ISOLATION OF A COMPOUND FROM ARTEMISIA DOUGLASIANA THAT IS CYTOTOXIC TOWARD BREAST CANCER CELLS
Gary Lai B.A., University of California, Davis, 2005
THESIS
Submitted in partial satisfaction of the requirements for the degree of
MASTER OF SCIENCE
in
CHEMISTRY (Biochemistry)
at
CALIFORNIA STATE UNIVERSITY, SACRAMENTO
SUMMER 2009 © 2009
Gary Lai ALL RIGHTS RESERVED
ii ISOLATION OF A COMPOUND FROM ARTEMISL4 DOUGLASIANA THAT IS CYTOTOXIC TOWARD BREAST CANCER CELLS
A Thesis
by
Gary Lai
Approved by:
-,Committee Chair D r. 'MryvcCarthy Hinte)1 Y Q
Second Reader Dr. Tom Savage I
, Third Reader Dr. Brad Baker
b/~ V n(::t Date
iii Student: Gary Lai
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.
,f////O Brad Baker, Graduate Coordinator Date
Department of Chemistry
iv Abstract
of
ISOLATION OF A COMPONENT FROM ARTEMISIA DOUGLASIANA THAT IS CYTOTOXIC TOWARD BREAST CANCER CELLS
by
Gary Lai
Artemisia douglasiana, also known as California mugwort or Douglas' sagewort,
is used commonly among Native California tribes for treating colds and infections as well
as premenstrual syndrome and dysmenorrhea. Previous work in this laboratory had
shown that California mugwort is cytotoxic towards the cultured breast cancer cell lines
MDA-MB-23 1 and BT-474. The purpose of this study was to isolate the chemical
constituents responsible for this cytoxicity. A. douglasiana leaves were collected and
prepared as ethanol extract. The EC50 values for the ethanol-soluble and hexane-soluble fractions were 1.8 1tg/ml and 0.053 jtg/ml for MDA-MB-23 1, respectively, and 1.2 [Ig/ml
and 0.025 jig/ml for BT-474, respectively. Various schemes were investigated to
separate the chemical components of the leaf extracts, including liquid-liquid extraction,
thin layer chromatography (TLC), flash column chromatography, and adsorption onto
lead tetraacetate. Cytotoxicity assays on MDA-MB-23 1 were used to guide the isolation.
High-performance liquid chromatography - charged aerosol detection, gas
chromatography - mass spectrometry, TLC, and nuclear magnetic resonance were used
to assess the purity of fractions. A method was developed to isolate a single cytotoxic
v compound from the plant leaf extract, but the amount obtained was too small to characterize and identify by chemical means or bioassay.
Committee Chair Dr. Mry McCarthy Hf tz' g
gFaed? Date/
vi ACKNOWLEDGMENTS
First, I would like to express my gratitude to Dr. McCarthy for her support and guidance throughout the project. I really appreciate Dr. Savage and Dr. Baker's valuable input and careful proofreading. At last, I want to thank Ursula Leyva Castro for sharing her ideas and helping in the laboratory. I am forever grateful to all of you.
vii TABLE OF CONTENTS Page
Acknowledgments...... vii List of Tables ...... ix List of Figures ...... x Chapter 1. INTRODUCTION...... 1 2. EXPERIMENTAL ...... 19 3. RESULTS ...... 28 4. DISCUSSIONS...... 64 5. CONCLUSIONS...... 75 References...... 77
viii LIST OF TABLES Page
1. Conditions for flash chromatography column ...... 25
2. Solvent systems tested for ability to separate constituents of A. douglasianaby TLC.36
3. Miscibility of different solvents...... 41
ix LIST OF FIGURES Page
1. Artemisia douglasiana Besser 7
2. Artemisia douglasianalocations in California 8
3. Artemisia douglasianalocations in the United States 9
4. Artemisia douglasianalocations throughout the world 9
5. Structure of Artemisinin I1
6. Structure of dehydroleucodin 14
7. Structure of vulgarone B 16
8. Structure of arglanine 16
9. Structure of douglanine 17
10. Cytotoxicity assay of extracts of dried leaf and fresh leaf in 95% ethanol, in MDA-MB-231 and BT-474 breast cancer cells 28
11. Cytotoxicity assay of 95% ethanol, water and hexane extracts in MDA-MB-231 and BT-474 breast cancer cells 29
12. Total ion chromatogram of hexane-soluble fraction 31
13. Total ion chromatogram of ethanol-soluble fraction 31
14. Total ion chromatogram of artemisinin standard 32
15. Mass spectrum of compound in ethanol-soluble fraction eluting
at 15.5 min 32
16. Mass spectrum of compound in hexane-soluble fraction eluting
atl5.5 min 33
17. Mass spectrum of an artemisinin standard eluting at 15.5 min 33
x 18. EC 50 determination of ethanol-soluble fraction in MDA-MB-231 and BT-474 breast cancer cells 34
19. EC50 determination of hexane-soluble fraction in MDA-MB-231 and BT-474 breast cancer cells 35
20. The effect of fractions from column 1 on MDA-MB-231 breast cancer cells 37
21. HPLC-UVD chromatograms at 210 nm: a) Hexane-soluble fraction 37 b) Fraction 3 from column 1 37 c) Fraction 5 from column 1 38 d) Fraction 6 from column 1 38 e) Fraction 9 from column 1 38 f) Fraction 10 from column 1 38
22. HPLC-CAD chromatograms: a) Hexane-soluble fraction 39 b) Fraction 3 from column 1 39 c) Fraction 5 from column 1 39 d) Fraction 6 from column 1 39 e) Fraction 9 from column 1 40 f) Fraction 10 from column 1 40
23. Cytotoxicity assay of LLE fractions of the hexane-soluble fraction on MDA-MB-231 42
24. Cytotoxicity assay of hexane-soluble fraction, the raffinate and extractant layer after acetonitrile LLE and hexane backwash 44
25. LLE scheme used on the hexane-soluble fraction 44
26. HPLC analysis of the acetonitrile layer after six n-hexane backwashes 45
27. Total ion chromatogram of acetonitrile layer 45
28. Cytotoxicity assay of the acetonitrile layer and the hexane-soluble and the hexane-insoluble fractions of the acetonitrile layer 46
xi 29. Total ion chromatograms of: a) Hexane soluble fractions of the acetonitrile layer from LLE 46 b) Hexane-insoluble fractions of the acetonitrile layer from LLE 47
30. Cytotoxicity assay of the fractions from column 3 48
31. HPLC chromatograms of fraction 1 from column 3 49
32. HPLC chromatograms of fraction 2 from column 3 49
33. NMR of fraction 1 from column 3 50
34. NMR of DhL provided by Dr. Luis Lopez from Universidad Nacional de Cuyo of Argentina 50
35. Cytotoxicity assay of the acetonitrile column fractions 51
36. Total ion chromatogram of fraction 1 from column 4 52
37. Total ion chromatogram of fraction 2 from column 4 52
38. Total ion chromatogram of fraction 3 from column 4 53
39. Cytotoxicity assay of the aqueous layer and chloroform washes after solvent extraction 54
40. Total ion chromatogram of aqueous layer from the DhL isolation protocol 55
41. Total ion chromatogram of chloroform layer after 1 chloroform solvent extraction wash 55
42 Total ion chromatogram of chloroform layer after 2 chloroform solvent extraction wash 56
43. Total ion chromatogram of chloroform layer after 3 chloroform solvent extraction wash 56
44. Cytotoxicity assay of the fractions collected from ethyl acetate column of chloroform layer 1 sample 57
45. Total ion chromatogram of column fraction 1 from ethyl acetate column 58 xii 46. Total ion chromatogram of column fraction 2 from ethyl acetate column 58
47. Total ion chromatogram of column fraction 3 from ethyl acetate column 59
48. Mass spectrum of the peak eluting at 10.5 min in GC-MS analysis of the ethyl acetate column fraction 1 59
49. Mass spectrum of the peak eluting at 10.5 min in GC-MS analysis of the ethyl acetate column fraction 2 60
50. Mass spectrum of the peak eluting at 10.3 min in GC-MS analysis of the hexane-soluble fraction of acetonitrile layer 60
51. Mass spectrum of the peak eluting at 10.3 min in GC-MS analysis of the hexane insoluble fraction of acetonitrile layer 61
52. Mass spectrum of the peak eluting at 10.3 min in GC-MS analysis of the chloroform layer after 1 chloroform solvent extraction wash 61
53. Mass spectrum of the peak eluting at 10.4 min in GC-MS analysis of the chloroform layer after 2 chloroform solvent extraction wash 62
54. Mass spectrum of the peak eluting at 10.5 min in GC-MS analysis of the chloroform layer after 3 chloroform solvent extraction wash 62
55. Mass spectrum of the peak eluting at 10.5 min in GC-MS analysis of the fraction 3 from column 4 63
xiii Chapter 1
INTRODUCTION
1.1 Introduction and Scope of Study
Artemisia douglasianaBesser, also known as A. vulgaris var. californicaBesser
(1), California mugwort, Douglas' sagewort, or Dream Plant, has been used for centuries to treat colds, flu, fevers, headaches, premenstrual syndrome and dysmenorrhea by different native Californian tribes (2, 3). Based on its uses for women's disease, and the use of other species in this genus for treating cancer, it was hypothesized that A. douglasiana Besser may have estrogenic activity and an effect on breast cancer cells.
Preliminary data showed that three different extracts of the leaves of A. douglasiana
Besser (aqueous, 95% ethanol, and hexane) exhibited cytotoxic activity towards MDA-
MB-23 land BT-474 breast cancer cells.
The hypothesis proposed is that A. douglasiana Besser contains active constituent(s) that have anti-proliferative effects (ability to inhibit growth), and/or cytotoxic effects (ability to kill cells). The active compound was isolated and characterized by bio-assay guided fractionation. A series of techniques including liquid- liquid extraction (LLE), thin layer chromatography (TLC), flash column chromatography
(CC), high-performance liquid chromatography - charged aerosol detection (HPLC-
CAD), gas chromatography - mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) were used to separate, isolate and characterize the active compound(s). 2
The biological activity of the different fractions was tested by bioassays on cultured human breast cancer cells.
1.2 Significance of Breast Cancer
Among the different types of cancer, breast cancer has been the main concern
for women. It is not only the fifth most common cause of cancer death worldwide, it
is also the most common cause of cancer death for women (4). Women in the United
States have the highest incidence of breast cancer in the world; they have a 1 in 8
lifetime chance to develop breast cancer and a 1 in 35 chance to die due to breast
cancer (5). In 2008, 182,460 new cases of invasive breast cancer were diagnosed and
about 40,480 women died from breast cancer in the US (5).
Early detection of breast cancer provides the best survival rate. Aside from
mammography, computer-aided detection and diagnosis (CAD), ultrasonography and
magnetic resonance imaging (MRI), doctors may remove a suspicious sample to be
checked under the microscope. After diagnosis, if cancer cells are present, lesions
will be further examined for over-expression of the estrogen receptor (ER),
progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2).
Estrogen and progesterone receptors, normally present on both healthy and
cancer cells, bind estrogen and progesterone that have traveled through the
bloodstream (6). When activated by estrogen, the ER signals the breast cell (normal
or cancerous) to grow. In the absence of estrogen, ER+ cancer cells, which over-
express ER, often stop growing. 3
HER2, another receptor that is over-expressed in cancer cells, sends growth
signals to the inside of the cell (7). In HER2+ breast cancer, the cancer cells have a
high number of HER2 proteins on the surface of the cells. This over-expression
causes the cancer cells to grow and divide rapidly (7).
