METHODS TO ISOLATE THE CHEMICAL CONSTITUENTS RESPONSIBLE FOR THE
CYTOTOXICITY OF LIGUSTICUM GRAYI TOWARDS BREAST CANCER CELLS
Ursula E. Leyva Castro
B.A., University of California, Davis, 2006
THESIS
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF SCIENCE
in
CHEMISTRY
(Biochemistry)
at
CALIFORNIA STATE UNIVERSITY, SACRAMENTO
FALL
2009
© 2009
Ursula E. Leyva Castro
ALL RIGHTS RESERVED
ii
METHODS TO ISOLATE THE CHEMICAL CONSTITUENTS RESPONSIBLE FOR THE
CYTOTOXICITY OF LIGUSTICUM GRAYI TOWARDS BREAST CANCER CELLS
A Thesis
by
Ursula E. Leyva Castro
Approved by:
__________________________________, Committee Chair
Dr. Mary McCarthy Hintz
__________________________________, Second Reader
Dr. Roy Dixon
__________________________________, Third Reader
Dr. Tom Savage
____________________________
Date
iii
Student: Ursula E. Leyva Castro
I certify that this student has met the requirements for format contained in the University
format manual, and that this thesis is suitable for shelving in the Library and credit is to
be awarded for the thesis.
______________________________________
Susan Crawford, Department Chair
Department of Chemistry
iv
___________________
Date
Abstract
of
METHODS TO ISOLATE THE CHEMICAL CONSTITUENTS RESPONSIBLE FOR
THE CYTOTOXICITY OF LIGUSTICUM GRAYI TOWARDS BREAST CANCER
CELLS
by
Ursula E. Leyva Castro
The taproot of the perennial herb Ligusticum grayi has been used by Native Americans
for many generations to treat common colds, respiratory infections, menstrual pain, and
many other ailments. More recently, it was also reported to exhibit cytotoxic properties
against breast cancer. An in vitro cell assay of hexane, ethanol, and aqueous root extracts
showed that both the hexane and ethanol extracts are cytotoxic towards both breast
cancer cells overexpressing estrogen and progesterone receptors (BT-474, LC50 = 14 + 4
g/mL) and those not overexpressing estrogen and progesterone receptors (MDA-MB231, LC50 = 15 + 5 g/mL). A hexane-soluble fraction was made by extracting the roots
with ethanol, drying the extract, then re-dissolving in hexane. This hexane-soluble
fraction was stored at -20 ºC and used for all further studies. In an attempt to isolate the
constituent(s) responsible for the cytotoxic properties, various separation techniques were
employed: Separation via column chromatography, liquid-liquid partitioning, preparative
thin layer chromatography, and liquid-liquid extraction followed by column
chromatography did not successfully isolate the cytotoxic compound(s). Although the
v
compounds were not successfully isolated, differences in peak abundance were observed
as a result of these separations, and elimination of non-cytotoxic constituents were
accomplished. The cytotoxicity assay had shown that oshá roots do not exhibit any
estrogenic activity, and chromatograms of GC and HPLC suggest one or a group of
compounds are responsible for the cytotoxicity. Of the methods attempted, liquid-liquid
extraction followed by column chromatography is the best scheme for the isolation of
cytotoxic constituents from L. grayi roots.
However, preparative HPLC with fraction collector is believed to be a better isolation
method as shown by an efficient separation of the chemical constituents and the ability to
collect fractions with relative close retention times.
___________________________________, Committee Chair
Dr. Mary McCarthy Hintz
___________________________________
Date
vi
ACKNOWLEDGMENTS
I would like to express my gratitude to all those who gave me the possibility to complete
this thesis. I want to thank the Department of Chemistry of California State University,
Sacramento for giving me permission to commence this thesis in the first instance, to do
the necessary research work and to use departmental data and equipment. I have
furthermore to thank my advisor Dr. McCarthy-Hintz who gave and confirmed this
permission and encouraged me to go ahead with my thesis. I really appreciate her
suggestions and help throughout this project, as well as being a great mentor.
I am deeply indebted to Professor Dr. Dixon and Dr. Savage for their help and
suggestions in the writing of this thesis.
I would like to thank my former colleague Gary Lai who supported my research work,
the rest of the research crew, and my sister Aurelia Leyva Castro and brother Luis A.
Leyva for all their help, support, interest and valuable hints.
Especially, I would like to give my special thanks to my parents whose patient love
enabled me to complete this work.
vii
TABLE OF CONTENTS
Page
Acknowledgments.................................................................................................................. vii
List of Tables ............................................................................................................................ix
List of Figures ........................................................................................................................... x
Chapter
1. INTRODUCTION .............................................................................................................. 1
2. EXPERIMENTAL ............................................................................................................ 16
3. RESULTS AND DISCUSSION ........................................................................................ 27
4. CONCLUSIONS................................................................................................................ 65
References ............................................................................................................................... 68
viii
LIST OF TABLES
Page
1. HPLC gradient profile...........................................................................................26
2. Percent peak area of GC-MS elution of the hexane-soluble fraction
before and after heat treatment.......................................................................32
3. Summary of HPLC-UVD analysis of the hexane-soluble fraction
and flash chromatography column 1 fractions C, D, E, F, G, H, L,
and M..............................................................................................................39
4. Summary of HPLC-CAD analysis of the hexane-soluble fraction and
flash chromatography column 1 fractions C, D, E, F, G, H, L, and
M....................................................................................................................40
5. Percent peak abundance from total ion chromatograms of fractions B
through E from flash column chromatography..............................................44
6. Solvent miscibility test...................................................................................45
7. Percent peak abundances of fractions Z1 and Z2 as shown by GC
analysis..........................................................................................................64
ix
LIST OF FIGURES
Page
1.
Ligusticum grayi. Plant (left), flower (upper right), and leave
(lower right)..............................................................................................10
2.
Oshála distribution map............................................................................11
3.
Oshála worldwide distribution map..........................................................11
4.
L.grayi distribution in the state of California...........................................12
5.
Cytotoxic activity of aqueous, 95% ethanol and hexane extracts on
MDA-MB-231 and BT-474 breast cancer cells using a
100 μg/mL concentration .........................................................................27
6.
Cytotoxic activity of the hexane soluble fraction and the ethanol
rinse of the hexane-insoluble residue on MDA-MB-231 cells at a
100 μg/mL concentration .........................................................................28
7.
Cytotoxicity assay of soil residues dissolved in 95% ethanol and
ethanolic oshála root extract on breast cancer cell line
MDA-MB-231 at a 100 μg/mL concentration.........................................30
8.
Cytotoxicity assay of the hexane-soluble fraction before and
after heat treatment, both at 40 μg/mL, on MDA-MB-231......................31
9.
LC50 determination of the hexane-soluble fraction of oshála on
MDA-MB-231 and BT-474 cells .............................................................33
10.
Cytotoxicity assay of flash chromatography column 1 fractions
of the hexane-soluble fractions (100 μg/mL) on breast cancer
cell line MDA-MB-231............................................................................36
11.
Representative chromatograms from HPLC analysis of flash
chromatography column 1 of the hexane-soluble fraction:
(from top to bottom) hexane-soluble fraction, fractions C, G,
and M........................................................................................................38
x
12.
Cytotoxicity assay of the collected column chromatography
fractions. The collected fractions were dried, re-dissolved
in ethanol, and tested on MDA-MB-231 cells at a
concentration of 40 μg/mL........................................................................41
13.
Total ion gas chromatograms of (from top to bottom) fractions
B, C, D, and E from the second chromatography separation ..................43
14.
Total ion gas chromatograms of representative liquid-liquid
extractions on the hexane-soluble fraction (raffinate) using
various acetonitrile as the extractant: (A) third acetonitrile
wash of hexane extract, (B) hexane layer after three acetonitrile
washes.......................................................................................................46
15.
Liquid-liquid extraction flow chart...........................................................48
16.
Cytotoxicity assay of fractions from liquid-liquid extractions
on the hexane-soluble fraction with acetonitrile: (A) Hexane
phase after 1 acetonitrile wash; (B) Acetonitrile wash 1;
(C) Acetonitrile wash 2; (D) Acetonitrile wash 3;
(E) Hexane phase after 3 acetonitrile washes on hexane
hexane-soluble fraction. All liquid phases were tested on cells
at a concentration of 40 μg/mL.................................................................48
17.
Total ion gas chromatograms of the hexane-soluble fraction
and liquid-liquid extraction fractions A, C, D, and E...............................49
18.
Liquid-liquid extraction flow chart...........................................................51
19.
Cytotoxicity assay of back-extractions of the first acetonitrile phase
resulting from an acetonitrile wash on the (hexane) hexane-soluble
fraction......................................................................................................51
20.
Total ion chromatograms of the raffinate and the extracts from
three liquid-liquid extraction hexane backwashes on
acetonitrile phases....................................................................................52
21.
Preparative TLC of the hexane-soluble fraction developed
with 100% dichloromethane.....................................................................53
xi
22.
Total ion gas chromatograms of selected bands A, B, D, G,
and O from preparative TLC separation of the hexane-soluble
fraction......................................................................................................55
23.
Flow chart illustrating LLE followed by FCC.........................................57
24.
Cytotoxcicity assay of the two column chromatography fractions,
tested at a 100 μg/mL concentration........................................................58
25.
HPLC- CAD/UVD chromatogram of two column chromatographic
fractions....................................................................................................60
26.
Cell assay of collected fractions Z1 and Z2 from a silica based DCM/
MeOH column with a 40 μg/mL concentration.......................................62
xii
1
Chapter 1
INTRODUCTION
1.1 Introduction and Scope of Study
Ligusticum grayi, commonly known as oshála (1), Gray’s lovage (2), Gray’s wild lovage
(2), Gray’s licorice root (3), Wild Plum (4), Sheep wild lovage (5), and kishwoof (6), has
been used for many years as a medicinal plant to treat common colds, respiratory
infections, and painful menstruations, among many other ailments (7). Its use as a
medicinal herb has raised interest in its chemical composition and the mechanism by
which it works.
The hypothesis proposed here is that Ligusticum grayi contains active constituent(s) that
kill or inhibit the growth of breast cancer cells. The following specific aim was addressed
in this project in accordance with the above stated hypothesis – to determine the best
method to isolate the constituent(s) in L. grayi responsible for killing or inhibiting the
growth of breast cancer cells.
A series of chromatographic techniques including flash column chromatography (CC),
liquid-liquid extractions (LLE), thin layer chromatography (TLC), gas chromatography mass spectrometry (GC-MS), and high performance liquid chromatography with charged
aerosol detection (HPLC-CAD) or ultraviolet spectrophotometer detector (HPLC-UVD)
2
were used to separate the bioactive constituent(s) from other extract components and
assess the efficacy of the separation methods. Determination of biological activity for the
active isolates was assessed as LC50 (50% lethality dose) values for a mammalian cell
assay using the human breast cancer cell line MDA-MB-231.
1.2 Significance of Breast Cancer
Worldwide, breast cancer is the second most common type of cancer and the fifth most
common cause of cancer death. The National Cancer Institute estimated that in 2008,
there would be 182,460 (female) and 1,990 (male) new cases and, from these, 40,480
(female) and 450 (male) deaths in the United States (8). Breast cancer usually originates
in the cells of the lobules (lobular carcinoma) or ducts (ductal carcinoma) of the milk
glands, or, much less commonly, in the stromal tissue (fatty and fibrous connective tissue
of the breast; 9). The term “breast cancer”, therefore, refers to cancer of the milk glands.
Similar to other cancers, breast cancer is caused by genetic abnormalities that are either
inherited, accounting for 5-10% of all cases, or occur as a result of de novo mutation
during aging accounting for over 90% of all cases (10).
