SEASONAL VARIATION OF THE CYTOTOXICITY OF Iva hayesiana ON THE
HUMAN BREAST CANCER CELL LINE MDA-MB-231
A Thesis
Presented to the faculty of the Department of Chemistry
California State University, Sacramento
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF SCIENCE
in
Chemistry
(Biochemistry)
by
Barbara J. Coulombe
SUMMER
2012
© 2012
Barbara J. Coulombe
ALL RIGHTS RESERVED
ii
SEASONAL VARIATION OF THE CYTOTOXICITY OF Iva hayesiana ON THE
HUMAN BREAST CANCER CELL LINE MDA-MB-231
A Thesis
by
Barbara J. Coulombe
Approved by:
_______________________________, Committee Chair
Dr. Mary McCarthy-Hintz
_______________________________, Second Reader
Dr. Katherine McReynolds
_______________________________, Third Reader
Dr. Roy Dixon
___________________________
Date
iii
Student: Barbara J Coulombe
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.
________________________________, Graduate Coordinator
Dr. Susan Crawford
Department of Chemistry
iv
_________________
Date
Abstract
of
SEASONAL VARIATION OF THE CYTOTOXICITY OF Iva hayesiana ON THE
HUMAN BREAST CANCER CELL LINE MDA-MB-231
by
Barbara J. Coulombe
Iva hayesiana, also known as San Diego poverty weed, is a California native
plant that grows in the San Diego coastal region and Baja California. I. hayesiana
contains the flavone hispidulin, which has been shown to be cytotoxic to human
pancreatic cancer cells. A previous screen indicated that an extract of the leaves and
stems of I. hayesiana is cytotoxic to the human breast cancer cell line MDA-MB-231.
However, with repeated extractions over several months, the cytotoxic properties
became negligible. The goal of this study was threefold: first, to optimize the extraction
conditions for I. hayesiana, second, to determine whether the cytotoxicity is seasonal,
and third, to determine whether hispidulin is responsible for the cytotoxicity towards
MDA-MB-231. Results showed that hexane extracts of fresh aerial parts of I.
hayesiana were the most effective against MDA-MB-231, with an IC50 of 56 ± 3
v
µg/mL. Vacuum distillation of the leaves and stems showed that the cytotoxic
constituents are not volatile; therefore, HPLC was used for chromatographic analysis.
Monthly extracts from February thru July showed that the cytotoxicity returned in April
and dissipated in June, when the plant flowered, which supports the hypothesis that the
cytotoxicity is seasonal and is therefore related to the life cycle of the plant. HPLC of
these extracts indicate that there are distinct chemical differences between the active
and inactive extracts. Most notably, there is one HPLC peak of interest that requires
further analysis. Lastly, it was determined that hispidulin is present in I. hayesiana
however; it was not found to be responsible for the cytotoxicity seen towards MDAMB-231. In fact the IC50 of the pure compound dissolved in ethanol was greater than
100 µg/mL, which was the highest concentration tested.
________________________________, Committee Chair
Dr. Mary McCarthy-Hintz
____________________________
Date
vi
ACKNOWLEDGEMENTS
There are many people to thank for helping me to complete this journey.
Although this thesis is the culmination of years of work on my part, there were many
people who directly and indirectly made this project possible. I would like to start by
thanking the Chemistry department here at Sacramento state for allowing me to pursue
this goal. Many of the faculty have had a hand in this project but none more so than my
research advisor Dr Mary McCarthy- Hintz. This project took seven years, with many
drastic changes along the way. Mary challenged me to persevere and encouraged and
supported me when I needed it most. This thesis could not have been completed
without her. I also want to thank my committee members Dr McReynolds and Dr
Dixon for the many hours of editing. Their unrelenting standards made this paper what
it is today.
Next I would like to thank a few of the research students who have shared this
journey with me. The friends I have made here, have made the time I spent in the
Masters Program a joy. Himali Somaweera was a member of my research group and
we frequently worked side by side, though on different projects. Soraya Ghasemiyeh
and I have shared many highs and lows during this process, including the race to our
defense dates, which were two weeks apart. Being able to share the stresses has made
them all more bearable! A special thank to Michelle Watterson who has been my friend
and confidant for years now. She helped me through the thesis writing process even
vii
though she finished several years ago and would have liked to never think about it
again.
My last thank you is the most important of all - to my family. My husband
Mark has put up with many years of late nights and weekends without me. His love and
support have made it possible for me to complete this thesis. My children, Maddy and
Jason who never complained about my absence but were always happy to see me when
I got home. I hope the completion of this project will show you that you can do
anything if you set your mind to it. I won't ever be able to truly express how much I
love you all! I also need to thank my mother, Pat , my grandmother, Regina and my
mother in law Julie for the love and support they gave me. All of you assisted in taking
care of my children in my absence for which I cannot thank you enough.
During the seven years of this journey I have achieved many goals and suffered
many losses. None so great as the loss of my father, Walt, just six months ago. While I
feel elated to have successfully completed my Masters degree, I also feel a little
melancholy that he wasn't here to see it. His loss reminded me that you have to live
every day like it's your last, and now that I am finished that is exactly what I am going
to do.
viii
TABLE OF CONTENTS
Page
Acknowledgements.........................................................................................................vii
List of Tables....................................................................................................................xi
List of Figures.................................................................................................................xii
Chapter
1.
INTRODUCTION......................................................................................................1
1.1 Breast Cancer Statistics..................................................................................1
1.2 What is Breast Cancer?..................................................................................1
1.3 Biomarkers for Breast Cancer........................................................................5
1.4 Breast Cancer Treatments...............................................................................6
1.5 Screening of California Native Plants..........................................................14
1.6 Botany and Taxonomy of Iva hayesiana......................................................16
1.7 Poverty Weed as Herbal Medicine...............................................................18
1.8 Previous Studies of Iva hayesiana and Related Plants.................................18
1.9 Proposal........................................................................................................19
2.
MATERIALS AND METHODS.............................................................................21
2.1 Abbreviations...............................................................................................21
2.2 Materials.......................................................................................................21
2.3 Instruments and Apparatus...........................................................................22
2.4 Extraction Methods......................................................................................22
2.4.1 Initial Extraction............................................................................22
2.4.2 Extraction Solvent Optimization...................................................23
2.4.3 Vacuum Distillation.......................................................................23
2.5 Cytotoxicity Assays......................................................................................24
2.5.1 Media Preparation.........................................................................24
2.5.2 Cell Line and Cell Culture.............................................................24
ix
2.5.3 Cell Viability Assay......................................................................24
2.5.4 Determination of IC50....................................................................26
2.6 HPLC Analysis.............................................................................................27
3.
RESULTS AND DISCUSSION...............................................................................29
3.1 Overview......................................................................................................29
3.2 Initial Plant Screening..................................................................................29
3.3 Determination of Optimum Extraction and Analysis Conditions................31
3.3.1 Extraction Solvent Optimization...................................................31
3.3.2 Determination of Volatility...........................................................32
3.4 IC50 Determination.......................................................................................33
3.5 Large Volume Extraction.............................................................................34
3.6 Investigating the Loss of Cytotoxicity.........................................................36
3.7 Evaluation of Seasonal Cytotoxicity............................................................39
3.8 HPLC Analysis of Monthly Extracts............................................................41
3.9 IC50 for Hispidulin........................................................................................46
3.10 HPLC Analysis of Hispidulin.....................................................................47
4.
CONCLUSIONS......................................................................................................50
5.
FUTURE WORKS...................................................................................................51
Appendix A. HPLC Chromatograms.............................................................................52
References.......................................................................................................................59
x
LIST OF TABLES
Tables
Page
1.
Less common forms of breast cancers and their occurrence rates........................4
2.
TNM staging of breast cancer...............................................................................5
3.
Retention times and peak areas from the HPLC chromatogram of I. hayesiana
extracted in April.................................................................................................42
4.
Evaluation of the 7 peaks of interest in the active extracts.................................45
5.
Re-evaluation of peaks 9 and 13 via HPLC........................................................45
6.
Evaluation of the HPLC retention times of hispidulin........................................49
xi
LIST OF FIGURES
Figures
Page
1.
Anatomy of the female breast................................................................................2
2.
Structure of Taxol..................................................................................................9
3.
Structure of the monoclonal antibody Herceptin.................................................11
4.
Enzyme mediated metabolism of Tamoxifen to Endoxifen................................11
5.
The Function of hormone therapy drugs Tamoxifen and Arimidex....................12
6.
The structure of the aromatase inhibitor Arimidex.............................................12
7.
The structure of Laetrile......................................................................................13
8.
Iva hayesiana.......................................................................................................17
9.
Distribution of Iva hayesiana by county.............................................................17
10.
The structures of the flavones hispidulin and axillarin.......................................18
11.
The cytotoxicity of six native California plants on the human breast cancer cell
line MDA-MB-231..............................................................................................30
12.
