RAPURU, SIVA KUMAR., M.S. Chemical Composition and Anti-proliferative Activity
of Several Medicinal Plants. (2008)
Directed by Dr. Nadja B. Cech. 61 pp.
Plants are valuable sources of medicinal compounds and their use for healing is
well known from ancient times. Natural drugs obtained from plants represent about 25%
of the prescription drug market in the United States. Plants have a long history of use in
the treatment of various cancer types. Currently, 60% of the anticancer agents available
in the market are derived from natural sources. Since phytoconstituents play a vital role
in the discovery of various anticancer drugs, they have been chosen as the area of focus
for our research. In this proposed study, four medicinal plants with reported anticancer
activity were selected (Hydrastis canadensis, Curcuma longa, Zingiber officinalis, and
Alpinia officinarum). All these plants were extracted by percolation and tested for antiproliferative activity against Dictyostelium cells. C. longa, Z. officinalis and A.
officinarum organic extracts all showed significant anti-proliferative activity in this
preliminary bioassay. Of the three active extracts, the turmeric extract was chosen for
further investigation because of its great historical significance and the promising results
of recent phase I clinical trials. Using flash chromatography, a total of nine fractions were
obtained from the complex C. longa organic extract. Curcumin in these fractions was
identified and quantified using high performance liquid chromatography – electrospray
ionization
mass
spectrometry
(HPLC-ESI-MS).
Other
active
components
(demethoxycurcumin, bisdemethoxycurcumin and ar-tumerone) were also characterized
using the same system. All these fractions were then tested for in vitro anti-proliferative
activity against MCF-7 cells using the XTT assay to determine whether activity correlates
with the presence of curcumin in the fractions, or whether other (perhaps unidentified)
compounds are involved. The results indicated that the major component curcumin was
responsible for the majority of the anti-proliferative activity of the complex turmeric
extract. Although no synergistic activity was seen for the various constituents present in
the complex extract in this case, a novel approach for probing potential synergistic or
additive effects was demonstrated. This approach could be applied to future
investigations of synergistic or additive activity of medicinal plants.
CHEMICAL COMPOSITION AND ANTI-PROLIFERATIVE
ACTIVITY OF SEVERAL MEDICINAL PLANTS
by
Siva Kumar Rapuru
A Thesis
Submitted to
The Faculty of The Graduate School at
The University of North Carolina at Greensboro
in Partial Fulfillment
of the Requirements for the Degree
Master of Science
Greensboro
2008
Approved by
Committee Chair
I dedicate my thesis to the most important people in my life.
To my parents Adi Lakshmi and Lakshmaiah Rapuru:
I thank you for giving me birth and taking all the pains to raise me into the person I am. I
attribute my academic and professional success to you both. The pain and courage you
showed in sending me abroad for my higher studies is indispensable. I am grateful to you
for providing me financial and emotional support through out my career inspite of
hardships you had in this journey. I love you so much.
To my lovely wife Rohini Devi:
You are the most lovable person in my life. I can never imagine my success without your
presence. You are the inspiration to my goals. Your love, affection and care towards me
always relieved me from the stress I had in completion of this project. I forget all my
miseries with your sweet smile. I can’t imagine life without you.
To my beloved brothers Narayana Rapuru and Anoj Rapuru:
You both are very special to me. I feel blessed to have you both in my family. My love
and care for you both will stay forever. You are so far away from me and I miss you the
most.
ii
To my close friends Kishore, Deepak, Kasyap, Chander, Srinivas and Vamsi:
Thank you all for everything. Without your help I would have not achieved all this in my
life. You all were always with me and I expect the same until the rest of my life. I really
thank God for having you all as my friends.
To my in laws Krishnakumari and Srinivasa Chary Shesham:
Thank you for your love and care you showered on me. I always feel secured with the
thought of having you in my life.
iii
APPROVAL PAGE
This thesis has been approved by the following committee of the Faculty of The
Graduate School at The University of North Carolina at Greensboro.
Committee Chair
Committee Members
Date of Acceptance by Committee
Date of Final Oral Examination
iv
ACKNOWLEDGEMENTS
This work was carried out at Department of Chemistry, University of North
Carolina at Greensboro (UNCG), NC during the years 2007-2008. Part of this work was
carried out at Department of Biology, UNCG, NC.
I sincerely express my deep sense of gratitude to my respectable teacher and
supervisor Dr. Nadja B. Cech. I thank her for giving me the opportunity to be a part of
this interesting research and hone my knowledge. She is instrumental in successful
completion of my dissertation. Her support and encouragement are invaluable. I am
especially grateful to her for solving various hurdles through out my years of graduation
and for sharing her wisdom with me. Her determination towards completion of my
project as my research advisor was indispensable. I really appreciate her patience and
belief in me. It was a great and wonderful experience working under her. Thank you for
everything.
I am thankful to Dr. Paul Steimle for agreeing to be a part of my project. Thank you
for your valuable support in carrying out the preliminary screening experiments in your
lab. I really appreciate your support, guidance and expert advice in completion of my
thesis. Thanks are also owed to Dr. Yashomati Patel who helped with training and
provided advice and protocols for culturing MCF-7 cells. Also, special thanks to Dr.
Brent Dawson for being in my thesis committee and for guiding me in the intricacies of
GCMS instrument. Thank you for answering all my questions and concerns. He is the
best teacher I ever had in my academic career.
v
I express my gratitude to Dr. Alice Haddy for being supportive through out my
MS. I am extremely grateful for guiding me in my course work and also for being
instrumental in providing me financial support. Her care towards me was really
appreciable during my initial days of masters in USA.
My special thanks to Kevin Spelman, Research Scientist for his fruitful
cooperation in carrying out the bioassays. Thank you for your kind help during my initial
days in our lab.
My sincere thanks to our research group, fellow graduates and staff for their
support throughout my course work and research.
Thank you one and all.
vi
TABLE OF CONTENTS
Page
LIST OF TABLES………………………………………………………………………..ix
LIST OF FIGURES……………………………………………………………………….x
ABBREVIATIONS……………………………………………………………………...xii
CHAPTER
I. INTRODUCTION AND RESEARCH GOALS………………………………..1
1.1 Natural products and their role in cancer treatment…………………….1
1.2 Selection of medicinal plants…………………………………………...2
1.3 Curcuma longa (Turmeric)……………………………………………..3
1.4 Anticancer activity of curcumin in vitro..................................................6
1.5 Methods used for the analysis of Turmeric extract……………………..7
1.5.1 HPLC-ESI-MS……………………………………………….8
1.6 Evaluation of biological activity (Anti-proliferative activity)………...10
1.6.1 Dictyostelium cells………………………………………….11
1.6.2 MCF-7 Human breast cancer cells……………………….....11
1.7 Measuring viability of cells using XTT assay………………………...12
1.8 Research Goals………………………………………………………..13
II. EXPERIMENTAL METHODS………………………………........................15
2.1 Extraction of Curcuma longa, Hydrastis canadensis, Zingiber
officinalis and Alpinia officinarum plants……………………………16
2.1.1 Procedure…………………………………………………..17
2.2 Identification of the active components from plant extracts using
HPLC-ESI-MS……………………………………………………….17
2.2.1 Instrumentation…………………………………………….18
2.3 Primary screening of plant extracts in vitro (Anti-proliferative
activity) using Dictyostelium cells.......................................................19
2.3.1 Procedure for screening with Dictyostelium……………….19
2.4 Separation and quantification of curcumin from turmeric extract…….20
2.4.1 Procedure…………………………………………………..21
2.5 Identification and quantification of ar-tumerone in the turmeric
fractions using HPLC-ESI-MS………………………………………22
vii
2.6 Testing turmeric fractions for anti-proliferative activity against
MCF-7 human breast cancer cells (XTT Assay)…………………….22
2.6.1 Preparation of Stock Solutions…………………………….23
2.6.2 Procedure…………………………………………………..24
III. RESULTS AND DISCUSSION……………………………………………..27
3.1 Extraction of medicinal plants………………………………………...27
3.2 Major components identified from plant extracts using
HPLC-ESI-MS………………………………………………………28
3.2.1 Analysis of Curcuma longa extract………………………..28
3.2.2 Analysis of Hydrastis canadensis extract………………….33
3.2.3 Analysis of Alpinia officinarum extract……………………33
3.3 Anti-proliferative activity of plant extracts on Dictyostelium cells…...38
3.4 Quantitation of curcumin in turmeric fractions……………………….42
3.5 Other curcuminoids identified in the turmeric fractions...……………44
3.6 Identification and quantitation of ar-tumerone in the turmeric
fractions..……………………………………………………………46
3.7 XTT assay results……………………………………………………...53
IV. CONCLUSIONS AND FUTURE SCOPE OF THIS RESEARCH………..60
REFERENCES…………………………………………………………………………..62
viii
LIST OF TABLES
Page
1. Amounts of ground plant material used for extraction ………...................................16
2. HL5 culture medium composition (1 liter)…………………………………………..20
3. Dissolved solid extracts produced ………………………………..............................27
4. Comparison of retention times of curcumin for both standard and
original extract ……………………………………………………………………..30
5. Dictyostelium cell growth in the presence of different concentrations
of C. longa organic extract …………………………………………………………39
6. Effect of different concentrations of A. officinarum organic extract
on Dictyostelium cell count ………………………………………………………...40
7. Dictyostelium cell growth in the presence of Z. officinalis organic extract …………41
8. Concentrations of curcumin from different fractions of C. longa organic
extract including the original extract ………………………………………………43
9. Comparison of retention times of ar-tumerone for both standard and
turmeric original extract ………………………………………………....................49
10. Concentrations of ar-tumerone present in fraction 1 and turmeric original
extract ………………………………………………………………………………52
11. MCF-7 cell growth in the presence of different concentrations of
standard curcumin at exposure time of 72h………………………………………...54
12. Cellular viability in the presence of different concentrations of turmeric
fractions, original extract and standard curcumin at 72 h exposure time
± SD………………………………………………………………………………...57
ix
LIST OF FIGURES
Page
1. Structures of curcuminoids found in turmeric extract ………………………………..5
2. Structures of main volatile oil components present in turmeric oil …………………..6
3. Schematic representation of an HPLC-ESI-MS hyphenated system ………………..10
4. Flow chart detailing various steps involved in the research ………………………...14
5. Base peak chromatogram of turmeric organic extract obtained
using HPLC-ESI-MS……………………………………………………………….29
6. Selected ion chromatogram of turmeric organic extract obtained using
HPLC-ESI-MS……………………………………………….……………………..30
7. Full scan mass spectra for (a) standard curcumin (molecular weight
