3. Shobha, et al.: Anti‑cancer activity of Momordica charantia
International Journal of Health & Allied Sciences • Vol. 4 • Issue 4 • Oct-Dec 2015 211
chronic inflammatory diseases. In recent years, research
on Momordica charantia (MC) has re‑emerged for its
anti‑oxidant and anti‑cancer properties.
Cervical cancer is leading cancer in the Indian women and
second most common cancer in women worldwide. The
worldwide incidence of cervical cancer is approximately
510,000 new cases annually, with approximately
288,000 deaths worldwide.[1]
Breast cancer is the second
most common cancer in the Indian women, the incidence
is more in urban than rural women and more prevalent in
higher socioeconomic groups. Over 100,000 new breast
cancer patients are estimated to be diagnosed annually in
India.[2]
A recent report by the Indian Council of Medical
Research predicts the number of breast cancer cases in
India to rise to 106,124 in 2015 and to 123,634 in 2020.[3]
People who are obese or overweight have a greater chance
of developing breast cancer than those who are normal
weight. In addition, women who consume high‑fat diet
have increased the risk of breast cancer. Although, early
diagnosis of both cancers often leads to complete cure of
disease in India, majority of cases present late in advanced
stages, in these cases therapies such as surgical intervention,
chemotherapy, and radiation are often not sufficient to
tackle the disease, thus needing other prevention‑related
or nonconventional therapeutic strategies. Hence, there
is a need for better options for therapy and prevention of
the disease.
MC or bitter melon is a tropical and subtropical creeping
plant, widely grown in Asia, Africa, and the Caribbean
for its edible fruit. The fruit is recommended in ancient
Indian and Chinese medicine for the prevention and
treatment of diabetes.[4,5]
Physiological benefits include
hypoglycemia, hypolipidemia, anti‑viral, anti‑bacterial,
immunomodulatory, and anti‑carcinogenic effects.[6]
The
anti‑cancer properties of MC are recently elucidated.
Many researchers have found that treatment of MC related
products in a number of cancer cell lines induces cell cycle
arrest and apoptosis without affecting normal cell growth.
MC derivative Alpha‑eleostearic acid act by decreasing
cell proliferation, G (2)‑M block in the cell cycle, increase
apoptosis on human breast cancer cell lines[7]
, and increase
apoptosis in leukemia and colon cancer cell lines.[8]
Oral
administrationof Momordicacharantiaextract (MCE)inhibits
prostate cancer progression in transgenic adenocarcinoma
of mouse prostate mice by interfering cell cycle progression
and proliferation as evidenced by reduced expression of
proliferating cell nuclear antigen, and poly (ADP‑ribose)
polymerase (PARP cleavage).[9]
MC treatment in head and
neck squamous cell carcinoma (HNSCC) cell lines Cal27,
JHU‑29, and JHU‑22 cells, showed reduced phosphoStat3,
c‑myc and Mcl‑1 expression, downstream signaling
molecules of c‑Met, modulated the expression of key cell
cycle progression molecules leading to halted cell growth
and also bitter melon feeding in mice bearing HNSCC
xenograft tumor showed inhibition of tumor growth and
c‑Met expression.[10]
Studies have used a part of MC like seed or pulp of the fruit
and, the extraction techniques and evaluation of the exact
concentration to be used is not clearly elucidated. Our aim
of the study is to estimate the total phenol content (TPC)
of the ethanolic extract of Momordica charantia (EEMC)
whole fruit in different percentage of ethanol (50%, 70%,
and 100%) and to estimate the cytotoxic activity of these
extracts against cell lines representing carcinomas of cervix
and breast (HeLa and MCF‑7).
MATERIALS AND METHODS
The study was conducted in the Centre of Excellence in
Molecular Biology and Regenerative Medicine, Department
of Biochemistry, JSS Medical College, JSS University,
Mysore.
Preparation of ethanolic extract of Momordica
charantia
MC fruit was purchased from a co‑operative horticulture
outlet and then washed in distilled water, weighed, and
paste was prepared of whole fruit using a regular household
mixer without adding any additional water. The paste
was then lyophilized using freeze‑dryer (Alpha 2‑4 LD
Plus from Christ, GmbH) and the powder obtained was
stored at −80°C in airtight plastic container. Different
percentage (50%, 70%, and 100%) of EEMC was obtained
using 50 g of this lyophilized powder.
