
H-20
22
16
OH
13
O
11
O 20
1
3
18
15
10
5
8
6
17
OH
O
19
Appendix 42: 1H-1H COSY Spectrum of Curcumrinol C (134)
158
22
16
OH
13
O
11
O 20
1
3
18
15
10
5
8
6
17
OH
O
19
Appendix 43: 1H-1H COSY Spectrum of Curcumrinol C (134) (Expansion)
159
22
16
OH
13
O
11
O 20
1
3
18
15
10
5
8
6
17
OH
O
19
160
Appendix 44: HMBC Spectrum of Curcumrinol C (134)
H-7
O
2'
7
2
O
4'
6'
6
4
H-2 to 6
H-3', 5'
H-2', 6'
H-4'
161
Appendix 45: 1H NMR Spectrum of Benzyl Benzoate (82)
O
2'
7
2
O
4'
6'
6
4
Appendix 46: IR Spectrum of Benzyl Benzoate (82)
162
C-2', 6'
C-3', 5'
C-2, 6
C-3, 5
C-4
C-4'
O
2'
7
2
O
4'
6'
C-1
6
4
C-7
C=O
C-1'
163
Appendix 47: 13C NMR Spectrum of Benzyl Benzoate (82)
O
All carbons
C=O
2'
C-1'
C-1
C-2', 6'
DEPT 45
C-4´
DEPT 90
7
2
O
4'
6'
6
4
C-2, 6
C-3, 5
C-3', 5'
C-4
DEPT 135
C-7
Appendix 48: 13C NMR and DEPT Spectra of Benzyl Benzoate (82)
164
O
2'
M
7
+
4'
6'
6
4
165
Appendix 49: EIMS Spectrum of Benzyl Benzoate (82)
2
O
O
7
1'
1
5
8
HO
9
O CH2
2'
CH2
3'
4'-21'
CH 2
CH2
CH3
n
3
OCH3
C-4´ to C-n-3'
C-n-1'
C-22'
C-n-2'
C-2'
C-3'
C-8
C-3 C-4
C-9
C-3 C-4
C-7
C6
C-1
C
C-1'
C-8 C-5
OCH3
C-2
166
Appendix 50: 13C NMR Spectrum of trans-Docosyl ferulate (137)
All carbons
C=O
C-3 C-4
C-1
C-8 C-5
DEPT 45
C-7
C6
C-2
DEPT 90
DEPT 135
C-22'
OCH3
C-1'
167
Appendix 51: 13C NMR and DEPT Spectra of trans-Docosyl ferulate (137)
C-n-1'
C-3'
C-4 to C-n-3', C-2'
C-n-2'
1463.89
65
2855.73
60
55
1045.15
70
1378.12
75
2100.98
2400.00
80
1711.69
1657.89
35
O
7
1'
1
5
15
8
HO
10
9
O CH2
2'
CH2
3'
4'-21'
CH 2
CH2
CH3
n
3
OCH3
5
4000
3500
3000
2500
2000
1500
668.96
20
755.51
25
2958.00
30
1215.37
40
2928.88
3019.22
45
3426.44
%T
50
1000
500
Wavenumbers (cm-1)
Appendix 52: IR Spectrum of trans-Docosyl ferulate (137)
168
H-2
4.212
4.195
4.178
6.281
6.321
6.934
6.913
7.091
7.070
7.045
7.597
7.637
H-7
H-5
H-8
H-1'
H-4' to H-n-1'
H-6
O
7
1'
1
5
8
HO
7.7
7.6
ppm
7.1
7.0
ppm
6.35
6.30
ppm
4.2
ppm
9
O CH2
2'
CH2
3'
4'-21'
CH 2
CH2
CH3
n
3
OCH3
OCH3
H-1'
169
Appendix 53: 1H NMR Spectrum of trans-Docosyl ferulate (137)
H-22'
H-3'
O
7
1'
1
5
8
HO
9
O CH2
2'
CH2
3'
4'-21'
CH 2
CH2
CH3
n
3
OCH3
170