Currently, two mutated genes are known to be responsible for the development of breast
cancer, BRCA1 and BRCA2 (11, 12). Both genes are tumor suppressor genes, which
control chromosomal repair and prevent uncontrolled proliferation (13). BRCA1
mutation carriers have a 54% risk of developing breast cancer by the age of 60 while
3
carriers of BRCA2 have a 71% risk (14). Although individuals expressing these
autosomal dominant genes have a high risk of developing breast cancer, only 2 to 5% of
all breast cancer cases are due to these mutations (15). To date, there are no medical
procedures known to benefit BRCA-positive patients, as a result, new studies have
focused on treatment options to reduce cancer risk (16).
Another gene largely linked to breast cancer is HER-2/neu. Amplification of HER-2/neu
is the most frequent oncogene amplification found in breast tumors (17). This gene
directs the production of proteins called HER-2 (human epidermal growth factor 2
receptors; 18). Cells that overexpress this gene (making excessive amounts of HER-2)
tend to grow faster and have an increased risk of spreading (19). About 20% of breast
tumors are associated with HER-2/neu protein over-expression (20, 21). Patients
expressing HER-2/neu receptors can be treated with therapies specifically targeting HER2/neu receptors (17).
Other receptors found to be overexpressed in breast cancer cells are estrogen and
progesterone receptors. About 75% of breast cancers are estrogen receptor positive
(ER+), and, of these, 65% are also progesterone receptor positive (PR+) (22). Although
breast cancers are mostly linked to the overexpression of estrogen receptors, not all breast
cancers exhibit this property. About 25% of all breast cancers are ER negative (ER-) and
PR negative (PR-) or of unknown status, 10% are ER+ and PR-, and 5% are ER- and PR+
(22). Because estrogen and progesterone receptors are expressed in a limited number of
4
cell types, hormonal therapy has the advantage of only targeting a small percentage of the
cells in the body. A person whose cancer cells express both receptors has a 70% chance
of responding to hormonal therapy (22). Patients expressing only one receptor (ER+/PRor ER-/PR+) have a 33% chance of response to such therapy, and patients whose receptor
status is unknown have a 10% chance (22). Hormonal therapy can work in multiple ways:
it can add, block or remove hormones. Breast cancer hormone therapy works by
inhibiting estrogen and progesterone from promoting breast cancer cell growth, or by
turning off the production of hormones from the ovaries (23). Some of the most common
breast cancer hormonal drugs include Tamoxifen, Faresto, Arimidex, Aromasin, Femara,
and Zoladex (23).
As little is known of the L. grayi chemical constituents and its behavior with breast
cancer cells, this project assessed the efficacy of this plant extract on ER-/PR- (MDAMB-231) and ER+/PR+ (BT-474) human breast cancer cells (24, 25, 26).
1.3 Plants as potential alternative treatment
For many years, herbs were used as the main remedy for various ailments. Willow bark,
for example, has been used as a medicinal plant for thousands of years by the ancient
Greeks, Assyrians, Sumerians, Egyptians, and Native Americans (27). Although widely
used, it was not until 1828 that the structure of the active compound, salicin, an analgesic,
anti-inflammatory drug, was discovered (28). Since the discovery of the active
5
constituent, improvements to the chemical structure have allowed for its use and
acceptance into Western medicine as what is now known as aspirin.
In the 1960’s, Moroe Wall and Mansukh Wani discovered that the bark of Taxus
brevifolia (Pacific yew) had cytotoxic activity against adenocarcinoma (cancer) cells
(29). In 1967, taxol was isolated and characterized as the active ingredient in the bark
(30). Since its discovery, taxol has been used as a chemotherapy agent (31) to treat
patients with lung cancer (32), ovarian cancer (33), breast cancer (34), head and neck
cancer (35), and advanced forms of Kaposi’s sarcoma (36). Taxol acts as a mitotic
inhibitor (37) that works by stabilizing microtubules and consequently interferes with the
normal breakdown of microtubules during cell division (38). In order to meet the
worldwide demand for this “wonder drug”, taxol, like aspirin, is now completely
chemically synthesized by pharmaceutical companies.
Aspirin and taxol are only two of many medicinal compounds derived from natural
products. As in the case of willow bark and Pacific Yew bark, identification of the
chemical constituents of other plants that possess medicinal properties led to the organic
synthesis and wide use of these compounds. Oshála root has been widely used for its
medicinal purposes by Native Americans in the Pacific Northwest of the United States to
treat sore throats from colds, coughing, and other ailments (1). Of most relevance to this
study is the use of oshála as women's medicine, particularly to bring on menses and as an
abortifacient. Preliminary in vitro studies of oshála on MDA-MB-231 and BT-474 cells
6
showed L. grayi to exhibit cytotoxic effects towards these two breast cancer cell lines.
This project sought to separate the chemical constituents of this plant in an attempt to
isolate the compound(s) that are cytotoxic towards breast cancer cells.
1.4 Statement of Problem
Although L. grayi has been used for generations as a medicinal herb, little is known of
the mechanism of action and the constituents responsible for its medicinal properties.
Assessment of the chemical properties of this plant could lead to the development of a
more natural form of anticancer treatment. This study seeks to evaluate various
separation methodologies for the isolation of the chemical constituent(s) responsible for
the cytotoxicity of L. grayi towards breast cancer cells.
1.5 Botany, taxonomy and identification
The genus Ligusticum is named after Liguria, an area in Italy where a closely related
genus (Levisticum) was first located (39). Ligusticum is a genus of about 25 species of
flowering plants in the family Apiaceae (40). The species name "grayi" was used to
honor an American botanist and Harvard professor who wrote Gray’s Manual of Botany
in 1848 (41).
7
The following lists the botanical name, synonyms, and classification of Ligusticum grayi:
Botanical name: Ligusticum grayi
Synonyms: oshála, Gray’s licorice-root, Gray’s lovage, Gray’s wild lovage
Plant Classification:
Kingdom: Plantae - plants
Subkingdom: Tracheobionta - vascular plants
Superdivision: Spermatophyta - seed plants
Division: Magnoliphyta - flowering plants
Class: Magnolipsida - dicotyledons
Subclass: Rosidae
Order: Apiales
Family: Apiaceae - carrot family
Genus: Ligusticum - licorice root
Species: Ligusticum grayi J.M. Coult. & Rose - Gray’s licorice root
1.5.1 The family Apiaceae
The Apiaceae family, made up of about 300 genera and more than 3000 species (42, 43),
is found all over the world but mainly in temperate areas (44). This family, also known as
Umbelliferae, consists of aromatic plants with hollow stems (45). The leaves are always
alternate (46), and are pinnately or palmately compound without stipules (47). Flowers
8
from these plants are typically small in umbels and present in different colors - white,
yellow or pink (48). Each flower is bisexual with five sepals, five petals and five stamens
(43).
This family includes members with different properties such as poison hemlock, water
hemlock, and fool’s parsley, which are well known for their toxic properties (43, 49);
carrot, celery, and parsnip (50), which are foods; anise, dill, coriander, caraway, and
cumin (50), which are spices; parsley and fennel, which are used as both food and spices
(51); and, finally, those used as herbal and folk remedies (7), such as wild carrot, which is
used for its estrogenic properties.
1.5.2
The genus Ligusticum
Ligusticum is a genus consisting of about 60 species found in Asia, Europe, and North
America, and, of these, 40 species (35 endemic) are found in China (52). This genus is
native to cool temperate regions of the Northern Hemisphere. Plants in this genus are
perennial herbs with cylindrical roots and thick or slightly inflated rootstocks. The stem is
erect, striated, and its base is usually clothed in fibrous remnant sheaths (53). The basal
and lower leaves are petiolate and the cauline leaves gradually reduce upward or are
absent (52). The roots of some of these species are used as medicinal herbs.
9
1.5.3 The species Ligusticum grayi
More commonly known as oshála, L. grayi is a more aromatic relative of true oshá,
Ligusticum porteri (54), a native perennial herb with aromatic taproots, 20 to 60 cm tall
stems, mostly basal leaves (55), and white to pink flowers in compound umbels (Figure
1). Although all have umbels of tiny white 5-petaled flowers, L. grayi is smaller and more
delicate than other relatives of the carrot family (39). Its flowers (7 to 14 per umbel) are
arranged in compound umbrella-like clusters as shown in Figure 1 (55). It has a unique
odor like a strong mix of carrot and celery. Its lack of spots on its stem and its distinctive
“spicy celery” odor allows for its distinction from water hemlock, Cicuta douglassi, an
extremely poisonous plant that has naturalized throughout California and can be confused
with L. grayi. L. grayi blooms from July to September.
10
Figure 1. Ligusticum grayi. Plant (left), flower
(upper right), and leaves (lower right; 1).
11
Figure 2. Oshála distribution map. Gray shades illustrate areas where L. grayi has been
observed (2).
Figure 3. Oshála worldwide distribution map. Gray areas (Alaska, Canada, U.S.
Mexico and India) represents areas where L. grayi has been observed (3).
12
L. grayi is native to mid- to sub-level elevations (55) in moist or dry open slopes (39). As
shown in Figure 2, L. grayi can be found in California, Nevada, Oregon, Washington,
Central Sierra, western Idaho and Montana (56). In addition to the U.S., L. grayi is also
found in Alaska, Canada, Mexico and India, where it may have been naturalized (Figure
3). More specifically, as shown in Figure 4, L. grayi communities in the state of
California are found mostly in alpine counties, including Yellow Pine forests, Red Fir
forests, Lodgepole Pine forests, subalpine forests, and wetland-riparian zones (57).
Figure 4. L.grayi distribution in the state of California. Darker blue indicates that there
is a specimen from this county in an herbarium, turquoise indicates a documented
observation, and purple indicates the presence of other (undocumented) observations.
Counties where L. grayi has been observed include: Alpine, Amador, Butte, Calaveras,
Del Norte, El Dorado, Fresno, Glenn, Humboldt, Lassen, Madera, Mendocino, Mono,
Modoc, Mariposa, Nevada, Placer, Plumas, San Bernardino, Shasta, Sierra, Siskiyou,
Tehama, Trinity, Tulare, and Tuolumne (4).
13
The Atsugewi tribe of California is one of many tribes that have traditionally used L.
grayi. Usage of L. grayi was so extensive that it was not only used for its medicinal
purposes, but also as a food supplement and for food capture. Some of its common uses
include the following (7):
Anesthetic: Roots used to avoid pain.
Cold Remedy: Infusion of root taken or roots chewed for colds.
Cough Medicine: Infusion of root taken or roots chewed for coughs.
Panacea: Infusion of root taken or roots chewed for ailments.
Substitution Food: Tender leaves soaked in water, cooked and used as a meat
substitute when acorns were eaten.
Vegetable: Tender leaves soaked in water, cooked and used for food.
Winter Use Food: Tender leaves soaked in water, cooked and stored for later use.
Hunting & Fishing Item: Pulverized root used for poisoning fish.
The Atsugewi reside in "the heavily wooded area between Mount Lassen and the Pit
River, particularly along Hat Creek" (58). They lived by hunting and gathering and lived
in small groups, speaking the Palaihnihan language. In the old times, they would occupy
villages between modern-day Burney and Cassel during the winter; through the summer
they would range from Black Butte to Mount Lassen (59), all areas where L. grayi was
readily available.