The cytotoxicity of four solvent extracts of I. hayesiana leaves and stems on the
human breast cancer cell line MDA-MB-231.....................................................32
13.
The cytotoxicity of extracts from the I. hayesiana distillate, filtered aqueous
suspension and 2-day extract from vacuum distillation on the human breast
cancer cell line MDA-MB-231............................................................................33
14.
IC50 determination for I. hayesiana extracted in hexanes on the human breast
cancer cell line MDA-MB-231............................................................................34
xii
15.
The IC50 determination for I. hayesiana extracted in hexanes (5mg/mL) on the
human breast cancer cell line MDA-MB-231.....................................................36
16.
The IC50 determination for I. hayesiana extracted in hexanes (10 mg/mL) on the
human breast cancer cell line MDA-MB-231.....................................................37
17.
The cytotoxicity of three new solvent extracts from fresh I. hayesiana (65 and
32.5 µg/mL) on the human breast cancer cell line MDA-MB-231.....................38
18.
HPLC investigation of the chemical differences of active and inactive extracts of
I. hayesiana..........................................................................................................39
19.
Results from the cell viability assay of monthly extracts of I. hayesiana
(65 µg/mL) on the human breast cancer cell line MDA-MB-231.......................40
20.
HPLC chromatogram of the April extract of I. hayesiana with the 14 peaks of
interest labeled, after comparison to the blank (ethanol) chromatogram............42
21.
HPLC chromatograms of I. hayesiana extracts from April 28th (red - active) and
Feb 28th (blue - inactive)....................................................................................44
22.
HPLC chromatogram of the April extract of I. hayesiana, diluted to a
concentration of 0.1 mg/mL, with peaks 9 and 13 labeled..................................45
23.
IC50 assay results for hispidulin dissolved in ethanol (10 mg/mL) on the human
breast cancer cell line MDA-MB-231.................................................................47
24.
Evaluation of the presence of hispidulin in I. hayesiana using HPLC................48
xiii
1
Chapter 1
INTRODUCTION
1.1 Breast Cancer Statistics
Breast cancer is a disease that affects approximately 1 out of every 35 women.1
It is the second most lethal cancer behind lung cancer in women. It is estimated that in
2012, 226,870 women will be diagnosed with an invasive form of breast cancer, and
another 63,000 women will be diagnosed with a non-invasive (in situ) form of breast
cancer. Men are also susceptible to breast cancer, and it is predicted that 2,190 will be
diagnosed in 2012. Some 39,510 deaths are expected in 2012 due to breast cancer or its
complications. It has been said that every fifteen minutes, five women will be
diagnosed with breast cancer and one woman will die.2 There are approximately 2.5
million women who are breast cancer survivors in the United State today.1 With
statistics like these, it is not hard to understand why so much research is being done to
find effective treatments, preventative measures, or a cure for this disease. Current
treatments usually involve a combination of surgery and an adjuvant therapy such as
radiation, chemotherapy, hormone therapy or immunotherapy. The types of therapies
used are based on the type and stage of the breast cancer the patient has.
1.2 What is Breast Cancer?
The definition of breast cancer is a malignant growth that inhabits the tissues of
the breast.3 The malignant cells form in the breast tissue when replication goes wrong
2
in one cell and that cell multiplies. When these cells build up, a lump or mass called a
tumor is formed. A mass in the breast can be benign (non-cancerous) or malignant
(cancerous). Benign masses in the breast are common, and are not life threatening,
while malignant masses can be life threatening, depending on the type and stage of the
cancer. Some of these malignant cells can invade the surrounding tissues, and then
spread (metastasize) to other parts of the body such as the lungs, liver and brain. Most
breast cancers form from the cells that line the milk ducts (Figure 1:A) or lobules
(Figure 1:B) of the breast, but a few do form in the fatty or connective tissues (Figure
1:E).4
Breast profile:
A. Ducts
B. Lobules
C. Dilated section of duct to hold milk
D. Nipple
E. Fat
F. Pectoralis major muscle
G. Chest wall/rib cage
I
II
III
Enlargement
I - Normal duct cells
II - Basement membrane
III - Lumen (center of duct)
Figure 1: Anatomy of the female
breast.3
There are fourteen types of breast cancer known to date, but only four are fairly
common. The four most common types of breast cancer are identified by the location
within the breast tissue and the invasiveness of the tumor. These four are: 1) ductal
3
carcinoma in situ (DCIS), 2) lobular carcinoma in situ (LCIS), 3) invasive ductal
carcinoma (IDC), and 4) invasive lobular carcinoma (ILC). Breast cancers are
determined to be invasive when they have infiltrated the surrounding tissues or chest
wall (Figure 1: E, F and G).4 Once infiltration has occurred, the cancer cells can then
metastasize to other parts of the body through the blood or lymph system. DCIS is the
most common form of non-invasive breast cancer. It forms in the ducts of the breast
and has not spread to the surrounding tissue (in situ). While this is the most commonly
diagnosed breast cancer; with one in every five new cases of breast cancer likely to be
DCIS, this form of cancer has a low mortality rate with early detection. LCIS is also a
non-invasive form of breast cancer. It begins in the cells of the lobules, which is where
milk is produced in the breast. This cancer does not usually progress to an invasive
form, but it does indicate a higher risk of developing some form of invasive breast
cancer later. IDC is the most common form of invasive breast cancer. This cancer
begins in the cells of the milk ducts, invades the surrounding tissue and can ultimately
become metastatic. ILC starts in the cells of the lobules of the breast and then
infiltrates the surrounding tissues, and can also become metastatic. There are ten
additional less commonly diagnosed types of breast cancer, which are listed in Table 1
below. The mortality rates of each of the cancers previously mentioned varies
depending on the type and stage (Table 2) of the cancer, as well as how aggressive it
is.4 The progression, or stage, of the cancer is also very important when determining
treatment protocols. Staging is done using a classification system such as the American
Joint Committee on Cancer (AJCC) classification system.
Each of these stages and
4
sub-stages (Table 2) has a different recommended treatment protocol that is dependent
on not only the stage of the cancer, but also on the biomarkers found within the cancer
cells.
Table 1: Less common forms of breast cancers and their occurrence rates.1 Occurrence
rates are based on the percentage of all diagnosed breast cancers. When occurrences are
called rare, it indicates a percentage below 1% that has not been calculated.
Type
Occurrence
Adenocystic Carcinoma
1%
Angiosarcoma
Rare
Inflammatory Breast Cancer
1-3%
Medullary Carcinoma
3-5%
Metaplastic Carcinoma
Rare
Mucinous Carcinoma
Rare
Paget Disease of the Nipple
1%
Papillary Carcinoma
>1-2%
Phyllodes Tumor
Rare
Tubular Carcinoma
2%
5
Table 2: TNM staging of breast cancer. This table correlates the stages of cancer to
tumor size (T), lymph node involvement (N), and metastasis (M), and the five-year
survival rates for each. Tumor classifications range from Tis (in situ) to T4 (tumor size
5 cm or greater). Lymph node involvement ranges from N0 (no involvement) to N3
(multiple lymph node involvement). Metastasis is rated M0 for no metastasis and M1
for metastasis.5
Stage
Tumor (T)
Node (N)
Metastasis (M)
5-Year Relative
Survival Rate3
Stage 0
Tis
N0
M0
100%
Stage I
T1
N0
M0
100%
Stage IIA
T0
N1
M0
92%
T1
N1
M0
T2
N0
M0
T2
N1
M0
T3
N0
M0
T0
N2
M0
T1
N2
M0
T2
N2
M0
T3
N1, N2
M0
T4
Any N
M0
Any T
N3
M0
Any T
Any N
M1
Stage IIB
Stage IIIA
Stage IIIB
Stage IV
81%
67%
54%
20%
1.3 Biomarkers for Breast Cancer
A breast cancer biomarker is a molecule found in the tumor, tissues or fluid of a
tumor, that can be used to predict how well the cancer will respond to a particular
treatment.5 Biomarkers, along with the type and stage of the cancer, are used to map
out a treatment protocol that will give the patient the best outcome possible. There are
currently three biomarkers used to determine the type of adjuvant therapy that will be
6
used for treatment. They are estrogen receptor (ER), progesterone receptor (PR), and
human epidermal growth factor receptor two (HER2). Sixty to eighty percent of all
breast cancer tumors will test positive for overexpression of ER.6 Cells that are positive
for ER and/or PR have receptor proteins that bind estrogen and progesterone,
respectively, in the nuclei of the cells. These cells usually need the respective hormone
to grow, and therefore respond well to treatments that block the binding sites or make
the hormone unavailable. HER2 is a protein found in both normal and cancerous cells.