Appendix 54: 1H-1H COSY Spectrum of trans-Docosyl ferulate (137)
O
7
1'
1
5
8
HO
9
O CH2
2'
CH2
3'
4'-21'
CH 2
CH2
CH3
n
3
OCH3
Appendix 55: HMBC Spectrum of trans-Docosyl ferulate (137)
171
M+
O
7
1'
1
5
8
HO
9
O CH2
2'
CH2
3'
4'-21'
CH 2
CH2
CH3
n
3
OCH3
Appendix 56: EIMS Spectrum of trans-Docosyl ferulate (137)
172
10
5
4000
20
3500
3000
HO
O
6
O
15
2500
1
5
4
O
2
3
OH
2''
4''
O
O
6''
2000
1500
1000
685.96
O
1070.50
7
1238.15
6'
667.44
1027.10
1178.00
1452.18
930.68
1585.07
2851.87
973.98
1115.41
1217.25
1317.12
1491.62
2'
1372.06
30
1602.61
4'
1516.03
2926.71
35
1654.63
40
1715.26
45
3018.85
50
711.88
1272.89
25
3435.32
%T
70
65
60
55
-0
Wavenumbers (cm-1)
500
Appendix 57: IR Spectrum of 6-Acetylzeylenol (68)
173
4'
2'
H-4, 5
H-3
H-6
OH
H-2
H-7
6'
HO
O
6
O
7
1
5
4
O
O
OH
2
3
4''
2''
O
6''
O
CH3 (OAc)
H-4, 5
Phenyl protons
H-6
H-3
H-7
OH
H-2
OH
174
Appendix 58: 1H NMR Spectrum of 6-Acetylzeylenol (68)
4'
2'
6'
HO
O
6
O
7
1
5
4
O
O
OH
2
3
4''
2''
O
6''
O
(M+1)+
Appendix 59: CIMS Spectrum of 6-Acetylzeylenol (68)
175
C-3
C-3'', 5''
C-3' , 5'
C-2
C-1
C-6
C-2'', 6''
C-2´ , 6´
C-7
C-4''
C-1'
C-1''
C-4'
C-5
C-4
4'
2'
6'
134
133
132
131
130
129
128
127
ppm
HO
O
6
O
7
1
5
4
O
O
75
OH
2
3
74
73
72
71
70
69
68
67
66
65
64
ppm
10
ppm
4''
2''
O
6''
O
CH3 (OAc)
C=O
(OAc)
190
180
170
C=O
(OBz)
160
150
140
130
120
110
100
90
80
70
60
40
30
20
176
Appendix 60: 13C NMR Spectrum of 6-Acetylzeylenol (68)
50
All carbons
C-1'
C-1''
C=O
C=O
OAc)
(OBz)
C-2'', 6''
C-2', 6'
C-4''
C-4'
DEPT 45
DEPT 90
4'
C-5
6'
HO
O
6
DEPT 135
O
7
1
5
4
C-3'', 5''
C-3', 5'
C-4
2'
C-1
C-3
C-2
C-6
O
O
OH
2
3
4''
2''
O
6''
O
CH3 (OAc)
C-7
177
Appendix 61: 13C NMR and DEPT Spectra of 6-Acetylzeylenol (68)
H-4, 5
H-3 H-6
H-7
H-2
OH
OH
OH
OH
4'
H-2
H-7
2'
6'
H-6
H-4, 5 H-3
HO
O
6
O
7
1
5
4
O
O
OH
2
3
4''
2''
O
6''
O
178
Appendix 62: 1H-1H COSY Spectra of 6-Acetylzeylenol (68)
4'
2'
6'
HO
O
6
O
7
1
5
4
O
O
OH
2
3
4''
2''
O
6''
O
179
Appendix 63: HMBC Spectrum of 6-Acetylzeylenol (68)
O
OH
180
Appendix 64: IR Spectrum of Benzoic Acid (138)
O
OH
181
Appendix 65: 1H NMR Spectrum of Benzoic Acid (138)
O
OH
182
Appendix 66: 13C NMR Spectrum of Benzoic Acid (138)