14
Ligusticum grayi is often confused with true oshá, Ligusticum porteri, one of the closest
relatives of oshála. As stated by its scientific name, oshá belongs to the same family and
genus as oshála, but it is known to have stronger medicinal properties (60). This perennial
herb grows from 50 to 100 cm tall or more (61). It is native to the Rocky/Occidental
Mountain chain of western North America from Wyoming to the states of Sonora and
Chihuahua, Mexico. Like L. grayi, the roots of L. porteri are prepared as tinctures,
infusions or tea, and used as medicine and food (62). Root preparations are taken
internally for catarrh, colds, coughs, bronchial pneumonia, flu and other respiratory
infections (39). They are also used to treat fever, diarrhea, gastrointestinal disorder,
hangover, sore throats (63) and rheumatism (62). The root is also used externally to treat
aches and pains (39), digestive problems (57), wounds and skin infections (45).
1.6
Isolation of Chemical Constituents from Related Plants
The discovery of multiple de novo drugs derived from natural products has caught the
attention of many researchers. A wide range of separation techniques have been reported
for the isolation of natural products, some of these include simple fractionation, such as
column chromatography, liquid-liquid partitioning/extractions, and thin layer
chromatography. Other more complex techniques involve the use of instrumentation
such as GC and HPLC. For example, Cegiela and colleagues isolated sabinyl acetate and
(Z)-ligustilide from the essential oil of L. porteri (64). Isolation of sabinyl acetate was
performed in two steps - an extraction of the dried plant using dichloromethane, followed
15
by silica gel column chromatography eluted with a toluene-ethyl acetate gradient.
Isolation of (Z)-ligustilide was a more complex process requiring of five steps - first,
extraction of the dried plant material with methanol; second, liquid-liquid partitioning of
the extract using water and dichloromethane (DCM); third, fractionation of the DCM
phase with Sephadex column chromatography; fourth, medium pressure liquid
chromatography; and fifth, silica gel column chromatography using cyclohexanedichloromethane-methanol gradient. Other fractionation techniques also include use of
filtration chromatography, alumina chromatography, preparative HPLC, and others.
Although many have reported separation methodologies for the isolation of specific plant
compounds, it is important to note the type of plant tissue being studied. For example,
salicylic acid, now commercialized as Aspirin, was found in the leaves and stem of
willow, Taxol on the other hand, was isolated from the bark of Pacific yew. For the
present study, the medicinal properties of L. grayi have been reported to come from the
root.
Although Ligusticum species have been studied and used for many generations, little is
known of the chemical composition of L. grayi. This study aims to expand on the
knowledge of cytotoxic properties of L. grayi by analyzing the ethanol extract of crushed
roots.
16
Chapter 2
EXPERIMENTAL
2.1 Chemicals
All solvents used were HPLC-UV grade (Pharmco Products Inc). Nanopure water was
obtained by filtration through an EASYpure LF compact ultrapure water system
(Barnstead). Media components included Improved Minimum Essential Media, Zinc
Option, 1X containing L-glutamine, 2 mg/L L-proline, and 50 μg/μL gentamicin sulfate
(IMEM, Invitrogen), 5,000 I.U./mL penicillin/streptomycin (Cellgrow), HEPES (4-(2Hydroxyethyl)-1-piperazineethanesulfonic acid; Sigma-Aldrich) and phenol red
(Invitrogen). Trypsin (0.25% with 0.1% EDTA in Hank's Balanced Salt Solution without
calcium, magnesium or sodium bicarbonate; Cellgro) was purchased from Invitrogen.
Trypan Blue (0.4 % in phosphate buffered saline), was purchased from Cellgro. Column
chromatography was performed using silica gel 60 with 0.063-0.200 mm beads (70-230
mesh ASTM, EM Science). Iodine crystals (Chemical Mfg Corp) were used for
visualizing compounds following thin layer chromatography (TLC).
2.1 Materials
Cells were grown in a T-25 or T-75 flask (VWR Scientific). Falcon tubes (15 and 50 mL,
VWR Scientific), parafilm (VWR Scientific) and 1.5 mL microcentrifuge tubes (USA
Scientific) were utilized for cell count and inoculation. Sterile filtration was
17
accomplished using a 25 mm diameter, 0.45 µm and 0.25 µm (Fisherbrand) pore sterile
syringe filters. Cytotoxicity was tested in individual wells of a Microtest Tissue Culture
96-well plate with flat bottom wells and a low evaporation lid (Becton Dickinson
Labware). Round bottom flasks (2 L, 1 L, 100 mL, 50 mL; Chemglass) were used to
rotary evaporate large sample volumes. Liquid-liquid extraction was carried out using a
30 mL or 60 mL separatory funnels (Chemglass). All glassware was soaked in a sodium
hydroxide/isopropanol bath (400 mL of 10% aqueous sodium hydroxide and 3.6 L
isopropanol) for 6 hours followed by 10 rinses with distilled water and 3 rinses with
acetone, prior to use. Thin Layer Chromatography analysis was performed on aluminum
plates pre-coated with 0.25 mm silica gel 60 UV254 (Whatman).
2.3 Instrumentation and equipment
Breast cancer cells were handled in a Purifier Class II Biosafety Cabinet (LABCONCO).
Cell material was thawed out using a 37 ºC water bath (Chicago Surgical & Electrical
Co.). Cells were counted using the quadrants of a Fisher Scientific hemacytometer under
a Swift compound microscope. Small volumes were measured with Fisherbrand
micropipettes. A vortex mixer (Thermolyne type 37600) and Du Pont Laboratory GLC
centrifuge (1.95 x 103 rev/min) were utilized to mix and pellet cell suspensions,
respectively. Cells were grown in a Nuaire IR autoflow CO2-water-jacketed incubator.
UV absorptions were recorded on a BioRad Microplate Reader (Model 680).
18
Column chromatographic separations were carried out using two Chemglass glass
columns, one 250 x 16 mm (i.d.) and the other 440 x 40 mm (i.d.). TLC results were
observed using a UV illuminator at 254 and 365 nm (Spectronics Corporation). Gas
chromatography was performed on a Hewlett Packard 5890 Series II GC-MS using a 5 %
phenyl silicone stationary phase (Rtx-5Sil MS) in a fused silica capillary column with a
0.25 mm (i.d.), 1 μm wall coating, and 30 m column length (Restek). HPLC (high
performance liquid chromatography) analysis was carried out on an Agilent 1100 Series
instrument fitted with a custom-built charged aerosol detector (CAD) (Abhyankar et al.,
2007) and an variable wavelength UV detector (VWD, Agilent) and an Agilent RP-C18
column (4.6 x 150 mm) with 5 μm diameter particles (Agilent), with a C18 guard
cartridge (Alltech).
2.4 Plant material
L. grayi roots were collected along Lyon’s Creek, El Dorado National Forest, in October
2007, under U.S. Department of Agriculture Forest Service Forest Product Free Use
Permit OMB No. 0596-0085. The roots were air dried at room temperature for 72 hours.
The dried roots (214.79 g) were soaked in 95% ethanol (2.7 L x 2) and stirred vigorously
at room temperature for 48 hours. The mixture was then filtered using a 0.45 µm with 25
mm diameter syringe filter to remove particulate matter. The filtrate was then transferred
to a pre-weighed 1000 mL round bottom flask and dried with a rotary evaporator. The
dried content was re-weighed, the extract weight calculated, and sufficient 100% n-
19
hexane was added to make a final concentration of 100 mg/mL and stored in a 5 L glass
jar at -20 °C.
2.5 Cytotoxicity Assay
Media preparation was done in a Purifier Class II Biosafety Cabinet. A 500-mL bottle of
IMEM (Improved Minimal Essential Media) was mixed with 50 mL
penicillin/streptomycin (5,000 IU/mL and 5,000 g/mL, respectively), 5 mL of 1.0 M
sterile HEPES, and 5 mL of 0.1% sterile phenol red. The sample was inverted several
times to mix and stored at -5 ºC for use as the media.
All cell handling took place in the Purifier Class II Biosafety Cabinet after it was exposed
to UV light for 30 minutes. Human breast cancer cell lines MDA-MB-231 and BT-474
were purchased from the American Type Culture Corporation and frozen in a cryogenic
chamber with liquid nitrogen. The cells were frozen in 2-mL cryogenic tubes (VWR
Scientific) with media supplemented with 10% dimethyl sulfoxide (DMSO) at about 1-2
x 106 cells/tube. When starting a new culture from frozen cells, cells were thawed in the
water bath (37 ºC) for 2 minutes, suspended in media, and centrifuged for 5 minutes. The
supernatant was discarded and the pellet was re-suspended in 5 mL of fresh media by
vortexing. The cells were then transferred to a T-25 cell culture flask and allowed to grow
(37 ºC incubator) for 3 to 6 days.
20
The media was aspirated from the T-25 cell culture flask, and the cells were released by
incubation with 1.5 mL of trypsin for 5 to 15 minutes. Media (5 mL) was then added to
wash down all cells, then transferred to a 15 mL Falcon tube and centrifuged for 5
minutes. The supernatant (media) was then aspirated and 5.0 mL of fresh media was
added to the pellet and mixed by vortexing. Trypan blue (10 μL) and cell suspension (10
μL) were mixed on a square of parafilm, and 10 μL of the mixture were loaded on each
side of the hemacytometer. This procedure was repeated for the second side of the
hemacytometer. The cells were counted under a Swift microscope and the concentration
of the cell suspension was calculated as follows:
1. The average of the number of cells from the eight quadrants was calculated.
2. The average was multiplied by 2 to account for the 1:2 dilution with trypan blue.
3. Because each quadrant contained 1 X 10-4 mL, the value was then multiplied by
10,000 to get the concentration of the cell suspension in cells/mL (C1).
4. C1V1 = C2V2 was used to determine the volume of cell suspension (V1) required
to get a final concentration (C2) of 5 x 104 cells/mL.
Media was added to this volume of stock cell suspension to make the final desired
volume (V2). A total of 9 mL of media with cells was prepared for each tissue culture
plate, so that V2 = (9 mL) X (number of plates). A multichannel pipette was used to
pipette 100 μL of the new dilution into each of the wells on the 96-well plate. The plate
was then incubated (37 ºC) for 48 hours.
21
Test media were prepared as follows: Test sample concentrations were determined using
C1V1=C2V2 equation, where C2 was calculated. The plant extract was sterile-filtered
through a 25 mm diameter, 0.2 m pore syringe filter. For each sample, 20 μL of a sterile
extract was added to 1980 μL of media in a centrifuge tube and mixed by vortexing. For
the control, 20 μL of sterile filtered ethanol (95%) replaced the plant extract. The cells
were transferred from the incubator to the Purifier Class II Biosafety Cabinet. The media
was then aspirated from each well and 100 μL of the sample was pipetted into each well
using a single-channel pipette. All tests (controls or samples) were performed in ten
wells. The inoculated plate was then incubated (37 ºC) for 48 hours.
After 48 hours, the media was aspirated from each well, and 100 μL of fresh media was
pipetted into each well. Cell titer 96 aqueous one solution reagent (20 μL) was then
pipetted into each well to measure the reduction of tetrazolium component (MTT) into an
insoluble formazan product, which is proportional to the number of viable cells. The plate
was placed in the incubator for one hour (for MDA-MB-231 cells) or two hours (for
BT474 cells). After incubation, the plate was removed from the incubator and placed in
the Microplate Reader; the absorbance was measured at 490 nm.
The standard deviation for each sample was calculated from ten individual tests, using the
following formula:
Standard deviation = [{X2 – n*(Blank mean)2}/{n-1}]1/2
X = Sum total of the raw absorbance for each blank
22
n = Number of blanks
Blank mean = X/n
The half lethal concentration was assessed on the hexane-soluble fraction and other
fractions of interest. Samples were further diluted by adding 20, 40, 60, or 80 µl 95%
ethanol to 80, 60, 40, or 20 µl stock extract, respectively. Twenty µL of the diluted
sample was mixed with 1980 µL of media to make “test media”. The media was
aspirated from the 96-well plate and 100 µL of test media was inoculated into each well.