of 368) and (b) the peak obtained at a retention time of 21.45 min
for turmeric organic extract………………………………………………………...32
8. Selected ion chromatogram of m/z 336 from goldenseal organic extract
obtained using HPLC-ESI-MS for three different concentrations
a) 1mg/mL b) 0.1mg/mL c) 0.01mg/mL…………………..…………......................34
9. Selected ion chromatogram of A. officinarum organic extract for m/z value
of 285 obtained using HPLC-ESI-MS..……………………………….....................35
10. Selected ion chromatogram of A. officinarum organic extract for m/z value
of 269 obtained using HPLC-ESI-MS..…………………………………………….36
11. Selected ion chromatogram of A. officinarum organic extract for m/z value
of 301 obtained using HPLC-ESI-MS……………………………………………...37
12. Effect of C. longa organic extract on Dictyostelium cell growth …………………...39
13. Effect of A. officinarum organic extract on Dictyostelium cell growth ……………..40
14. Effect of Z. officinalis organic extract on Dictyostelium cell growth ……………….41
15. Calibration curve for curcumin ……………………………………………………...42
16. Concentrations of curcumin in the turmeric extract fractions
x
determined by HPLC-ESI-MS ………………………………..................................44
17. Relative amounts of demethoxycurcumin present in the turmeric
fractions …………………………………………………………………………….45
18. Relative amounts of bisdemethoxycurcumin present in the turmeric
fractions …………………………………………………………………………….45
19. Total ion chromatogram (mass range 216-220) of the turmeric original
extract obtained using positive mode HPLC-ESI-MS.…………………..................46
20. Selected ion chromatogram of m/z value of 217 obtained using positive
mode HPLC-ESI-MS for three different concentrations of turmeric
fraction 1. a) 1mg/mL b) 0.1mg/mL c) 0.01mg/mL.……………………………….48
21. Selected ion chromatogram of turmeric original extract obtained for
an m/z value of 217 using positive mode HPLC-ESI-MS………………………….49
22. Full scan mass spectra of (a) standard std-tumerone (molecular
weight of 216) and (b) the peak at a retention time of 24.62
min for turmeric original extract……………………………………………………50
23. Calibration curve for ar-tumerone obtained by plotting log of concentration
vs log of peak area of the selected ion chromatogram for the ion of
m/z 217……………………………………………………………………………...51
24. Concentrations of ar-tumerone in turmeric fraction 1 and original
extract determined by positive mode HPLC-ESI-MS ……………………………..52
25. Effects of different concentrations of standard curcumin on MCF-7
cell proliferation.……………………………………………………………………54
26. MCF-7 cell viability in the presence of different concentrations of
turmeric fractions, original extract and standard curcumin………………………...58
27. The percent reduction in MCF-7 cell viability at 30µM for all the
turmeric fractions including the original extract and standard
curcumin.……………...............................................................................................59
xi
ABBREVIATIONS
MTT: 3-(4, 5- Dimethylthiazol-2-yl)-2, 5- Diphenyltetrazolium Bromide
MCF: Michigan Cancer Foundation
MF: Molecular Formula
MW: Molecular Weight
C. longa: Curcuma longa
A. officinarum: Alpinia officinarum
Z. officinalis: Zingiber officinalis
et al;: and others
HPLC-ESI-MS: High Performance Liquid Chromatography - Electrospray Ionization Mass Spectrometry
GCMS: Gas Chromatography Mass Spectrometry
C18: covalently bonded octadecylsilane
min: minutes
mL/min: milliliter per minute
µm : micrometer
µL: micro liter
m/z: mass to charge ratio
V : volts
kV : kilovolts
DMSO : dimethyl sulfoxide
NaCl : sodium chloride
xii
KCl : potassium chloride
Na2HPO4: disodium hydrogen phosphate
KH2PO4: potassium dihydrogen phosphate
AX: axenic
cm: centimeter
rpm: rotations per minute
h : hours
M : molar
mM : millimolar
CO2: carbon dioxide
EDTA: ethylene diamine tetra acetic acid
nm : nanometer
µM : micromolar
Avg : average
µg/mL : microgram per milliliter
Conc : concentration
Wt% : weight percent
Std. Dev : standard déviation
Org ext : original extract
Abs : absorbance
SD : standard déviation
approx : approximately
xiii
rpm : rotations per minute
xiv
CHAPTER I
INTRODUCTION AND RESEARCH GOALS
1.1 Natural products and their role in cancer treatment
Natural products are valuable sources of medicinal compounds and their use for
healing is well known from ancient times. They still continue to provide new remedies to
human kind in treating various diseases. Though significant development has been made
in the combinatorial chemistry field, drugs derived from natural products still play an
important role in the drug discovery process1. Today, natural drugs obtained from plants
still represent about 25% of the prescription drug market in the United States2.
Furthermore, according to the World Health Organization, 80% of the population in
developing countries depends on traditional medicine for their primary health care, and
85% of traditional medicine is derived from plant extracts2.
Cancer, a dreadful disease, is considered to be a leading cause of death in the
United States, second only to heart disease3. Over the past 25 years, the United States
government, through the National Cancer Institute (NCI), has expended billions of
dollars to combat cancer2. In the process of development of drugs to treat cancer, natural
products played an important role as contemporary cancer chemotherapeutic agents.
Plants have a long history of use in the treatment of various cancer types4. Natural
product chemists collect plants throughout the world, prepare their extracts and subject
1
them to various biological screening processes, which finally lead to the isolation and
characterization of active compounds. Over 35,000 plant samples had been collected
world wide by the National Cancer Institute (Natural Products Branch) and had screened
around 114,000 extracts for antitumor activity5. As a consequence of extensive research,
a number of clinically useful and market approved drugs are now available for use. It is a
known fact that 60% of currently used anticancer agents are derived from natural
sources6. Since plant constituents constitute an important source of cancer drugs, they
have been chosen as the area of focus for this research.
1.2 Selection of medicinal plants
In this study, the first step was to select the medicinal plants which were shown to
possess anticancer activity. To date, over 3000 species of plants had been reported to
possess anticancer properties7. So, correct choice of plant material was necessary. Hence,
in order to narrow the list of plants the following factors were considered:
•
Plants that showed success with in vitro and/or in vivo studies
•
Plants whose exact mechanism of action was not fully understood
•
Plants that are native to North Carolina
Finally, four plants were selected and subsequently obtained to begin the
preliminary research. Of these chosen plants, goldenseal is the only plant native to North
Carolina.
1) Goldenseal (Hydrastis canadensis) – Rhizome
2) Turmeric (Curcuma longa) – Rhizome
2
3) Ginger (Zingiber officinalis) – Rhizome
4) Lesser galangal (Alpinia officinarum) – Rhizome
Of all the plants selected, turmeric, ginger and lesser galangal extract all showed
activity in our preliminary bioassay. Of the three active extracts, the turmeric extract was
chosen for more detailed studies given its traditional and clinical significance8, 9.
Turmeric (Curcuma longa) has been used for thousands of years as a healing agent for
variety of illnesses. Turmeric was found to be a vital source of active components that
have strong anti-inflammatory and anticancer activities, including most notably the
compound curcumin10. Based on several research studies, curcumin can be considered as
a promising tool for cancer therapy and also has undergone a number of phase I human
trials11. Although several cellular and molecular mechanisms of action for curcumin have
been proposed the precise mode of anti-proliferative activity of curcumin has not been
fully elucidated12, 13. Since turmeric extract is a mixture of several components, it is of
interest to study how the presence of these multiple components affects the overall antiproliferative activity of the complex turmeric extract. Taking all these facts into
consideration, the turmeric plant was chosen as the plant of focus for this research.
1.3 Curcuma longa (Turmeric)
Curcuma longa or turmeric, a perennial herb belonging to ginger family, is a
tropical plant native to southern and southeastern tropical Asia. The rhizome part of the
turmeric plant is considered to be the main source of active components. The dried
rhizome of turmeric contains the flavonoids curcumin, demethoxycurcumin and
3
bisdemethoxycurcumin, which are commonly known as curcuminoids, as well as volatile
oils (mostly terpenoids) such as tumerones, atlantones and zingiberene14,
15
(Figures
1&2). The most extensively studied component is curcumin (diferuloylmethane) which
comprises 2-5% of the total components in turmeric. Over 2000 papers have been
published on turmeric over the last 50 years16.
Curcumin possess various biological activities like anti oxidant, anti inflammatory,
anti platelet, anti viral, anti fungal, and anti bacterial activities and also has been used in
the treatment of Alzheimer’s disease, multiple sclerosis and rheumatoid arthritis.
Moreover, there is extensive literature which indicates the potential of curcumin in the
prevention and treatment of various cancers7. Of all the curcuminoids, curcumin was
found to possess the most potent anti-proliferative activity (e.g. human ovarian cancer
cell line Ho-8910 inhibition by MTT assay17). In one study, it was shown that
demethoxycurcumin is a better anti-proliferative agent than the other two curcuminoids
(MCF-7 human breast cancer cell line inhibition18). Bisdemethoxycurcumin also showed
more significant cytotoxic activity than the other two
19
. It was also found that all three
curcuminoids exhibited similar anti-proliferative activity against various cell lines
(Jurkat, KBM-5, and A549 cell lines16). Among the volatile oils, ar-tumerone exhibited
potent cytotoxic effect on cancer cell lines like K562, L1210, U937 and RBL-2H3 by
MTT assay20.
4
O
O
MeO
HO
OMe
Curcumin
MF- C21H20O6
MW- 368.38g/mol
O
OH
O
OMe
HO
Demethoxycurcumin
MF- C20H18O5
MW- 338.35g/mol
O
HO
OH
O
Bisdemethoxycurcumin
M.F- C19H16O4
M.W- 308.32g/mol
OH
Fig 1 - Structures of curcuminoids found in turmeric extract
5
H
H
O
Ar-tumerone
MW-216
O
O
α -tumerone
MW-218
β -tumerone
MW-218
OH
O
Curlone
MW-218
Curcumene
MW-202
α-tumerol
MW-218
Fig 2- Structures of main volatile oil components present in Turmeric oil
1.4 Anticancer activity of curcumin in vitro
Curcumin, an active constituent of C. longa extract, has the ability to induce
apoptosis in different cancer cells. This property was used in developing curcumin as a
universal cancer prophylactic agent21. Curcumin has been shown to suppress
transformation, proliferation and metastasis of tumors through various mediators like
transcription factors, growth factors, growth factor receptors, inflammatory cytokines,
protein kinases, various enzymes and genes regulating apoptosis and proliferation22. It
also inhibits proliferation of cancer cells by arresting them in various phases of the cell
6
cycle and by inducing apoptosis, indicating its potential in the treatment of various
cancers. In vitro studies conducted on curcumin have shown to inhibit the growth of large
number of human cancer cell lines, like leukemia, prostate, lung, renal, colon, melanoma,
breast23, hepatocellular and ovarian carcinomas.