Fifty grams lyophilized powder of MC was mixed with 50%
ethanol till the whole powder were completely covered with
the solvent, in stoppered container and kept overnight at 4°C
and were subjected to maceration using magnetic stirrer the
next day. After 3 h of magnetic stirring, the solvent mixture
was centrifuged, and the supernatant was collected in a
brown bottle. The pellet is remaining at the bottom of the
centrifuge tube, i.e., marc was again collected into another
stoppered container and again subjected for extraction using
magnetic stirrer. This extraction procedure was carried
out for 3 times with 50% solvent. Finally, the complete
solvent mixture was filtered using Whatmann’s filter paper
number‑1 and stored at −20°C. This was followed by
sequential and gradient extraction with 70% ethanol and
finally 100% absolute ethanol. The extracts were preserved
in a stoppered brown bottle and stored at −20°C until used
for further analysis.
4. Shobha, et al.: Anti‑cancer activity of Momordica charantia
International Journal of Health & Allied Sciences • Vol. 4 • Issue 4 • Oct-Dec 2015212
The obtained different percentage solutions of EEMC were
thenconcentratedusingRotovaporR‑215 (Buchi,Switzerland)
which was later filtered using Whatmann’s filter paper
number‑1 and stored at −20°C covered with aluminum foil.
The concentrated extracts were subjected for lyophilization
using freeze‑dryer (Alpha 2‑4 LD Plus from Christ, GmbH)
which was done by dehydrating all the 50%, 70%, and 100%
concentratedextractscompletelyatreducedpressureafterbeing
frozenat −80°C.Oncecompletelydehydrated,theconcentrated
lyophilized powder extracts were preserved at −80°C.
For further analysis, EEMC stock was prepared by
dissolving the lyophilized powder in phosphate buffer
saline.
Estimation of total phenol content of ethanolic
extract of Momordica charantia using
Folin–Ciocalteu method
TPC of the extracts were measured using
Folin–Ciocalteu (F‑C) method.[11]
All samples and standards
were prepared and measured in triplicate. Gallic acid (Sisco
Research Laboratories Pvt. Ltd.) was used as a standard.
Working stock standard range was fixed at 5 ug –60 µg/µl.
50%, 70% and 100% EEMC were taken in the volume of
100 µl and made up to 1 ml with absolute ethanol. To this,
1 ml of F‑C reagent (Merck Specialities Private Limited) and
0.8 ml of 4% ‑ NaHCO3
was added and incubated along with
standards for 30 min in dark at room temperature. Finally,
absorbance maxima recorded at 760 nm using UV‑Visible
Spectrophotometer (Eppendorf India Ltd.). TPCs of 50%,
70%, and 100% ethanolic extracts were expressed in terms of
Gallic acid equivalents (GAEs) from the standard calibration
curve and percentage total phenol were obtained by back
calculating for dried powdered plant materials and expressed
as percentage gram weight (% w/w).
Cell culture
Cervical and breast cancer cell lines (HeLa and MCF‑7,
respectively,) were procured from National Center for Cell
Sciences, Pune, India. Cells were cultured in Dulbecco’s
modified eagle medium (DMEM) media supplemented
with 10% fetal bovine serum, 1% glutamine, and 1%
Penicillin‑Streptomycin (Gibco®
by Life Technologies) in
an adherent tissue culture plate at 37°C in a humidified
incubator containing 5% CO2
. 96 wells microtiter plate
were seeded with 5 × 103
cells per well and incubated again
in a humidified atmosphere with 5% CO2
at 37°C in an
incubator. When the seeded plates achieved confluency,
the cells were treated with different percentage of EEMC
in different concentrations.
Treatment of cells with ethanolic extract of
Momordica charantia
Based on the TPC serial dilutions were done to get
concentrations ranging from 25 µg/ml to 0.195 µg/ml.
Diallyl disulfides (DADS) – 1 mM dissolved in 4% dimethyl
sulfoxide was used as positive control, DMEM media
without cells as a media blank and DMEM media with
cells as cell blank. Treated microtiter plates were incubated
for 24 h, 48 h, and 72 h in a humidified atmosphere with
5% CO2
at 37°C. All treatments were done in triplicates.
Evaluation of anti‑cancer activity by
sulforhodamine‑B assay
The cytotoxic effect of the extracts in different concentrations
was evaluated by sulforhodamine‑B (SRB) assay.[12]
Fixing
of viable cells was done using 100 µl of 10% trichloro‑acetic
acid at 4°C for 1 h. After fixation, plates were washed with
distilled water in order to remove excessive fixative and
dead cells and kept overnight at 4°C. Once the plate was
dried, 100 µl of 0.4%‑SRB (Sigma‑Aldrich, St. Louis, USA)
was added, and plates were incubated for 30 min at room
temperature. Plates were then washed with 1% acetic acid
for 3 times to remove unbound SRB dye. Viable cells take up
SRB dye and stain pink. Plates were allowed for air drying
and finally 100 µl of 10 mM ‑ Tris base [tris (hydroxymethyl)
aminomethane] was added. Plates were kept over rotor for
5 min for complete mixing of bound dye with tris base, and
absorbance was recorded at 490 nm using microtiter plate
reader Bio‑Rad Laboratories, Inc. Percentage inhibition (I%)
was determined by the equation: I (%) = (A control − A
sample/A control) ×100, where A control is the absorbance
of the positive control with 1 mM ‑ DADS, and A sample is
the absorbance of the extract in different concentrations. The
IC (50) value for the two different cell lines was calculated
from the plot of inhibition (%) in dose‑ and time‑dependent
manner using GraphPad PRISM software.