The LC50 value was calculated by graphing absorbance as percent of control vs extract
concentration and performing sigmoidal curve fit, where y = 50 (50% lethal
concentration) and x = LC50 value. The standard error was also calculated for each LC50
value, using the following formula:
Square root ((100*Standard Deviation of Test 1/Mean (OD) of Test1)^2 + (100*Standard
Deviation of Control/Mean (OD) of Control)^2)
2.6 Separations
2.6.1 TLC
TLC analysis was performed on aluminum plates pre-coated with silica gel 60 UV254
(0.25 mm thickness). Several solvents used were assessed for their ability to fractionate
23
the chemical constituents in the starting material. The mobile phase consisted of ethyl
acetate:n-hexane (0:1, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 1:0), ethyl acetate: ethanol
(7:3), butyl acetate:n-hexane (1:0, 2:8, 4:6, 5:5, 6:4, 7:3, 8:2, 0:1), 100% chloroform,
100% dichloromethane, 100% methanol, or 95% ethanol. To facilitate mobile phase
migration, blotting paper (Whatman 3mm) was allowed to soak and equilibrate for 20 to
30 minutes previous to TLC runs. Detection was carried out by UV light at 254 and 365
nm, and with iodine crystals.
Preparative TLC plates were prepared for larger sample analysis. To prepare a 1.0 mm
thick preparative TLC plate, twenty-five grams of silica gel 60 (0.063-0.200 mm mesh)
were suspended in 50 mL of deionized water, shaken vigorously in an Erlenmeyer flask
for 45 seconds. The resultant slurry was poured on a 15 x 15 cm glass plate and spread
evenly with a glass rod until the surface was even. The plate was allowed to dry for
several hours and then placed in an oven at 120 ºC for 2 hours to activate.
2.6.2 Flash Column Chromatography
Five different column chromatographic fractionations were performed using different
column dimensions and elution gradients. For the first column, 20 mL of the hexanesoluble fraction (100 mg/mL) was fractionated using silica gel 60 (0.063-0.200 mm
mesh) in a 440 x 40 mm glass column. As the eluent, 700 mL of a mixture of n-hexane
with increasing amounts of butyl acetate was used (n-hexane:butyl acetate - 1:0, 200 mL;
24
7:3, 200 mL ; 5:5, 100 mL ; 0:1, 200 mL) with a final 95% ethanol rinse (350 mL).
Individual 1 mL fractions were combined based on TLC band similarities to yield a new
set of fractions (A through R). A second column partitioned 1 mL of hexane-soluble
fraction (100 mg/mL) using the same column conditions as for column number 2, except
that ethyl acetate was used in place of butyl acetate. As the eluent, 400 mL of a mixture
of n-hexane with ethyl acetate was used (n-hexane:ethyl acetate – 1:0, 100 mL; 7:3, 250
mL; 5:5, 50 mL; 0:1, 50 mL), followed by a 95% ethanol rinse (100 mL).
2.6.3 Liquid-liquid extractions
A series of liquid-liquid extractions were performed on the hexane soluble fraction. Prior
to partitioning, the miscibility of various solvents was tested. The tested solvents include:
DMSO, n-hexane, dichloromethane, methanol, ethanol, diethyl ether, acetone, toluene,
acetonitrile, 2-propanone, and water. Separatory funnels were used and the partitioning of
the sample was analyzed via GC-MS and HPLC-CAD/UVD.
2.6.4 Liquid-liquid Extraction followed by Column Chromatography
A series of liquid-liquid extractions were performed on the hexane-soluble fraction using
acetonitrile as the extractant. A 25 mL separatory funnel was used to partition 10 mL of
the hexane soluble fraction (100 mg/mL). After addition of the extractant (acetonitrile),
the separatory funnel was inverted a couple times and allowed to settle; samples were
25
collected. After one acetonitrile rinse of the hexane crude extract, the acetonitrile phase
was concentrated and fractionated via column chromatography. A 250 x 16 mm glass
column was packed with silica gel 60 (0.063-0.200 mm) and 1 mL acetonitrile extract
was eluted using an acetonitrile:ethanol gradient (1:0, 40 mL; 1:1, 20 mL; 0:1, 40 mL).
Fractions were analyzed via GC-MS.
A second LLE followed by CC was performed. Five hundred milligrams of the hexane
soluble fraction was dried, re-dissolved in MeOH, and partitioned with H2O:CH2Cl2
(1:1). The CH2Cl2 phase was collected and partitioned using silica gel 60 (0.063-0.200
mm) in a 250 x 16 mm column chromatography with a CH2Cl2: MeOH (7:3) mobile
phase. Fractions were analyzed via HPLC-UVD/CAD.
2.6.5 Gas Chromatography-Mass Spectrometry
Samples were dried with nitrogen gas and re-dissolved in 10 μL to 2 mL of 100%
dichloromethane, depending on the initial sample volume. Exact sample concentrations
were not calculated due to small sample volumes and high dichloromethane volatility.
From the newly redissolved dichloromethane sample, 1 μL was injected into the GC-MS
column. Injector temperature was 250 ºC. The analysis was performed with the following
temperature gradient: 40 ºC for 1 min, 20 º/min to 150º, 30 º/min to 280 º, hold for 2 min.
Electron Impact MS (1329 V) was used for detection.
26
2.6.6 High Performance Liquid Chromatography
An Agilent Liquid Chromatography Series 1100 equipped with a charged aerosol
detector (CAD) and a UV detector (UVD) was used to determine the purity of each
fraction. All samples were filtered and dissolved in 95% ethanol to a concentration of 10
mg/mL, prior to each run. An RP-C18 Agilent column was used (4.6 x 150 mm, 5 μm)
with a guard column. The mobile phase consisted of nanopure water (solvent A) and
acetonitrile (solvent B) run with an oven temperature of 40 ºC. A 2 μL sample volume
was analyzed using the following gradient profile.
Time (min)
0
9
38
43
44
45
% solvent B
40
43
95
95
40
40
Flow Rate (mL/min)
1
1
1
1
1
1
Max Pressure (psi)
400
400
400
400
400
400
Table 1. HPLC gradient profile.
A CAD and a UVD (210 nm) chromatogram were acquired after each analysis. Data
analysis was carried out on the Agilent chemstation.
27
Chapter 3
RESULTS AND DISCUSSION
3.1 Cytotoxicity of aqueous, ethanol and n-hexane extracts
To assay for cytotoxicity towards breast cancer cells, the peeled and crushed roots were
extracted with water, ethanol, or hexane at room temperature for 48 hours. The three
different extracts were tested on MDA-MB-231 and BT-474 breast cancer cells. As
shown in Figure 5, both hexane and ethanol extracts were toxic towards both cell lines.
The lack of cytotoxicity from the aqueous extract indicates that the polarity of the
cytotoxic compounds is not as polar as water, and thus an aqueous extract was not used
Mean OD (% of control)
for further investigations.
180
160
140
120
100
80
60
40
20
0
MDA-MB-231
BT-474
Water
Control
Ethanol
Control
Water
Ethanol
Hexane
Type of Extract
Figure 5. Cytotoxic activity of aqueous, 95% ethanol and hexane extracts on MDA-MB231 and BT-474 breast cancer cells using a 100 μg/mL concentration.
28
The cytotoxicity assay results indicated that both ethanol and hexane extracts had strong
cytotoxicity against cancer cells. Ethanol was chosen as the initial extractant for
preparative extractions for its lower volatility relative to n-hexane, which allowed for a
larger sample collection due to less evaporation after 48 hours of root extraction.
Because the hexane extract exhibited substantial cytotoxicity against both cancer cell
lines, the ethanol extract was dried and the non-polar components in the ethanolic extract
were solubilized in a smaller volume of hexane. Dissolution in hexane served as a first
means of partitioning where the more non-polar compounds were selected. As not all of
the plant extract dissolved in hexane, the hexane-insoluble residue was dissolved in
ethanol. A cell assay of the hexane-soluble and hexane-insoluble fractions was
performed on MDA-MB-231 cells to determine if the cytotoxic constituents were
successfully solubilized with hexane (Figure 6).
Mean OD (% of control)
120
100
80
60
40
20
0
Ethanol Control
Hexane-soluble
Fraction
Hexane-insoluble
Fraction
Figure 6. Cytotoxic activity of the hexane soluble fraction and the ethanol rinse of the
hexane-insoluble residue on MDA-MB-231 cells at a 100 μg/mL concentration.
29
The hexane–insoluble fraction did not inhibit breast cancer growth compared to the
control (Figure 6; p = 0.14 compared to the control). The cytotoxic constituents were
extracted with hexane (p = 4.41 x 10-5 compared to the control). The hexane-soluble
fraction was concentrated to 100 mg/mL and stored at -20 ºC for all further
investigations.
3.2 Assessment of Soil for Cytotoxic Activity
Because the roots contain traces of soil, the possibility that compounds from the soil,
rather than the roots, were responsible for the observed cytotoxic activity was addressed.
Trace amounts of soil collected from the roots were dissolved in ethanol and tested on
MDA-MB-231 cells. As shown in Figure 7, soil alone did not inhibit the growth of
breast cancer cells (p = 0.81 compared to the control), indicating that any inhibitory
properties were solely due to the root constituents.
30
Mean OD (% of control)
140
120
100
80
60
40
20
0
Ethanol Control
Hexane-soluble
Fraction
Soil Exract
Figure 7. Cytotoxicity assay of soil residues dissolved in 95% ethanol and ethanolic
oshála root extract on breast cancer cell line MDA-MB-231 at a 100 μg/mL
concentration.
3.3 Heat Lability
A heat assay was performed to determine the heat lability of the cytotoxic activity in the
hexane-soluble fraction. A small amount of the hexane-soluble fraction was immersed in
boiling water (100 ºC) in a glass capped vial for 10 minutes, allowed to cool, and tested
on the cells at a concentration of 40 μg/mL. As shown in Figure 8, heating decreased the
cytotoxic activity on breast cancer cell line MDA-MB-231.
31
Mean OD (% of control)
120
100
80
60
40
20
0
Ethanol Control
Hexane-soluble
Fraction
Heat-treated Hexanesoluble Fraction
Figure 8. Cytotoxicity assay of the hexane-soluble fraction before and after heat
treatment, both at 40 μg/mL, on MDA-MB-231.
The composition of the hexane-soluble fraction and the heat-treated hexane-soluble
fraction were also analyzed by GC-MS. Table 2 shows the percent peak abundance of
the two samples. Peak areas that differ by two-fold or greater are shown in bold in Table
2.
32
Retention Time (min)
5.07
9.26-9.28
9.37-9.39
9.89
10.3
10.7
11.10-11.12
11.73-11.75
12.11-12.15
12.24-12.28
12.35-12.42
12.44-12.49
12.7
12.80-12.89
13.1
Percent Peak Area
Hexane-soluble fraction
Heat-Treated Hexanesoluble fraction
1.50
1.40
0.20
1.60
0.20
1.00
2.20
4.10
6.10
2.00
25.9
6.60
9.20
22.8
13.7
0.00
3.40
1.00
0.20
0.65
0.30
1.70
6.80
8.10
3.10
18.6
9.80
10.7
20.9
14.0
Table 2. Percent Peak Area of GC-MS elution of the hexane-soluble fraction before and
after heat treatment.