1.5 Methods used for the analysis of Turmeric extract
The main aims of natural product research are - qualitative and quantitative analysis
of medicinal plant constituents and isolation of biologically active purified plant extract
fractions or compounds. So, considering the fact that a single plant can have several
thousand constituents, the above goals can be achieved by employing rapid and high
performance separation methods. A major task for a phytochemist in the analysis of
natural compounds is the characterization of compounds with minimal sample
preparation. Usually compounds present in the plant extracts can be separated rapidly
using chromatography. A liquid chromatographic method with spectrophotometric
detection was developed for the first time in 1953 to separate and quantify
curcuminoids24. Later on, various methods such as paper or thin layer chromatography25,
26
(TLC), capillary electrophoresis27 (CE), gas chromatography28 (GC), high performance
liquid chromatography (HPLC), and its coupling to photodiode array detection (HPLCDAD29) and mass spectrometry (LC-MS30) were used to analyze the chemical
constituents of turmeric extracts. Xian-Guo He et al. (1998) were the first researchers to
employ online high performance liquid chromatography-electro spray ionization-mass
spectrometry (HPLC-ESI-MS) for the identification of turmeric constituents30. Not only
7
individual curcuminoids but also a number of essential oil constituents like ar-tumerone
and curlone could be identified from a single HPLC analysis. Hongliang Jiang et
al.(2006) and Kuo-Yi Yang et al.(2007) validated HPLC coupled with tandem mass
spectrometry (LC-MS/MS) to identify curcuminoids in turmeric extract31, 32.
The aroma of turmeric is due to the presence of several volatile oils. These volatile
oils are usually a mixture of sesquiterpene ketones and alcohols. Malingre (1975) for the
first time reported volatile oil constituents’ ar-tumerone, tumerone, curlone, curcumene
and other sesquiterpene alcohols from Curcuma longa33. Phan et al. (1987) for the first
time reported the GC-MS analysis of turmeric oil34. Richard Hiserodt et al. (1996)
employed direct thermal desorption-gas chromatography-mass spectrometry (DTD-GCMS) for the analysis of various volatile and semi-volatile compounds in powdered
turmeric35. GC-MS serves as a convenient and sensitive analytical technique for
analyzing essential oils, and remains one of the most common methods applied for this
purpose. And also online HPLC-ESI-MS technique was used to analyze various
sesquiterpenoid volatile components present in turmeric extract30, 35.
1.5.1 HPLC-ESI-MS
Chromatographic separation coupled with mass spectrometric detection provides a
pathway for the rapid and sensitive characterization of active compounds from plant
extracts. LC-MS, which couples liquid chromatography (LC) with mass spectrometry
(MS), has gained attention as a convenient method for identification, structural
determination, and quantitative analysis of various bioactive compounds in complex
8
extracts. LC-MS is considered to be a major breakthrough in the analysis of natural
products possessing significant biological activities. Of the various LC-MS techniques,
high performance liquid chromatography coupled to electro spray ionization-mass
spectrometry (HPLC-ESI-MS) is an ideal technique for the analysis of a wide variety of
analytes36. This soft ionization technique coupled to a single quadrupole mass
spectrometer provides the molecular weight of compounds, and further structural details
can be obtained with HPLC-MS-MS systems (those with triple quadrupole or ion trap
mass analyzers), which provide information on the characteristic fragmentation pattern
typical of a compound. In contrast to other ionization techniques, multiply charged
analytes can easily be formed with ESI, making it possible to analyze larger molecules.
Small polar to medium polar analytes are also readily ionized with this technique, making
HPLC-ESI-MS useful in phyto-chemical investigation37. In most quantitative HPLC-ESIMS methods, low detection limits and reproducible results are the desirable features. A
schematic of an HPLC-ESI-MS system is shown in Figure 3. A typical HPLC-ESI-MS
system has mainly three different regions; the HPLC system, the ESI ion source and the
mass spectrometer. An HPLC-ESI-MS system similar to that shown in Figure 4 was
employed for this study to analyze the plant extracts.
9
Flow split
Analytical column
ESI ion source
Injection loop
Mass spectrometer
Binary gradient pump
Mass spectrum
Fig 3 - Schematic representation of an HPLC-ESI-MS hyphenated system
1.6 Evaluation of biological activity (Anti-proliferative activity)
The isolation of anticancer natural products needs an appropriate bioassay to guide
fractionation at each step. Many bioassay systems are available for anticancer drug
discovery. A screening bioassay is applied to large number of initial samples to identify
those with desired bioactivities. The purpose of using this bioassay is to discard inert
materials and identify active fractions. A monitoring bioassay is used to guide
fractionation of crude materials towards isolation of the pure active components. Modern
analytical instrumentation may be most effective in bioassay-directed isolation. In this
research, Dictyostelium cells and MCF-7 human breast cancer cells have been selected to
test for anti-proliferative activity of fractions and constituents of C. longa.
10
1.6.1 Dictyostelium cells
In this study, the initial screening of all the plants extracts was done using the
model system Dictyostelium discoideum. Dictyostelium was used as a powerful
eukaryotic model system for identifying and characterizing the basic mechanisms which
control the cell division38, cell migration39, multicellular development processes40 and
other vital cellular processes that play significant roles in various diseases. When
compared to other cell types, this cell type is considered to be less complicated (i.e. easy
to grow, handle and manipulate). Several studies on Dictyostelium have provided useful
information on how the various cellular processes were been altered during the
uncontrolled cell multiplication seen in tumor growth41, 42. This model system allows
identifying a specific gene involved in this process and also serves as a model for
processes that occur in mammalian cells, since it has some of the complex features that
resemble mammalian cells. Perhaps most importantly, Dictyostelium discoideum is an
extensively studied model organism for which a great deal of genome sequencing work
has been completed43.
1.6.2 MCF-7 Human breast cancer cells
In addition to Dictyostelium cells, the MCF-7 Human breast cancer cell line was
employed in order to identify the fractions of C. longa with significant anti-proliferative
activity. The MCF-7 cell line, initially developed by Soule et al. (1973), was used
universally for various in vitro and in vivo studies on potent compounds used in the
11
therapy of breast cancer as it relates to the vulnerability of the cells to apoptosis21. This
cell line is the best characterized of all the existing estrogen responsive lines. Since MCF7 cells are mammalian cells, the data derived from these cell lines will be used as a first
step towards gaining insight into the possible anti-tumor activity of the components of
interest. Follow up experiments would be necessary to confirm the in vivo relevance of
any anti-proliferative effects observed in vitro. Curcumin has already been shown to
suppress cell proliferation of MCF-7 human breast cancer cells by targeting several
molecules and pathways involved in cancer pathogenesis44, 45.
1.7 Measuring viability of cells using XTT assay
In the process of screening various cytotoxic agents, determination of viable cell
number is often used to measure the cell proliferation rate. The XTT assay, a colorimetric
method, is a valuable method for screening of plant extracts for anti-proliferative activity.
The XTT method is a simple, fast, rapid and accurate assay and yields reproducible
results46. In this assay, the XTT reagent (tetrazolium salt) combines with mitochondrial
dehydrogenase enzyme of viable cells yielding orange colored formazan crystals. The
amount of formazan formed indicates the degree of cytotoxicity caused by the test
samples (i.e. the amount of formazan resulted from XTT is proportional to the total
number of living cells in the sample wells). The resulting orange colored product was
quantified by measuring the absorbance spectrophotometrically. The percentage of cells
surviving after the sample treatment is determined by comparing the absorbance of
treated cells with that of the control cells. Since dead cells are unable to metabolize
12
tetrazolium salts, the colorimetric assay like XTT can be used to measure cell
viability/cell proliferation rate. Apoptosis, normal cell death, requires metabolism of
cells. Hence for this reason colorimetric assay (XTT), which can not measure cellular
damage, can detect cell death only at later stages of apoptosis when the cellular
metabolism is reduced.
1.8 Research Goals
The main objective of this study is to separate and quantify phytoconstituents from
Curcuma longa (Turmeric) plant extract and then test the bioactivity of isolated
compounds. Turmeric extract is a complex extract with several components which have
significant biological activities
16, 12 & 47
. Though significant research was done on all the
individual components of the turmeric extract, still the relative importance of all these
components for the overall anti-proliferative activity is not clear. It is also not known
whether there are compounds other than curcuminoids and volatile components that are
important in the anti-proliferative activity of C. longa. The major goal of this study is to
address this gap in the knowledge base. The main aim of this research is to study how the
various components present in the complex turmeric extract contribute to its antiproliferative activity. To accomplish this goal, isolation, identification and quantification
of constituents from the turmeric extract will be accomplished using HPLC-ESI-MS.
Further, preliminary screening experiments will be conducted on the plant extracts
obtained using Dictyostelium cells to test for anti-proliferative activity. Once the complex
turmeric extract is separated into individual fractions or compounds, these individual
13
fractions will again to be tested for anti-proliferative activity against MCF-7 human
breast cancer cells using the XTT assay. The flow chart (Figure 4) shown below
represents an outline of this research-
Percolation
Characterize
extracts with
HPLC-ESI-MS
Bioassay
Separation
Extraction
Medicinal
plants
Flash
chromatography
Extracts
Fractions
Bioassay
Bioassay
Pure
constituent (s)
Fig 4 – Flow chart detailing various steps involved in the research
14
CHAPTER II
EXPERIMENTAL METHODS
The following section gives a brief overview of the experimental procedures used in
the research. In order to identify and quantify active components from plant extracts the
first step would be to extract these components using a suitable extraction process.
Percolation utilizing suitable solvents was employed to obtain a complex extract of all
four plants- Hydrastis canadensis, Curcuma longa, Zingiber officinalis, and Alpinia
officinarum. These extracts were analyzed using HPLC-ESI-MS to identify major
constituents of the extracts. The anti-proliferative activity of these extracts was then
tested using the Dictyostelium cell model. Flash chromatography was used to further
separate the complex turmeric extract into fractions. The concentrations of the active
components in the fractions obtained were also determined using HPLC-ESI-MS. Finally,
the turmeric fractions were tested against MCF-7 human breast cancer cells to identify
fractions with significant anti-proliferative activity and to correlate the HPLC-ESI-MS
data with biological efficacy.