1.3 Current Drug Treatments
Currently, approximately 75% of breast cancers are ER+, and about 65% of ER+ breast cancers are also PR+ (6). Only about 5% of breast cancers are ER- and PR+, and approximately 20% of breast cancers are HER2+ (7). For ER+ and PR+ cancer cells, hormonal therapies have the best advantages, as the drug restricts the hormones from forming or binding to the receptor site. Zoladex, Femara, and Tamoxifen are some common medications available for hormonal therapies.
Zoladex blocks the signal between the pituitary gland and the ovaries to stop the production of estrogen, which in turn decreases the growth of ER+ cells (8). Some of the common side effects include hot flashes, decreased libido, vaginal dryness, breast swelling and tenderness (8). Femara is an inhibitor of the enzyme aromatase, which converts androgens into estrogens (8). Similar side effects such as hot flashes, stomach pain and vomiting may present (9). Femara is only used for postmenopausal patients (8).
Instead of stopping estrogen production, another hormonal therapy method is to block the ER in cancer cells. Tamoxifen is the most common drug available today that is a selective estrogen receptor modulator (SERM), meaning that it can stimulate or block estrogen-like behavior to the selected tissue type (8). Tamoxifen, the best selling SERM 4 today, binds to and inactivates the ER and, thus, reduces growth of the cancerous cell (6).
Tamoxifen is currently used for the treatment of both early and advanced ER+ breast cancer in pre- and post-menopausal women (8). It is also approved by the FDA for the prevention of breast cancer in women at high risk of developing the disease (10). Some side effects of Tamoxifen include serious problems such as liver cancer, blood clots, stroke, and impotence (8, 10).
With HER2+ breast cancer, the cancer cells tend to be much more aggressive and fast-growing. Herceptin, a humanized monoclonal antibody which binds to domain IV of the extracellular segment of HER2 receptor, has been shown to dramatically reduce the risk of recurrence (1 1). Cells treated with herceptin undergo arrest during the GI phase of the cell cycle, and cell proliferation will be reduced (11). Relative to chemotherapy, herceptin has fewer immediate side effects. However, there is a small but real risk of heart damage and lung damage (12). Scientists are still studying how long women should take herceptin for the greatest benefit. Other drugs targeting the HER2 protein are also being developed.
For cancer victims that are not able to receive hormonal therapy or herceptin, such as many ER- patients, taxol is an alternative. Unlike hormonal therapy, taxol inhibits microtubule function in a cell, making the cell unable to divide (8). Although this sounds promising, taxol kills all fast growing cells, meaning that both cancer and fast growing healthy cells are killed. This can result in loss of body hair, serious infections caused by low white blood cell production, anemia due to low red blood cell production, 5 unusual bruising or bleeding, difficulty swallowing, dizziness, shortness of breath, severe exhaustion, numbness and tingling in the hands and feet (13).
Due to the different side effects and limitations of current drug therapies, discovery of new anticancer drugs with minimal side effects is a necessity.
1.4 Statement of Problem
This study investigates the efficacy of A. douglasianafor its cytotoxic effects
on breast cancer cells. Specifically, separation and analytical methodology was
developed to isolate the active compound(s) from the leaves of A. douglasiana,thus
documenting and giving validity to its medicinal use as an anti-cancer agent.
1.5 Botany, taxonomy and identification of Artemisia douglasiana Besser
Artemisia, belonging to the family Asteraceae, is a large genus consisting of 200-
400 species. This genus is most well known for its volatile oils. Throughout history,
Artemisia species have been used as food seasonings, insect repellants, cold medicines, and hallucinogens. Some of the most common species are wormwood, mugwort, and sagewort. Mugwort and sagewort are common names for the herbaceous members of this genus, and "wort" is an archaic term for any herbaceous plant (14).
The following lists the botanical name and the classification of Artemisia douglasiana:
Botanical Nomenclature: Artemisia douglasianaBesser 6
Plant classification:
Kingdom: Plantae - Plants
Subkingdom: Tracheobionta- Vascular plants
Superdivision: Spermatophyta - Seed plants
Division: Magnoliophyta - Flowering plants
Class: Magnoliopsida- Dicotyledons
Subclass: Asteridae
Order: Asterales
Family: Asteraceae - Aster family
Genus: Artemisia Linnaeus - Sagebrush
Species: Artemisia douglasiana Besser
Named after David Douglas (1798 - 1834), a Scottish botanist, A. douglasiana
Besser is a hexaploid species whose origin was attributed by Keck as a natural hybrid between Artemisia suksdorfii Piper (diploid) and Artemisia ludoviciana Nutt (tetraploid) by chromosomal analysis and physical characteristics, since A. ludoviciana, A. douglasianaand A. suksdorfii are the only species in the Artemisia genus in California that contain imperfect flowers and horizontal rhizomes (1, 15, 16). However, the hybrid relationship between A. suksdorfii (2n = 9) and A. ludoviciana (2n = 18) has been questioned (17). Artemisia species have compatible chromosomes that pair up regularly, and chromosomal analysis alone cannot disprove or confirm that A. douglasiana (2n
27) is a hybrid between the two species (17). 7
About 3 - 7 feet tall, A. douglasiana is a small perennial that has a strong sage odor (18). It flourishes near water and tolerates sandy soils and seasonal flooding (1, 19).
As shown in Figure 1, the stems are brownish to gray-green colored, and the leaves, approximately 1 - 2 inch long, are evenly spaced with 3-5 lobes near the tips and are dark green on top and silver-toned light green underneath (1, 20). Lower leaves are darker green, larger and cleft, and small white flowers flourish and spread their pollen between the June and October (1, 18).
Figure 1: Artemisia douglasianaBesser (from website http://www.baynatives. com/plants/Artemisia-douglasiana/). 8
Mainly located in California (Figure 2), A. douglasianacan be found from the west coast to the western slopes of the Rockies, and its range extends into
Washington, Oregon, Nevada, Idaho, and as far south as Baja California, Mexico
and even Argentina (Figures 3 and 4; 1, 21, 22). Due to the invasiveness and water tolerance of A. douglasiana, it is highly possible that the plant travelled throughout the coastline and expanded from California to other regions and countries in South
America (M. Baad, personal communication).
Figure 2: Artemisia douglasianalocations in California. Blue indicates a county where A. douglasianahas been reported while white indicates no reports (from website http://www.calflora.org/cgi-bin/speciesquery.cgi?where- calrecnum=708). 9
Figure 3: Artemisia douglasiana locations in the United States. The green color indicates A. douglasianahad been located while the white color indicates the plant's absence (from website http://plants.usda.gov/j ava/profile?symbol=ARD03).
Figure 4: Artemisia douglasiana locations throughout the world (from website http://zipcodezoo.com/Plants/A/Artemisia-douglasiana/). 10
1.6 Phytochemical analysis of related plants
Artemisia ludoviciana, also known as A. vulgaris sspp. ludoviciana, silver wormwood, white sagebrush, and gray sagewort, is native to North America, where it is widespread coast to coast, but many subspecies are found only in the western United
States (1, 16, 23, 24, 25). This plant was used by many Native American groups for a variety of medicinal, veterinary, and ceremonial purposes. A. ludovicianawas used to treat gastrointestinal tract problems, and the bitterness of the plant provides gastric simulation (16). A. ludovicianahas also been shown to inhibit HSV (herpes simplex virus) type I and 11 (16). Chemicals present in A. ludoviciana include sesquiterpenes
(achillin, anthemidin, artedouglasia oxide, douglanine, ludovicin, tanaparthin-Q-peroxide, and tanapartholide b), monoterpenes (borneol, camphor, chrysanthemol, transchrysanthenol, and a-pinene), flavonoids (butein, isoliquiritigenin, isorhamnetin, and quercetin), and coumarins (lacarol and scopoletin) (16, 23, 24, 25, 26).
Artemisia suksdorfii, also known as A. vulgaris var. litoralis Suksdorfii, coastal mugwort, coastal wormwood, and Suksdorf sagewort, is native to coastal western North
America and has a similar habitat to A. douglasianain coastal drainage and around the ocean. Not much information is available on A. suksdorfii, although scientists have tried to isolate different compounds hoping to locate compounds like artemisinin. Ahmed et al were able to isolate five new polyol monoterpenes and seven new sesquiterpene lactones, but no anticancer or antimicrobal tests were performed after isolation (27).
Artemisia vulgaris, also known as common mugwort, European mugwort, or common wormwood, is native to Britain, Germany, France, Norway, the Netherlands, I1
Poland, Sweden and the eastern US (28). Similarly to A. douglasiana,A. vulgaris has been used for fevers, female troubles, nervous disorders, stomach ulcers and indigestion, and the plant oil also contains similar components, such as cineole, camphor and thujone
(29). One record has stated that A. douglasianais shown to be five to ten times more potent against stomach ulcers and indigestion than A. vulgaris (30).
Another similar plant in the Artemisia genus is Artemisia annua, also known as
Sweet Wormwood, Sweet Annie, Sweet Sagewort, Annual Wormwood and qinghdo (31).
Since ancient times, the Chinese have used it to treat fever (32). Due to its native location and uses, many considered this plant the "Chinese mugwort". In 1971, scientists discovered its anti-malarial activity and extracted the active ingredient, artemisinin (C15
H22 05; Figure 5; 33).
K A~
Figure 5: Structure of Artemisinin (from website https://scifinder.cas.org/scifinder/view/link_v I /substance j sfrl=t7c6OyhXV6uzZ klgPcio5NEXaly4gR9x 1WZKxCAmvjwdTIpcS6BgXBNK7-KINqNu).
Much research has been done on artemisinin. Not only is artemisinin a common treatment for malaria, but it also has been shown to have anti-cancer properties against leukemia, colon cancer, melanomas, breast cancer, prostate cancer and renal cancer (34). 12
Interestingly, Lai et al discovered that the ability of artemisinin and its derivatives to selectively kill cancer cells was due to the higher iron content in cancer cells (35).
1.7 History, uses, and current studies of Artemisia douglasiana Besser
The Chumash, a Native California tribe, used A. douglasianafrequently for treating premenstrual syndrome and dysmenorrhea, as it increases blood circulation to the pelvic area and the uterus (2, 36). Chumash women would drink tea made from mugwort stems for a few days to relieve premenstrual syndrome and chew mugwort seeds for the treatment of dysmenorrhea (2).
Aside from the Chumash, other Native Americans have used mugwort for colds, flu, bronchitis and fever (3). Of the California tribes, the Miwok stuffed mugwort leaves in their nostrils to cure headaches, the Yurok used fresh leaves to soothe arthritis, and the
Kawaiisu used different parts of the plant to cure women's cramps (37, 38). In
Argentina, A. douglasianaleaves are popularly known as "matico" in traditional medicine and are used as a cytoprotective agent against peptic ulcer and as an antimicrobial agent in the external treatment of sores (39).
In the present day, non-Native herbalists recommend a tea made from California mugwort to promote menstruation and to treat nervous disorders and in baths to treat aches and pains due to arthritis (29). The oil can also aid the entire gastrointestinal tract by increasing stomach acid and bile production; thus, it will ease gas and bloating and improve digestion (40). It is also said to cure poison oak (19). 13
Previous research has shown that A. douglasiana produces a variety of compounds. Monoterpenes such as cineole, camphor, linalool, isothujone and thujone are the best known compounds from A. douglasiana (41). In addition, artemisia ketone, artemisia alcohol and hexanol were also present in A. douglasianaoil (40). Cineole, linalool and isothujone are considered pain relieving compounds and may contribute to the relief of premenstrual syndrome (2). Thujone can be addictive and can cause convulsions if taken in high dosage (2). There is only a small amount of thujone in mugwort, but long-term use of a concentrated alcoholic plant extract can still be a concern (40, 42).