The increase in abundance of selected peaks (eluting at 9.3 9.4, and 10.3 minutes) could
be a result of degradation products whose abundance increases after heat exposure. The
decrease in abundance of other peaks (eluting at 5.1, 9.9, and 10.7 minutes) could be due
to the breakdown of cytotoxic compounds. Although the percent peak area provides a
good means of comparison, it is important to note that the integration of both
chromatograms was not identical, thus it is possible that the variation seen between the
two chromatograms is due to error inherent in the integration.
33
3.4 LC50 of the hexane-soluble fraction
The 50% lethal concentration (LC50) of the hexane-soluble fraction was assessed on
MDA-MB-231 and BT-474 cells. As shown in Figure 9 and also seen in Figure 5, the
root’s chemical constituents are equally cytotoxic towards ER+/PR+ cells and ER-/PRcells, expressing nearly equal LC50’s (14 + 4 μg/mL and 15 + 5 μg/mL on BT-474 and
MDA-MB-231, respectively). This indicates that the cytotoxicity is not dependent on the
present hormone receptors. All following experiments were performed on MDA-MB231 cells (ER-/PR-), because of their faster doubling time (23 hr vs. 72 hr) and easier
handling compared to BT-474 cells.
Mean OD (% of control)
120
100
80
60
40
MDA-MB-231
20
BT-474
0
-20
0
10
20
30
40
50
60
Extract Concentration (micrograms/mL)
Figure 9. LC50 determination of the hexane-soluble fraction of oshála on MDA-MB-231
and BT-474 cells.
34
LC50 determinations were repeated on MDA-MB-231 cells one year after the hexanesoluble fraction was prepared. In two different tests of the same extract, the LC50 value
increased to 35 + 7 μg/mL and 37 + 7 μg/mL. The observed increase in LC50 value could
be attributed to the loss or decomposition of cytotoxic compounds as a result of handling
the hexane-soluble fraction at room temperature for long periods of time. Although the
increase in LC50 value could be a result of volatile compound loss, all fractions were
rotary-evaporated prior to cytotoxicity assays, and as a result, most volatile compounds
would have been removed early in the process. Therefore the decrease in cytotoxicity is
most likely due to compound breakdown. After this test, cell assays were performed
using an extract concentration of 40 μg/mL, to account for possible increase in LC50
value due to compound degradation. In earlier assays (i.e., Figure 10), using this lower
extract concentration could have facilitated elimination of the less cytotoxic fractions.
3.5 Isolation of cytotoxic compounds from the hexane-soluble fraction
After assessment of the properties and behavior of the hexane-soluble fraction under
different conditions, the goal then was to isolate individual cytotoxic constituents. To
achieve this goal, a series of separation techniques were used on the hexane-soluble
fraction.
35
3.5.1 Column Chromatography
The first fractionation was performed using flash column chromatography on silica gel
60. The mobile phase was chosen based on preliminary TLC analysis of the hexanesoluble fraction. For the first flash column chromatographic fractionation, two grams of
plant extract were partitioned using a 440 x 40 mm column packed with silica resin, with
a hexane/butyl acetate mobile phase gradient. Eighteen fractions (labeled A through R)
were collected and assayed for cytotoxicity. As shown in Figure 10, the first two
fractions (A and B, eluted with hexane as the mobile phase) and last two fractions (Q and
R, eluted with ethanol as the mobile phase) did not inhibit cell growth. Fractions eluted
with a hexane/butyl acetate gradient (7:3, then 1:1) all exhibited high levels of
cytotoxicity, and thus it can be concluded that the cytotoxic compounds are of
intermediate polarity between hexane and butyl acetate. (Because this assay was
performed before the LC50 value was determined, the fractions were tested on the cells at
a concentration of 100 g/mL.)
36
Mean OD (% of control)
120
100
80
60
40
20
Et
ha
no
l
R
Q
P
O
N
M
L
K
J
I
H
G
F
E
D
C
B
Co
nt
ro
l
A
0
Fraction
Figure 10. Cytotoxicity assay of flash chromatography column 1 fractions of the hexanesoluble fractions (100 μg/mL) on breast cancer cell line MDA-MB-231.
HPLC-UVD/CAD analysis of the active fractions showed the presence of both UVabsorbing compounds, detectable by UVD, and compounds of low volatility, detectable
by CAD (Figure 11). As shown by the hexane-soluble fraction, compounds with
retention times of 9.6, 13.2, 22.4, 35.3, and 42.9 minutes are detected by both UVD and
CAD. (A 0.1 minute lag time is observed between the UVD and CAD chromatograms,
as a result of a slightly longer distance from the splitter to the CAD.) The detection of a
larger number of compounds via UVD indicates that many L. grayi constituents are good
UV-absorbers which are either not highly abundant in the root extracts or are volatile and
thus not detected via CAD. As observed by CAD, fractions D through H all contain a
prominent non-volatile molecule with a 36 minute retention time, which was either not
prominent or fully absent in fractions C, L, and M. The presence of a second CAD-
37
detected molecule with a 22.3 minute retention time is observed in later eluting fractions
L and M. Multiple closely eluting UV-absorbing compounds are observed in fractions C
through H. The observed decrease in percent peak abundance of these UV-absorbing
compounds with increasing butyl acetate ratio implies that their polarity is closer to that
of hexane. Fractions C through F contain two prominent peaks with retention times of
16.2-16.4 and 17.3-17.5 minutes. Although their abundance is shown to decrease with
increasing butyl acetate, its early elution in both normal phase and reverse phase
chromatography implies that these two molecules are likely to contain both polar and
non-polar groups. A third molecule eluting at 29 minutes is observed in fractions C
through F, whose decrease in abundance implies its polarity is closest to that of hexane.
Lastly, fraction L and M exhibit a molecule with a 22 minute retention time that is not
present in previous fractions. Its large abundance in the later eluting fractions implies its
polarity is closest to that of butyl acetate.
38
Figure 11. Representative chromatograms from HPLC analysis of flash chromatography
column 1 of the hexane-soluble fraction: (from top to bottom) hexane-soluble fraction,
fractions C, G, and M.
Hexane-soluble fraction (UVD210)
Hexane-soluble fraction (CAD)
ADC1A, ADC1CHANNELA(URSULA_B\DEF_LC2009-01-0711-55-24\041-0101.D)
VWD1A, Wavelength=210nm(URSULA_B\DEF_LC2009-01-0711-55-24\041-0101.D)
23.751
24.851
25.054 24.624
25.657
26.279
100
500
400
16.451
80
42.977
39.675
40.40.625929
35.362
37.542
20.993
9.787
100
0
22.560
200
1.5381.857
2.5212.178
29.429
40.359
25.352
22.390
23.463
12.492
6.110
3.804
20
9.596
17.505
1.998
40
300
13.206
60
13.397
mV
mAU
0
5
5
10
15
20
25
30
35
10
15
20
25
30
35
20
25
30
35
20
25
30
35
30
35
min
40
min
40
Fraction C (CAD)
Fraction C (UVD210)
ADC1A, ADC1CHANNELA(URSULA\032108000002.D)
VWD1A, Wavelength=210nm(URSULA\032108000002.D)
mV
16.275
350
200
40.075
40.983
mAU
250
25.943
300
17.309
150
43.10542.686
0
250
200
0
42.530
150
38.640
50
29.147
24.520
24.738 24.312
25.302
100
100
50
5
10
15
20
25
30
35
40
Fraction G (UVD210)
5
min
10
15
min
40
Fraction G (CAD)
VWD1A, Wavelength=210nm(URSULA\032108000006.D)
ADC1A, ADC1CHANNELA(URSULA\032108000006.D)
mV
23.499
24.401 24.596
25.411
mAU
1600
1400
35.200
0
8000
24.812
1200
1000
6000
800
2000
0
39.540
200
37.946
22.755 23.154
6.390
6.808
7.033
11.770
12.296
12.600
400
25.089
26.012
4000
600
0
0
5
10
15
20
25
30
35
40
min
5
10
15
min
40
Fraction M (CAD)
Fraction M (UVD210)
ADC1A, ADC1CHANNELA(URSULA\032408000006.D)
VWD1A, Wavelength=210nm(URSULA\032408000006.D)
22.233
22.384
mV
mAU
250
8000
200
41.142
37.986
39.394
20.789
40.986
36.263
37.831
36.106
26.013
26.957
22.992
17.229
2000
27.113
0
9.554
9.854
50
18.794
19.770 20.028
20.627
4000
2.225
6000
100
3.7304.166
4.920
5.948
150
0
0
5
10
15
20
25
30
35
40
min
5
10
15
20
25
40
min
39
Retention
Time
(min)
6.40-6.81
8.5-8.8
11.6-11.8
12.26-12.7
15.98-16.4
17.059
17.3-17.48
18.4-18.8
19.7-20.0
20.3-20.6
20.86
21.12-21.21
21.729
22.0-22.24
22.58-23.2
23.3-23.5
24.21-24.31
24.41324.415
24.59-24.64
24.8-24.93
25.07-25.11
25.24-25.55
25.87-26.1
Hexanesoluble
fraction
0.604
1.99
1.88
5.44
6.98
3.42
0.302
0.878
0.331
0.403
0.360
0.331
0.403
3.167
1.04
12.2
13.9
10.8
Percent of total signal in each fraction (VWD)
C
D
E
F
G
H
L
39.0
3.08
18.8
3.03
7.75
17.7
9.58
4.62
2.60
1.23
3.00
2.93
2.97
4.61
3.32
2.37
3.57
7.06
10.30
14.30
2.50
15.8
19.7
21.6
24.2
20.6
21.7
6.46
9.71
4.62
12.5
22.6
9.87
12.1
5.72
17.4
17.3
14.7
11.0
5.44
19.4
9.59
14.6
8.81
2.69
19.1
10.6
68.3
7.21
3.31
3.57
4.39
6.28
9.18
10.1
55.2
7.97
11.6
8.32
3.70
13.8
9.73
3.09
M
8.27
7.62
23.9
9.99
26.9
2.76
1.79
2.71
Table 3. Summary of HPLC-UVD analysis of the hexane-soluble fraction and
flash chromatography column 1 fractions C, D, E, F, G, H, L, and M.
3.29
40
Retention
Time
(min)
2.00-2.30
19.0-20.8
22.1-22.4
22.5-22.6
34.8-35.0
35.1-35.2
35.3-35.4
37.0-38.0
38.8
39.4-39.9
40.8-40.9
42.6-42.9
Percent of total signal in each fraction (CAD)
Hexane
-soluble
fraction
73.6
1.64
C
D
E
F
G
H
3.00
48.5*
20.8
79.2
*
48.5
19.3
5.25
2.04
2.58
M
17.4
8.56
4.17
2.15
L
15.1
26.5
35.5
35.5
34.3
16.7
22.8
20.1
40.4
28.9
38.5
48.2*
13.2
100*
57.0*
22.7
20.3
Table 4. Summary of HPLC-CAD analysis of the hexane-soluble fraction and flash
chromatography column 1 fractions C, D, E, F, G, H, L, and M. (*) are off-scale and
therefore might not represent actual percent total signal.
The presence of the same compounds in all active fractions, as shown in Figure 11 and
Tables 3 and 4, indicates the possibility of column overload. To address this problem,
column height, column diameter, retention factor, and sample size are taken into
consideration. Still (65) suggests, this column can be loaded with 2.3 grams of sample
before it is considered overloaded. Because the mass used here was 2 grams, the
possibility of column overload exists. The presence of multiple closely eluting UVabsorbing compounds (24.5-26.1 minutes) was seen in cytotoxic fractions C through H
(Figure 11). Elution of this group of compounds could be a result of high structural
and/or polarity similarities that cause the compounds to elute closely in multiple fractions
or as mentioned earlier a result of column overload. It was concluded that this column
41
did not provide sufficient separation for the isolation of the cytotoxic constituent(s).