15
2.1 Extraction of Curcuma longa, Hydrastis canadensis, Zingiber officinalis and
Alpinia officinarum plants
Two crucial stages involved in the isolation of phytoconstituents are the selection of
plant material and the procedure of extraction. This includes grinding of the plant
material, extraction of the constituents, and concentration and drying of the extracts. In
this research, all the medicinal plants selected were extracted by percolation, which is a
simple maceration process. Maceration is the most commonly used preparative extraction
method by most of the phytoanalysts48. Using a laboratory grinder, the dried plant
material was ground to fine powder to create as much surface area as possible. The
amounts of the ground plant material taken for the purpose of extraction are given in
Table 1.
Table 1 – Amounts of ground plant material used for extraction
Plant
Amount of ground plant material
Goldenseal (Hydrastis canadensis)
500g
[Pacific botanicals, Lot no.- 0906M-TCP]
Turmeric (Curcuma longa)
390g
[Horizon Herbs, Lot no. – 4360]
Ginger (Zingiber officinalis)
416.5g
[Horizon Herbs, Lot no. – 4361]
Lesser Galangal (Alpinia officinarum)
312g
[Horizon Herbs, Lot no.- 4362]
16
2.1.1 Procedure
The ground plant material was initially submerged in the solvent mixture of
dichloromethane – methanol (1:1) completely. The plant material was then left in contact
with the solvent mixture overnight and the solvent was drained off the next morning.
Next, the extracted plant material was covered in methanol and a second extraction was
performed in order to rinse off the residual dichloromethane (approx. 6h). Then the
methanol solvent was removed by suction filtration. Next, the dichloromethane –
methanol (1:1) solubles were combined with the methanol solubles and then dried by
rotary evaporation. Finally, sufficient water was added to cover the remaining plant
extract, making a second extract (aqueous), which was then stored at -20◦C. These
procedures resulted in an organic solvent extract and aqueous extract for each of the four
plants that could then be further analyzed using HPLC-ESI-MS and tested for bioactivity.
2.2 Identification of the active components from plant extracts using HPLC-ESI-MS
Once the final crude extracts are obtained, they have to be chemically screened for
the presence of active constituents. The characterization of active metabolites in complex
plant extracts requires sophisticated hyphenated techniques, which should provide good
sensitivity and selectivity as well as structural information on the constituents of interest.
For this study, the HPLC-ESI-MS technique was employed to identify the bioactive
components of the plants extracted.
17
Initially, three concentrations (1mg/mL, 0.1mg/mL, and 0.01mg/mL) of both the
organic and aqueous extracts were prepared using methanol solvent. Using a 0.2 µm
nylon filter, all the samples prepared were filtered prior to analysis.
2.2.1 Instrumentation
Analyses were conducted using an Agilent 1100 series HPLC instrument with a
C18 reversed phase column (Alltech, 50mm x 2.1mm, 3µm particle size) coupled with an
ESI-ion trap mass spectrometer (LCQ Advantage, ThermoFinnigan). A 0.5µm precolumn filter (Mac Mode Analytical) attached to the column inlet was used to remove
any residual particulate present in the HPLC solvents or samples. A gradient elution (0-4
min - 90% A, 4.00-30.00 min -90-0% A, 30.10-36.00 min – 90%A) was run with mobile
phase solvents A (1% acetic acid in nanopure water) and B (HPLC grade acetonitrile)
with a flow rate of 0.2 mL/min and injection volume of 10µL. The mass spectrometer
was operated with a scan range of 50-2000 m/z, a capillary temperature of 275°C, and a
sheath gas pressure of 20 (arbitrary units). Two analyses were carried out for each
sample, one in the positive ion mode and one in the negative ion mode. The source,
capillary, and tube lens voltages for the positive ion mode analyses were 4.5 kV, 3V and 60V, respectively, and for the negative ion mode analyses were 4.5 kV, -10 V and -50 V,
respectively. The total analysis time was 36 min.
18
2.3 Primary screening of plant extracts in vitro (Anti-proliferative activity) using
Dictyostelium cells
Once the extracts were prepared and characterized, they were initially screened for
anti-proliferative activity using Dictyostelium cells. The purpose of this bioassay was to
identify the crude plant extracts with significant inhibitory effects on cell growth in vitro.
Three concentrations (2 mg/mL, 1 mg/mL and 0.5 mg/mL) of both the organic and
aqueous extracts of all the plants were prepared using sterile technique in DMSO and
phosphate buffered saline (PBS) solution [NaCl (81%) + KCl (2%) + Na2HPO4, dibasic
(14%) + KH2PO4, monobasic (3%)] of pH 7.4. These experiments were accomplished
under the direction of Dr. Paul Steimle and were performed by his student Erica Fields in
the Department of Biology at the University of North Carolina, Greensboro.
2.3.1 Procedure for screening with Dictyostelium
Dictyostelium discoideum AX2 strain49, an axenic mutant, was used for this in vitro
cell inhibition assay. These cells were grown to confluence in 15 cm Petri dishes
containing HL5 culture medium and were harvested by centrifugation. These cells were
then used to prepare 5 x104 cells/mL cultures which were transferred into Falcon tubes.
These cell cultures were then treated with different plant extract samples. All these
cultures were grown with shaking at 185 rpm at 21°C. A positive control (lacking plant
extract sample) for normal cell growth was also grown. All the cultures prepared
contained 0.7% DMSO. An aliquot of each culture was removed at 24 h interval (up to
120 h) and the cells were counted using a hemacytometer. Growth rates for different
19
cultures were then plotted as cell number versus time. The composition of the HL5
culture medium is given below (Table 2).
Table 2 - HL5 culture medium composition (1liter)
1.)
5g Protease peptone
2.)
5g Thiotone E peptone
3.)
10g Glucose
4.)
5g Yeast extract
5.)
0.35g Na2HPO4. 7H2O
6.)
0.35g KH2PO4
All the ingredients were mixed to a final volume of 1 liter. The pH of the medium
was adjusted with HCl to 6.4 - 6.6 and was then finally autoclaved.
2.4 Separation and quantification of curcumin from turmeric extract
The main goal of this experiment was to separate curcumin from the C. longa
organic extract and further quantify the turmeric fractions containing various amounts of
curcumin using suitable methods. All the fractions obtained must then be screened using
a suitable assay of anti-proliferative activity.
20
2.4.1 Procedure
Flash chromatography over silica gel (Column dimensions, 15 × 1.5 cm) was
employed to separate the complex C. longa organic extract. Using a binary solvent
mixture of hexane and diethyl ether, a total of nine fractions of turmeric extract was
obtained. The fractions were run in the increasing amounts of diethyl ether (10%, 20%,
30%, 40%, 50%, 60%, 70%, 85% and 100%). Each of the fractions obtained was
aliquoted (1 mL) into a pre weighed centrifuge tube and the solvent was removed under
vacuum to determine the dry weight. The dried samples were redissolved and diluted
using methanol solvent to make three concentrations of each fraction (1mg/mL,
0.1mg/mL and 0.01mg/mL, expressed as mg dissolved solids per mL of solvent). Similar
samples for the original turmeric extract were also prepared.
The concentration of curcumin present in the turmeric fractions were quantified
using a standard of curcumin (Acros organics, New Jersey, USA). Stock solutions of this
standard were prepared in methanol solvent at concentrations of 0.01M and 0.03M and
then serially diluted to make standards of concentrations 1.0 × 10-4 M, 1.0 × 10-5 M, 1.0 ×
10-6 M, 1.0 × 10-7 M, 3.0 × 10-5 M and 3.0 × 10-6 M. Finally, all of the standards, along
with the turmeric fraction samples and the original extract samples were analyzed using
HPLC-ESI-MS method (negative ion mode scanning) with the same conditions as
described in Section 2.2.
21
2.5 Identification and quantification of ar-tumerone in the turmeric fractions using
HPLC-ESI-MS
The complex turmeric extract has several volatile oil components besides
curcuminoids. Among the volatile oil constituents, ar-tumerone, α-tumerone and βtumerone have been shown to induce apoptosis on various cancer cell lines16. Of these
three volatile oils ar-tumerone is the major component and is the most extensively studied
compound. So, the next step in this research would be to identify and quantify artumerone present in the turmeric fractions obtained. For this purpose HPLC-ESI-MS was
utilized with the same parameters mentioned in Section 2.2. Three different
concentrations (1mg/mL, 0.1mg/mL and 0.01mg/mL) of all the nine fractions including
the original extract were made using methanol solvent. In order to quantify ar-tumerone
present a standard ar-tumerone (Chromadex, Santa Ana, California, USA) was used. A
stock solution of 0.01 M ar-tumerone was prepared in methanol and then serial dilutions
of this solution were performed to prepare solutions with concentrations of 3.0 × 10-3 M,
3.0 × 10-4 M, 3.0 × 10-5 M, 3.0 × 10-6 M and 1.0 × 10-6 M. All these samples prepared (the
fractions and the standards) were then analyzed using HPLC-ESI-MS in the positive ion
mode.
2.6 Testing turmeric fractions for anti-proliferative activity against MCF-7 human
breast cancer cells (XTT assay)
Once the major bioactive components present in the turmeric fractions were
identified they had to be tested for in vitro anti-proliferative activity using a suitable cell
22
proliferation assay. For this purpose, the MCF-7 human breast cancer cell line was
selected. The anti-proliferative activity of the complex turmeric extract, the turmeric
fractions and the standard curcumin was tested against the MCF-7 human breast cancer
cell line using the XTT assay in a 96 well plate format50. The purpose of using MCF-7
cells for this cell viability assay is that these cells are more amenable to the 96 well plate
set up and also the results obtained from these cells should be more closely relevant to
humans.
The first task in this assay was to determine the LD50 (median lethal dose) for pure
curcumin which was determined by testing different concentrations of standard curcumin
(1µM, 3µM, 10µM, 30µM and 100µM of curcumin in 0.5% DMSO in medium) on
MCF-7 cells. Once the LD50 of curcumin was determined, turmeric fractions including
the original extract containing varying concentrations of curcumin were tested against
MCF-7 cell line, with the ultimate goal of determining which constituents of the fractions
contributed to their anti-proliferative activity.
2.6.1 Preparation of Stock Solutions
For determining the LD50 of pure curcumin a stock solution of 24,000µM (100%
DMSO) of standard curcumin was prepared. This stock solution was further diluted with
media (without phenol red) to make a solution of 1200µM (5% DMSO) of standard
curcumin. From this stock, serial dilutions were further made using media (5% DMSO)
to make solutions of five different concentrations (1000µM, 300µM, 100µM, 30µM, and
10µM). Finally, 20µL of each of these concentrations when present in a final well
23
volume of 200µL represents a final concentration of 100µM, 30µM, 10µM, 3µM, and
1µM of standard curcumin (0.5% DMSO) in each well of the 96 well plate.