When HER2 is overexpressed, the cancer is considered HER2+. This protein plays an
important role in cell growth and division, and overexpression causes the cells to grow
and divide more rapidly, therefore these cancers tend to be more aggressive and are
more likely to re-occur. Twenty to forty percent of all breast cancers test positive for
HER2 overexpression.7 A breast cancer can be positive for a combination of these
biomarkers, such as ER+ and PR+, or negative for all three, which is called a triple
negative. Ten to fifteen percent of all breast cancers are triple negative, with a high
occurrence in patients that are positive for the breast cancer 1 gene (BRCA1) mutation.
These cancers are called basal-like because they express basal cell-type cytokeratins
(proteins).8 They tend to be very aggressive and have a poor survival rate. There are
no target-specific therapies for hormone insensitive cancers.9
1.4 Breast Cancer Treatments
Treatment protocols for breast cancers in stages 0 - III are targeted to removal of
the cancer and then prevention of recurrence. This is usually accomplished with a
7
combination of surgery and adjuvant therapies such as radiation, chemotherapy,
hormone therapy, complimentary medicine, and alternative medicine. Surgery is
usually the first step in cancer treatment. The patient can have either a lumpectomy or
mastectomy along with the removal of one or more of the lymph nodes. A lumpectomy
involves the removal of the cancerous mass, while leaving as much of the surrounding
breast tissue as possible. This procedure is usually used for smaller masses and is
coupled with radiation to ensure that all of the cancer cells are eradicated. A simple
mastectomy is the complete removal of all of the breast tissue, and, except in later
stages, radiation is not needed. A lymph node close to the tumor is removed (sentinel
dissection) and checked for cancerous cells. If the node is positive, additional nodes in
the armpit are removed and tested as well (axillary dissection). There is a risk of
bleeding or infection with surgery, and arm swelling with lymph node removal. The
tumor size and lymph node involvement are determined with surgery, and then used to
stage the cancer for further treatment. The excised mass is tested for biomarkers and
aggressiveness to give a complete picture.4
Radiation therapy is usually recommended if the tumor is five centimeters or
larger, there is chest wall infiltration or multiple lymph node involvement, or the patient
has had a lumpectomy. Radiation is also used for inflammatory breast cancer. There
are two types of radiation therapy - external radiation and internal radiation. External
radiation is delivered to the breast from outside of the body. Internal radiation uses
radioactive seeds that are implanted in the affected breast tissue. While radiation is
effective at reducing the risk of recurrence, it is not without issues. The treatment takes
8
six to seven weeks to complete, and treatments occur daily. Patients may experience
fatigue and skin irritation, and there is an increased risk of secondary cancers, as well as
lung or heart damage. If radiation is recommended, treatment usually begins three to
six weeks after surgery. If chemotherapy is recommended in addition to radiation, it is
usually completed before radiation begins.
Chemotherapy drugs ("chemo") are used to kill fast-growing cells. Chemo is
usually recommended for patients with cancers that have metastasized, are re-occurring,
or are likely to re-occur. The treatments can last from three to six months in one to
three week cycles. The schedule usually includes breaks in treatment to allow for
recovery time. Chemotherapy is very effective at killing fast growing cancer cells, but
cancer cells are not the only fast growing cells in the body. Hair follicles and nails are
examples of other fast growing cells that are killed, which is why many patients who
undergo chemo lose their hair.3 Paclitaxel (Taxol) is one of many chemo drugs
currently in use. It comes from a class of drugs called taxanes, which disrupt cell
division. Taxol (Figure 2A) was originally isolated from the bark of the pacific yew
tree (Taxus brevifolia) in 1971.10 The Pacific yew tree is a very slow growing tree that
produces very little taxol. Today, the main source of taxol is a semisynthesis from
precursors such as baccatin III (Figure 2B), which is found in the needles of the yew
tree. The side effects from this drug include nausea, vomiting, diarrhea, decreased
blood counts, muscle aches, and hair loss.
9
A
B
Figure 2: Structure of Taxol11 (A), and the precursor baccatin III12 (B) used in the semisynthesis of Taxol.
Another form of adjuvant treatment for breast cancer is hormone therapy.
Hormone therapy is used for breast cancers that have a positive biomarker for HER2,
ER, or PR. Cancers that are ER+, PR+, or ER+/PR+ use the body's estrogen and/or
progesterone to promote cell growth. To interfere with the cell cycle of the cancer,
drugs are given to block either the production of these hormones or the binding of these
hormones to the receptor. Either strategy slows the growth of the cancer, as well as
reduces the possibility of recurrence. For cancers that are HER2 positive, there is a
targeted form of hormone therapy which specifically affects cancer cells. The drug
trastuzumab (Herceptin; Figure 3) is a monoclonal antibody produced by recombinant
DNA technology.13 Herceptin binds to the HER2 receptor on the surface of the cancer
cell and blocks the signals for cell growth and division. This drug is frequently given in
combination with taxol. Side effects include headache, nausea, vomiting, weakness,
10
breathing difficulty, and skin rashes. There is also an increased risk of congestive heart
failure. Herceptin only works for cancers that are HER2+. Other targeted hormone
therapy drugs are available for cancers that are ER+ and/or PR+. Tamoxifen (Figure 4)
is one of the most commonly used hormone therapy drugs when a cancer is ER+. It is a
selective estrogen receptor modulator (SERM) that interferes with the estrogen
receptors in the cancer cells (Figure 5A). In the body, tamoxifen requires the action of
the cytochrome P450 enzyme family. Specifically, cytochrome CYP-2D6 and
cytochrome CYP3A4/5 metabolize tamoxifen to endoxifen (Figure 4). Endoxifen has
greater binding affinity for the estrogen receptor and is considered the activated form of
tamoxifen. Both cytochromes occur naturally in the human body; however, some
patients have flawed versions or take a medication that interferes with their function.14
When this occurs, tamoxifen is no longer effective and an aromatase inhibitor may be
used instead.
Aromatase inhibitors (AI) such as anastrozole (Arimidex; Figure 5B, and 6),
block the enzyme aromatase, which converts androgens into estrogens.15 While AI's
reduce the amount of estrogen the body produces, they do not completely stop estrogen
production in the ovaries; therefore, there is significant debate about their effectiveness
in premenopausal women. The course of treatment for both types of hormone therapy
lasts up to five years. The side effects for tamoxifen are hot flashes, increased risk of
cataracts, blood clots, uterine cancer, and osteoporosis. AI are a fairly new breast
cancer therapy, so long term side effects are still not known. Short-term side effects
11
include hot flashes, joint pain, muscle aches, vaginal dryness, and increased risk of
osteoporosis.
Figure 3 : Structure of the monoclonal antibody Herceptin.13
O
N
CH3
O
O
N
CH3
CH3
CH3
CYP2D6
CYP3A4/5
OH
Tamoxifen
4-hydroxytamoxifen
Figure 4: Enzyme mediated metabolism of Tamoxifen to Endoxifen.14
H
N
CH3
OH
Endoxifen
12
A
B
Figure 5: The Function of hormone therapy drugs Tamoxifen and Arimidex. A.
Endoxifen binds to an estrogen receptor. This path leads to the inhibition of gene
transcription. B. Arimidex blocks the final step in the production of estradiol. The
estrogen receptor remains unbound and transcription cannot occur.
Figure 6: The structure of the aromatase inhibitor Arimidex.16
A final class of treatments for breast cancer falls outside of conventional
medicine. Complementary and alternative medicine (CAM) is practiced by providers
outside of the standard medical community. Some examples of CAM providers are
chiropractors, herbalists, and acupuncturists. Complimentary medicine consists of nonconventional treatments that are used along with traditional medicine. These treatments
13
are usually used to treat the side effects caused by conventional medicines such as
chemo. Today there are many medical professionals who offer a combination of
conventional and complimentary (integrated) treatments. Alternative medicine is nontraditional medicine that is used instead of standard medicine. As such, alternative
medicine is generally frowned upon within the standard medical community in the
United States. Many standard medical treatments and practices currently in use were
once considered CAM. The use of acupuncture to treat nausea from chemo is a good
example of this: With years of scientific proof of the effectiveness of this treatment, it
has become an accepted practice in standard medicine.17 Other treatments used as
alternative medicine, such as laetrile, were believed to have anti-cancer properties, but
were proven to be ineffective and even harmful. Laetrile (Figure 7) is a chemically
altered form of amygdalin, which is a glycoside found in the fruit pits and raw nuts.
Laetrile was patented in the US in the 1950’s for cancer treatment. Testing later proved
that laetrile had no anticancer properties, and that the drug was potentially lethal. An
enzyme in the small intestines of humans catalyzes the release of cyanide from laetrile,
causing cyanide poisoning.18 There are many natural products used today that are
believed to have anticancer properties that still need to be tested.
Figure 7: The structure of Laetrile.19
14
While there are many chemotherapeutic drugs in use today, serious side effects
are a problem. Some of the side effects are nausea, vomiting, hair loss, blood clots,
lung and heart damage, congestive heart failure and even secondary cancers. Targeted
therapies can limit some side effects, but there are cancers, such as ER-, that do not
respond well to the current treatments available. There are also treatments that become
ineffective with prolonged use. The side effects and sometimes ineffectiveness of
current treatments indicate the need for novel therapies effective against breast cancer.