Further fractionation was performed on the hexane-soluble fraction.
Flash column chromatography run was repeated using a mobile phase consisting of
hexane and ethyl acetate, which is slightly more polar than the hexane and butyl acetate
used in the previous column. A cell assay of the collected fractions once again showed
the presence of multiple cytotoxic fractions (Figure 12). Similarly to previous results, the
cytotoxic compounds eluted with hexane/ethyl acetate, thus exhibiting intermediate
Mean OD (% of control)
polarity between hexane and ethyl acetate.
120
100
80
60
40
20
0
Ethanol
Control
Hexanesoluble
fraction
A
B
C
D
E
Fraction
Figure 12. Cytotoxicity assay of the collected column chromatography fractions. The
collected fractions were dried, re-dissolved in ethanol, and tested on MDA-MB-231 cells
at a concentration of 40 μg/mL.
42
GC analysis of the fractions (Figure 13) showed that separation of individual constituents
was not complete, as individual chromatograms once again exhibited the presence of
multiple peaks. Table 5 summarizes the percent peak areas from the various collected
fractions. The abundance of individual peaks varied from fraction to fraction, but no
single peak was found to correlate with cytotoxicity.
Fractions B, C, and D all contain the presence of the following peaks: 9.35 minutes,
12.49 minutes and 12.96 minutes (Figure 13). Although these were the most abundant
peaks present in all active fractions, the presence of these same peaks at the same or
higher abundance in non-active fraction E indicate that these compounds are not
responsible for the cytotoxic properties. Instead, the cytotoxicity could be a result of a
group of compounds (i.e., multiple eluting compounds from 11.79 to 13.20 minutes from
fraction B), compounds with low abundance not easily seen in the presence of the more
abundant ones, nonvolatile compounds that cannot be analyzed by GC, or different
compounds that elute in the different fractions (i.e., the peak at 5.16 minutes in fraction C
and the peak at 10.78 minutes in fraction D).
43
TIC: B659.D
Abundance
12.49
1600000
12.96
1400000
13.20
1200000
1000000
800000
12.55
12.78
12.19
600000
400000
11.79
9.33
200000
0
Time-->
6.00
8.00
12.35
10.00
12.00
14.00
16.00
Retention Time (min)
TIC: C659.D
Abundance
9.35
25000
20000
15000
12.96
10000
5.16
12.49
5000
9.55 10.34
0
Time-->
6.00
8.00
10.00
12.60
13.22
13.29
12.80
13.90
14.03
12.00
14.00
16.00
Retention Time (min)
TIC: D659.D
Abundanc e
1800000
9.35
1600000
1400000
1200000
1000000
800000
600000
10.78
10.31
400000
200000
0
Time-->
12.49
13.00
13.25
9.49
6.00
8.00
10.00
12.00
14.00
16.00
Retention Time (min)
TIC: E659.D
Abundanc e
9.33
200000
150000
100000
50000
12.46
10.33 11.30
12.95
13.22
12.24
11.61
12.80
0
Time-->
6.00
8.00
10.00
12.00
14.00
16.00
Retention Time (min)
Figure 13. Total ion gas chromatograms of (from top to bottom) fractions B, C, D, and E
from the second chromatographic separation.
44
Retention
Time (mins)
9.33-9.35
9.48-9.55
10.3
10.8
11.3
11.61-11.79
12.19-12.24
12.35
12.46-12.49
12.55-12.60
12.78-12.80
12.96-13.00
13.2
Percent Peak Area
D
B
C
1.50
39.6
2.50
2.30
2.70
6.30
1.90
24.4
8.70
9.20
24.3
20.9
57.6
3.40
8.70
10.6
E
45.9
4.80
5.30
4.30
4.90
8.50
3.90
2.30
22.0
6.50
9.3
1.00
0.80
6.60
3.70
18.2
2.70
8.80
5.10
Table 5. Percent peak abundance from total ion chromatograms of fractions B through E
from flash column chromatography.
Other column chromatographies were performed using different column dimensions and
mobile phase solvents (data not shown). Solvent gradients using hexane with butyl
acetate or ethyl acetate provided the best fractionation. However, it is concluded that
column chromatography alone did not prove to be an efficient method for the isolation of
individual L. grayi root constituents.
3.5.3 Liquid-Liquid Extractions
Because previous fractionation methods (i.e., column chromatography) exhibited a
change in the abundance of compounds in the presence of mobile phase solvents of
different polarities, liquid-liquid extractions were performed on the hexane-soluble
45
fraction. First, solvent miscibility tests were performed to identify highly immiscible
solvents (Table 6); the hexane-soluble fraction was then only partitioned with these
solvent combinations.
Solvent 1
Dichloromethane
Acetone
Methanol
Acetonitrile
Dichloromethane
Toluene
Diethylether
Dimethyl sulfoxide
Hexane
Polarity
Index
3.1
5.1
5.1
5.8
3.1
2.4
2.0
7.2
0.0
Solvent 2
Methanol
Hexane
Hexane
Hexane
Water
Water
Water
Hexane
Water
Polarity
Index
5.1
0.0
0.0
0.0
9.0
9.0
9.0
0.0
9.0
Polarity
Difference
2.0
5.1
5.1
5.8
5.9
6.4
7.0
7.2
9.0
Miscibility
Miscible
Miscible
Immiscible
Immiscible
Immiscible
Immiscible
Immiscible
Immiscible
Immiscible
Table 6. Solvent miscibility test.
Each phase was collected separately, concentrated to dryness, and re-dissolved in 100%
dichloromethane. The two liquid phases were assessed by GC analysis. Of the various
solvent combinations used, hexane and acetonitrile exhibited the best partitioning, in that
the abundance of selected peaks was greatly reduced (Figure 14). The changes in percent
peak abundances brought about by extraction with solvents of different polarities allowed
for conclusions to be made about the nature of compounds in individual GC peaks. For
example, the large abundance of peaks 2, 7, and 10 in the hexane phase relative to the
acetonitrile phase indicates that these three compounds are the most non-polar,
preferentially partitioning into the hexane phase after three acetonitrile rinses. However,
compounds 7 and 10 are also present in the acetonitrile phase, indicating that these
compounds are of intermediate polarity between acetonitrile and hexane. Compounds 4,
46
5, 6, 8, 9, and 11 are more polar, as they partition better into acetonitrile than into hexane.
No selectivity was observed for the other peaks.
A.
A
b u n d a n c
7
e
T
I C :
A
3 . D
3 0 0 0 0 0 0
10
2 8 0 0 0 0 0
9
2 6 0 0 0 0 0
2 4 0 0 0 0 0
8
5
2 2 0 0 0 0 0
2 0 0 0 0 0 0
4
1 8 0 0 0 0 0
11
1 6 0 0 0 0 0
1 4 0 0 0 0 0
6
2
1 2 0 0 0 0 0
1 0 0 0 0 0 0
3
8 0 0 0 0 0
6 0 0 0 0 0
1
4 0 0 0 0 0
2 0 0 0 0 0
T
0
im e - - >
6 . 0 0
8 . 0 0
B.
1 0 . 0 0
1 2 . 0 0
1 4 . 0 0
1 6 . 0 0
1 8 . 0 0
Retention Time (min)
T IC: F3 .D
Abundanc e
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
T im e -->
6 .0 0
8 .0 0
1 0 .0 0
1 2 .0 0
1 4 .0 0
1 6 .0 0
1 8 .0 0
Retention Time (min)
Figure 14. Total ion gas chromatograms of representative liquid-liquid extractions on the
hexane-soluble fraction (raffinate) using various acetonitrile as the extractant: (A) third
acetonitrile wash of hexane extract, (B) hexane layer after three acetonitrile washes.
47
Based on these results, three acetonitrile extractions were performed on the hexanesoluble fraction, as shown in Figure 15. A cytotoxicity assay was performed on the
various LLE fractions. As shown in Figure 16, all LLE fractions exhibit cytotoxic
activity on MDA-MB-231 cells. The increase in cytotoxicity observed in fractions B and
C indicate that most of the cytotoxic compounds either preferentially partition into the
acetonitrile phase or that more of the non-cytotoxic compounds preferentially partition
into the hexane phase. The decrease in cytotoxicity of fractions D and E, derived from
the final extraction of the hexane layer, support the idea that the cytotoxic compound(s)
preferentially partition into acetonitrile, since the acetonitrile phase appears to have a
stronger cytotoxic effect compared to the hexane phase throughout the whole experiment.
48
Hexane
B. ACN
A. Hexane
C. ACN
Hexane
D. ACN
E. Hexane
Figure 15. Liquid-liquid extraction flow chart.
120
100
Mean OD (% of control)
80
60
40
20
0
Ethanol
Control
Hexanesoluble
A
B
C
D
E
Collected Fractions
Figure 16. Cytotoxicity assay of fractions from liquid-liquid extractions on the hexanesoluble fraction with acetonitrile: (A) Hexane phase after 1 acetonitrile wash; (B)
Acetonitrile wash 1; (C) Acetonitrile wash 2; (D) Acetonitrile wash 3; (E) Hexane phase
after 3 acetonitrile washes on hexane hexane-soluble fraction. All liquid phases were
tested on cells at a concentration of 40 μg/mL.
49
A. Hexane-soluble fraction
A
b
u
n
d
a
n
c
e
T
9
e
+
0
7
8
.
5
e
+
0
7
8
e
+
0
7
7
.
5
e
+
0
7
7
e
+
0
7
6
.
5
e
+
0
7
6
e
+
0
7
5
.
5
e
+
0
7
5
e
+
0
7
4
.
5
e
+
0
7
4
e
+
0
7
5
e
+
0
7
3
e
+
0
7
5
e
+
0
7
2
e
+
0
7
5
e
+
0
7
1
e
+
0
7
0
0
0
0
0
3
.
2
.
1
5
i m
A
b
e
.
0
C
:
O
S
H
A
C
O
n
d
a
n
c
e
+
0
. 5
e
+
0
7
8
e
+
0
7
7
. 5
e
+
0
7
7
e
+
0
7
6
. 5
e
+
0
7
6
e
+
0
7
5
. 5
e
+
0
7
5
e
+
0
7
. 5
e
+
0
7
4
e
+
0
7
3
. 5
e
+
0
7
3
e
+
0
7
2
. 5
e
+
0
7
2
e
+
0
7
. 5
e
+
0
7
1
e
+
0
7
0
0
0
0
0
1
5
b
.
0
0
6
.
0
0
7
.
0
0
8
.
0
0
9
n
0
d
a
n
c
9
.
8
.
7
.
6
.
5
.
4
.
3
.
2
.
1
.
0
5
e
+
0
7
9
e
+
0
7
5
e
+
0
7
8
e
+
0
7
5
e
+
0
7
7
e
+
0
7
5
e
+
0
7
6
e
+
0
7
5
e
+
0
7
5
e
+
0
7
5
e
+
0
7
4
e
+
0
7
5
e
+
0
7
3
e
+
0
7
5
e
+
0
7
2
e
+
0
7
5
e
+
0
7
1
e
+
0
7
0
0
0
0
0
5
e
d
a
t
a
.
m
s
.
0
0
1
0
.
0
0
1
1
.
0
0
1
2
.
0
0
:
U
4
. D
\
d
a
t a
. m
1
1
. 0
0
1
2
. 0
0
s
. 0
0
6
. 0
0
7
. 0
0
8
. 0
0
9
. 0
0
1
0
. 0
0
Retention Time (min)
e
5
i m
\
- - >
T
T
D
Retention Time (min)
I C
C. Fraction B
u
.
12.65
5
A
L
7
8
4
e
O
e
9
i m
R
- - >
T
T
T
11.8
B. Fraction A
u
N
8.0
5
T
I
.