In a case study, A. douglasiana leaf oil was shown to be effective against chronic bladder infection in a paraplegic youth (40). The leaf oil was then tested against various microbes including Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. It was shown that the leaf oil had antimicrobial activity in vitro, but the role of the oil in vivo was still unclear (40).
In Argentina, where herbalists use A. douglasianato treat peptic ulcers and external sores, scientists isolated a bioactive sesquiterpenic lactone of the guaianalide type, dehydroleucodine (DhL, C 15H1603; Figure 6). In several different experiments, A. douglasianaextracts and DhL have shown antioxidant capacity and have successfully prevented gastric ulcers induced by different necrotizing agents. The results are more pronounced in the extract, which indicates that other compounds in the extract, besides just DhL, are responsible for this activity. The capacity of these compounds to scavenge oxygen free radicals may be important to prevent peptic ulcer development (43). 14
C.
0
Figure 6: Structure of dehydroleucodin (from website https://scifinder.cas.org/scifinder/view/linkvi /substance j sfl=t7c6OyhXV6vUo 9czc4QEwkrwEpPgmeqB 1WZKxCAmvjw9KzCLdrOTR-lZ1FxLFXBd).
The effect of DhL was further tested on gastric acid secretion in rats, and the results indicated that DhL did not inhibit gastric acid secretion (44). This suggested that the anti-ulcerogenic effects can be attributed to its action on the mucosa defense factors
(44). The researchers also tested the anti-inflammatory activity of DhL, and DhL inhibited both chronic and acute arthritis induced by Freund's adjuvant carrageenan (44).
In a recent study, DhL was screened for antidiarrheal effects (45). DhL significantly reduced intestinal transit and intraluminal accumulation of fluid in mice, suggesting that DhL produces an inhibitory action on gastrointestinal functions, motility and secretion, and that this effect is mediated through the c 2-adrenergic system (45).
Although the exact mechanism is not known, this study has provided important information on the many uses of A. douglasiana.
Many sesquiterpene lactones have been shown to have strong anti-protozoal activity (46). In a study by Brengio, an inhibitory effect of DhL on the growth of
Trypanosoma cruzi was observed, and the effect was irreversible (47). Interactions of 15 sesquiterpene lactones and DNA have been reported (48). DhL, as a non-polar compound, can easily pass through the plasmalemma of parasites and inhibit the replication of DNA but not the synthesis of mRNA (47).
To prove the importance of DhL, the gastric cytoprotective effect of sesquiterpene lactones with similar structures from different plants inside and outside of the Artemisia genus were studied (49). Interestingly, the results indicated that not all guaianolides showed cytoprotective activity against ulcers, but the ones that had activities required the presence of a-methylene-y-lactone functional group (49). Another key finding of this study was that the presence of a P-substituted or -unsubstituted cyclopentenone ring was not a structural requirement for cyctoprotective activity, contrary to its requirement for antitumor (48) and antimicrobial properties (50).
Beside DhL, the only other study of bioactive compounds derived from A. douglasiana sesquiterpenes was that of vulgarone B (C15 H22 0; Figure 7). Its molluscicidal activity against invasive pests in the Philippines, such as Pomacea canaliculataand Planorbellatrivolvis, was measured and compared. The data indicated that vulgarone B was not toxic to crops at concentrations that caused 100% mortality of the pest, signifying that vulgarone B could be an effective molluscicide (51). 16
O
Figure 7: Structure of vulgarone B (from website https://scifinder.cas.org/scifinder/view/link_vi /substance j sfl=t7c6OyhXV6vyn3 9 El mHtHz4ttEK-xtYI 1WZKxCAmvj zEnlfhZpiej mbMv3 aMLzMS)
Two other sesquiterpene lactones were found in A. douglasiana. Arglanine
(C 15H1 804 , MP = 207 'C), also known as 11,13-dehydrovulgarin, was found in both A. douglasiana and in A. ludoviciana (Figure 8; 25, 52). Douglanine (CI5H2003, MP = 115 -
117 'C), which has a very similar structure to arglanine, was found in the dried aerial parts of A. douglasianain the spring (Figure 9; 53). No current reports on the uses of these sesquiterpene lactones can be located at this time.
M e~0~~~~~ N,f'rn
Figure 8: Structure of arglanine (from website https://scifinder.cas.org/scifinder/view/link_v I /substance j sf?l=t7c6OyhXV6vd BfrGcFsuOH 1a-Otzwmm 1WZKxCAmvjzzI9Lea-_OIvITV7VuAWbr). 17
Mm HO
Figure 9: Structure of douglanine (from website https://scifinder.cas.org/scifinder/view/link_v l/substance j sf?l=t7c6OyhXV6sR wrmGbx8rJh-xYdCi6ZPT1 WZKxCAmvjyJpiuiUlemOr3U5Oc7iAm2).
Since A. douglasianais used differently around the world, it is not surprising that plants of this species collected from different locations contain different chemical constituents. Plants from Argentina produce eudesmanolides in the spring and
guainaolides in the autumn (54), whereas in California, these chemicals were not found, but longipinene and nerolidol derivatives were present (55, 56). In plants collected in
Oregon, guaianolides were discovered, but they were not identical to those found in
Argentinian plants (54, 55, 56). Besides finding different chemicals in the roots, leaves
and stems of seedlings, fresh and air-dried materials also provided different DhL yields.
The largest amounts of DhL were from leaves, and fresh materials were reported to have higher yields compared to air-dried materials (56).
1.8 Study Contribution
In this study, a cytotoxic compound was isolated from the leaves of A. douglasiana. This study further extends the current knowledge about A. douglasianaby providing insight into the chemical differences of plants of this species located in 18 different regions of the world and contributing to the body of knowledge about the bioactive constituents in this important medicinal herb. The bioactive compound isolated can be further studied as a possible anti-cancer agent.
I 19
Chapter 2
EXPERIMENTAL
2.1 Chemicals and reagents
Ethanol (190 proof, 95%, ACS/USP grade) was purchased from Pharmco Products,
Inc. Artemisinin standard was purchased from Sigma Chemical Company. All other chemicals were general use HPLC-UV grade from Pharmco Products, Inc. Nanopure water was collected through an Easypure LF Compact Ultrapure Water System
(Barnstead). Glassware was cleaned by soaking in a NaOH/isopropanol bath (400 ml
10% aqueous NaOH + 3.6 L isopropanol) for at least 6 hours followed by 10 rinses with distilled water and 3 rinses with acetone.
2.2 Plant Extracts
Artemisia douglasianaleaves were obtained from the riverbank approximately 20 yards north of the Guy West Bridge along the east bank of the American River in
Sacramento in April 2006. Leaves were diced and extracted with the selected solvent by stirring for 48 hours at room temperature, and the extracts were then centrifuged in a
Sorvall GLC-2B (DuPont Instruments) at 1.95 X 10 revolutions per minute with a 5.5 angular velocity for 5 minutes. The supernatants were collected, evaporated to dryness using a rotary evaporator (Yamato Scientific Company), weighed and dissolved in 95% ethanol. For cell proliferation assays, extracts were filter-sterilized through a 0.20 Elm syringe filter (Fisherbrand). 20
a. Fresh vs. dried leaves
Both fresh (10 g) and air-dried (10 g) A. douglasianaleaves were diced and incubated with 250 ml of 95% ethanol, with stirring, for 48 hours to extract chemical components. After centrifugation, the extracts were then concentrated to 0.25 mg/ml, filter-sterilized and frozen for further testing.
b. Comparison of ethanol, hexane and water extracts
Air-dried leaves (10 g) were diced and incubated with 250 ml of 100% n-hexane,
95% ethanol, or 100% water for 48 hours to extract chemical components of differing polarities. Upon concentration and sterile filtration as described above, the extracts were stored for future use.
c. Hexane-soluble and ethanol-soluble fraction
Air-dried leaves (200 g) were diced and incubated with 5000 ml of 95% ethanol, with stirring for 48 hours to extract chemical components. The extract was then centrifuged, dried and dissolved in 500 ml n-hexane three times ("hexane-soluble fraction"), and the residue on the flask was further dissolved in 95% ethanol ("ethanol- soluble fraction"). Both the hexane-soluble and ethanol-soluble fractions were concentrated to 140 mg/ml, as described above, and stored in the freezer for future use. 21
2.3 Cell Proliferation Assay
a. Cell culture
Two human breast cancer cell lines, MDA-MB-23 1 and BT-474, obtained from the
American Type Culture Corporation, were stored in growth media (Improved MEM Zinc
Option, Richter's Modification IX, with L-Glutamine, L-Proline 2 mg/L, L-Gentamicin
Sulfate at 50 [ig/ml (Gibco) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin/streptomycin (5000 jtg/ml, Cellgro), 1% HEPES (Sigma Chemical Co.), and
1% phenol red (Sigma Chemical Co.)) with 7% dimethyl sulfoxide (DMSO) in liquid nitrogen. Prior to use, a Purifier Class II Biosafety cabinet (LABCONCO) was irradiated with ultraviolet (UV) light for 30 minutes. All cell solutions were thawed in a 37 'C water bath (Chicago Surgical & Electrical Co.) and the tubes were sprayed with a 70% ethanol solution before being placed in the hood. Cultures were maintained in growth media in a T-25 cell culture flask (VWR Scientific). Cells were cultured at 37 'C in 5%
C02 /95% air and 100% humidity in a Nuaire IR autoflow CO2 water-jacketed incubator.
Cells were harvested or split 1 to 5 when they reached approximately 90% confluency.
b. Cell plating
Media was aspirated from the cell culture flask, and 1 mL trypsin (0.25% with
0.1% EDTA in Hank's Balanced Salt Solution without calcium, magnesium or sodium bicarbonate; Cellgro) was added to detach the cells from the flask and each other. (BT-
474 required 30 minutes for complete release by trypsination and MDA-MB-23 1 required
10 minutes.) Upon trypsination, 5 mL fresh growth media was placed in the flask to
I 22
inactivate the trypsin, and the cell suspension was then transferred to a 15 mL sterile
centrifuge tube (Fisher Scientific) and centrifuged for 5 minutes at 1000 rpm. Upon
centrifugation, the supernatant was discarded and the cell pellet was re-suspended in 5
mL growth media. Ten AL of cell suspension and 10 itL Trypan Blue (0.4% solution in
phosphate buffered saline, Cellgro) were thoroughly mixed on Parafilm (VWR
Scientific), and 10 ,uL of this mixture was placed into each side of a hemacytometer
(Fisher Scientific). The cells, located in the four quadrants on both side of the
hemacytometer, were then counted under a Swift compound microscope. The average of
the number of cells from the eight (1 X 10-4 ml) quadrants was calculated, and this
number was multiplied by 2 to account for the 1:2 dilution with trypan blue. This value
was then multiplied by 10,000 to get cells/ml, and CIVI = C2V2 was used to determine the cell suspension volume required to get a final concentration of 5 x 104 cells/ml. Cell
suspension (100 [tL) was placed in each of the middle 60 wells of a sterile 96-well plate
(Becton Dickinson Labware). (The perimeter wells were not used.) The plate was
0 incubated in a Nuaire IR autoflow CO2 water-jacketed incubator at 37 C for 48 hours.
c. Cell treatment
Prior to mixture preparation, phytochemical fractions were sterile filtered using
0.2 ptm syringe filters. Samples were prepared by mixing 1980 p.L growth media and 20
p.L sample of interest. A control using 20 pL of 95% ethanol or 100% water was also
prepared in the same manner. When the samples were ready, the media was aspirated
from each of the middle 60 wells of the 96-well plate using a fresh, sterile pasteur pipet 23
(Fisherbrand) for each row. Each row contained a different sample, with 100 pLL of sample pipetted into each well. The plate was then incubated for another 48 hours.
d. Determination of Relative Cell Number
After 48 hours, the media was aspirated from each of the wells using a fresh sterile pasteur pipet for each row, and 100 jtL fresh media added. Fresh growth media was also added to the first row (where the wells were initially empty) to treat as blanks.