In the search for new chemotherapies, plants are proving to be an excellent source for
natural therapies that may have less adverse side effects. Thus, there is a renewed
interest in investigating plant natural products that might be used against breast cancer.
1.5 Screening of California Native Plants
For centuries, plants have been used for medicinal purposes by different cultures.
In the search for new anti-cancer compounds, these plants represent a vastly underinvestigated resource. There are several different ways to identify plant targets for
breast cancer research.
One method targets plants that are used in traditional
indigenous or folk medicine against cancer. One such plant is Vernonia amygdalina,
which is used to treat cancer in South Africa. Gresham et al. tested the plant for
anticancer activity and found it very effective against ER+ breast cancers.20 A second
method targets plants used as traditional medicines, but not necessarily for cancer.
Plants that are used to kill fast-growing organisms such as bacteria or helminthes are
possible targets for killing fast growing cancer cells. One such study completed by
15
Reddy et al. found that Hedychium spicatum, which is used medicinally to treat
stomach ailments and other disorders, had good cytotoxic effects against breast, lung,
colon and skin cancer.21 A third method is random screening of for cytotoxicity. This
method is usually used by pharmaceutical companies and research institutes. An
example of this is a study completed by the National Cancer Institute, in which seventyfive hundred South African plants were screened; fifty showed moderate anticancer
activity.22 Lastly, screening multiple plants for anticancer activity can also be done in a
more targeted fashion. Ramirez-Erosa et al. screened fifteen plants from the Asteraceae
family, some members of which are known for anticancer activity, with very positive
results. Out of fifteen plants tested, five were very toxic to ER- breast cancer cells.23
The McCarthy research group has also had considerable success with targeted
screening.
The McCarthy group has screened over 75 plants for anticancer activity in a
targeted manner. The area of focus for this group is California native and naturalized
plants that are, or were, used medicinally (in traditional, folk, or modern herbal
medicine). Chris Hobbs performed most of the initial research and screening. Of all of
the plants screened, six showed moderate to good cytotoxicity against triple negative
breast cancer cell line MDA-MB-231: yellow star thistle (Centaurea solstitialis), yellow
pond lily (Nuphar luteum), Indian hemp (Apocynum cannabinum), prickly pear cactus
(Opunita ficus-indica), American dogwood (Cornus sericea) and San Diego poverty
weed (Iva hayesiana). In the current study, a cytotoxicity assay was performed using
water extracts of the medicinal parts of these six plants, as well as ethanol extracts of C.
16
solstitialis, O. ficus-indica, N. luteum. The aqueous extracts of I. hayesiana roots and
C. sericea, N. luteum, C. solstitialis, and O. ficus-indica, and the ethanol extract of C.
solstitialis were not cytotoxic against the triple negative-cancer cell line MDA-MB-231.
However, results of this screening confirmed the previous results that showed
cytotoxicity for aqueous extracts of I. hayesiana leaves and A. cannabinum, and the
ethanol extract of N. luteum. Of these, the cytotoxic compounds in A. cannabinum24,
and N. luteum25 have already been isolated and identified. Only I. hayesiana remains to
be investigated.
1.6 Botany and Taxonomy of Iva hayesiana
Iva hayesiana (Figure 8), also known as San Diego poverty weed, belongs to
the family Asteraceae, the genus Iva, and the tribe Iveae.26 There are 27 species in the
genus Iva throughout the United States, Mexico and British Columbia. Common plant
names for the Iva genus include Jesuit's bark, copperweed, marshelder, poverty weed,
and sumpweed. I. hayesiana is a fast growing evergreen shrub that grows primarily in
the San Diego coastal region as well as in Baja California (Figure 9). The plant prefers
an alkaline environment below an elevation of 1000 feet. It is highly adaptive and has
become a popular plant for ground cover and erosion control. The shrub flowers from
April to September with very small green flowers which are nearly invisible. The plant
has a strong odor due to a sesquiterpene lactone (exact structure unknown), which deer
and other herbivores do not like.27 The plant also contains two flavones, which were
17
identified as hispidulin (Figure 10A) and axillarin (Figure 10B), both of which have
been identified in other plant species in the Asteraceae family.28
Figure 8: Iva hayesiana29
Figure 9: Distribution of I. hayesiana
by county.30
18
A
B
Figure 10: The structures of the flavones hispidulin31 (A) and axillarin32 (B).
1.7 Poverty Weed as Herbal Medicine
Plants within the Asteraceae family have been shown to have many medicinal
uses. Native American cultures use or used plants from the Asteraceaea family to treat
ailments such as stomach and intestinal ailments, skin problems, female problems and
cancers. Within the Iva genus specifically, plants such as I. axillaris, which is a close
relative of I. hayesiana, were used by the Shoshone Indians to treat stomach aches,
cramps, diarrhea, and colds. The Paiute used it for sores, rashes and itching, and the
Mahuna used it as an abortifacient and for birth control.33 The flavone hispidulin,
which can be found in I. hayesiana, as well as other plants in the Asteraceae family, is
also used in Chinese medicine to treat inflammatory diseases.34
1.8 Previous Studies of Iva hayesiana and Related Plants
To date, very little research has been documented regarding I. hayesiana. The
flavones hispidulin and axillarin were both isolated from I. hayesiana and identified by
Herz et al. in 1969.28 There was also a sesquiterpene lactone found that has not been
19
completely characterized due to its polymerization. Axillarin was originally isolated
from I. axillaris in 1966. The flavone hispidulin was originally isolated from Ambrosia
hispida and identified in 1964. Since then it has been found in other plants within the
Asteraceae family. Hispidulin extracted from Artemisia vesitita has been shown to be
effective in killing pancreatic cancer cells as well as ovarian cancer cells. The fact that
one of the chemical constituents of I. hayesiana has shown cytotoxicity against several
types of cancer cells is promising.
1.9 Proposal
In the search for new chemotherapies for breast cancer, plants are proving to be
an excellent source for natural therapies, as they may have less adverse side effects.
Although no documentation was found that I. hayesiana was used for medicinal
purposes, it does contain hispidulin, which has been found to have anticancer
properties. I. hayesiana was originally screened with ten other plants for cytotoxicity
against the human breast cancer cell line MDA-MB-231 for the McCarthy group by
Chris Hobbs in 2004. Based on those positive results, the six plants that showed
moderate to good cytotoxicity were re-tested to choose the best target. The results of
both screenings coupled with the lack of previous research on I. hayesiana made it a
good candidate for study. The presence of a chemical constituent (hispidulin) known to
be cytotoxic to pancreatic cancer also makes I. hayesiana a good target. The original
objective of this thesis was to identify the cytotoxic constituents of I. hayesiana. While
the initial extracts of I. hayesiana showed anticancer activity, some subsequent extracts
20
showed no activity. Upon review of the data, it was noted that the inactive extracts
were obtained from leaves harvested at different times of the year than the initial active
extracts. Due to these findings, the objectives of this thesis were changed. The new
objectives are threefold 1) to determine the best extraction method for future extractions
of I. hayesiana, 2) to determine whether the cytotoxic constituent(s) of I. hayesiana are
seasonal and 3) to determine whether hispidulin is responsible for the cytotoxicity of I.
hayesiana on the human breast cancer cell line MDA-MB-231. Therefore, the
hypothesis for this study is that the cytotoxicity of I. hayesiana is related to seasonal
variation in the chemical constituents of the plant.
21
Chapter 2
Materials and Methods
2.1 Abbreviations
[4-(2-hydroxyethyl)-1-piperazine]ethanesulfonic acid, (HEPES); Reverse phase
high performance liquid chromatography, (RP- HPLC); Electrical Aerosol Size
Analyzer, (EAA).
2.2 Materials
Three I. hayesiana plants were purchased from Village Nursery in Sacramento,
California. The plants were shipped in from the nursery's San Diego, California,
location. Plants were then grown in the greenhouse located at California State
University, Sacramento. Sterile flat bottom tissue culture treated 96 well plates with
360 µL well volume (Corning; Corning, NY), Minimum Essential Media (IMEM) with
zinc option containing 2 mg/L L-glutamine, 2 mg L-proline, 50.0 µg/mL L gentamicin
sulfate (Mediatech, Inc; Herndon, VI), heat inactivated fetal bovine serum (Anexia
Biologix; Dixon, CA), trypsin-EDTA (Mediatech, Inc; Herndon, VI) and CellTiter 96*
AQueous One Solution Cell Proliferation Assay (Promega; Madison, WI) were used for
all cytotoxicity and IC50 assays. All other reagents were purchased through Fisher
Scientific or VWR.