0
0
6
.
0
0
7
.
0
0
8
.
0
I
C
:
U
1
.
D
\
0
d
9
a
t
.
0
a
.
m
s
0
1
0
.
0
0
1
1
.
0
0
1
2
.
0
0
1
1
.
0
0
1
2
.
0
0
1
1
. 0
0
1
2
. 0
0
1
1
. 0
0
1
2
. 0
0
- - >
Retention Time (min)
D. Fraction C
A
b
u
n
d
a
n
c
e
T
9
.
8
.
7
.
6
.
5
.
4
.
3
.
2
.
1
.
5
0
5
e
+
0
7
9
e
+
0
7
5
e
+
0
7
8
e
+
0
7
5
e
+
0
7
7
e
+
0
7
5
e
+
0
7
6
e
+
0
7
5
e
+
0
7
5
e
+
0
7
5
e
+
0
7
4
e
+
0
7
5
e
+
0
7
3
e
+
0
7
5
e
+
0
7
2
e
+
0
7
5
e
+
0
7
1
e
+
0
7
0
0
0
0
0
5
T
i m
e
.
0
0
6
.
0
0
7
.
0
0
8
.
0
I
C
:
U
b
u
n
d
a
n
c
7
. 5
7
e
+
0
7
6
. 5
e
+
0
7
6
e
+
0
7
5
. 5
e
+
0
7
5
e
+
0
7
4
. 5
e
+
0
7
4
e
+
0
7
3
. 5
e
+
0
7
3
e
+
0
7
. 5
e
+
0
7
2
e
+
0
7
. 5
e
+
0
7
1
e
+
0
7
0
0
0
0
0
1
5
0
e
+
0
9
b
u
n
d
a
n
c
7
e
+
0
7
6
. 5
e
+
0
7
6
e
+
0
7
5
. 5
e
+
0
7
5
e
+
0
7
4
. 5
e
+
0
7
4
e
+
0
7
3
. 5
e
+
0
7
3
e
+
0
7
2
. 5
e
+
0
7
2
e
+
0
7
1
. 5
e
+
0
7
0
1
e
+
0
7
0
0
0
0
0
5
e
.
0
a
.
m
s
0
1
0
.
0
0
I C
:
U
2
. D
\
d
a
t a
. m
s
. 0
0
6
. 0
0
7
. 0
0
8
. 0
0
9
. 0
0
1
0
. 0
0
Retention Time (min)
e
5
im
t
- - >
T
T
a
Retention Time (min)
F. Fraction E
A
d
7
5
e
\
e
2
im
D
0
T
T
.
- - >
E. Fraction D
A
1
. 0
0
6
. 0
0
7
. 0
0
8
. 0
0
I C
:
U
3
. D
\
d
9
a
t a
. 0
0
. m
s
1
0
. 0
0
- - >
Retention Time (min)
Figure 17. Total ion gas chromatograms of the hexane-soluble fraction and liquid-liquid
extraction fractions A, B, C, D, and E.
50
GC analysis of these LLE fractions further eliminates compounds that were believed to
be cytotoxic. As shown in Figure 17, compounds eluting at 8.0 and 12.65 minutes were
also abundant in fraction E, the least active fraction, indicating that cytotoxicity is not a
result of either of these compounds. The compound eluting at 11.8 minutes in the
original hexane-soluble fraction is absent in all of the cytotoxic fractions, indicating that
this compound is not responsible for cytotoxicity. Because of the presence of multiple
compounds in each cytotoxic fraction, it is possible that the cytotoxic properties of L.
grayi could be a result of more than one cytotoxic compound. Peak to peak ratio was
compared between fraction C (most active) and fraction E (least active), of these, peaks
eluting at 9.00, 9.40, and 9.65 minutes displayed a five-fold or larger increase in peak
areas. The stronger cell inhibitory properties from fraction C could be a result of these
peaks.
A second LLE was performed, where the hexane backwashes were analyzed, as shown in
Figure 18. All collected phases exhibited cytotoxic activity on MDA-MB-231 cells
(Figure 19). The observed increase in activity from the last two phases could be
attributed to the removal of most non-cytotoxic compounds, and, thus, at this point, only
the cytotoxic compounds are partitioning into both hexane and acetonitrile phases. The
two most active fractions were in acetonitrile, while the least active fraction was in
hexane. From the gas chromatograms, it can be noted that fraction D contained two
additional peaks eluting at 11.2 and 11.8 minutes. These more polar compounds might
contribute to the stronger cytotoxic effect of fraction D.
51
Hexane
ACN
ACN
ACN
D. ACN
Hexane
A. Hexane
B. Hexane
C. Hexane
Figure 18. Liquid-liquid extraction flow chart.
Mean OD (% of control)
120
100
80
60
40
20
0
Ethanol
Control
Hexanesoluble
Fraction
A
B
C
D
LLE Fractions from Hexane backw ashes on
Acetonitrile phase
Figure 19. Cytotoxicity assay of back-extractions of the first acetonitrile phase resulting
from an acetonitrile wash on the (hexane) hexane-soluble fraction. The following
fractions were analyzed: (A) hexane phase after one hexane backwash, (B) hexane phase
after two hexane backwashes, (C) hexane phase after three hexane backwashes, (D)
acetonitrile phase after 3 hexane backwashes. All extracts were tested using a 40 μg/mL
concentration on MDA-MB-231 cells.
52
Fraction A (Least Active)
A
b u n d a n c e
T
I C :
U
5 . D
\ d a t a . m
s
9 . 5 e + 0 7
9 e + 0 7
8 . 5 e + 0 7
8 e + 0 7
7 . 5 e + 0 7
7 e + 0 7
6 . 5 e + 0 7
6 e + 0 7
5 . 5 e + 0 7
5 e + 0 7
4 . 5 e + 0 7
4 e + 0 7
3 . 5 e + 0 7
3 e + 0 7
2 . 5 e + 0 7
2 e + 0 7
1 . 5 e + 0 7
1 e + 0 7
5 0 0 0 0 0 0
5 . 0 0
T
im
6 . 0 0
7 . 0 0
8 . 0 0
9 . 0 0
1 0 . 0 0
1 1 . 0 0
1 2 . 0 0
e -->
Retention Time (min)
Fraction B (2nd most active)
A
b u n d a n c e
T
I C :
U
7 . D
\ d a t a . m
s
9 . 5 e + 0 7
9 e + 0 7
8 . 5 e + 0 7
8 e + 0 7
7 . 5 e + 0 7
7 e + 0 7
6 . 5 e + 0 7
6 e + 0 7
5 . 5 e + 0 7
5 e + 0 7
4 . 5 e + 0 7
4 e + 0 7
3 . 5 e + 0 7
3 e + 0 7
2 . 5 e + 0 7
2 e + 0 7
1 . 5 e + 0 7
1 e + 0 7
5 0 0 0 0 0 0
5 . 0 0
T
im
6 . 0 0
7 . 0 0
8 . 0 0
9 . 0 0
1 0 . 0 0
1 1 . 0 0
1 2 . 0 0
e -->
Retention Time (min)
Fraction D (Most Active)
A
b u n d a n c e
T
I C :
U
8 . D
\ d a t a . m
s
9 . 5 e + 0 7
9 e + 0 7
8 . 5 e + 0 7
8 e + 0 7
7 . 5 e + 0 7
7 e + 0 7
6 . 5 e + 0 7
6 e + 0 7
5 . 5 e + 0 7
5 e + 0 7
4 . 5 e + 0 7
4 e + 0 7
3 . 5 e + 0 7
3 e + 0 7
2 . 5 e + 0 7
2 e + 0 7
1 . 5 e + 0 7
1 e + 0 7
5 0 0 0 0 0 0
5 . 0 0
T
im
6 . 0 0
7 . 0 0
8 . 0 0
9 . 0 0
1 0 . 0 0
1 1 . 0 0
1 2 . 0 0
e -->
Retention Time (min)
Figure 20. Total ion chromatograms of the raffinate and the extracts from three liquidliquid extraction hexane backwashes on acetonitrile phases.
53
3.5.2. Preparative TLC
Preparative TLC was performed using 8 x 6 inch aluminum plates pre-coated with silica
gel 60 UV254 (Figure 21). The hexane-soluble fraction (1.5 g) was applied as a line and
then was developed with 100% dichloromethane for 2 hours. Individual bands were
excised, eluted with dichloromethane, filtered, concentrated with nitrogen gas, then
analyzed using GC (Figures 22); exact concentrations were not determined.
Figure 21. Preparative TLC of the hexane-soluble fraction developed with 100%
dichloromethane.
54
Although visibly distinct bands were seen with preparative TLC (Figure 21),
fractionation was not efficient, as observed by the presence of a mixture of compounds in
each band (Figure 22). For instance, peaks with 4.8 and 5.07 minute retention times were
seen in all bands, peaks with a 9 minute retention time are exhibited in bands A and B,
and peaks with a retention time of 12 minutes are seen in bands G and O.
55
Band A
T IC:
B A N D A .D
A b u n d a n c e
1 0 0 0 0 0
9 0 0 0 0
8 0 0 0 0
7 0 0 0 0
6 0 0 0 0
5 0 0 0 0
4 0 0 0 0
3 0 0 0 0
2 0 0 0 0
1 0 0 0 0
0
T im e -->
6 .0 0
8 .0 0
Band B
1 0 .0 0
1 2 .0 0
1 4 .0 0
1 6 .0 0
14.00
16.00
Retention Time (min)
T IC:
BAN D B.D
Abundanc e
500000
450000
400000
350000
300000
250000
200000
150000
100000
50000
0
T ime-->
6.00
8.00
10.00
12.00
Retention Time (min)
Band D
T IC:
BAN D D .D
Abundanc e
120000
100000
80000
60000
40000
20000
0
T ime-->
6.00
8.00
10.00
Band G
12.00
14.00
16.00
14.00
16.00
14.00
16.00
Retention Time (min)
T IC: BAN D G.D
Abundanc e
200000
150000
100000
50000
0
T ime-->
6.00
8.00
10.00
12.00
Retention Time (min)
Band O
T IC:
BAN D O.D
Abundanc e
500000
450000
400000
350000
300000
250000
200000
150000
100000
50000
0
T ime-->
6.00
8.00
10.00
12.00
Retention Time (min)
Figure 22. Total ion gas chromatograms of selected bands A, B, D, G, and O from
preparative TLC separation of the hexane-soluble fraction.
56
The apparent absence of chromatographic peaks from certain bands (i.e. Figure 22D and
others whose data is not shown) is most likely due to a lower sample concentration, as
can be inferred from the low intensity of the most abundant peaks. In addition, early
eluting peaks with 4.8 and 5.07 minutes could possibly represent contaminants. Due to
the small volume of samples collected, further fractionation and cell assays could not be
performed.
Compound identifications using the MS library, nbs75K, were 72% confident or less and
therefore not reliable. Although the inability to find a match could, in some cases, be
attributed to a low signal to noise ratio, there were some peaks with substantial signals
that did not have a match in the database. This points out the need for a larger MS
library, such as nbs300K. Because so many peaks were present in the active fractions, it
was deemed prudent to narrow the number of compounds of interest before attempting to
determine their structures.
3.5.4 Liquid-Liquid Extraction with Column Chromatography
With previous attempts to isolate the cytotoxic constituents yielding multiple cytotoxic
and non-cytotoxic compounds in each fraction, a different approach was used where
isolation of non-cytotoxic compounds was targeted in order to eliminate some candidates
as the cytotoxic compound(s). In this study, the hexane-soluble fraction was partitioned
with acetonitrile four times, and the resultant hexane layer was collected and further
57
fractionated with hexane/ethyl acetate/ethanol (1:0:0, 6:4:0, 0:0:1) via flash column
chromatography (Figure 23).