CellTiter 96Aqueous One Solution (20 piL; Promega) was then added into each well that contained media. This reagent allows quantification of the number of actively growing cells. The plate was incubated at 37 'C for 1-3 hours and placed in the Microplate reader
(BioRad Model 680) to measure the absorbance at 490 nm. (There was a longer wait time for BT-474 due to its slower growth rate compared to MDA-MB-23 1).
The standard deviation for each sample was calculated from ten individual tests, using the following formula:
2 2 Standard deviation = [f{X - n*(Blank mean) }/{n- 1}] 1/2
X = Sum total of the raw absorbance for each blank
n = Number of blanks
Blank mean = X/n
e. Half Maximal Effective Concentration (EC 50)
Similar to the preparation of cell treatment samples, the stock extract sample was first sterile filtered. Further dilution samples were prepared by adding 20, 40, 60, or 80 24
1il 95% ethanol to 80, 60, 40, or 20 tll stock extract, respectively. Twenty [I of the stock extract or diluted samples were added to 1980 ~il media and mixed. The rest of the experiment followed the cell treatment protocol by aspirating the media of the 96-well plate and adding in 100 pd sample into each well. After 48 hrs, the number of cells in each well was determined as described above.
2.4 Thin Layer Chromatography
Thin layer chromatography (TLC) analysis was carried out on aluminum plates pre-coated with silica gel UV254 (250 gim, Whatman). The solvent consisted of n- hexane:ethyl acetate (55:45), hexane, dichloromethane, chloroform, or acetonitrile and direct detection was in UV light at 254 and 365 nm.
2.5 Solvent Extraction
The hexane-soluble and ethanol-soluble fractions were partitioned with different solvents in a separatory funnel (Chernglass). Prior to liquid-liquid partitioning, all solvents were tested for miscibility. Tested solvents included n-hexane, methanol, ethanol, water, chloroform, dichloromethane, toluene, diethyl ether, acetronitrile, ethyl acetate and butyl acetate. Initial volumes ranged from 1 ml to 20 ml, and wash volumes were one or three times the initial volume. All fractions were saved and were evaluated for their biological activities in cell proliferation assay and for their constituents by TLC and/or GC-MS.
I 25
2.8 Flash column chromatography
Flash column chromatography was performed using Silica Gel 60 (0.063 - 0.200 mm; 70 - 230 mesh, EM Science) and different elution gradients. Sample (2 ml to 10 ml) was chromatographed in different column dimensions (440 x 40 mm or 250 x 16 mm; Column Kontes Glass Co.) using increasingly polar solvent mixtures. The eluting systems are indicated in Table 1.
Column 1 2 3 4 5 Sample n-hexane Fraction 6 ACN layer ACN Chloroform extract from n- after LLE layer after wash layer (10 ml) hexane (1 ml) LLE (1.5 1in EtOAc extract (2 ml) (1 ml) Ml) Column 440x40 250X 16 250x 16mm 250x 16 250x 16 dimension mm mm mm mm Solvent 1 Hexane Hexane (20 ACN (45 ml) ACN (60 EtOAc (55 (200 ml) ml) ml) ml) Solvent 2 Hexane: Hexane: ACN: EtOH ACN: EtOAc: BuOAc 1:1 EtOAc 3:2 (35 ml) EtOH 3:2 EtOH 3:2 (140 ml) 5.5:4.5 (50 (40 ml) (40 ml) Ml) Solvent 3 BuOAc (160 EtOAc (25 EtOH (20 EtOH (25 EtOH (20 ml) ml) ml) ml) ml) Solvent 4 EtOH (200 EtOH (25 None None None ml) ml)
Table 1: Conditions for flash chromatography columns.
Fractions were monitored by TLC, and identical fractions were pooled. The fractions were rotary-evaporated, brought to equal concentration by dissolving in ethanol, and then tested in a cell proliferation assay.
I 26
2.8 Gas chromatography - mass spectrometry
All samples were evaporated to dryness and dissolved in dichloromethane prior to
injection into an HP model 5890 gas chromatograph coupled with a HP 5890 mass
spectrometer (GC-MS). The extracted compounds were separated on a 5 %
phenylsilicone stationary phase in a fused silica capillary column (30 m X 0.25 mm i.d., 1
Elm; Restek). The carrier gas was helium (Praxair) at a flow rate of 1.0 ml/min. A 1.0 pl
volume of the sample was injected using the splitless mode. The data acquisition system
was controlled by MS ChemStation (Agilent Technologies). The column temperature
program was 40 'C for 1 min, 20 'C/min to 150°, 30'C/min to 280'C, hold 2 minutes.
Electron impact MS was used for detection.
2.8 High performance liquid chromatography with ultraviolet and charged aerosol
detection
Prior to HPLC injection, all samples were dried, re-dissolved in ethanol to a
concentration of 1.00 mg/ml, and sterile filtered. These samples (1 ml each) were
prepared and placed in HPLC ambersnap vials with caps (Hewlett Packard) for analysis.
An Agilent 1100 HPLC system with an Agilent Eclipse XDB-C18 column (4.6 x 150
mm, 5 ptm) and guard cartridge (7.5 x 4.6 mm Benson Carbohydrare BC-I00, Alltech)
coupled with ultraviolet (Agilent) and custom-built charged-aerosol detector (Thermal-
System Inc.) were used. The injection volume was 1 jl, and the solvent gradient was
water: acetonitrile, 80:20 to 20:80, with a flow rate of 1 ml/min under ambient
temperature and maximum pressure of 200 bar. Each run required 20 minutes. Detection
I 27
was by UV at 210 nm and by charged aerosol chromatograms. The sample was run
following the program listed below, with linear solvent gradients between each event
time.
Time (min) % ACN 0 20 2 20 10 80 14 80 15 20
2.9 'H Nuclear magnetic resonance spectroscopy
All samples were dried, re-dissolved in 0.5 ml of deuterochloroform containing
0.03 % tetramethylsilane (TMS) and placed in a clean, dry NMR tube. A Bruker
Avance-300 NMR spectrometer with xwinnmr spectrometer operating system was used
to produce free induction decay (FID) signals, and Nuts was used to process the data.
2.10 Lead tetraacetate preparation
Thirty grams of pre-dried lead tetroxide (Pharmco Products Inc.) was slowly
added into a 1-liter, three-neck round bottom flask with 55 ml glacial acetic acid
(Pharmco Products Inc.) and 17 ml acetic anhydride (Pharmco Products Inc.) at 40 'C.
The solution was continuously mixed and filtered through a Buchner funnel with filter
paper 1 (Whatman, 9.0 cm) covered with celite, the supernatant was discarded and
precipitate was rewashed with glacial acetic acid, dried and saved in a container. 28
Chapter 3
RESULTS
3.1 Fresh vs. dried leaves
Both fresh and air-dried A. douglasianaleaves were extracted with 95% ethanol
and concentrated to 0.25 mg/ml. The fresh leaf extract had a bright green and clear
appearance, whereas the dried leaf sample was dark green and cloudy. The samples were
tested on both MDA-MB-23 1 and BT-474 cancer cells for inhibitory or cytotoxic effects.
The results indicated both samples had cytotoxic properties, but the dried leaf extract
tended to have a stronger effect (Figure 10). Similar results were obtained with both
cancer cell lines. Based on these results, air-dried leaves were used for all further
extracts.
120 -- - 100 ......
-g60 S° 60 tX0+ ___~.~..>...... _.~.... .
°40 -t0|0| MDA-MB-231Gus ~~ S
i_ * * UBT-474
Ethanol Control Dry leaf ethanol Fresh leaf extract ethanol extract Samples
Figure 10: Cytotoxicity assay of extracts of dried leaf and fresh leaf in 95% ethanol, in MDA-MB-23 1 and BT-474 breast cancer cells. OD is plotted as percent of control. * indicates the sample is significantly different from the control (p<0.05). Error bars denote the standard deviation of ten individual tests. 29
3.2 Comparison of ethanol, hexane and water extracts
To compare the cytotoxicity of extracts using solvents of different polarities, air- dried leaves were extracted with 100% n-hexane, 95% ethanol or 100% water. Both the water and ethanol extracts exhibited a dark green color whereas the hexane extract had a much lighter green tone. After concentration and sterile filtration, the extracts were tested on both cancer cell lines. The cytotoxicity assay results indicated that both ethanol and hexane extracts had similar cytotoxicity against cancer cells, whereas the water extract had a much weaker effect [p = 2.28 x 10-1 and 6.11 x 10-1 for MDA-MB-231 and
BT-474, respectively compared the ethanol and hexane extract] (Figure 11). The two different cell lines gave similar results.
0120
ooo -T-C a - , 8 0 ...... 0 2 80 'i_ I_ A MDA-MB-231 CZ M BT-474 20-lA_*__*
0 Ethanol Water Ethanol Water Hexane control control extract extract extract Samples
A~~~~~~~~~~~~~~~~~~~~.... .--- ...... _. - l ...... -.._...... --
Figure 11: Cytotoxicity assay of 95% ethanol, water and hexane extracts in MDA-MB- 231 and BT-474 breast cancer cells. OD is plotted as percent of control. * indicates the sample is significantly different from the control (p<0.05). Error bars denote the standard deviation of ten individual tests. 30
3.3 Hexane-soluble, ethanol-soluble fractions and artemisinin
Based on the results from preliminary studies, the cost, and the lower volatility and toxicity of ethanol compared to hexane, ethanol was the solvent of choice for extraction. Two hundred grams of air-dried leaves were extracted with ethanol. After rotary evaporation to remove the solvent, the extract was washed three times with n- hexane to dissolve all hexane-soluble compounds. (This sample is called the hexane- soluble fraction throughout the rest of the study.) The remaining residue was then fully dissolved in 95% ethanol. (This sample is called the ethanol-soluble fraction throughout the rest of the study.) Gas chromatography analysis of both hexane-soluble (Figure 12) and ethanol-soluble (Figure 13) fractions was performed after aliquots of these fractions were dried down and redissolved in dichloromethane. The two earliest-eluting peaks, with retention times of 5.0 and 5.2 min, are due to contaminants in the dichloromethane
(data not shown), so these peaks are not considered in any of the GC analyses. Three main peaks were observed in both fractions, eluting at 9.1, 10.8 and 15.5 min, respectively. The hexane-soluble fraction had an additional major peak eluting at 14.1 min. Because the retention time of artemisinin, a well-known sesquiterpenic lactone from A. annua, nearly matched that of one of the peaks from the experimental samples, mass spectra were compared for the peaks at 15.5 min (Figure 15, 16, 17). The mass spectra showed completely different patterns between artemisinin and the two A. douglasianaextracts, clearly showing that artemisinin was not present in the A. douglasianaextracts. 31
TIC: HEXEXTF.D Abundance
800000
600000
400000 oR~~o
200000 -
Time-->Tm-::o0 0t _ .J ,.-Am,1 ' 6.00 8.00 10.00 12.00 14.00 16.00
Figure 12: Total ion chromatogram of hexane-soluble fraction.