22
2.3 Instruments and Apparatus
A Labconco (Kansas City, MO) class IIA biosafety hood was used for all cell
culture procedures. A Du Pont Instruments (Cincinnati, OH) Sorvall GLC-4B general
laboratory centrifuge was used for centrifugation. Cells were kept in an IR Auto Flow
Incubator manufactured by Nuaire (Plymouth, CA). A BioRad (Hercules, CA)
microtiter plate reader, model 680, was used for all cytotoxicity assays.
Samples were analyzed using an Agilent (Santa Clara, CA) 1100 HPLC
equipped with a binary pump operated using Chem Station software. The system is also
equipped with a variable wavelength Agilent UV detector in series with a custom built
in-house charged aerosol detector (CAD). The CAD uses the same basic principles as
in instruments described by Dixon and Peterson and Gamache et al.35-36 However, it
was modified to use nebulization to cause particle charging and is described in more
detail by Abhyankar.37 The column used was a Phenomenex (Torrance, CA) C12
Synergi 4 µm diameter MAX-RP 80A with the dimensions 150 x 4.60 mm.
2.4 Extraction Methods
2.4.1 Initial Extraction
All glassware was cleaned in an alcoholic sodium hydroxide bath (10% sodium
hydroxide (w/v) in isopropanol ) and rinsed with DI water, until pH of the water was
neutral, prior to use. One gram of fresh I. hayesiana leaves and stems were ground in a
blender and extracted with 25 mL of hexanes (ACS grade). Extraction was performed
at room temperature with stirring over a two day period. The hexane extract was first
23
filtered with a large Buchner funnel and 9.0 cm qualitative grade 1 filter paper
(Whatman) to remove large particulate matter. The extract was then filtered with a 0.45
µm sterilizing polyethersulfone (PES) bench top filter (150 mL). Extracts were next
concentrated using a rotary evaporator (Sorvall). The residue was dissolved in ethanol
using a volume calculated to bring the sample concentration to 10.0 mg/mL. All
extracts were then stored at -20 ºC.
2.4.2 Extraction Solvent Optimization
To determine the best solvent for extracting I. hayesiana, a series of solvents
varying from non-polar to polar were tested. The solvents used were water, diethylether
(ethylether anhydrous, ACS grade), hexanes (ACS grade) and ethanol (ACS grade).
Each solvent was used to extract both a fresh sample of I. hayesiana leaves and stems,
as well as a dried sample using 1 g of plant material and 25mL of solvent. For dried
samples, Iva leaves and stems were collected and dried at room temperature for 3-4
days. All extracts were performed at room temperature with stirring over a two day
period. Samples were then filtered and stored as described previously.
2.4.3 Vacuum Distillation
Vacuum distillation was performed using standard vacuum distillation
apparatus. One gram of fresh whole I. hayesiana leaves and stems was placed in a
round bottomed flask (100 mL) with 5 mL of water. The distillate was collected in a
dry flask kept at approximately 0ºC. The I. hayesiana leaves and stems began to distill
24
at 40ºC and continued until the temperature reached 60ºC. At 60ºC the temperature
began to drop rapidly. The distillate was extracted three times using equal volumes of
hexanes and dried using a rotary evaporator. The leftover aqueous suspension was
further extracted with hexanes at room temperature with stirring for 48 hours. This 2
day extract extract was finally filtered and dried using a rotary evaporator and stored as
described previously.
2.5 Cytotoxicity Assays
2.5.1 Media Preparation
Growth media was prepared with the addition of 50 mL of fetal bovine serum, 5
mL of 1M HEPES and 5 mL of penicillin/streptomycin to 500mL of IMEM-zinc option.
2.5.2 Cell Line and Cell Culture
Human breast cancer cells from the cell line MDA-MB-231 were purchased
from American Type Culture Corporation (ATCC) Rockville, Maryland. The cells
were grown in growth media in T-25 and T-75 cell culture flasks. Cells were cultured
for 48 hours at 37ºC under a 100% humidified 95%:5% mixture of air and CO2.
2.5.3 Cell Viability Assay
A cell viability assay was used to assess the cytotoxicity of all initial extracts on
the human breast cancer cell line MDA-MB-231. The plant extracts tested were
25
C. solstitialis, N. luteum, A. cannabinum, O. ficus-indica, C. sericea and I. hayesiana.
These extracts tested resulted from the initial plant screening, solvent optimization and
vacuum distillation. Using sterile technique, MDA-MB-231 cells were detached from a
T-25 flask by incubating with trypsin:EDTA (0.05%:0.2%). The cells were washed
down and collected with 5 mL of warm media and transferred to a 10 mL Falcon tube.
The cells were then pelleted by centrifugation at 1409 X g. The supernatant was
removed and replaced with 5 mL of fresh media and the pellet was resuspended with
vortexing. Each well of a 96 well plate was inoculated with 100 µL of cell suspension
with a cell density of 10,000 cells/mL. The plate was incubated for 48 hours at 37ºC
under a 100% humidified 95%:5% mixture of air and CO2. Extracts of interest were
normalized at a concentration of 10 mg/mL, and were prepared using 1987 µL of
growth media and 13 µL of sample to create a working concentration of 65µg/mL. The
negative control contained 13 µL of 95% ethanol and 1987 µL of growth media. The
media was aspirated from a set of ten wells (ten replicates) designated for the test, and
aliquots of 100 µL from each test sample were added to each well. After 48 hours of
incubation, the media was aspirated from wells and replaced with 100 µL of fresh
growth media and 20 µL of CellTiter. A blank row also received 100 µL of fresh
growth media and 20 µL CellTiter. The plate was incubated for 1-2 hours at 37ºC, then
the relative number of cells was quantitated using a microplate reader measuring the
absorbance at 490nm. Cell viability was expressed as % of control, which was
calculated from the following formula (Equation 1). Cell viability was then plotted to
26
compare the effectiveness of various extracts. The standard deviation was next
calculated using Equation 2.
Equation 1
𝑚𝑒𝑎𝑛𝑂𝐷
Cell Viability (% Control) = (𝑚𝑒𝑎𝑛𝑂𝐷 𝑠𝑎𝑚𝑝𝑙𝑒 ) x 100%
𝑐𝑜𝑛𝑡𝑟𝑜𝑙
𝑀𝑒𝑎𝑛 𝑂𝐷𝑠𝑎𝑚𝑝𝑙𝑒 = Mean Absorbance of 10 sample wells at 490 nm
𝑀𝑒𝑎𝑛 𝑂𝐷𝑐𝑜𝑛𝑡𝑟𝑜𝑙 = Mean Absorbance of 10 control wells at 490nm
Equation 2
Standard Deviation = (% 𝑜𝑓 𝐶𝑜𝑛𝑡𝑟𝑜𝑙) ∗ (((
𝑠𝑡𝑑 𝑑𝑒𝑣𝑂𝐷𝑠𝑎𝑚𝑝𝑙𝑒 2
𝑚𝑒𝑎𝑛 𝑂𝐷𝑠𝑎𝑚𝑝𝑙𝑒
) ) + ((
𝑠𝑡𝑑 𝑑𝑒𝑣𝑂𝐷𝑐𝑜𝑛𝑡𝑟𝑜𝑙 2
𝑚𝑒𝑎𝑛𝑂𝐷𝑐𝑜𝑛𝑡𝑟𝑜𝑙
0.5
) ))
2.5.4 Determination of IC50
Using sterile technique, MDA-MB-231 cells were plated using the same
protocol as the cell viability assay. Plates were incubated for 48 hours at 37ºC under a
100% humidified 95%:5% mixture of air and CO2. Working solutions of six
concentrations (100, 80, 60 40, 20, 0 µg/mL) for each extract were prepared as follows:
100 µL stock extract in 9.9 mL media, 80 µL extract/20 µL ethanol in 9.9 mL media, 60
µL extract/40 µL ethanol in 9.9mL media, 40 µL extract/60 µL ethanol in 9.9 mL
media, 20 µL extract/80 µL ethanol. A negative control using 100 µL ethanol in 9.9
mL media was also prepared. An aliquot of 100 µL of each working solution was
added to ten designated replicate wells. The plate was then incubated for another 48
hours at 37ºC under a 100% humidified 95%:5% mixture of air and CO2. The Celltiter
27
procedure and percent control calculations (Equation 1) were completed using the
previously defined protocols. Data was graphed in a scatter plot with percent control
vs. concentration with error bars to represent the standard deviation (Equation 2). The
absorbance data for each of the ten replicate measurements per concentration was then
used to calculate the half maximal effective concentration (IC50) using the ED50Plus
v1.0 Excel worksheet developed by Dr. Mario H. Vargas at Instituto Nacional de
Enfermedades Respiratorias.38
2.6 HPLC Analysis
An Agilent 1100 HPLC was used to analyze the chemical constituents present in
active and inactive extracts. All extracts were diluted in 95% ethanol to a concentration
of 1 mg/mL and filtered using a 0.22 µm sterile syringe filter (13mm, Fisherbrand) prior
to each run. The injection volume was 5 µl and flow rate was 1 mL/min. The oven
temperature was set at 30ºC and the UV detection wavelength was 210 nm. The EAA
voltage was set to -0.150 mV. The mobile phase was a binary gradient consisting of
nanopure water and acetonitrile. The starting solvent gradient was a 90:10 ratio of
water to acetonitrile, which was isocratic for the first five minutes. The gradient then
ramped up linearly from 90:10 to a 0:100 ratio of water to acetonitrile over the next five
minutes. Following this, the gradient was isocratic at 0:100 for ten minutes, ending
with a linear return to 90:10 over the next six minutes. The total run time for each
sample was 26 minutes. Chromatograms of the active extracts were overlayed with the
chromatograms of the inactive extracts using the Chem Station software. These
28
overlays were evaluated to compare peaks and retention times between the active and
inactive fractions.