Stock solutions for all the turmeric fractions (except fraction 1) including the
original extract were prepared based on the curcumin content present in the solid extracts.
Initially a stock solution of 7000µM (100% DMSO) of curcumin for all the turmeric
fractions including the original extract was prepared. This stock was further diluted with
media (without phenol red) to make a 350µM curcumin (5% DMSO) stock solution.
From this stock solution, serial dilutions were made with media (5% DMSO) to make
solutions of two different concentrations (300µM and 30µM of curcumin). Finally, 20µL
of each of these concentrations in a final well volume of 200µL yields a final
concentration of 30µM and 3µM of curcumin (0.5% DMSO). For fraction 1, which has
only ar-tumerone but not curcumin, the stock solution was prepared based on the amount
of ar-tumerone present in the solid extract. All these solutions were prepared under sterile
conditions.
2.6.2 Procedure
MCF-7 human breast carcinoma cells (ATCC), grown as a monolayer in 75cm2
canted neck flask (Corning), were maintained in 500 mL Dulbecco’s Modified Eagle
Medium (DMEM) (GIBCO) supplemented with 57 mL of 10% fetal bovine serum (FBS)
(GIBCO), 5.8 mL of 1mM sodium pyruvate (Sigma-Aldrich), 5.8 mL of 2mM Lglutamine (Sigma-Aldrich), 5.0 mL non essential amino acids (Sigma-Aldrich), 500 µL
insulin (Sigma-Aldrich) and 5.0 mL of 100mM penicillin/streptomycin (Sigma-Aldrich)
24
in a humidified atmosphere of 5% CO2 at 37°C. Once they have reached 70-80%
confluence, the culture medium was removed and the adherent cells were washed with
phosphate buffered saline (PBS, pH-7.4) (GIBCO) solution. To loosen the cells from the
flask surface, 0.5% Trypsin-EDTA (3mL) (GIBCO) was added and was diluted with 4
mL DMEM medium (with phenol red). From this stock culture of cells, cells of density
1×105cells/mL were prepared using DMEM medium (without phenol red) and were
plated into each well (180µL) in a 96 well plate (Costar 3599, Corning, NY, USA). These
cells were then incubated (at 37°C and humidified 5% CO2 atmosphere) for 2-3 days
until they reached 70% confluence. Subsequently 20µL of each sample dissolved in 0.5%
DMSO in medium was added to each well (in triplicate). DMSO (0.5%) was used as
vehicle control. Wells containing cell culture medium alone, cells in medium without
sample (negative control), and the samples in medium alone at each concentration to be
tested (blank control) were used as additional controls. The plate was shaken using a
rocking shaker (Lab-Line Instruments Inc, Melrose Pk, IL, USA) with a speed of 4 rpm
to ensure that the samples were thoroughly mixed into the media. Cells were then
incubated for 72 h at 37°C and humidified 5% CO2 atmosphere. Finally, 50µL of XTT
reagent (Sigma Aldrich) (sodium salt of 2, 3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2Htetrazolium-5-carboxyanilide + 1% phenazine methosulfate (PMS), 5mg in serum vial)
dissolved in cell culture medium with out phenol red and serum (5mL) was added to each
well of the 96 well plate and then incubated for 6 h to allow the XTT to be metabolized.
Using a rocking shaker (speed of 4 rpm) the plate was shaken to evenly distribute the dye
in the wells. The final volume of each well was 250µL. The XTT reagent combines with
25
the mitochondrial dehydrogenase enzyme present in the viable cells to form orange
colored formazan crystals. The absorbance of the orange colored formazan solution in
each well of the 96 well plate was then spectrophotometrically measured at a wavelength
of 490nm using a plate reader (POLARstar Optima, BMG LABTECH, Durham, NC).
The percentage of cells surviving and the concentration at which 50% of the cells remain
viable after 72h sample treatment (LD50) were determined. Each sample was tested in
triplicate and their mean values were taken to calculate the cell viability and LD50. Cell
viability (%) = [(As - Ab)/Av×100] where As = Absorbance of test sample well (well with
sample+cells+media), Ab = Absorbance of blank control well (well with sample+media
but no cells), Av = Absorbance of vehicle control well (well with cells+DMSO of 0.5%).
26
CHAPTER III
RESULTS AND DISCUSSION
3.1 Extraction of medicinal plants
The main aim of the preparative extraction method is the identification of the major
or bioactive compound(s) from a specific plant. The concentrations (mg of dissolved
solids per mL of solvent) of the extracts produced are shown in Table 3.
Table 3- Dissolved solid extracts produced
Plant
Organic extract
Aqueous extract
Goldenseal
219mg/mL
38.7mg/mL
Turmeric
568.8mg/mL
44mg/mL
Ginger
13.4mg/mL
39.8mg/mL
Lesser galangal
29.9mg/mL
25.4mg/mL
These weights were obtained by transferring 1mL of extracts into eppendorf tubes
and then drying them using a speedvac evaporator. All these extracts were transferred to
a suitable glass container and then stored in a refrigerator (4◦C) for subsequent analysis.
27
3.2 Major components identified from plant extracts using HPLC-ESI-MS
The organic and aqueous extracts of all the four medicinal plants were analyzed
using reversed phase high performance liquid chromatography on a C18 column and
electrospray ionization mass spectrometry method. Significant peaks were observed in
only organic extracts and were absent in aqueous extracts.
3.2.1 Analysis of Curcuma longa extract
The base peak chromatogram of C. longa organic extract shows a significant peak
at a retention time of 21.45 min (Figure 5). Figure 6 shows a series of selected ion
chromatograms for different concentrations of turmeric (Curcuma longa) organic extract
carried out in the negative ion mode. Since the phenolic group in the structure of the
major component curcumin can be easily deprotonated, the negative ion mode scanning
was selected. In selected ion chromatograms, ions with selected m/z values (those that
correspond to the masses of particular components of the samples) are plotted as a
function of time. This type of monitoring increases the selectivity for individual analytes
and improves the detection limit by decreasing the background noise. In these
chromatograms, an m/z value of 367 was selected since the molecular mass of neutral
curcumin was 368. The major molecular ion present has an m/z ratio of 367
(deprotonated curcumin) and has been identified as curcumin with reference to
literature30. In addition, the retention time was found to be similar to that of the standard
curcumin which is shown in Table 4.
28
RT: 0.00 - 36.79
NL:
1.88E7
Base Peak
F: MS
tmog
10ppmneg
19.15
755.20
100
95
90
85
80
75
21.45
366.99
70
65
R e la tiv e A b u n d a n c e
60
55
18.98
725.26
21.18
336.98
50
0.91
400.90
0.96
40
400.87
45
23.74 25.00
25.90
818.34 818.36
818.39
35
23.36
818.37
1.08
400.87
30
26.95
818.47 27.75
818.46
29.08
818.43
25
20
1.29
400.93
15
10
11.88
405.08
5
1.77
3.24
400.86 1911.26
0
0
2
29.26
818.38
16.80
367.02
4
12.89
343.02
11.50
8.59
1390.09 375.11
6
8
10
12
14
29.84
818.43
16.59
337.13
16
18
20
Time (min)
22
24
26
28
30
33.94
714.66
33.81
818.30
31.21
999.15
32
34.12
408.78
34
36
Fig 5 – Base peak chromatogram of turmeric organic extract obtained using HPLC-ESIMS
29
Table 4 – Comparison of retention times of curcumin for both standard and original
extract
Curcumin
Avg. retention time
Std. Dev (N= 3)
Standard
20.63 min
0.049
Extract
20.55 min
0.055
O
O
OMe
MeO
100
21.45
366.99
O
OH
NL:
1.28E7
m/z=
366.80-367.80
1mg/ml
80
60
a)40
20
0
22.20
366.99
16.80
367.02
1.11
367.31
2.53
366.82
6.93
367.07
10.48
367.76
14.53
366.97
22.45
366.95
17.18
367.04
16.54
366.93
25.57
366.82
26.95
366.98
31.16
367.68
100
NL:
4.69E6
m/z=
366.80-367.80
0.1mg/ml
80
b)
60
40
20
0
1.05
366.90
2.55
366.89
7.58
367.68
9.65
366.82
15.42
366.99
16.90
367.11
21.76
366.95
21.06
366.85
23.85
367.33
30.66
367.13
60
22.00
367.10
22.48
367.10
40
7.46
367.78
10.94
366.86
14.15
19.68
367.07
21.14
367.25
32.04
367.19
34.22
366.85
NL:
4.32E5
m/z=
366.80-367.80
0.01mg/ml
21.82
367.02
80
20
27.88
367.03
21.70
366.91
100
c)
35.33
367.63
21.49
367.00
27.98
367.10
28.73
366.97
32.45
367.07
36.75
367.71
0
Fig 6 – Selected ion chromatogram of turmeric organic extract obtained using HPLCESI-MS. Three concentrations were analyzed, a) 1mg/mL b) 0.1mg/mL and c) 0.01mg/mL
(expressed as mg dissolved solids/mL solvent). The m/z value of 337 was plotted, which
corresponds to mass of deprotonated curcumin (see structure in chromatogram a.)
30
It can be concluded from the above chromatogram that the peak size increases with
the increasing concentration of the extract (i.e. 0.01 mg/mL to 1 mg/mL), as would be
expected.
And also the presence of the compound curcumin was confirmed by comparing the
MS-MS spectrum of standard curcumin with that of the peak at a retention time of 21.45
min (Figure 7).