TIC: ETOHNEWC.D Abundance VI)
450000
400000
350000
300000
250000 00
200000
150000 o
100000
50000-
Time--> -o 10.UU 8.uu 1U.UU 12.uu
Figure 13: Total ion chromatogram of ethanol-soluble fraction. 32
TIC: ARTEMF.D m Abunrdana ce
2600000
2400000
2200000
2000000
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
Time- X0 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00
Figure 14: Total ion chromatogram of artemisinin standard.
Scan 492 (15.451 min): ETOHNEVVC.D Abtrunda rice 53
35000-
30000-
25000 91
20000 .5 77
1 5000
10000 K1011 10 o o o o 131 24* H0 X 1l 14 13 isa 945 5000 IL175 20 I~~~i 217~8923 iea 204 231 ~~U .,.Ill ,H "1, , ~~~~~_'5 2 282I 60 80 1i00 1i20 140 160 180 200 220 240 260 280
Figure 15: Mass spectrum of compound in ethanol-soluble fraction eluting at 15.5 min. 33
Scan 493 (15 474 min): IEX->)EXTF.D Alb kind an ce
45000 -
,40000 -
35000 - 91
30000 -
-77 25000 -
20000-
15000- I I 9
1 C72-16 1 0000
1 751 938 2j0.4 23l1 5000-
5 2 ~~d H, 25227281 11 - ,1 II.11111 ... l bldh lllinllll ...... 1..1 .LL..... 1 1 ." 'I"' i i -° ' I 4.0. hjl.Jiii 60 80 100 120 1i40 150 180 200 220 240 260 280
Figure 16: Mass spectrum of compound in hexane-soluble fraction eluting at 15.5 min.
Scan 484 (15.274 min): ARTrEMVF.D Aba. ad aan ce 55 69 137151 166
80000 l
70000 96
60000
50000 81
40000
1 09 30000 1 23
20000 21118
193 2 9 1 0000 239 2~ 0257
Figure 17: Mass spectrum of an artemisinin standard eluting at 15.5 min. 34
3.3.1 EC-50
EC50 determinations for both ethanol and hexane-soluble fractions were performed (Figures 18 & 19). For the ethanol-soluble fraction, the EC50 for MDA-MB-
231 and BT-474 were 1.8 Rg/ml and 1.2 Ftg/ml, respectively. For the hexane-soluble fraction, the EC50 for MDA-MB-231 and BT-474 were 0.053 ,ug/ml and 0.025 jig/ml, respectively. Due to the higher growth inhibition exhibited by the hexane-soluble fraction, the rest of the experiment mainly focused on the hexane-soluble fraction. For the rest of the study, only MDA-MB-231 was used for the cytotoxicity assay because it grows faster and is easier to handle.
18O 160 14 6.0 ...... ---I...... -....-... _ _ ...... I......
0 ° 1000......
0 g 80 > ~~~~ ~...... 9...... - A MDA-MB-231 0 60 T -- BT-474 ,4 0 .o..... f...... t. 20 .. _ .. . __ .A ...... _ ......
-20 0 0.5 1------1.5 - 2-. 2 5.... . 3 Extract concentration (pg/mi) ...... -...... I...... -..".' I ...... - - -., I ...... - .1 111 1 .11...... -- ...... l
Figure 18: EC 50 determination of ethanol-soluble fraction in MDA-MB-231 and BT-474 breast cancer cells. OD is plotted as percent of control. Error bars denote the standard deviation of ten individual tests. 35
1 7 0 l ------...... 150 . ... . 130
110 '
970 MDA-MB-231
co - -- BT-474B :E 30 \ 10 0 0.. 0. . .00. 00.2 0.4 0.6 0.8 Extract concentration (pg/ml)
Figure 19: EC50 determination of hexane-soluble fraction in MDA-MB-23 1 and BT-474 breast cancer cells. OD is plotted as percent of control. Error bars denote the standard deviation of ten individual tests.
3.3.2 Separations
a. Flash column chromatography
A series of TLC separations were performed to identify an ideal solvent system for separating the compounds in the extract via column chromatography. Table 2 lists the various solvent systems and their separation outcomes. 36
Solvent 1 Solvent 2 Ratio Separation of (Solvent 1 to compounds Solvent 2) by TLC Chloroform N/A N/A Acceptable Dichloromethane N/A N/A Acceptable Ethyl acetate N/A N/A Excellent Ethanol N/A N/A No Butyl acetate N/A N/A No Acetone N/A N/A Acceptable Toluene N/A N/A Acceptable Ethyl acetate Hexane 1:1 Acceptable Ethyl acetate Ethanol 1:1 Acceptable Chloroform Ethanol 1:1 Acceptable Ethyl acetate Hexane 3:7 Excellent Ethyl acetate Hexane 4:6 Excellent Ethyl acetate Hexane 4.5:5.5 Excellent Table 2: Solvent systems tested for ability to separate constituents of A. douglasiana by TLC.
Based on the results from TLC, 10 ml of the hexane-soluble fraction was separated using flash column chromatography, as described in Table 1 (column 1). The fractions were monitored by TLC, and identical fractions were pooled. Eleven fractions were collected and tested against MDA-MB-23 1 breast cancer cells. Traces of green were detected in all fractions and were more prominent in the active fractions. Figure 20 displays the results of the cytotoxicity assay of the different fractions from this column, showing that fractions 5, 6 and 9 exhibited cytotoxicity. These fractions were further examined by HPLC-UVD/CAD; figure 21 and 22 present the chromatograms of the different fractions. 37
160 .---.--.--...... --...- ...... 160
4 01 1 20 ._...... ~~~....._
U100 0 n 80 | l-- ____
E) 40''''"'""""-1 * 11 20 ______
ErOH 1 2 3 4 5 6 7 8 9 10 I1 Control
Fraction
Figure 20: The effect of fractions from column 1 on MDA-MB-23 1 breast cancer cells. OD is plotted as percent of control. * indicates the sample is significantly different from the control (p<0.05). Error bars denote the standard deviation of ten individual tests.
a. Hexane-soluble fraction
b. Fraction 3 38
e. Fraction 9
f. Fraction 10 Figure 21: Chromatograms of the hexane-soluble fraction of A. douglasiana (a) and fractions 3(b), 5 (c), 6 (d), 9 (e) and 10 (f) from column 1, using HPLC-UVD at 210 nm. 39
ADC' A. ADC CHANNELA(GARWY'3140B00000010.)
2000e
0 2.6 -
- a~~~0 . 10 12.8 5 T a. Hexane-soluble fraction
b. Fraction 3
AD01 A. *D01 CUANEUACOARY)03100SS000004.0)
20 6 6 10 106 16 ITm d. Fraction 6 40
e. Fraction 9
C] ADC A. AD.1 CHANNEL.A.(...31 .0)00000)
I'd
520
2.5 7.5 10 12.5 15 1.5m f. Fraction 10
Figure 22: HPLC-CAD chromatograms of the hexane-soluble fraction of A. douglasiana (a) and fractions 3 (b), 5 (c), 6 (d), 9 (e) and 10 (f) from column 1.
Both fraction 5 and 6 showed high cytotoxicity in the cytotoxicity assay and
similar HPLC chromatograms. Fraction 6 was further concentrated and a second flash
column (column 2) was performed as described in Table 1. TLC analysis of the fractions
indicated this attempt at fractionation did not successfully separate the compounds (data
not shown). Every fraction contained dark green compound and two or more additional
compounds.
b. Liquid-liquid extraction
Various solvents were tested for miscibility with each other. Table 3 lists the
conditions and miscibility of the solvents.
I 41
Solvent 1 Solvent 2 Solvent 3 Miscibility DMSO Hexane N/A No DMSO Methanol N/A Yes Dichloromethane Water N/A No Methanol Hexane N/A No Diethyl ether Water N/A No Acetonitrile Water N/A Yes Acetone Hexane N/A Yes Toluene Water N/A No Ether Hexane N/A Yes Acetonitrile Hexane N/A No Hexane Ethanol N/A No Acetonitrile Ethanol N/A Yes Acetonitrile Water Hexane No Acetonitrile Ethanol Hexane No Table 3: Miscibility of different solvents.
Although DMSO and toluene displayed miscibility consistent with good separation systems, they were discounted for liquid-liquid extraction due to their high boiling points (189 'C for DMSO and 111 C for toluene), which was impractical for rotary evaporation. Acetonitrile, a solvent with similar polarity to DMSO, was then the best solvent choice, as it has a lower melting point (82 0C), which could allow the solvent to evaporate easily so that the sample residue could be re-dissolved in ethanol for cytotoxicity assays.
The hexane-soluble fraction was further fractionated by partitioning with water, acetonitrile or 95% ethanol. One ml of hexane-soluble fraction was washed with an equal volume of the solvent of choice three times; both layers were then collected, dried, 42 concentrated and re-dissolved into ethanol. The samples were further tested in cytotoxicity assays (Figure 23). Acetonitrile is miscible with ethanol, so partitioning was performed with mixtures of 7:3 and 9:1 acetonitrile:ethanol and were also tested on breast cancer cells (Figure 23). The cytotoxixity assay indicated that LLE with ethanol and acetronitrile provided the best results, while water LLE also showed distinct difference between the two layers. LLE using acetonitrile:ethanol (9:1 and 7:3) mixtures did not
show any improvement in cytotoxicity against breast cancer cells compared to 100% acetonitrile LLE.
120
°. 100 C 0 U 80 4.4 0 g 60 o 40 C 4' 20
0
Sample
Figure 23: Cytotoxicity assay of LLE fractions of the hexane-soluble fraction on MDA- MB-23 1. OD is plotted as percent of control. * indicates the sample is significantly different from the control (p<0.05). Error bars denote the standard deviation of ten individual tests. 43
To improve the removal of chlorophyll, hexane-soluble fraction (2 ml) was partitioned with acetonitrile (2 ml) once; the denser acetronitrile layer was collected and backwashed with hexane (6 ml) six times. The hexane backwash was able to remove most of the green color, leaving the acetonitrile layer yellowish. Further backwash yielded a light yellow hexane layer and a yellow acetonitrile layer. Cytotoxicity assays indicated that the hexane layer showed no cytotoxicity against cancer cells while the acetonitrile layer displayed effective cytotoxicity towards breast cancer cells (Figure 24).
The acetonitrile layer was further examined by HPLC-CAD and UVD, and results
are shown in figure 26. Three main peaks were detected at 210 nm (7.4, 10.1, 12.6 min), while two closely eluting peaks were detected around 12.6 min by the CAD. TLC also yielded similar results, with three peaks present on the plate (data not shown).
The acetonitrile layer was then evaporated to dryness, and the residue was only
partially soluble in hexane. The hexane-soluble fraction of the acetonitrile layer was
returned to dryness, and the two samples (hexane-soluble and hexane-insoluble fractions
of the acetonitrile layer) were re-dissolved in ethanol and tested in a cytotoxicity assay
(Figure 28). The two samples had similar cytotoxicity against breast cancer cells, and the
GC analysis also suggested similar volatile composition for both samples (Figure 29). 44
140 t 120 C Uo 100 o 80 . 60 ° 40 C s 20 0...... T ...... 0
Sample
Figure 24: Cytotoxicity assay of hexane-soluble fraction, the raffinate and extractant layer after acetonitrile LLE and hexane backwash. OD is plotted as percent of control. * indicates the sample is significantly different from the control (p<0.05). Error bars denote the standard deviation of ten individual tests.
iLLE 1x ACN
LLE 6x i hexane
Figure 25: LLE scheme used on the hexane-soluble fraction. * indicates cytotoxic effect on breast cancer cells. 45
ADC1 A A)C1 Ca-NA(GRW04140M=.D) nt-
40W
Eo wiF; $tX 'Ffi °8ZiJ
l ) 25 5 7.5 10 125 15 17.5 mr '\ND1 A V\ h2Orm(GWW408Maq n~U 140J 12)-
00
10D25 5 7.5 10 125 15 175 nr
Figure 26: HPLC analysis of the acetonitrile layer after six n-hexane backwashes (CAD- top, UVD- bottom).