Statistical analysis
The experiment was performed in triplicate. The results
obtained were analyzed using one‑way ANOVA method.
A difference was considered statistically significant if
P ≤ 0.05.
RESULTS
Total phenol content of different percentage of
ethanolic extract of Momordica charantia
The data obtained showed that the 50% ethanolic extract
had the highest phenol content (0.029%) followed by the
70% (0.0098%) and 100% (0.0022%) ethanolic extracts,
respectively, [Figure 1] (P ≤ 0.0001) using Gallic acid as
5. Shobha, et al.: Anti‑cancer activity of Momordica charantia
International Journal of Health & Allied Sciences • Vol. 4 • Issue 4 • Oct-Dec 2015 213
standard (R2
= 0.9236). Since a gradient extraction was
followed, most of the water‑soluble fractions of the phenols
were found in 50% extracts than ethanol soluble.
Cytotoxic effect of ethanolic extract of
Momordica charantia on HeLa cells
The data obtained from the cytotoxicity assay on HeLa cells
showed that in 50%, 70%, and 100% EEMC, there was a
dose‑dependent decrease in cell inhibition from the highest
to lowest concentration [Figures 2‑4]. The percentage of
cell inhibition was highest at 48 h duration in 50% and
70% EEMC and 100% EEMC did not show any significant
difference.
Cytotoxic effect of ethanolic extract of
Momordica charantia on MCF‑7 cells
The data obtained from the cytotoxicity assay on the
MCF‑7 cells showed that in 50%, 70%, and 100% EEMC
there was a dose‑dependent decrease in cell inhibition
from the highest to lowest concentration [Figures 5‑7]. The
percentage of cell inhibition was the highest at 48 h duration
in 50% and 70% EEMC, and 100% EEMC did not show
any significant difference.
Calculation of IC (50)
The data obtained from the cytotoxicity assay of HeLa
cell lines showed that the 50% (12.31 µg/ml) EEMC
has lowest IC (50) value than 70% (16.72 µg/ml) EEMC
and 100% (15.67 µg/ml) EEMC at 48 h incubation
[Figure 8 and Table 1].
The data obtained from the cytotoxicity assay of MCF‑7
cell lines showed that the 50% (0.769 µg/ml) EEMC has
lowest IC (50) value than 70% (1.029 µg/ml) EEMC
and 100% (0.846 µg/ml) EEMC at 48 h incubation
[Figure 9 and Table 2].
Different percentage of EEMC
%oftotalphenol
50
70
100
0.00
0.01
0.02
0.03
0.04
Figure 1: Percentage of total phenol content in 50%, 70%,
and 100% ethanolic extract of Momordica charantia
concentration of EEMC in g/ml
%inhibition
0.000000.195000.390000.780001.560003.125006.2500012.5000025.00000
1.00000
-100
-50
0
50
100
24
48
72
1mM DADs
Figure 2: Time‑ and dose‑dependent percentage inhibition of
HeLa cell lines during treatment with 50% ethanolic extract
of Momordica charantia
concentration of EEMC in g/ml
%inhibition
0.000000.195000.390000.780001.560003.125006.2500012.5000025.00000
1.00000
-40
-20
0
20
40
60
80
24
48
72
Figure 3: Time‑ and dose‑dependent percentage inhibition of
HeLa cell lines during treatment with 70% ethanolic extract
of Momordica charantia
concentration of EEMC in g/ml
%inhibition
0.000000.195000.390000.780001.560003.125006.2500012.5000025.00000
1.00000
-400
-300
-200
-100
0
100
24
48
72
Figure 4: Time‑ and dose‑dependent percentage inhibition of
HeLa cell lines during treatment with 100% ethanolic extract
of Momordica charantia
6. Shobha, et al.: Anti‑cancer activity of Momordica charantia
International Journal of Health & Allied Sciences • Vol. 4 • Issue 4 • Oct-Dec 2015214
DISCUSSION
In MC, primary metabolites found are common sugars,
proteins, and chlorophyll, while the secondary metabolites
commonly seen are alkaloids, flavonoids, phenols, tannins,
etc.[13]
MC is a good source of phenolic compounds mainly