31
stdcur1 #861 RT: 22.16 AV: 1 NL: 1.02E9
T: - c ESI Full ms [ 50.00-2000.00]
367.08
1000000000
950000000
900000000
850000000
800000000
a)
750000000
700000000
650000000
Intensity
600000000
550000000
500000000
450000000
400000000
350000000
300000000
250000000
200000000
150000000
337.44
217.30
100000000
173.23
50000000
325.54
158.26
0
200
396.56 501.07 674.49 734.33
400
600
902.35 986.93
800
1000
m/z
1100.82
1295.77
1200
1390.22
1571.09
1663.17
1400
1600
1307.44 1391.71
1592.50
1822.53
1938.92
1800
2000
tmog 10ppmneg #821 RT: 21.43 AV: 1 NL: 1.19E7
T: - c ESI Full ms [ 50.00-2000.00]
367.01
11500000
11000000
10500000
10000000
9500000
9000000
8500000
8000000
Intensity
b)
7500000
7000000
6500000
6000000
5500000
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
336.96
216.95
172.99
149.00
325.49
413.26
545.33
737.22
860.15
1054.68
1140.35
1724.11 1835.09
1989.90
0
200
400
600
800
1000
m/z
1200
1400
1600
1800
2000
Fig 7 - Full scan mass spectra for (a) standard curcumin (molecular weight of 368) and
(b) the peak obtained at a retention time of 21.45 min for turmeric organic extract
32
3.2.2 Analysis of Hydrastis canadensis extract
For the analysis of goldenseal (Hydrastis canadensis) organic extract, carried out in
the positive ion mode scanning, the major molecular ion observed has a m/z ratio of 336
which was been identified as berberine with reference to literature51. The berberine
molecule, which has a quaternary nitrogen atom, exists as a cation. Hence the positive ion
mode was used. A series of selected ion chromatograms for three different concentrations
of goldenseal are shown in Figure 8. Note that without comparison to a standard,
identification of berberine is only tentative.
3.2.3 Analysis of Alpinia officinarum extract
The major molecular ions present in the selected ion chromatograms for lesser
galangal (Alpinia officinarum) organic extract (Figures 9, 10 and 11) have m/z ratios of
285, 269 and 301. These ions were tentatively identified as kaempferol, galangin, and
quercetin, respectively, with reference to literature52. This analysis was performed in the
negative ion mode.
Zerumbone53, the major component of ginger (Zingiber officinalis), could not be
detected as a single molecular ion peak using HPLC-ESI-MS because it cannot easily be
protonated or deprotonated. It can be analyzed using GCMS.
33
O
RT: 0.00 - 36.79
15.07
336.12
100
N
O
+
15.76
336.11
80
OMe
60
a)
40
20
1.58
5.78 10.25 11.91
336.02 336.98 336.28 336.20
b)
Relative Abundance
0
100
16.46
336.09
17.41 20.23
336.15 336.11
16.29
336.15
80
26.47 28.50
336.12 336.12
34.33
336.13
NL:
1.09E8
m/z=
336.00337.00 F:
MS
gsog1ppm
40
20
1.69
4.82
336.11 336.31
0
100
17.86 20.66
12.16 15.71
336.13 336.12
336.13 336.14
16.40
336.11
80
c)
OMe
16.80
336.10
60
NL:
4.00E8
m/z=
336.00337.00 F:
MS
gsog0pt1ppm
28.27
336.18
34.45
336.16
NL:
3.18E7
m/z=
336.00337.00 F:
MS
gsog10ppm
16.66
336.13
60
40
1.76
336.42
20
0
0
5.68
336.66
5
13.56 15.72
336.03 336.03
10
15
17.69
21.87
336.14 336.13
20
Time (min)
28.15
336.20
25
30
34.53
336.24
35
Fig 8 – Selected ion chromatogram of m/z 336 from goldenseal organic extract
obtained using HPLC-ESI-MS for three different concentrations a) 1mg/mL b)
0.1mg/mL c) 0.01mg/mL. The m/z value of 336 correlates with that of berberine
(structure shown in chromatogram a.)
34
80
15.9
285.1
4
9
60
a)
O
O
15.86
285.15
RT: 0.00 - 36.79
100
40
OH
19.11
285.15
20
0.20
285.37
0
5.21
284.83
10.72
285.10
10.00
285.03
20.00
285.10
24.32
285.24
26.37 28.71
285.33 284.98
33.15
285.63
16.08
285.16
Relative Abundance
100
15.01
285.26
OH
OH
O
36.73
284.94
MS
80
19.30
285.11
19.2
285.1
0
9
60
b)
40
5.16
3.43
285.74
285.04
20
8.74
285.64
10.72
284.96
25.76
285.21
22.17
285.18
28.30
285.05
15.93
285.40
29.21
285.11
0
24.29
284.94
100
31.18
285.15
36.27
285.15
30.88
284.93
80
60
c)
40
0.97
285.52
3.73
285.63
6.64
285.30
20
18.3
16.21 285.0
9
10.37 13.05
285.17 7
285.30
284.87
19.53
285.09
24.02
285.29
25.18
284.95
28.72
285.27
28.49
285.79
29.82
285.04
36.01
284.97
0
0
5
10
15
20
25
30
35
Time
(min)
Fig 9 – Selected ion chromatogram of A. officinarum organic extract for m/z value of
285 obtained using HPLC-ESI-MS. a) 1mg/mL b) 0.1mg/mL c) 0.01mg/mL. The m/z value
of 285 corresponds to the mass of deprotonated kaempferol
35
O
O
18.74
269.19
100
80
a)
OH
60
OH
40
20
0
O
19.72
269.17
0.41
268.95
5.4
8
268.9
7
9.24
269.07
10.56
269.27
15.63
269.77
20.27
269.12
17.12
269.16
22.38
269.16
25.99
269.17
27.60
269.31
30.36
269.10
26.53
269.07
29.61
269.11
32.21
269.45
36.55
269.08
18.92
269.17
100
80
b)
60
40
19.37
269.19
20
0
0.10
268.95
2.76
269.07
6.56
269.3
2
10.22
269.14
13.32
269.38
14.52
269.07
21.54
269.03
18.04
268.95
25.61
269.58
31.74
269.37
19.11
269.23
100
80
c)
60
40
19.53
269.19
20
1.71
268.84
4.40
269.08
8.66
268.87
10.01
269.30
12.21
269.71
15.39
18.85
269.04
23.77
269.12
27.54
269.1
6
28.39
269.55
30.80
269.29
35.51
268.87
0
Fig 10 – Selected ion chromatogram of A. officinarum organic extract for m/z value of
269 obtained using HPLC-ESI-MS. a) 1mg/mL b) 0.1mg/mL c) 0.01mg/mL. The m/z value
of 269 corresponds to the mass of deprotonated galangin
36
O
O
19.11
301.05
100
a)
0.57
301.64
5.40
300.97
8.92
300.87
20.82
301.13
14.30
301.15
10.62
301.13
17.30
301.04
0
Relative Abundance
OH
80
26.87
301.28
28.16
300.89
30.36
301.41
36.66
300.97
19.50
301.07
19.25
301.24
60
23.50
301.72
40
0.57
301.67
22.56
300.91
19.30
301.17
100
20
O
OH
20.35
301.06
40
b)
OH
20.02
301.34
60
20
OH
19.23
301.17
80
3.93
301.25
5.86
300.86
9.60
300.96
11.98
301.10
15.39
300.98
0
18.47
301.28
25.11
301.77
19.58
301.20
100
30.29
300.82
32.11
301.25
36.71
301.78
26.85
301.63
80
60
c)
40
0.23
301.65
3.36
301.76
7.98
300.87
14.71
301.17
17.00
300.94
19.50
301.18
19.69
300.97
23.42
301.23
33.98
301.79
36.50
300.83
20
0
Fig 11 – Selected ion chromatogram of A. officinarum organic extract for m/z value of
301 obtained using HPLC-ESI-MS. a) 1mg/mL b) 0.1mg/mL c) 0.01mg/mL. The m/z value
of 301 corresponds to the mass of deprotonated quercetin
37
3.3 Anti-proliferative activity of plant extracts on Dictyostelium cells
Organic and aqueous extracts of all the four medicinal plants were tested for antiproliferative activity using Dictyostelium cells. All of the aqueous extracts and the
goldenseal organic extract showed no inhibition of cell growth. Organic extracts of
Curcuma longa, Zingiber officinalis, and Alpinia officinarum demonstrated significant
concentration dependent anti-proliferative activity as shown in Tables 5, 6, and 7 and
Figures 12, 13, and 14, respectively. From the data obtained, it was found that the
organic extracts of C. longa, A. officinarum, and Z. officinalis have shown similar antiproliferative activity profiles with Dictyostelium cells.
38
Table 5 – Dictyostelium cell growth in the presence of different concentrations of C.
longa organic extract
CELL NUMBER
Days
Turmeric 17
µg/mL
Turmeric 8.5
µg/mL
Turmeric 4.2
µg/mL
DMSO
Control
0
5
5
5
5
1
6
7
9
7
4
3
19
95
325
5
17
7
210
400
4
Cell Density (cells/mL×10 )
450
400
350
300
Turmeric 17µg/mL
250
Turmeric 8.5µg/mL
200
150
Turmeric 4.2µg/mL
DMSO Control
100
50
0
0
2
Days
4
6
Fig 12 – Effect of C. longa organic extract on Dictyostelium cell growth
39
Table 6 – Effect of different concentrations of A. officinarum organic extract on
Dictyostelium cell count
CELL NUMBER
Days Alpinia off. 17
µg/mL
Alpinia off.8.5
µg/mL
Alpinia off.4.2
µg/mL
DMSO
Control
0
5
5
5
5
1
4
6
11
18
2
4
5
22
23
3
9
7
24
52
4
3
8
110
240
4
Cell Density (cells/mL×10 )
300
250
200
Alpinia off. 17µg/mL
Alpinia off. 8.5µg/mL
150
Alpinia off. 4.2µg/mL
100
DMSO Control
50
0
0
2
Days
4
6
Fig 13- Effect of A. officinarum organic extract on Dictyostelium cell growth
40
Table 7 – Dictyostelium cell growth in the presence of Z. officinalis organic extract
CELL NUMBER
Ginger 17
µg/mL
Ginger 8.5
µg/mL
Ginger 4.2
µg/mL
DMSO Control
0
5
5
5
5
1
3
1
16
18
2
1
24
32
23
3
3
38
53
52
4
3
100
220
240
Days
4
Cell Density (cells/mL×10 )
300
250
200
Ginger 17µg/mL
Ginger 8.5µg/mL
150
Ginger 4.2µg/mL
100
DMSO Control
50
0
0
2
Days
4
6
Fig 14 – Effect of Z. officinalis organic extract on Dictyostelium cell growth
41
3.4 Quantitation of curcumin in turmeric fractions
Given the promising results and therapeutic significance of turmeric (C. longa), this
plant was chosen as the subject of further study. The C. longa extract was fractionated
over silica gel using a binary solvent mixture of hexane and diethyl ether (see Methods
Section 2.4) and the resulting fractions were analyzed for concentration of curcumin.
From the data obtained, it was evident that all the turmeric fractions contained curcumin
(ion with an m/z ratio of 367) except fraction 1. The concentration of this component in
each fraction was determined from the calibration curve shown in Figure 15. This
calibration curve was plotted as log of concentration versus the log of peak area of the
selected ion chromatogram for the ion with m/z 367 and retention time 20.60 min.