29
Chapter 3
Results and Discussion
3.1 Overview
The goals of the work presented in this thesis was to determine if I. hayesiana is
cytotoxic to the human breast cancer cell line MDA-MB-231 and to identify the
chemical constituents responsible for the cytotoxicity. These goals were derived from
the fact that many of the plants in the Asteraceae family, of which Iva is a member,
have been used medicinally for centuries. Furthermore, I. hayesiana contains the
flavone hispidulin, which is known to be cytotoxic to pancreatic cancer cells. As with
all research, this study morphed over the life of the project and the goals changed. As
work progressed towards this goal, the cytotoxicity of the extracts disappeared and the
focus shifted to determining the cause. This study showed that the cytotoxic
compounds in I. hayesiana are seasonal and related to seasonal variation in the chemical
constituents of the plant.
3.2 Initial Plant Screening
The extracts of six plants collected and processed by Chris Hobbs in 2004, C.
solstitialis, N. luteum, A. cannabinum, O. ficus-indica, C. sericea and I. hayesiana, were
screened for cytotoxicity against the ER- cell line MDA-MB-231 using the procedure
described in Materials and Methods. The results showed that three plants killed more
30
than 50% of the breast cancer cells (Figure 11). These plants are: I. hayesiana, A.
cannabinum, and N. luteum. A literature search indicated that the cytotoxic compounds
in both A. cannabinum and N. luteum had been previously isolated and identified.24-25
Releative Number of Cells (% of Control)
Therefore, I. hayesiana was chosen for this study.
350
300
250
200
150
100
50
0
Plant Species
Figure 11: The cytotoxicity of six native California plants on human breast cancer cell
line MDA-MB-231. The bars represent the percentage of cells alive compared to the
control (cells treated with vehicle only). All extracts used were prepared by Chris
Hobbs in 2004. Samples 1-7 were extracted in water and 8-10 in ethanol. Error Bars
represent one standard deviation from the average of ten samples.
31
3.3 Determination of Optimum Extraction and Analysis Conditions
3.3.1 Extraction Solvent Optimization
To determine the optimum extraction method, a series of solvents was used to
extract both fresh and dry samples of I. hayesiana leaves and stems. The solvents used
were water, ethanol (ACS grade), diethyl ether (ethylether anhydrous, ACS grade), and
hexanes (ACS grade). All extracts were then tested against human breast cancer cell
line MDA-MB-231 using a cell viability assay (Figure 12). The aqueous extracts of
fresh and dry I. hayesiana leaves and stems were not cytotoxic. The ethanol extracts of
the dry and fresh samples were somewhat cytotoxic. The most cytotoxicity occurred
with both the fresh and dry samples extracted in diethyl ether and hexanes. Due to the
fact that the cytotoxicity was so poor in the more polar solvents, coupled with the strong
cytotoxicity in hexanes, along with the fact that hexanes are easier to work with, it was
determined that hexanes would be the best solvent for all future extracts.
Relative Number of Cells
(% control)
32
160
140
120
100
80
60
40
20
0
-20
Solvent
Figure 12: The cytotoxicity of four solvent extracts of I. hayesiana leaves and stems
(65 µg/mL) on the human breast cancer cell line MDA-MB-231. The bars represent the
percentage of cells alive compared to the control (cells treated with vehicle only). The
error bars represent one standard deviation from the average of ten samples.
3.3.2 Determination of Volatility
To determine the volatility of the cytotoxic compound(s) found in I. hayesiana,
an aqueous vacuum distillation was performed. Using liquid – liquid extraction, the
distillate was extracted into hexanes and then concentrated to a concentration of 10 mg/
mL. The remaining water from the aqueous suspension was poured off and filtered for
further testing, and the remaining plant material from the aqueous extract was then
extracted with hexanes using the initial extraction method discussed in Materials and
Methods. These extracts, along with the (filtered) water from the aqueous suspension,
were then tested for cytotoxicity against the human breast cancer cell line MDA-MB231 using the cell viability assay. The results from the assay showed that all of the
33
cytotoxicity existed in the aqueous suspension as well as the 2 day extract of the
aqueous suspension (Figure 13). The distillate killed approximately 15% of the cells,
compared to over 70% cell death from the aqueous suspension and the 2-day extract.
These results indicate that the compounds of interest are not volatile. Therefore, all
future extracts were performed using the initial extraction method, and HPLC (rather
Relative # of Cells (% Control)
than gas chromatography) was used for all future chromatographic analysies.
140
120
100
80
60
40
20
0
I. hayesiana Distillate
Aqueous Suspension 2-Day Hexane Extract
of Aqueous Suspension
Sample
Figure 13: The cytotoxicity of extracts from the I. hayesiana distillate, filtered aqueous
suspension and 2-day extract from vacuum distillation on the human breast cancer cell
line MDA-MB-231. The bars represent the percentage of cells alive compared to the
control (cells treated with vehicle only). The error bars represent one standard deviation
from the average of ten samples.
3.4 IC50 Determination
IC50 stands for the half maximal inhibitory concentration, which in this case
means the concentration which causes half the cells to die. This method is commonly
34
used to determine the potency of a drug. To determine the best concentration for future
cell viability assays, an IC50 assay was completed on I. hayesiana. The IC50
concentration of the hexane extract on the human breast cancer cell line MDA-MB-231
Relative Number of Cells
(% Control)
was calculated to be 56 ± 3 µg/mL (Figure 14).
170
150
130
110
90
70
50
30
10
-10 0
20
40
60
80
100
Concentration (µg/mL)
Figure 14: IC50 determination for I. hayesiana extracted in hexanes on the human
breast cancer cell line MDA-MB-231. The error bars represent one standard deviation
from the average of ten samples.
All work to this point was completed to address the optimization of future extractions
and assays. From here, a large scale extraction was completed to increase the amount
of available extract for analysis.
3.5 Large Volume Extraction
To increase the volume of extract available for further analysis, a new extract
was prepared using 5 g of chopped fresh I. hayesiana leaves, stems and flowers in 125
35
mL of hexanes (ACS grade), as described in the Materials and Methods section. The
extract was brought to a concentration of 5 mg/mL in ethanol and then diluted to
working concentrations of 50, 40, 30, 20, and 10 µg/mL in growth media using the IC50
protocol. The concentration change from 10 mg/mL to 5 mg/mL was completed to try
to work in a concentration range closer to the calculated IC50 value. The working
solutions were then used in an IC50 assay to inoculate the human breast cancer cell line
MDA-MB-231. A solution consisting of growth media and 100 µL of ethanol served as
the control. The IC50 results were plotted to determine the potency of the extract
(Figure 15). The results showed almost no activity. At the highest concentration
(50 µg/mL) the cytotoxicity was approximately 21%. At concentrations below 50
µg/mL, the cells actually thrived, with cell growth above 120% of the control cells.
Due to the lack of activity, the IC50 value for this assay was not calculated. It was
noted that there were three obvious differences between the initial extract and the new
extract: (1) Flowers were used in addition to the leaves and stems used in the first
extraction. (2) The hexane extract used in the first solvent screening was extracted from
a plant harvest in September of 2010, whereas the scaled-up extraction used new
material from the same plant, but harvested in November of 2010. (3) There was also
an oily residue in the flask after rotary evaporation of the sample to remove the hexanes.
Based on the first difference, a new extraction was performed using only the leaves and
stems. This new extract was tested with a cell viability assay, and it also showed no
activity (data not shown). This process of completing new extractions and performing
36
cell viability assays was completed numerous times before it was determined that
Relative Number of Cells
(% Control)
something within I. hayesiana had changed as evidenced by a lack of cytotoxicity.
140
120
100
80
60
40
20
0
0
10
20
30
40
50
60
Concentration µg/mL
Figure 15: The IC50 determination for I. hayesiana extracted in hexanes (5 mg/mL) on
the human breast cancer cell line MDA-MB-231. The error bars represent one standard
deviation from the average of ten samples.