TIC: IT09ACN.D Ab L ndance 2600000
2400000
2200000
2000000
1800000
1600000
1400000
1200000
1 000000
800000
600000 6) 400000
200000
0 Time--- O 6. 10.00 12.00 14.00 16.00 Figure 27: Total ion chromatogram of acetonitrile layer. 46
1 2 0 -- - ...------......
=100 - ' ___E ''''----'---''''I--'-"'------I------'-A'-
o 80 40
40
20 _ _ _ .. * ______. _ _ _ . _ .. .. ______. _ _ _ . E 60
O~~~~~~~~ I_ 0 EtOH control ACN Flexane-soluble liexane-insoluble
Sample
Figure 28: Cytotoxicity assay of the acetonitrile layer and the hexane-soluble and the hexane-insoluble fractions of the acetonitrile layer. OD is plotted as percent of control. * indicates the sample is significantly different from the control (p<0.05). Error bars denote the standard deviation of ten individual tests.
TIC: H.D Abundance T H, 1000000-
800000-
600000
400000
200000-
Time--> nn s, fi 1A nn .1 n U.UU O.UU IU.UU le-UU 1-+.UU I 'U. ULF
a. Hexane-soluble fraction of acetonitrile layer .47
TIC: A.D Abundance
300000-
250000- m
200000 tr
150000-
100000-
50000_
Time-- I 6.00 8.00 10.00 12.00 14.00 16.00
b. Hexane-insoluble fraction of acetonitrile layer
Figure 29: Total ion chromatograms of (a) the hexane-soluble and (b) hexane-insoluble fractions of the acetonitrile layer from LLE.
To further purify the cytotoxic compounds, 2 ml of hexane-soluble fraction was partitioned with an equal volume of acetonitrile; the acetonitrile layer was backwashed with n-hexane six times, then concentrated and prepared for flash column chromatography. The column was eluted with 45 mL acetonitrile, then 35 mL acetonitrile:ethanol (3:2), then 20 mL ethanol (column 3). Two distinct bands were observed by UV illumination, so two fractions were collected. The first eluting band showed a strong cytotoxic effect, whereas the second band appeared to have no effect on the breast cancer cells (Figure 30). TLC indicated one band in the first fraction and two distinct bands in the second fraction. HPLC-UVD analysis showed a large peak eluting at 10.2 min in the first fraction that was absent in the second fraction (Figure 31, 32). 48
NMR analysis of the first fraction from the acetonitrile layer (Figure 33) was performed and compared with the NMR spectrum of DhL, which was kindly provided by Luis
Lopez from Universidad Nacional de Cuyo, Argentina (Figure 34).
140
1 2 0 .I. ------...... ----.... ------...------.
0 s-100 * _
8 0 ......
0 60 ......
0 40
20 .+...... *----__
EtOH Control Fraction 1 from ACN Fraction 2 from ACN
Sample
Figure 30: Cytotoxicity assay of the fractions from column 3. OD is plotted as percent of control. * indicates the sample is significantly different from the control (p<0.05). Error bars denote the standard deviation of ten individual tests. 49
ADC1 A, ADC1 CHANNIEL A (GAR'AO40609Da -04-09 10-42-1604909000001.D)
350i
300- 250 0 4N "W.'9-:==z = 20D
150-
150- 5D
0 25 5 7.5 10 12.5 15 17.5 rin Fgr D1 A, V3: 2 10 nm (GA of09090-42-16\040990 fracti fromD) 3C -oW1. 20D-~~~~~~~~~~~~~~~~~~~C rrAU
3,90
253- 200 153- 100
o 25S 5 7.5 110 12.5 15 1715 mnn Figure 3 1: HPLC chromatograms of fraction 1 from column 3 (CAD - top, UVD - bottom).
Figure 32: HPLC chromatograms of fraction 2 from column 3 (CAD - top, UVD - bottom). 50
Figure 33: NMR of fraction 1 from column 3.
q
i -kg . '. il" ,:I- : I s '. 11-11-::.-11-.- V i
1 ^ MUMA~~~~~c.
~~~I ' ' i.., I" -l4
14 1 :. 0 a : :11 S.: .I . 0I "M 0 7.' , : , t 5
Figure 34: NMR of DhL provided by Dr. Luis Lopez from Universidad Nacional de Cuyo of Argentina.
To further investigate this method, the procedure was repeated with a larger sample volume. Ten mL hexane-soluble fraction was prepared for solvent extraction with 10 ml acetonitrile. Upon partitioning, the acetonitrile layer was saved and further partitioned six times with hexane of equal volume to remove the chlorophyll and other 51 unwanted compounds. The acetonitrile layer was then further concentrated and run through an acetonitrile column (column 4). Three fractions were collected, concentrated to 1.0 .tg/ml, tested on cells, and examined under gas chromatography-mass spectroscopy
(Figure 35, 36, 37 and 38). Fraction 3 was the only fraction displaying strong cytotoxicty against breast cancer cells.
180 160 I ° 140 T C o 120 o 100 80 ET...... " " ,, ...... ~~~~~~~~~~~~~~~~~~~~~~~~~~...... |El-..--1EEl1 oC 60 W 40 ...... lmTov-i * 20 -.....- ...... - _ _ ._ 0 EtOH Control ACN column ACN column ACN column fraction 1 fraction 2 fraction 3
Sample
Figure 35: Cytotoxicity assay of the acetonitrile column fractions. OD is plotted as percent of control. * indicates the sample is significantly different from the control (p<0.05). Error bars denote the standard deviation of ten individual tests. 52
TIC: ACrCCF1 F.D AtbLEnCldnce
800000-
750000-
700000-
650000-
600000 -
550000-
500000-
450000
400000-
350000
300000-
250000
200000
150000
100000
50000
Timk-e__ 0
Figure 36: Total ion chromatogram of fraction 1 from column 4.
TIC: ACNCCF2F.D Abundance 1400000
1 300000
1 200000
1 1 00000
1000000
900000
800000
700000
600000
500000
400000
300000
200000
1 00000
Time- ->0 6.00 8.00 10.00 12.00 14.00 16.00
Figure 37: Total ion chromatogram of fraction 2 from column 4. 53
TIC: ACNCCF3F.D AbUndance
65000( -3 600001
550004 -3 500001
450001 2 - 400001 2- 350001
30000(2-
250004 2~ ~~~~~~~~~W 20000i2_
15000(D2
1 0000( D2
5000(
Tirne-->( 6.00 8.00 10.00 12.00 14.00 16.00
Figure 38: Total ion chromatogram of fraction 3 from column 4.
c. DhL isolation protocol
The next extraction method was based on the work of Giordano et al (49). Ten mL of ethanol-soluble fraction was added to 1O ml of 4% aqueous lead tetraacetate, then the solution was filtered through a celite-covered filter paper and partitioned with chloroform three times. The raffinate and extractant layers were both dried, concentrated to 0.1 jIg/mil and re-dissolved in ethanol to test on breast cancer cells (Figure 39) and were also examined by gas chromatography (Figure 40, 41, 42 and 43). The aqueous layer appeared as a dark orange color; the first chloroform wash sample had a brown- greenish appearance, the second chloroform wash sample had a light yellowish color, and the third chloroform sample appeared colorless. Due to the large color difference, the three chloroform wash samples were tested separately in cell assays. A chloroform 54 control was prepared by using clean 95% ethanol to follow the protocol of the DhL isolation; the chloroform layer was collected, dried and tested on cells. The three
chloroform wash samples displayed strong cytotoxicity against breast cancer cells, with chloroform wash 1 and 2 showing the strongest cytotoxicity (p = 1.74 x 10-6 and 2.48 x
10-6 compared to chloroform wash 3), whereas the non-chloroform layer displayed no
inhibitory effects on breast cancer cells.
180 -m_- --- .__.....- 160 - -... _.. _.
VO0 120 .... __.
40
EtO II Chiloroform Aqueous Chloroform Chloroform Chloroform Control Control Layer Wash Layer Wash Layer Wash Layer 1 2 3 Sample
Figure 39: Cytotoxicity assay of the aqueous layer and chloroform washes after solvent extraction (Trial 5). OD is plotted as percent of control. * indicates the sample is significantly different from the control (p<0.05). Error bars denote the standard deviation of ten individual tests. 55
TIC: NMS1.D Abundancie
40000 -
35000 -
30000 -
25000 -
20000 -
15000 -
1 0000 -
5000 -
Tir-ne--_-- 6.00 8.00 10.00 12.00 14.00I 16.00 Figure 40: Total ion chromatogram of aqueous layer from the DhL isolation protocol.
kr)
TIC: NMFI.D tr AbfLndance
800000
750000
700000
650000
600000
550000
500000
450000
400000
350000
300000
250000
200000 It 150000
100000
50000
0
Figure 41: Total ion chromatogram of chloroform layer after 1 chloroform solvent extraction wash. 56
TIC: NMC2.D Abrud ancce
350000
300000
250000
200000
150000 If Lr~ C 1 00000
50000
10.00 12.00 14.00 16.00 Figure 42: Total ion chromatogram of chloroform layer after 2 chloroform solvent extraction wash.
TIC: NMC3.D Abundance
550000-
500000 -
450000 -
400000 -
350000-
300000-
250000-
200000
150000-
100000 - '1) < It\' 50000-
Time--> - 1 .I 1 .I I I 6.00 8.00 10.00 12.00 14.00 16.00
Figure 43: Total ion chromatogram of chloroform layer after 3 chloroform solvent extraction wash. 57
Chloroform layer 1 was dried, dissolved in ethyl acetate, and further separated by a column using ethyl acetate (column 5, Table 1). Three fractions were collected, concentrated to 1.0 ptg/ml, and tested in a cytotoxicity assay (Figure 44). Fractions 1 and
2 displayed strong cytotoxicity while fraction 3 showed a weak but observable cytotoxic activity against the cancer cells [p = 8.12 x 10-3 (fractionl), 7.53 x 10-3 (fraction 2), and
2.32 x 10-1 (fraction 3) compared with the chloroform control]. The fractions were then analyzed using gas chromatography (Figure 45, 46 and 47). The chromatograms of fractions 1 and 2, which are most cytotoxic, both contain a prominent peak with a retention time of 10.5 min., whereas this peak is almost nonexistent in the chromatogram of fraction 3. Figure 48 and 49 display the mass spectra of the 10.5 min peak of column fraction 1 and 2, respectively. Figure 50 - 55 displayed the mass spectra of the different samples at 10.5 min.
120 1 2 0 ...... -......
o o 80 10 ...... -- c 60
60 ...... *- 8 0 * E
E2 0O ...... *...... -
EtOH Control Chloroform EtOAc coluttmn EtOAc colu111nEtOAc coIlum n Control fraction 1 fraction 2 fraction 3
Sample
Figure 44: Cytotoxicity assay of the fractions collected from ethyl acetate column of chloroform layer 1 sample. OD is plotted as percent of control. * indicates the sample is significantly different from the control (p<0.05). Error bars denote the standard deviation of ten individual tests. 58
TIC: ETOACFF1 .D Abundance
350000
300000
250000
200000
150000
00 100000- ir~
0n 50000- A ^_X\ A LJ Kk. A T ime -- I I .6.00 8.00 10.00 12.00 14.00 16.00 Figure 45: Total ion chromatogram of column fraction 1 from ethyl acetate column.