-7.5
-6.5
-5.5
-4.5
8.5
8
7.5
7
6.5
6
5.5
5
log plot peak area
y = 0.9429x + 12.593
R2 = 0.9982
9.5
9
-3.5
log conc. (M)
Fig 15 - Calibration curve for curcumin
42
From the calibration curve, the concentrations of the turmeric fractions collected
were obtained. Fraction 8 has the highest concentration of curcumin, whereas fraction 6
has the least curcumin concentration (Figure 16). Table 8 shows the concentrations of
curcumin in the turmeric fractions collected. Standard deviations were calculated from
statistical analysis of the calibration curve data.
Table 8 – Concentrations of curcumin from different fractions of C. longa organic extract
including the original extract (Wt% is mg curcumin per mg dissolved solids in extract ×
100)
Fraction no.
Conc. (mg/mg) +/- Std. Dev.
Wt% +/- Std. Dev
2 (20% Diethyl Ether)
0.076 ± 0.021
7.6 ± 2.1
3 (30% Diethyl Ether)
0.114 ± 0.031
11.4 ± 3.1
4 (40% Diethyl Ether)
0.034 ± 0.009
3.4 ± 0.9
5 (50% Diethyl Ether)
0.049 ± 0.014
5.0 ± 1.4
6 (60% Diethyl Ether)
0.021 ± 0.006
2.1 ± 0.6
7 (70% Diethyl Ether)
0.200 ± 0.055
20.0 ± 5.5
8 (85% Diethyl Ether)
0.418 ± 0.117
42.0 ± 12.0
9 (100% Diethyl Ether)
0.151 ± 0.041
15.1 ± 4.1
Original extract
0.118 ± 0.032
12.0 ± 3.2
1 (10% Diethyl Ether)
43
wt% of curcumin
60.000
50.000
40.000
30.000
20.000
10.000
0.000
1
2
3
4
5
6
7
8
Fraction no.
9
org
ext
Fig 16 - Concentrations of curcumin in the turmeric extract fractions determined by
HPLC-ESI-MS
3.5 Other curcuminoids identified in the turmeric fractions
After determining the curcumin concentrations in the turmeric fractions, the next
step in this phytochemical screening would be to identify and quantify the other two
curcuminoids present. Demethoxycurcumin (MW - 338) and bisdemethoxycurcumin
(MW - 308) were identified in all the turmeric fractions except in fraction 1. The relative
amounts of these curcuminoids in the various fractions were then determined based on
the peak areas obtained for the selected ion chromatograms for the ions with m/z values
of 337 and 307 (Figure 17 and Figure 18). Absolute quantification of these compounds
was not possible given that standards were not commercially available. As with
curcumin, fraction 8 has the highest concentration of the curcuminoids.
44
40000000
35000000
30000000
25000000
Plot Peak Area 20000000
15000000
10000000
5000000
0
1
2
3
4
5
6
7
8
9
Fraction no.
org
ext
Fig 17 – Relative amounts of demethoxycurcumin present in the turmeric fractions
20000000
18000000
16000000
14000000
12000000
Plot
Peak Area 10000000
8000000
6000000
4000000
2000000
0
1
2
3
4
5
6
7
8
9
org
ext
Fraction no.
Fig 18 – Relative amounts of bisdemethoxycurcumin present in the turmeric fractions
45
3.6 Identification and quantitation of ar-tumerone in the turmeric fractions
All the turmeric fractions including the original extract were analyzed for the
presence of volatile oil constituents using positive mode HPLC-ESI-MS method.
Significant peaks were observed only in fraction 1 and original extract samples. Volatile
oil components like ar-tumerone, curlone and α-tumerone were all identified with
reference to literature35 (Figure 19).
RT: 0.00 - 36.79
NL:
1.95E8
m/z=
216.00220.00 F:
MS
ORGEXT1
MG
26.04
219.00
100
curlone
(MW – 218)
24.62
216.96
24.54
216.99
95
90
85
80
ar-tumerone
(MW – 216)
75
70
26.23
219.02
R elative Abundance
65
60
α-tumerone
(MW – 218)
26.36
219.03
55
50
45
40
35
30
25
20.68
217.06
20
20.32
217.07
15
10
5
1.77
2.74
216.03 216.83
0
0
2
4
9.99 11.21
216.84 217.02
5.98
218.84
6
8
10
12
14.40
217.00
14
15.39
216.99
16
20.17
217.02
18
20
Time (min)
20.87
217.07
21.18
217.05
22.62
217.12
22
27.06
219.01
27.35
218.96
27.94
29.22
217.03 219.04 31.12
217.01
24
26
28
30
32
34.87
218.71
34
36
Fig 19 – Total ion chromatogram (mass range 216-220) of the turmeric original extract
obtained using positive mode HPLC-ESI-MS. Three volatile oil components of m/z values
of 217 and 219 were been identified
46
The peak with an m/z value of 219 corresponds to the presence of two volatile oil
constituents curlone and α-tumerone. Since they have the same m/z values they co-elute
together with curlone being the first and α-tumerone the latter35. Since ar-tumerone was
the compound of interest, selected ion chromatogram of this compound (MW-217) was
plotted. Figures 20 and 21 represent the selected ion chromatograms of m/z value of 217
for three different concentrations of fraction 1 and original extract samples. The peak
with an m/z value of 217 represents the protonated form of neutral ar-tumerone35. The
presence of this compound ar-tumerone was also confirmed by comparing the retention
time with that of the standard ar-tumerone (Table 9). The tentative structure for
protonated ar-tumerone compound is shown in Figures 20 and 21.
47
RT: 0.00 - 36.80
NL:
2.08E8
m/z=
216.50217.50 F:
MS f11mg
24.71
216.94
+
100
OH
80
a)
60
40
20.52
217.08
20
1.83
2.83
217.09 216.96
0
5.97
7.99
9.36
216.92 217.09 216.94
12.20
216.95
14.52
216.98
17.35 18.83
216.90 216.96
21.08
216.99
R e la tive A b u n d a nce
80
24.98
216.94
60
26.24
216.98
40
20
2.28
6.39
5.20
216.67 216.98 217.12
0
9.25 11.17
216.93 216.79
26.52
216.94
20.45 20.91
14.45 16.69 18.81 217.07 217.01
216.98
216.96 216.81
28.66 31.52
216.92 216.94
34.84
217.34
NL:
1.53E6
m/z=
216.50217.50 F:
MS
f1001mg
24.60
216.98
100
c)
NL:
1.71E7
m/z=
216.50217.50 F:
MS f101mg
24.65
216.92
100
b)
26.05
26.74 28.63 31.71 34.77 36.76
217.04
216.98 216.98 217.10 216.87 216.94
24.89
216.90
26.18
216.92
80
60
26.47
216.92
26.60
216.93 28.69
217.00
40
20
5.21
5.90
9.34
216.92 217.00 216.77
2.44
216.59
11.87
216.88
14.08 15.76
216.79 216.78
20.71
19.17 217.01 21.36
216.80
216.98
31.91
216.74
34.87
217.29
0
0
2
4
6
8
10
12
14
16
18
20
Time (min)
22
24
26
28
30
32
34
36
Fig 20 – Selected ion chromatogram of m/z value of 217 obtained using positive mode
HPLC-ESI-MS for three different concentrations of turmeric fraction 1. a) 1mg/mL b)
0.1mg/mL c) 0.01mg/mL. The m/z value of 217 corresponds to the mass of protonated
form of ar-tumerone
48
RT: 0.00 - 36.79
+
100
24.62
216.96
OH
NL:
1.52E8
m/z=
216.50217.50 F:
MS
ORGEXT1
MG
80
a)
24.96
216.96
60
40
20
15.39
7.32
10.36 11.34 14.40 216.99
216.72 217.33 217.01 217.00
1.80
2.74
216.98 216.83
0
20.17
217.02
20.68
217.06 21.18
217.05
24.61
216.93
100
R e la tive A b u n d a n c e
b)
24.53
216.92
80
NL:
9.17E6
m/z=
216.50217.50 F:
MS
orgext01mg
24.84
216.94
60
26.29
216.95
40
20
1.62
216.94
0
c)
26.15
26.74 28.64 30.48 34.87 36.77
217.10
217.02 216.99 217.04 216.93 216.88
4.49
216.93
15.42
14.44 216.98
216.98
10.04
217.06
6.78
217.02
20.70
20.26 217.03 21.20
217.13
216.95
26.42
216.97 28.41 30.41
216.90 216.95
34.83
216.88
24.54
217.03
100
NL:
6.47E5
m/z=
216.50217.50 F:
MS
orgext001m
g
24.85
217.02
80
24.43
216.89
60
40
2.10
216.90
20
3.29
216.89
6.26
216.94
12.35
9.36 11.67
217.16
217.14 216.93
15.75
216.96
18.39
216.97
20.65
217.07
25.99
217.03
34.78
217.34
26.30
217.03
27.89 29.23
217.04 216.86 32.34 34.68
217.45
217.03
24.27
217.22
0
0
2
4
6
8
10
12
14
16
18
20
Time (min)
22
24
26
28
30
32
34
36
Fig 21 – Selected ion chromatogram of turmeric original extract obtained for an m/z
value of 217 using positive mode HPLC-ESI-MS. Three different concentrations of a)
1mg/mL b) 0.1mg/mL c) 0.01mg/mL were analyzed. The m/z value of 217 corresponds to
the mass of protonated ar-tumerone
Table 9 – Comparison of retention times for standard ar-tumerone and the ion with m/z
value of 217 in the turmeric original extract
Ar-tumerone
Avg. retention time
Std. dev (N= 3)
Standard
22.86 min
0.046
Turmeric extract
22.90 min
0.098
49
Figure 22 shows the MS-MS spectra of standard ar-tumerone and the peak obtained
at a retention time of 24.62 min for turmeric original extract.
stdar-tum1mg #931 RT: 24.53 AV: 1
T: + c ESI Full ms [ 50.00-2000.00]
216.95
NL: 4.14E8
400000000
380000000
360000000
340000000
320000000
300000000
280000000
Intensity
a)
260000000
240000000
220000000
200000000
180000000
160000000
140000000
432.64
120000000
100000000
80000000
60000000
40000000
20000000
0
161.02
233.78
200
336.00
450.10
400
564.12
758.37
600
800
941.22
1517.79
1306.60
1070.45
1000
m/z
1200
1400
1598.94
1600
1811.07 1901.35
1800
2000
orgext1mg #933 RT: 24.56 AV: 1 NL: 1.35E8
T: + c ESI F ull ms [ 50.00-2000.00]
216.96
135000000
130000000
125000000
120000000
115000000
110000000
105000000
100000000
95000000
90000000
b)
85000000
80000000
Intensity
75000000
70000000
65000000
60000000
55000000
50000000
45000000
40000000
35000000
30000000
25000000
20000000
15000000
10000000
5000000
716.28
203.17
233.98
432.53
586.98
758.33
985.36
1126.55
1356.46
0
200
400
600
800
1000
m/z
1200
1400
1503.24
1706.23 1826.14
1600
1800
1952.25
2000
Fig 22 – Full scan mass spectra of (a) standard ar-tumerone (molecular weight of 216)
and (b) the peak at a retention time of 24.62 min for turmeric original extract
50
Further, the concentration of ar-tumerone in fraction 1 and original extract was
determined from the calibration curve of standard ar-tumerone as shown in Figure 23.