3.6 Investigating the Loss of Cytotoxicity
To rule out a concentration issue, the initial extraction volume and methods
were used to create a new extract. This extract was brought to a stock concentration of
10 mg/mL in ethanol and then diluted to working concentrations of 100, 80, 60, 40 and
20 µg/mL in growth media using the IC50 protocol. A solution consisting of growth
media and 100 µL of ethanol served as the control. The working solutions were then
used in an IC50 assay to inoculate the human breast cancer cell line MDA-MB-231, this
being the concentration used for the first IC50 assay in which I. hayesiana showed
37
strong cytotoxicity. The return to the higher concentration used in the cell viability
assay only marginally improved the cytotoxicity (Figure 16). A repeat of the solvent
screening assay using new plant material for the extractions also showed almost no
Relative Number of Cells
(%Control)
cytotoxicity for any of the resultant extracts (Figure 17).
140
120
100
80
60
40
20
0
0
20
40
60
80
100
Concentration (µg/mL)
Figure 16: The IC50 determination for I. hayesiana extracted in hexanes (10 mg/mL)
on the human breast cancer cell line MDA-MB-231. The error bars represent one
standard deviation from the average of ten samples.
Relative # of Cells
( % Control)
38
160
140
120
100
80
60
40
20
0
65 µg/mL
Hexane
32.5 µg/mL
Hexane
65 µg/mL
Ether
32.5 µg/mL
Ether
65 µg/mL
Ethanol
Solvent
Figure 17: The cytotoxicity of three new solvent extracts from fresh I. hayesiana (65
and 32.5 µg/mL) on the human breast cancer cell line MDA-MB-231. The bars
represent the percentage of cells alive compared to the control (cells treated with
vehicle only). The error bars represent one standard deviation from the average of ten
samples.
To investigate the chemical differences between the new, inactive extracts and
the old, active extracts, both the new and old extracts were compared via HPLC. The
chromatograms indicated that there were some differences between the two extracts
(Figure 18). This, coupled with the fact that the extractions were performed during
different seasons or plant cycles, led to the hypothesis that the observed cytotoxicity is
seasonal.
39
N orm.
500
400
300
200
100
0
8
10
12
14
16
18
20
22
24
min
Figure 18: HPLC investigation of the chemical differences of active and inactive
extracts of I hayesiana. The HPLC chromatogram of the original active extract
(extracted September 2010) shown in blue, overlayed with an inactive extract (extracted
November 2010) shown in red.
3.7 Evaluation of Seasonal Cytotoxicity
To determine whether the cytotoxic compound(s) in I. hayesiana are seasonal,
extractions were performed monthly on the same plant, beginning February 28th, 2011
and recurring on the 28th of every month through July 2011. Extractions were
performed using 1 g of I. hayesiana leaves and stems and 25 mL of hexanes (ACS
grade), following the initial extraction protocol. Extracts were then evaluated monthly
using the cell viability assay, at a concentration of 10 mg/mL. The results shown in
Figure 19 indicate that cytotoxicity returned in April and was present until June. Both
April and May extracts showed almost 100% cell death, while the June extract resulted
in approximately 50% cell death. The plant flowered at the end of June. Upon review
of previous data, it was noted that the original extract, which showed cytotoxic activity,
40
was completed in September, one month prior to the plant flowering. The second
extract was completed in November, after the plant had flowered. This information,
coupled with the cell viability assay data from April and May, suggests that cytotoxic
constituents are present prior to the flowering of the plant, and that once the plant
flowers, these constituents are either no longer present, or are no longer present at high
enough concentrations to have an effect on the cancer cells. The data from June, the
month the plant flowers, shows a 50% reduction in cytotoxicity, as the compound(s) of
interest decreases just before the plant flowers. All active and inactive extracts were
evaluated further using HPLC.
Relative Number of Cells
(%Control)
135
115
95
75
55
35
15
-5
February
March
April
May
June
July
Month of Extraction
Figure 19: Results from the cell viability assay of monthly extracts of I. hayesiana (65
µg/mL) on the human breast cancer cell line MDA-MB-231. The extracts from April
and May show 100% cytotoxicity and the extract from June shows ~50% cytotoxicity.
All other months are inactive. The bars represent the percentage of cells alive
compared to the control (cells treated with vehicle only). The error bars represent one
standard deviation from the average of ten samples.
41
3.8 HPLC Analysis of Monthly Extracts
All seven monthly extracts were analyzed using an Agilent 1100 HPLC.
Extracts of I. hayesiana were prepared using the method described in the initial
extraction section of Materials and Methods at a concentration of 10 mg/mL. Samples
were further normalized to 1mg/mL and filtered using a 0.22 µm sterile syringe filter
(Fisherbrand) prior to injection. For evaluation purposes, each of the chromatograms of
the active extracts was first overlaid with the ethanol control chromatogram. Peaks
present in the ethanol control chromatogram were excluded from analysis for all of the
extracts. This left 14 distinct peaks that represent the HPLC profile of I. hayesiana and
required further analysis (Figure 20). Chromatograms of all monthly extracts are
available in Appendix A. The retention times and peak areas for these peaks are shown
in Table 3.
Figure 20: HPLC chromatogram of the April extract of I. hayesiana with the 14 peaks
of interest labeled, after comparison to the blank (ethanol) chromatogram.
42
Table 3: Retention times and peak areas from the HPLC chromatogram of I. hayesiana
extracted in April. Fourteen distinct peaks were identified for further analysis.
Peak #
Time
(Min)
Area
(mV)
1
14.391
1031.6
2
14.641
4454.3
3
15.584
75.1
4
15.761
2674.3
5
17.316
2095.2
6
19.246
862.9
7
20.055
416.8
8
20.411
126.2
9
20.846 29950.9
10
21.197
3105.3
11
21.934
2312.9
12
22.100
780.6
13
22.846 22252.5
14
25.077
2386.1
Once the ethanol peaks were eliminated from further consideration, the
remaining peaks were evaluated by comparing retention times of the 14 peaks of
interest in the chromatograms of the active samples with those of the inactive samples.
Each active sample chromatograms were overlaid with the inactive sample
chromatograms for analysis (for example, Figure 21). Any peaks present in the
inactive extracts at similar concentrations (peak areas) to those seen in the active
extracts were ruled out. This left 7 peaks of interest for further evaluation. Of these,
only peaks 8 and 9 were present exclusively in the active extracts. The 5 remaining
43
peaks (4, 7, 10, 11, and 13) were present in both active and inactive extracts, but the
concentration of these constituents was significantly lower in the inactive extracts.
Figure 21: HPLC chromatograms of I. hayesiana extracts from April 28th (red active) and Feb 28th (blue - inactive). The overlay shows 7 peaks of interest (4, 7, 8, 9,
10, 11 and13).
To complete the analysis, the three chromatograms of the active extracts were
individually overlaid with each other. The data from the cell viability assays showed
that the extracts from April and May had 100% cytotoxicity while the extract from June
had approximately 50% cytotoxicity. This indicates that the concentration of the
cytotoxic constituent(s) should show a significant decrease, which should be visible in
the peak area of the constituent of interest. Based on this information, the peak areas of
the 7 remaining peaks of interest were compared, to determine whether a decrease in
concentration was visible for the June extract (Table 4). Peaks 8 and 10 were ruled out
44
due to large increases in concentration in the month of June. Peaks 4, 7 and 11 were
ruled out because the concentration in June, when activity was at 50%, was
approximately the same as the concentration in April, when activity was 100%. Peak 9
was not present in any of the inactive extracts (February, March and July) and had offscale peak areas (not quantifiable) for the active extracts. Peak 13 was present in small
amounts in the inactive extracts from February and March but not in July. It was not
excluded because the concentrations for April, May and June were off scale, and could
not be quantified. To further elucidate peaks 9 and 13, the extracts from April, May
and June were diluted to 0.1 mg/mL and re-evaluated via HPLC. The resulting
chromatograms (Figure 22) show that peaks 9 and 13 were on scale for analysis (Table
5). Based on the new data, peak 13 is ruled out because the concentration for June
(50% activity) is higher than the concentration for May (100% activity). Peak nine
appears to be the remaining peak of interest. The areas for peak nine decrease
significantly from May to June which indicates a concentration decrease during the time
when activity is decreasing. To determine whether the peak of interest might be
hispidulin, further HPLC analysis was conducted.
45
Table 4: Evaluation of the 7 peaks of interest in the active extracts . Peak areas (mV)
of the 7 peaks of interest seen in the HPLC chromatograms of three active extracts
(April, May and June) of I. hayesiana (* off scale peaks)
Peak #
Peak 4
Peak 7
Peak 8
Peak 9
Peak 10
Peak 11
Peak 13
Peak Areas
4/28/2011 5/28/2011 6/28/2011
2674.3
2992.6
2629.2
416.8
505.1
437.9
126.2
179.4
505.9
*29950
*28120
*32157
3105.3
3290.3
4340.2
2312.9
2390.0
3090.5
*22252
*21981
*24161
Figure 22: HPLC chromatogram of the April extract of I. hayesiana, diluted to a
concentration of 0.1 mg/mL, with peaks 9 and 13 labeled.