TIC: 2ARR.D Abundance
200000
150000- o
100000
50000
:,o L ~T- -.~ - . -II- . -. .. f---- T _ime--> 6.00 8.00 10.00 12.00 14.00 16.00
Figure 46: Total ion chromatogram of column fraction 2 from ethyl acetate column. 59
TIC: 3AFRRR.D Abundance
200000 -
150000
100000.
50000
| . I . . . . I I I I . . . I . , Time->0 . . ! . I ...... 1 6.00 8.00 10.00 12.00 14.00 16.00
Figure 47: Total ion chromatogram of column fraction 3 from ethyl acetate column.
Scan 265 (10.413 min): ETOACFF1 .D A\bLrndance 55 600 -
550-
500 -
450- 67 400-
350
71 300- 51 79 95 250
11 1 200 154 99 150 83 1 08 121126 100
50
rn/z-->° I T nrn rn Tnri Ti Ti Tn 7T*n .f .n -inn iI.1T I 0 io V ov , VIo v . -- . - .- -- - .-- .- .-- Figure 48: Mass spectrum of the peak eluting at 10.5 min in GC-MS analysis of the ethyl acetate column fraction 1. 60
Scan 270 (10.525 min): 2AFR.D AbtznClance 71 55 1 0000 87 9000 58 e
8000
7000
6000 -
5000 67 1 09 el 4000 12
3000
2000 I 1 t 3 5
1000
40 50 60 70 80 90 100 110 120 130 140 150 160
Figure 49: Mass spectrum of the peak eluting at 10.5 min in GC-MS analysis of the ethyl acetate column fraction 2.
scan 200 (10.304 -in): "I.D tv-cIni eiariceB
1 1000 - 1
1 0000 10000
83000 Ei
7000 aooo 8 C.000
S000~ ~ ~ ~ 10
.4000
3000
2000
1000 R
om21~ l3l7| | | i | z5 1 26e0 .40t-1o 0 E30 100 I120 1.40 ISO0 1 SC) ;'00 ;220~ Z2,0 2ao Figure 50: Mass spectrum of the peak eluting at 10.3 min in GC-MS analysis of the hexane-soluble fraction of acetonitrile layer. 61
Scan 260 (10.303 min): A.D Abundance 55
8000 - 71 7000 -
6000 87
5000
4000 109 81 126 3000 74
2000 51
1000
0 119 4SOO--> 404010 0 50 60 70 80 90 100 110 120 130 140 150 160
Figure 51: Mass spectrum of the peak eluting at 10.3 min in GC-MS analysis of the hexane-insoluble fraction of acetonitrile layer.
~~~~~OCIC>~~1( 3~4 r-I.) .I~~ Z
eooo~~~~~~~~~~~~~-
M!500
M.~Oo
I ~ ~ ~ :2 I C 0 m z
Figure 52: Mass spectrum of the peak eluting at 10.3 min in GC-MS analysis of the chloofom lyerafter 1 chloroform solvent extraction wash. 62
265r (<0.435, -ir.): NAC-(c2.ID A Lt-J -ll a c
2200
2000
I Boo
1 G00
1 400 S9 l 1200
1000
Boo
4600
1 0O 12(3 .400 ... I''..... 200 ...... 1111.11, 2 1 ...... ~~I I, 11111 11 '1 1$'''3''To, T' ...... I'J, I-llllllllllll - .. ... I" ... ' ...... I ...... I ,40 ..11-. . 60' ' . . .oO.. . . 100.. I 120.. . . 10 180 100 200 220 Figure 53: Mass spectrum of the peak eluting at 10.4 min in GC-MS analysis of the chloroform layer after 2 chloroform solvent extraction wash.
s 0 . --. e I l O .f .-. -fl - r- J N I . r
---
I-
I- --
I- --
I
---
---
---
---
---
---
I -- Izr% .,I I i1 -1 iI_` I'l II I I I I - , c3,, I 6 6Ce-6e ,- - e - - ' -66 Figure 54: Mass spectrum of the peak eluting at 10.5 min in GC-MS analysis of the chloroform layer after 3 chloroform solvent extraction wash.
I 63
Mass spectral differernce (263--) (-) Abundan ce 71
1 1000 -
1 0000 - 1 26
9000 - I1 1 8000-
7000- 55
6000
5000-
4000 - 93 I I 83 67 3000- 2000- DE 1 27 1000 - T7. 137. 14Eh53 1 6-7173 47II I - ii I 1 , . , I 40Ii-- 50 60 70 80 90 100 ilo 120 130 1i40 150 160 17' i0 Figure 55: Mass spectrum of the peak eluting at 10.5 min in GC-MS analysis of the fraction 3 from column 4.
I I 64
Chapter 4
DISCUSSIONS
This purpose of this study was to investigate the efficacy of A. douglasianafor its
cytotoxic effects on breast cancer cells and to develop separation methodology to isolate
the active compound from the leaves of A. douglasiana. A single cytotoxic compound
was successfully isolated.
4.1 Fresh vs. dried leaves
To determine the best way to extract cytotoxic compound(s) from A. douglasiana,
both fresh and dried leaf extracts were prepared. Not only did fresh leaves contain more
water content, but they also had a stronger, fresh odor, whereas dried leaves were more
concentrated due to the lack of water. The cytotoxicity assay results showed that the
dried leaf sample had stronger inhibitory effects (Figure 10). This suggested that the
compounds responsible for the plant's aroma were not responsible for cancer cell death.
In a previous study, Pestchanker et al reported that a larger amount of DhL, the
sesquiterpenic lactone with antioxidant capacity, was obtained from a fresh A.
douglasianaleaf sample compared to a dried sample (56). In another study, Ferreira et al
stated that a higher amount of artemisinin, which has antimalarial activity, was obtained
from indoor air-dried A. annua leaf sample compared to a fresh sample (57). In this
study, the dried leaf sample showed greater cytotoxicity against breast cancer cells. This
result suggests that the cytotoxic agent is neither DhL nor artemisinin.
I 65
4.2 Comparison of ethanol, hexane and water extracts
As indicated in the cytotoxicity assay (Figure 11), ethanol and hexane extracts displayed the best cytotoxicity towards breast cancer cells, with over 90% cancer cell death, whereas the aqueous extract killed approximately 60% of the cancer cells. This
indicated the compound(s) responsible for cancer cell death had intermediate to low polarity, between ethanol and hexane. It could be either that water was too polar to extract the bioactive compound(s) well or that water extracts a second bioactive
compound that is not as active or is not as concentrated. Brengio et al stated that DhL
was insoluble in water (47), so the bioactive compound in the more nonpolar extracts could be DhL, whereas the bioactive compound in water could be a polar DhL derivative
or an unrelated compound. Artemisinin is poorly soluble in both oils and water, but the
artemisinin precursor has also yielded other derivatives such as oil-soluble artemether
and arteether and water-soluble artesunate and artelinic acid (58). Therefore, although it
is unlikely that the bioactive compound is artemisinin, it is possible that the bioactive
compound is related to artemisinin.
4.3 Hexane-soluble, ethanol-soluble fractions and artemisinin
Based on the similar cytotoxicity of the hexane and ethanol extracts, it was
difficult to decide which solvent to use for the initial extraction. The cost of ethanol was
much more convincing and attractive when compared to the price of hexane, especially because a large volume (5 L) was needed for the initial extraction. Secondly, hexane was
volatile and therefore harder to work with than ethanol. Thirdly, mammalian cells can 66 tolerate a small amount of ethanol in the medium, whereas hexane is toxic to cells.
Hence, ethanol was the ideal solvent to start this study.
Five L. of fresh ethanol was used to prepare an extract from 200 g of dried mugwort (A. douglasiana)leaves. After rotary evaporation of the ethanol, the solvent- free extract was extracted with n-hexane. The remaining hexane-insoluble residue was then dissolved in ethanol. Since the initial extraction was done by ethanol, any compounds isolated during the experiment should be soluble in ethanol and be able to be used in cell cytotoxicity assay. Hexane could pull out the less polar compounds from the original extract. The gas chromatograms of both ethanol-soluble and hexane-soluble fractions showed similar results with additional signals at 14.06 min for the hexane- soluble fraction (Figure 12 and 13). Based on its molecular weight (246), the large peak at 15.5 min (in both the ethanol-soluble and hexane-soluble fractions) might be DhL.
Artemisinin standard was also examined by GC-MS (Figures 14). As seen in the three chromatograms (Figures 12, 13, and 14), the retention time of the peak with the highest percent signal in all three fractions was 15.5 min. Although the retention times were alike, it was possible that compounds with similar volatility and polarity could elute within the same time range. In this study, the mass spectra of the peaks eluting at 15.5 min from both ethanol-soluble (Figure 15) and hexane-soluble (Figure 16) fractions appeared to be the same compound, whereas the artemisinin standard (Figure 17) showed a completely different spectrum. This data strongly suggests that artemisinin is absent in both A. douglasiana leaf extracts. 67
4.3.1 EC50
Both MDA-MB-231 and BT-474 breast cancer cell lines exhibited dose- dependent inhibition by A. douglasiana leaf extracts. The hexane-soluble fraction displayed higher cytotoxicty toward both cancer cell lines (Figure 19), but MDA-MB-231 required a higher concentration to achieve this effect (Figure 18 and 19). MDA-MB-23 1, which lacks the estrogen and progesterone receptors, was a faster growing cancer cell line, with a doubling time of 23 hours. BT-474, which expresses the estrogen and progesterone receptors, grew much slower, with a 72-hour doubling time. Both ethanol- soluble and hexane-soluble fractions of A. douglasianahad a greater cytotoxicity (lower
EC 5o) in BT-474 than in MDA-MB-23 1, suggesting that A. douglasianamay have estrogenic activity. This hypothesis is supported by the use of mugwort to treat women's disease in many Native Californian tribes. Interestingly, both hexane-soluble and ethanol-soluble fractions in BT-474 showed a steady slope of growth inhibition whereas the extracts tested in MDA-MB-231 showed an increase of cancer growth at low
concentrations and gradually killed cells at higher concentrations. This may be due to the
faster growth rate of MDA-MB-23 1, as low concentrations of plant extracts would
provide nutrients which allow the cells to grow more rapidly. Another factor to consider
was the time of the cytotoxicity assay; CellTiter was added to the cells after 48 hours of
different testing conditions, so MDA-MB-231 cancer cells would have time to reproduce
themselves twice while BT-474 cells would not even finish one cycle of multiplying. It
is possible that even greater differences in EC5 0 values would be seen if BT-474 cells
were treated with sample for a time corresponding to two doublings. 68
Due to its faster growth rate and reaction time, MDA-MB-23 1 was used for the rest of the experiment. (As mentioned in the Results section, the slower growth of BT-
474 meant that the time necessary for it to react with CellTiter was over twice the time necessary for MDA-MB-23 1 to react.) The possible estrogenic constituents of mugwort were not investigated in this study, but warrant further investigation.