Hexane
Hexane
Hexane
Hexane
Hexane
ACN
ACN
ACN
ACN
FCC Hex/EtOAc
(6:4)
Fraction 1
FCC EtOAc
(100%)
EtOH (100%)
Fraction 2
Figure 23. Flow chart illustrating LLE followed by FCC.
58
A 250 x 16 mm column was packed with silica resin suspended in 100% n-hexane. A
small amount (90 mg) of the hexane sample was fractionated, and two bands were
collected, one eluting with n-hexane/ethyl acetate (6:4) and the other with 95% ethanol.
Because of the limited ethanol solubility of the first band, a cytoxicity assay could only
be performed on the ethanol soluble constituents of this fraction, leaving behind
compounds that were not ethanol soluble. As shown in Figure 24, only the first band
exhibited cytotoxicity. In addition, although the cytotoxic constituents partitioned
preferentially into more polar solvents, not all cytotoxic compounds were removed from
the non-polar hexane phase after four acetonitrile washes. The large cytotoxic effects
observed from non-polar fraction 1 indicate the presence of non-polar cytotoxic
compounds in L. grayi.
Mean OD (% of control)
120
100
80
60
40
20
0
Ethanol Control
Fraction 1
Fraction 2
Figure 24. Cytotoxcicity assay of the two column chromatography fractions, tested at a
100 μg/mL concentration.
59
HPLC was also used to assess the separation (Figure 25). Fraction 1, which showed
cytotoxic activity, exhibited multiple peaks, including two sets of closely-eluting
compounds - one group of UV-absorbing compounds with retention times from 24 to 30
minutes, which are either volatile or present in low abundance, as they are not detected
by CAD; and one group with retention times from 39 to 43 minutes, which are detected
by both UVD and CAD, indicating that they are non-volatile UV-absorbing compounds.
Three peaks are observed for Fraction 2: a non-volatile UV-absorbing compounds with a
23 minute retention time as shown by both CAD and UVD, and two non-volatile and
non-absorbing or non-volatile low abundance compounds with retention times of 34.6
and 41.8 minutes, visible only via CAD. The non-volatile UV-absorbing compounds at
23 minutes is only present in non-cytotoxic fraction 2. Based on these results, it can be
concluded that compounds represented by these peaks at 23.3, 34.7, and 41.8 minutes do
not have any cytotoxic activity.
60
Fraction 1 (UVD)
VWD1A, Wavelength=210nm(URSULA\SHUTDOWN2009-05-0111-05-19\050109000001.D)
40.309
mAU
80
20
29.447
23.779
24.653
24.880
25.36925.086
25.683
26.289
40
38.904
39.115
39.601
40.736
41.182
41.484
41.879
42.661
60
0
0
5
10
15
20
25
min
30
35
40
30
35
40
min
30
35
40
min
Retention Time (min)
Fraction 1 (CAD)
ADC1A, ADC1CHANNELA(URSULA\SHUTDOWN2009-05-0111-05-19\050109000001.D)
42.827
mV
90
70
50
20.198
1.524
30
37.044
40
40.260
40.906
60
44.812
41.683
42.577 42.128
80
20
0
5
10
15
20
25
Retention Time (min)
Fraction 2 (UVD)
VWD1A, Wavelength=210nm(URSULA\0108092009-01-0914-28-28\010909B000001.D)
1.230
mAU
30
23.148
25
20
15
1.353
10
5
0
0
5
10
15
20
25
Retention Time (min)
Fraction 2 (CAD)
ADC1A, ADC1CHANNELA(URSULA\0108092009-01-0914-28-28\010909B000001.D)
34.661
3.636
mV
100
1.867
120
41.772
23.334
60
1.400 1.538
80
40
20
5
10
15
20
25
30
35
40
min
Retention Time (min)
Figure 25. HPLC- CAD/UVD chromatogram of two column chromatographic fractions.
61
3.5.5 LLE followed by CC 2
A second LLE followed by column chromatography was performed following CegielaCarlioz and colleagues’ procedure for Z-ligustilide isolation from oshá, with slight
modifications (64). A small amount (500 mg) of the hexane soluble fraction was dried,
re-dissolved in MeOH, and partitioned with H2O:CH2Cl2 (1:1). The CH2Cl2 phase was
collected and separated using silica-based CC with a CH2Cl2: MeOH (7:3) mobile phase.
Two fractions were collected, Z1 and Z2. Cytotoxicity assays and GC analysis were
performed (Figure 26 and Table 7, respectively). As shown in Figure 26, the first
fraction (Z1) had little to no cytotoxic properties while the second fraction (Z2) was very
cytotoxic, although not as potent as the original hexane-soluble fraction. Even though
fraction Z2 exhibited large cytotoxicity, no additional inhibition was observed from this
fraction relative to the hexane-soluble fraction (starting material). The decrease of
cytotoxicity from fraction Z2 can be attributed to possible compound loss during column
chromatography, compound degradation as a product of harsh solvents, and/or functional
group loss.
Mean OD (% of control)
62
140
120
100
80
60
40
20
0
Ethanol
Control
Hexanesoluble
Fraction
Z1
Z2
Figure 26. Cell assay of collected fractions Z1 and Z2 from a silica based DCM/ MeOH
column with a 40 μg/mL concentration.
GC analysis exhibited the presence of multiple peaks from both fractions (Table 7). As
shown by their large peak abundances in both cytotoxic and non-cytotoxic fractions Z1
and Z2, it can be concluded that the closely eluting compounds with retention times
between 9.19 and 9.84 minutes are not likely for cytotoxicity. Compounds that have a
two-fold increase in abundance in active fraction Z2 in comparison to Z1 could be the
compounds to target, and are shown in bold in Table 7.
As shown in Table 7, five main peaks can be assigned as possibly cytotoxic.
Nonetheless, as discussed earlier, the peak with a 7.99 minute retention time is most
likely representative of a breakdown product shown to be largely abundant in noncytotoxic fractions (Figure 25), therefore cytotoxicity is believed to be a result of one or
more peaks with the following retention times: 8.43, 8.51, 9.07, and 11.7 minutes.
63
Removal of the molecules that elute at 9.19-9.84 minutes on GC would facilitate the
study of less abundant compounds believed to be the ones exhibiting cytotoxic properties
on MDA-MB-231 breast cancer cells.
64
Retention Time
(mins)
5.53
5.61
6.21
6.44
6.70
6.91
7.15
7.62
7.99
8.20
8.03
8.43
8.51
8.75
9.01-9.02
9.07
9.19
9.29
9.34
9.40
9.48
9.58-9.60
9.64-9.66
9.70-9.72
9.82-9.84
9.91
10.08
10.19
10.35
10.42-10.47
10.50-10.56
10.84
10.88
10.96
11.05
11.72
12.32
12.38
12.54
12.65
Z1
0.98
0.35
0.28
0.25
0.89
0.87
0.58
0.20
0.35
0.16
0.22
0.28
3.54
6.39
3.04
19.7
5.53
0.85
9.35
5.68
15.7
13.8
0.38
0.37
0.30
0.42
0.39
0.21
1.07
0.44
1.50
0.65
Percent Peak Abundance
Z2
0.44
1.58
0.24
0.87
0.25
5.01
0.58
8.43
4.02
19.8
7.08
1.29
10.5
6.49
15.7
15.3
0.31
0.24
0.26
0.41
1.39
0.99
2.36
0.63
Table 7. Percent peak abundances of fractions Z1 and Z2 as shown by GC analysis.
65
Chapter 4
CONCLUSION
The cytotoxic constituents in L. grayi were not successfully isolated through this study.
Instead, elimination of non-cytotoxic constituents was accomplished. Further
fractionation of specific compounds is required to identify the cytotoxic constituents.
From the ten experiments presented here, the following conclusions were drawn:
-
Both ethanol and hexane extract the cytotoxic constituents from L. grayi roots, but
water does not.
-
L. grayi extracts do not exhibit estrogenic activity, as demonstrated by equal half
lethal concentrations (LC50) on both ER+/PR+ (BT-474) and ER-/PR- (MDA-MB231) breast cancer cells.
-
The cytotoxic compound(s) in L. grayi are of intermediate polarity between that of
hexane and butyl acetate. This is demonstrated by the cytotoxicity of fractions
eluting from FCC with a mobile phase consisting of a mixture of these two solvents.
-
L. grayi’s cell inhibitory properties may be due to a single compound or a group of
compounds as observed by the presence of different compounds in active fractions of
varying polarities.
-
Through GC analysis, cytotoxicity is believed to be a result of one or more of the
compounds with the following retention times: 8.43, 8.51, 9.07, 11.72 minutes, or a
compound that is not detectable by GC-MS.
66
-
Lack of detection of cytotoxic compounds via HPLC-CAD indicated that either the
cytotoxic compound(s) are either strong UV absorbers or volatiles and therefore not
detected by CAD.
-
The single fractionation methods evaluated (FCC, TLC, and LLE) are not sufficient
for the isolation of individual L. grayi compounds.
-
Of the analytical methods used here (HPLC, GC-MS, and TLC), HPLC exhibited the
best separation of individual L. grayi components. Whereas GC-MS detects volatile
compounds, HPLC-CAD detects nonvolatile compounds. Therefore, these two
methods are complementary when one does not know the volatility of the compound
of interest.
6. Future Work
Based on these conclusions, preparative HPLC using a C-18 column and a fraction
collector is believed to be the best fractionation method for the isolation of individual
cytotoxic constituents from an L. grayi extract. Thus, it is recommended that future work
employ the following fractionation scheme: First, L. grayi root should be extracted with
ethanol, then this extract should be dried and re-extracted with hexane. The isolation of
cytotoxic constituents may be facilitated by subjecting this hexane-soluble fraction to
liquid-liquid extraction using acetonitrile followed by methanol/dichloromethane silica
based flash column chromatography of the acetonitrile LLE phase. Finally, the chemical
constituents should be further fractionated using preparative HPLC. Unfortunately, a
67
preparative C-18 column is not available in this laboratory. Therefore, extraction and
identification of the cytotoxic constituent(s) in oshála root cannot be accomplished at this
time.
Separation efficiency can be further improved by considering a set of factors. First, the
possibility of overloading can be eliminated by performing careful calculations before
loading a column. Second, contaminants and background noise can be discarded by
adding blank runs in between samples for both HPLC and GC analysis. Third, volatile
and non-volatile compounds can be further separated by steam distillation, vacuum
chamber, or freeze drying studies, where volatile compound composition can then
detected by GC-MS while the non-volatile compound can be detected by HPLC-CAD,
thus narrowing the detection methods and the number of compounds under study.
68
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Figure References:
1. Figure 1. Illustration of Ligusticum grayi. CalPhotos Photo Database (2009).
Ligusticum grayi; Gray's Licorice-root Coult. & Rose. Retrieved January 12, 2009 from
http://calphotos.berkeley.edu/cgi/img_query?query_src=photos_index&enlarge=0000+00
00+0305+0706.
2. Figure 2. Oshála distribution map. Southwest School of Botanical Medicine.
www.swsbm.com/Maps/Ligusticum_grayi.gif.
3. Figure 3. L. grayi worldwide distribution. ZipCodeZoo. (2009). Ligusticum grayi
(Gray’s Licorice-Root). Retrieved March 2, 2009 from
http://zipcodezoo.com/Plants/L/Ligusticum_grayi/.
4. Figure 4. L. grayi distribution in the state of California.
http://www.calflora.org/cgi-bin/species_query.cgi?where-calrecnum=4796.