.
Table 5: Re-evaluation of peaks 9 and 13 via HPLC. Peak areas (mV) of the two
peaks of interest (Peaks 9 and 13) seen in the HPLC chromatograms of the three active
extracts (April, May and June) diluted to 0.1 mg/mL.
Peak #
Peak 9
Peak 13
4/28/2011 5/28/2011 6/28/2011
3216.6
4485.9
941.0
771.8
537.8
611.3
46
3.9 IC50 for Hispidulin
To determine whether hispidulin might be the cytotoxic compound responsible
for the activity seen in the active I. hayesiana extracts, an IC50 assay was completed
using the protocol described in the Materials and Methods section. Commercially
obtained hispidulin was brought to an initial concentration of 10 mg/mL before
beginning the assay dilutions (100, 80, 60, 40, and 20 µg/mL). A solution consisting of
growth media and 100 µL of ethanol served as the control. The working solutions
along with the control were used to inoculate the human breast cancer cell line MDAMB-231. This concentration range was chosen based on an IC50 of 200 µmol/L reported
by Lijun He et al. to be effective against pancreatic cancer cells.34 The IC50
concentration of the hispidulin sample on the human breast cancer cell line MDA-MB231 was greater than 100µg/mL (Figure 23). At the highest concentration of hispidulin
(100µg/mL), only 25% of the cells were killed. This minimal level of cytotoxicity
effectively rules out the possibility that hispidulin could be the constituent responsible
for the cytotoxicity seen in I. hayesiana. The hispidulin sample was further evaluated
using HPLC to determine whether hispidulin could be identified in the I. hayesiana
extracts.
47
Relative Number of Cells
(% Control)
140
120
100
80
60
40
20
0
0
20
40
60
80
100
Concentration (µg/mL)
Figure 23: IC50 assay results for hispidulin dissolved in ethanol (10 mg/mL) on the
human breast cancer cell line MDA-MB-231. The error bars represent one standard
deviation from the average of ten samples.
3.10 HPLC Analysis of Hispidulin
For the purposes of HPLC, hispidulin was diluted in ethanol and brought to a
concentration of 1mg/mL. This sample was sterile filtered using a 0.22µm PTFE
syringe filter (FisherBrand). Sample was injected using the method previously
described in the HPLC section of Materials and Methods. Solvent peaks were ruled out,
which left two remaining peaks, either of which could be hispidulin (Figure 24A). The
chromatogram of hispidulin was then overlaid with the chromatogram of the active
extract of I. hayesiana (extracted 4/28/11). A comparison of the retention times (Table
6) of the two peaks present in the hispidulin sample with the peaks from the I.
hayesiana extract showed that only one of the peaks (peak 2 in Figure 24B) was
present in the I. hayesiana extract. The fact the two peaks were identified as possible
hispidulin peaks indicates that the sample was not pure or that there were
48
decomposition products present in the hispidulin sample. The data indicates that
hispidulin is present in I. hayesiana, as was reported by Hertz et al. (1951), although it
has been ruled out as the constituent responsible for the cytotoxicity seen with MDAMB-231.28
A
B
Figure 24: Evaluation of the presence of hispidulin in I. hayesiana using HPLC. (A)
Chromatogram of hispidulin in ethanol (1 mg/mL) with the hispidulin peaks labeled.
(B) Overlay of Active Extract (4/28/11)of I. hayesiana (blue) with hispidulin (red).
Peak 2 represents the only peak with a retention time that matches either of the two
peaks identified in hispidulin.
49
Table 6: Evaluation of the HPLC retention times of hispidulin. Retention Times of
peaks 1 and 2 represented in Figure 24A and 24B. The difference in retention times for
peak one indicate that peak 1 is not hispidulin.
Peak 1
Peak 2
Hispidulin
14.268 min
17.310 min
I. hayesiana
14.391 min
17.316 min
50
Chapter 4
Conclusions
The purpose of this research was three-fold: 1) To determine the best extraction
method for extractions of the cytotoxic component(s) I.hayesiana, 2) To determine
whether the cytotoxic constituent(s) occur seasonally in I. hayesiana, and 3) To
determine whether hispidulin is responsible for the cytotoxicity of I. hayesiana on the
human breast cancer cell line MDA-MB-231. The results showed that the cytotoxic
compound(s) in I. hayesiana are non-polar and non-volatile. Therefore, hexanes was
determined to be the best solvent for future extractions, and HPLC was the best method
for purity analysis. Analysis of monthly extractions showed that the cytotoxicity was
seasonal and related to the flowering of the plant. Cytotoxicity appeared approximately
two months before the plant flowered and disappeared rapidly once flowering occured.
The IC50 for an active hexane extract of I. hayesiana was determined to be 56 ± 3
µg/mL. It was also determined that hispidulin was present in I. hayesiana; however, it
was not responsible for the cytotoxicity seen in the extracts from April through June.
51
Chapter 5
Future Work
Based on the results from this study, a number of recommendations can be made
for the extension of this project. This study was focused on the cytotoxicity of I.
hayesiana extracts towards the triple negative breast cancer cell line MDA-MB-231.
To determine whether I. hayesiana is in fact a viable target for future anticancer
research, I. hayesiana must be tested against normal human cells. I. hayesiana should
also be tested against other breast cancer cell lines such as MCF-7 and BT-474 to
determine whether it might also be effective against ER/PR+ cell lines.
Now that the seasonal nature of the cytotoxicity has been established, large scale
extractions can been performed during the appropriate season. Samples from these
larger scale extractions can then be used to isolate and identify the cytotoxic
constituent(s) present in I. hayesiana, starting with the peak labeled as # 9 in this
research. It would also be advisable to attempt to determine the chemical fingerprint
(LC/MS) of I. hayesiana, which is different during the seasonal cytotoxic period.
HPLC Chromatogram of Iva hayesiana Extracted 2/28/11 (1 mg/mL)
21.21
-21.99
22.88
25.08
466.5
-161.4
403.2
195.5
APPENDIX A
Time Area
14.39 362.5
14.64 2337
--110.7
15.76
17.31 1290
19.27 475.5
---
HPLC Spectrum
Peak
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
52
HPLC Chromatogram of Iva hayesiana Extracted 3/28/11 (1 mg/mL)
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Time
14.39
14.64
15.59
15.76
17.32
19.27
---21.21
--
Area
756.2
3985
118.1
754.4
2440.1
647.0
---383.5
--
22.88
25.07
683.0
369.9
53
HPLC Chromatogram of Iva hayesiana Extracted 4/28/11 (1 mg/mL)
Peak
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Time
Area
(Min)
(mV)
14.391 1031.6
14.641 4454.3
15.584
75.1
15.761 2674.3
17.316 2095.2
19.246
862.9
20.055
416.8
20.411
126.2
20.846 29950.9
21.197 3105.3
21.934 2312.9
22.100
780.6
22.846 22252.5
25.077 2386.1
54
HPLC Chromatogram of Iva hayesiana Extracted 5/28/11 (1 mg/mL)
#
Time
Area
1
14.39
1138
2
14.64
4843
3
15.58
91.6
4
15.76
2993
5
17.32
2397
6
19.24
868.3
7
20.05
505.1
8
20.41
204.6
9
20.84 28120
10
21.19
3290
11
21.93
2390
12
22.1
986.7
13
22.84 21982
14
25.08
477.2
55
HPLC Chromatogram of Iva hayesiana Extracted 6/28/11 (1 mg/mL)
#
Time
Area
1
14.39
685.0
2
14.64
2709.3
4
15.76
2629.2
5
17.31
2211.2
6
19.24
809.5
7
20.10
437.9
8
20.41
505.9
9
20.82 32157.2
10
21.18
4340.2
11
21.92
3090.5
12
22.09
2035.7
13
22.82 24161.6
14
25.08
3
432.5
56
HPLC Chromatogram of Iva hayesiana Extracted 7/28/11 (1 mg/mL)
#
Time
Area
1
--
--
2
--
--
3
--
--
4
15.76
5
17.32 1104.1
6
19.22
285.7
7
20.04
147.6
8
--
--
9
21.03
88.9
10
21.19
474.3
11
21.95
447.3
12
--
--
13
22.85
614.1
14
25.10
428.2
81.7
57
HPLC Chromatogram of Iva hayesiana Extracted 4/28/11 (0.1 mg/mL)
#
Time
Area
1
--
--
2
--
--
3
--
--
4
15.76
81.7
5
17.32
1104
6
19.223
285.7
7
20.04 147.6
8
--
--
9
21.03
88.9
10
21.19 474.3
11
21.95 447.3
12
--
--
13
22.85 614.1
14
25.1 428.2
58
59
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