4.3.2 Separations a. Flash column chromatography
Based on TLC results using different solvent systems (Table 2), flash column chromatography was first performed using a step gradient with hexane, hexane:butyl
acetate (1:1), butyl acetate, and ethanol (See Table 1). All column fractions were very
green, probably due to a high concentration of chlorophyll, an unwanted compound. It was difficult to tell if any compounds might have been hidden within the large green spot
on the TLC. Upon analyzing the HPLC data of the hexane-soluble fraction and the active
fractions (3, 5, 6, 9, and 10), it was determined that the ultraviolet signals that
corresponded to biological activity in the different fractions eluted around 7.2, 8.0, 10.1
and 12.1 min, and the charged aerosol signals that corresponded to biological activity
eluted around 7.3, 8.2, and 12.2 min.
The chromatogram for fraction 6 showed the fewest extraneous peaks, other than
those that corresponded to biological activity. Therefore, fraction 6 was further run in a
flash column under conditions described in Table 1. The dark green band continued to
appear throughout all eluate fractions, along with two or more other compounds as 69
indicated by TLC. This method was discarded due to inefficient separation, and no flash
chromatography data were further analyzed. A way to remove the green color from the
sample became the essential element to successfully isolate the active compound. It
might be that, once these goals were achieved, flash chromatography might be useful.
b. Liquid-liquid extraction
Protocols to remove chlorophyll from the sample were based on a previous report
of successful isolation of the natural product taxol, a unique taxane diterpene amide that
possesses anti-tumor and anti-leukemic properties, from the Pacific yew (Taxus
brevifolia). The bark of the Pacific Yew was extracted with methanol and water and then
partitioned with dichloromethane to remove the chlorophyll and other unwanted
compounds (59). In the process, taxol molecules would move from the aqueous
methanol to the more hydrophobic dichloromethane. Although this exact method was not
used in this study, it served as a guide for the extraction scheme.
Dichloromethane is a carcinogen, so it was not a great solvent choice to use in this
study. Acetone, acetonitrile and dimethyl sulfoxide (DMSO) have similar polarity to that
of dichloromethane and are also polar aprotic in nature. In miscibility tests with the
hexane-soluble fraction, acetonitrile and DMSO displayed promising separation results
(Table 3). Due to the high boiling point of DMSO (189 'C), it was discounted, as it was
impractical for rotary evaporation. The hexane-soluble fraction was partitioned with
water, ethanol or acetonitrile, and both raffinate and extractant layer were collected and
tested in the cell cytotoxicity assay (Figure 23). Both ethanol and acetonitrile showed
I 70 great separation of cytotoxic constituents, with cytotoxicty in the extractant layer and no significant activity in the hexane layers.
In a previous study, El Sohly prepared a large scale isolation of artemisinin from
A. annua using liquid-liquid extraction with 20% aqueous acetonitrile (60). This study served as a model to compare the effect of polarity difference on extraction of the cytotoxic constituents. Hence, the hexane-soluble fraction was partitioned with mixtures of ethanol and acetonitrile. (Water was not considered for these mixtures because its boiling point (100 'C) made rotary evaporation difficult.) Two mixtures of ethanol and acetonitrile (3:7 and 1:9) were used to partition the hexane extract. The cytotoxicity assay indicated that extraction with the new mixtures provided weaker cytotoxic effect on the cells, as compared to extraction with acetonitrile alone. Because this can be attributed to a decrease in the desired nonpolar compound(s) entering the extractant layer, it can be concluded that the bioactive compound(s) is (are) intermediate in polarity between hexane and acetonitrile. Based on these results, extraction of the hexane layer with 100% acetonitrile was considered the best choice for further experimentation.
To further examine the effectiveness of partitioning with acetonitrile, the hexane extract was partitioned with acetonitrile, which was then collected and backwashed with hexane. The backwash was performed to remove any compounds that were soluble in both phases and had been carried through the extraction. Chlorophyll is a substance with intermediate polarity, so it could easily have traveled to both hexane and acetonitrile layers. The hexane backwash was a crucial~part of this experiment, as chlorophyll and other unwanted compounds migrated back to the hexane layer. At least three backwashes I
71
were necessary to remove most of the green color from the acetonitrile layer. Further
backwashing yielded light yellow hexane layers and a yellow acetonitrile layer, allowing
the acetonitrile layer to be free of chlorophyll.
The acetonitrile layer was further examined by HPLC; three peaks were detected
by UVD and two peaks were detected by CAD (Figure 26). Three bands were also
observed in TLC, and the distances between the three bands were enough to warrant
further separation with column chromatography. Before running a column, the sample
was evaporated to dryness and dissolved in hexane. The sample was only partially
soluble in the small volume of hexane used, so both the hexane-soluble sample and the
hexane-insoluble sample of the acetonitrile layer were dissolved in ethanol and tested in
the cell cytotoxicity assay and by GC-MS analysis (Figures 28 and 29). Both fractions
showed biological activity. Additionally, according to GC-MS, the same compounds
were present in the two samples, but the peak areas clearly showed that there was a
difference in the quantity of the compounds in the samples. In the hexane-soluble sample
of the acetonitrile layer, the peak at 5 min was relatively intense compared to the same
peak in the hexane-insoluble fraction of the acetonitrile layer. Similarly, the hexane
insoluble fraction of the acetonitrile layer had a larger signal at 14.7 min compared to the
hexane-soluble fraction of the acetonitrile layer. However, it is clear that this method did
not efficiently separate compounds.
Solvent extraction proved to be an effective primary step, so the experiment was
repeated and the bioactive acetonitrile layer was collected. The sample was then
concentrated and separated by flash column chromatography. Two fractions were 72 collected: fraction 1 eluted with acetonitrile and fraction 2 eluted with ethanol. Based on solvent choice, fraction 2 contained the more polar compounds, and it also showed no cytotoxicity against breast cancer cells (Figure 30). The bioactive acetonitrile fraction had three major peaks (7.3, 10.3, and 12.7 min) using HPLC-UVD (Figure 31), and the signal at 10.3 min was exclusively shown on fraction 1 and absent in fraction 2 (Figure
32). Fraction 2 contained the other signals such as that at 7.3 min. Fraction 1 was further examined under NMR (Figure 33) and compared with a DhL standard (Figure 34), and the NMR spectra were similar. These results indicate that DhL might be the compound of interest, but there are also other compounds in the fraction, and these might be the bioactive compound(s). DhL is the most abundant compound in the mixture, so it contributes the most to the NMR spectrum.
Due to the small volume used in the last trial, only 2 column fractions were collected and no gas chromatography could be performed. In this trial, a larger volume of sample was used at every step. The acetonitrile column yielded three distinct fractions, of which only fraction 3 displayed cytotoxicity (Figure 35). In GC analysis, both 10.5 and 15.5 min peaks were present in column fraction 3 (Figure 38). Column fraction 2 also contained a small peak at 15.5 min (DhL), but no peak at 10.5 min was observed (Figure 37). Neither the 10.5 min nor the 15.5 min peak was present in column
fraction 1 (Figure 36). From these results, it can be concluded that the compound corresponding to the peak at 10.5 min was very likely to be responsible for the cytotoxic activity against breast cancer cells and that DhL (the peak at 15.5 min) was not. 73 c. DhL isolation method
With similar NMR spectra from DhL and acetonitrile column fraction 1, DhL might be the compound of interest. Giordiano et al developed a method for isolating
DhL from A. douglasianausing lead tetraacetate and chloroform extraction (49), and the method was modified in this study to better suit our lab conditions. Upon mixing of ethanol-soluble fraction and aqueous lead tetraacetate, the solution was filtered through celite-covered filter paper and partitioned with chloroform three times. Based on the large color differences, the three chloroform wash layers were collected separately and examined by cytotoxicity assays and GC analysis. The three chloroform-layer chromatograms each displayed very distinctive results, but all three layers showed strong cytotoxicity (Figure 39). In GC analysis, the first chloroform layer showed major peaks at 9.1, 10.5 and around 14 -16 min (Figure 41). The second chloroform layer showed major peaks at 10.5, 12 - 13 min, and a much weaker 15 - 16 min group (Figure 42).
The third chloroform layer, which showed the weakest cytotoxicity, had strong 12 - 13 min peaks, a weak 10 -1 1 min peak and a weak 15.4 min group (Figure 43). Three key points can be concluded from this experiment: [1] The cytotoxic activity might be due to the presence of DhL, which could be represented by the peaks at around 15.4 - 15.5 min
in GC, as all three samples contained this peak with abundance corresponding to
cytotoxicity. [2] Following the same logic, the cytotoxicity might also be due to the peak
at 10.5 min. [3] The peaks around 12 - 13 min in both chloroform layer 2 and 3 should
not have any cytotoxicity because chloroform layer 3 had a higher percent of these peaks
present in the sample, and it had the lowest cytotoxicity. I
74
To further confirm that DhL was not the cytotoxic component, flash column
chromatography was used to further separate the compounds in chloroform layer 1.
Since chloroform layer 1 (Figure 41) contained a large concentration of DhL, it is the
ideal sample to determine if DhL has cytotoxic properties. Because the sample was not
soluble in hexane, an intermediate-polarity solvent, ethyl acetate, was used. Of the three
column fractions collected, fractions 1 and 2 clearly displayed strong cytotoxicity against
breast cancer cells (Figure 44). Not only did both samples contain a large amount of the
compound that eluted at 10.5 min in GC, but fraction 2 did not have any peaks located at
15.5 min, indicating that DhL was not responsible for the cytotoxicity of breast cancer
cells (Figures 45 and 46). Both a chloroform control and fraction 3 displayed similar
effects towards the cells (Figure 44), so it could be concluded that fraction 3 had no
cytotoxic activity against breast cancer cells. The results of this experiment further
demonstrated that the compound at 10.5 min was a cytotoxic compound, whereas DhL,
which eluted at 15.5 min, was not responsible for the death of breast cancer cells.
All cytotoxic fractions of A. douglasianahad peaks with similar mass spectra at
10.3 - 10.5 min. Due to the small volume sample obtained from the experiment, the
compound can only be tentatively characterized using the mass spectra obtained from the
GC-MS. From the mass spectra, it is indicated that the structure has at least one double
bond or ring structure, an ether, and a 4-carbon chain. Further mass spectra analysis is
necessary. 75
Chapter 5
CONCLUSIONS
In this study, a cytotoxic compound was successfully isolated from the ethanol extract of Artemisia douglasiana leaf. From the experimental results, the following conclusions can be made:
1. Dried A. douglasianaleafs has greater cytotoxicity compared to fresh leafs.
2. Artemisinin is not present in A. douglasiana.
3. The cytotoxic compound in A. douglasiana has intermediate polarity (which explains
the weaker cytotoxicity of the water extract).
4. A. douglasianamay have estrogenic activity.
5. Chlorophyll removal is an important first step for separation of natural products.
6. Liquid-liquid extraction is a great initial step for separating the chemical components
of A. douglasiana.
7. DhL is not cytotoxic to breast cancer cells.
After a series of trials, it was determined the best method to isolate the cytotoxic compound from A. douglasianawas to mix equal volumes of ethanol extract and 4% aqueous lead tetraacetate together, filter the resulting mixture through a filter paper covered with celite, partition three times with chloroform and perform ethyl acetate flash column chromatography using the chloroform layers from liquid-liquid extraction. One of the column fractions yielded a clean, active compound as assessed by gas chromatography and bioassays. 76
To obtain enough of this compound to analyze, characterize, and identify it, this
isolation method must be scaled up. In a large scale isolation, additional chloroform
partitioning might be necessary to purify the sample extract further before column
chromatography. A thicker and longer column is also another essential factor for scale- up of this chemical isolation. Additional column chromatography might be necessary to
increase the purity of the compound. 77
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