10.5
y = 0.9009x + 12.459
2
R = 0.9997
10
log plot peak area
9.5
9
8.5
8
7.5
7
6.5
-6.5
-6
-5.5
-5
-4.5
-4
-3.5
-3
-2.5
-2
log conc. (M)
Fig 23 – Calibration curve for ar-tumerone obtained by plotting log of concentration vs.
log of peak area of the selected ion chromatogram for the ion of m/z 217
Based on the calibration curve data, the concentrations of ar-tumerone in fraction 1
and turmeric original extract were calculated as shown in Table 10 and Figure 24.
51
Table 10 – Concentrations of ar-tumerone present in fraction 1 and turmeric original
extract (wt% is mg of ar-tumerone per mg of dissolved solid extract × 100)
Sample
Conc. (mg/mg) +/- Std. Dev.
Wt% +/- Std. Dev
Fraction 1
0.078 ± 0.004
7.8 ± 0.4
Turmeric original extract
0.028 ± 0.001
2.8 ± 0.1
9.00
wt% of ar-tumerone
8.00
7.00
6.00
5.00
4.00
3.00
2.00
1.00
Fraction#1
Original
extract
0.00
Fig 24 – Concentrations of ar-tumerone in turmeric fraction 1 and original extract
determined by positive mode HPLC-ESI-MS
52
3.7 XTT assay results
In the present study, the anti-proliferative activity of all the turmeric fractions
including the original extract was evaluated using MCF-7 human breast cancer cells. This
was accomplished by measuring the viability of cells using the XTT assay (see Methods
Section 2.6).
Table 11 and Figure 25 demonstrate the MCF-7 cell growth in the presence of
different concentrations of standard curcumin for 72h duration. The relative absorbance
at 490nm (% of vehicle control) was plotted against concentration of curcumin. Only
viable cells metabolize the XTT reagent and produce orange colored formazan crystals
which absorb at 490nm. Therefore, the absorbance measured is proportional to the total
number of viable cells. Standard curcumin inhibited the MCF-7 cell proliferation in a
dose dependent manner. The LD50 of curcumin was found to be 10µM.
53
Table 11 – MCF-7 cell growth in the presence of different concentrations of standard
curcumin at exposure time of 72h
Concentration (µM)
Cell viability (%) ± SD
0
100 ± 5.3
1
74.7 ± 3.8
3
61.2 ± 4.3
10
51.9 ± 3.2
30
25.0 ± 8.3
100
22.3 ± 6.0
Relative Absorbance
(% of control)
120
100
80
60
40
20
0
0
20
40
60
80
100
120
Conc. of curcumin (µM)
Fig 25 – Effects of different concentrations of standard curcumin on MCF-7 cell
proliferation. Relative Absorbance (% of vehicle control) vs. Concentration of curcumin
was plotted to determine the LD50 of curcumin which was found to be 10µM
54
Once the LD50 for curcumin against the MCF-7 cells had been determined, two
different concentrations (3 and 30µM curcumin) of all the turmeric fractions were tested
against MCF-7 cells using the XTT assay. The original extract and standard curcumin
were also included in the assay. The approach used for testing these fractions was
different than that typically employed for anti-proliferative screening of natural products.
Typically, fractions were tested according to their dry weight, i.e. at concentrations of 2
and 20µg/mL. This approach is very useful for identifying the most active fraction from
amongst a set of fractions (traditional bioassay guided fraction). However, it is not
particularly useful for situations such as this one where all of the fractions contain a
major active component (in this case curcumin) and the desired result is information
about whether other minor constituents contribute also to the activity. To address the
question of whether or not constituents other than curcumin contributed to the activity of
the extract and fractions, a novel approach was employed. Relying on the quantitative
analysis of curcumin in the isolated fractions (Table 8), each fraction (except fraction 1)
was normalized to the same concentration of curcumin. Because fraction 1 did not
contain curcumin, this fraction was tested at concentrations of 4 and 40µM of artumerone, instead of as concentration of curcumin. By testing the fractions all with the
same concentration of curcumin, it is possible to directly compare the results obtained for
the fractions to those for standard curcumin alone at the same concentration. Any
fractions that contain constituents that contribute (either additively or synergistically) to
the activity of the fractions will show greater activity than curcumin alone at the same
concentration. Any fractions that contain compounds that suppress the activity of the
55
curcumin will show less activity than curcumin alone at the same concentration. Finally,
if the activity of the fractions is due solely to the presence of curcumin, these fractions
will exhibit the same dose-response behavior as curcumin alone.
The results of the MCF-7 cell assay with the individual C. longa fractions are
shown in shown in Table 12 and Figure 26. Our data indicate that all the turmeric
fractions and the complex turmeric extract containing curcumin exhibited similar potency
(Figure 27). All the turmeric fractions (excluding fraction 1) including the original
extract showed anti-proliferative activity similar to that of the standard curcumin.
Fraction 1, which contained ar-tumerone and other volatile constituents but not curcumin,
was found to be less effective (since the concentrations of this fraction used were 4 and
40µM of ar-tumerone). The complex turmeric extract which contains all the
curcuminoids and ar-tumerone showed activity similar to that of the commercially
available curcumin compound. All these results suggest that for this assay there is no
major synergistic or additive activity observed for the various components present in the
turmeric samples. Our results also suggest that the overall anti-proliferative activity of the
turmeric extract is mainly due to the presence of the major component curcumin. Fraction
1 of the turmeric extract contained no curcumin but still had anti-proliferative activity;
therefore, ar-tumerone and/or other volatile constituents may contribute somewhat to the
anti-proliferative activity of C. longa. However, at the two concentrations of the complex
extract tested, ar-tumerone was only present at concentrations of 9µM and 0.9µM.
Therefore, its concentration was likely too low to result in significant activity.
56
Table 12 – Cell viability in the presence of different concentrations of turmeric fractions,
original extract and standard curcumin at 72h exposure time ± SD
% Cell viability ± SD
3µM
30µM
Fraction 1
91.9 ± 0.3
78.7 ± 4.9
Fraction 2
87.1 ± 5.1
54.7 ± 0.1
Fraction 3
84.6 ± 3.4
57.0 ± 6.5
Fraction 4
86.7 ± 3.9
56.5 ± 7.2
Fraction 5
83.8 ± 0.6
55.1 ± 0.4
Fraction 6
79.8 ± 3.1
56.5 ± 9.7
Fraction 7
85.0 ± 7.3
54.4 ± 0.8
Fraction 8
77.4 ± 4.1
43.5 ± 11.7
Fraction 9
86.4 ±3.8
55.8 ± 0.2
Original extract
85.6 ± 4.6
55.7 ± 1.5
Std curcumin
83.3 ± 9.2
55.6 ± 2.5
57
120
R e la tiv e a bs orba nc e
(% of c ontrol)
100
F1
F2
80
F3
F4
60
F5
F6
40
F7
F8
20
F9
Org ext
0
Std curc
0
3
6
9
12
15
18
21
24
27
30
33
Conc. of curcumin (µM)
Figure 26 – MCF-7 cell viability in the presence of different concentrations of turmeric
fractions, original extract and standard curcumin
58
% r ed u c tio n in ce ll via b ility at 30 u M
70
60
50
40
30
20
10
0
F1
F2
F3
F4
F5
F6
F7
F8
F9
Org
ext
Std
curc
Figure 27 – The percent reduction in MCF-7 cell viability at 30µM for all the turmeric
fractions including the original turmeric extract and standard curcumin. % Cell viability
reduction = [100 - (As – Ab)/Av×100%] where As = Absorbance of test sample well (well
with sample+cells+media), Ab = Absorbance of blank control well (well with sample +
media but no cells), Av = Absorbance of vehicle control well (well with cells+DMSO of
0.5%)
59
CHAPTER IV
CONCLUSIONS AND FUTURE SCOPE OF THIS RESEARCH
One of the major goals of our research group is to determine how the complex
mixtures of plant compounds contribute to the over all biological activity of the plant
extracts. In the case of this research, an approach was developed that allowed for
investigation of the contributions of multiple, potentially bioactive compounds to the
anti-proliferative activity of C. longa. A simple and rapid HPLC-ESI-MS method was
employed for the phytochemical characterization of extracts and fractions from this plant,
and was successfully applied for both qualitative and quantitative analysis of several
major constituents. A novel approach relying on the quantitative analysis of the fractions
was then used to test these fractions for anti-proliferative activity against the MCF-7
human breast cancer cell line. With this approach, it was possible to quickly determine
that curcumin alone was primarily responsible for the overall anti-proliferative activity of
the complex turmeric extract. Some anti-proliferative activity was also observed for a
fraction containing ar-tumerone and other volatile constituents, but these constituents did
not appear to contribute significantly to the overall anti-proliferative activity of the
complex C. longa extract. While specific synergistic or additive interactions were not
identified for C. longa in this study, the novel approach developed here could certainly be
employed in the future for the investigation of other medicinal plants with potentially
synergistic or additive activity.
60
This is an exciting area of research which our research group has focused on. The
main purpose of this study was to identify the isolated turmeric fractions that have shown
significant in vitro anti-proliferative activity. This is not the end of this research. There is
still more investigation that needs to be done. Further studies must be designed to gain
deeper understanding of the actual content in the isolated turmeric fractions. Fractions
that showed significant activity will be subjected to further isolation using flash
chromatography and/or preparatory scale HPLC. Ultimately, we seek this experimental
approach to determine which specific fractions of turmeric are responsible for its antiproliferative activity. Once the isolated active components of the extract are identified,
they have to be further studied using Dictyostelium cell model in order to gain more
insight into the mechanism of action. Since the exact mechanism of anti-proliferative
activity of curcumin is not clearly understood, the outcomes of this Dictyostelium cell
assay would prove highly beneficial.
61
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