Gallic acid that may have the potential as antioxidant and
antimutagen.[14]
In this study, we have tried to evaluate the phenolic
content in MC by F‑C method. F‑C reagent, a mixture of
phosphotungstic and phosphomolybdic acids, is reduced
to blue oxides of tungsten and molybdenum during phenol
oxidation which occurs under alkaline condition provided
by sodium carbonate. The intensity of blue color reflects the
quantity of phenolic compounds, which can be measured
using a spectrophotometer.[15]
concentration of EEMC in g/ml
%inhibition
0.000000.195000.390000.780001.560003.125006.2500012.5000025.00000
1.00000
-50
0
50
100
24
48
72
Figure 5: Time‑ and dose‑dependent percentage inhibition of
MCF‑7 cell lines during treatment with 50% ethanolic extract
of Momordica charantia
concentration of EEMC in g/ml
%inhibition
0.000000.195000.390000.780001.560003.125006.2500012.5000025.00000
1.00000
-50
0
50
100
24
48
72
Figure 6: Time‑ and dose‑dependent percentage inhibition of
MCF‑7 cell lines during treatment with 70% ethanolic extract
of Momordica charantia
concentration of EEMC in g/ml
%inhibition
0.000000.195000.390000.780001.560003.125006.2500012.5000025.00000
1.00000
-50
0
50
100
24
48
72
Figure 7: Time‑ and dose‑dependent percentage inhibition
of MCF‑7 cell lines during treatment with 100% ethanolic
extract of Momordica charantia
50
70
100
0
20
40
60
50
70
100
Different percentage of EEMC
IC(50)valueinµg/ml
Figure 8: IC (50) of ethanolic extract of Momordica charantia
on HeLa cell lines
Table 1: IC (50) of EEMC on HeLa cell lines
HeLA
IC (50)
Mean (SE)
24 h 48 h 72 h
50% 40.98 µg/ml (0.37) 12.315 µg/ml (0.41) 19.19 µg/ml (0.005)
70% 50.13 µg/ml (0.16) 16.72 µg/ml (0.23) 23.72 µg/ml (0.18)
100% 31.80 µg/ml (0.51) 15.67 µg/ml (0.30) 19.63 µg/ml (0.34)
P 0.001 0.005 0.001
SE: Standard error, P: Probability of chance, EEMC: Ethanolic extract
of Momordica Charantia, IC (50): Half maximal inhibitory concentration,
HeLa: Henrietta lacks
Table 2: IC (50) of EEMC on MCF‑7 cell lines
MCF‑7
IC (50)
Mean (SE)
24 h 48 h 72 h
50% 1.47 µg/ml (0.06) 0.769 µg/ml (0.12) 6.82 µg/ml (0.59)
70% 10.92 µg/ml (0.4) 1.029 µg/ml (0.15) 20.13 µg/ml (0.78)
100% 4.32 µg/ml (0.30) 0.846 µg/ml (0.04) 7.75 µg/ml (0.59)
P 0.004 0.370 0.001
SE: Standard error, P: Probability of chance, EEMC: Ethanolic extract
of Momordica Charantia, IC (50): Half maximal inhibitory concentration,
MCF‑7: Michigan cancer foundation-7
7. Shobha, et al.: Anti‑cancer activity of Momordica charantia
International Journal of Health & Allied Sciences • Vol. 4 • Issue 4 • Oct-Dec 2015 215
In a study by Koffi et al., showed that ethanol was better
than acetone, water or methanol for the extraction
of polyphenolic components from the Ivorian plants.
The average of TPCs of ethanolic, acetone, aqueous,
and methanolic extracts was 9000, 2500, 2000, and
1000 mg GAE per 100 g dry weight in decreasing order,
respectively.[16]
Badu et al., have shown that ethanol
is usually preferred for the extraction of antioxidant
compounds from plant extract due to its toxicity and good
extraction efficacy.[17]
Based on these studies, we have used
ethanol as a solvent for extraction.
The MCE and juices have been found to treat different
diseases or clinical symptoms. The stem and leaf
of bitter melon is also used in cancer treatment, in
viral infections (HIV, Epstein–Barr, herpes, influenza,
hepatitis, and measles), in bacterial infections (Salmonella,
Staphylococcus, and Streptococcus), as a digestive aid (for
dyspepsia and sluggish digestion) and the most well‑known
usage in diabetes.[18,19]
Wu and Ng et al. and Kubola
and Siriamornpun et al. independently investigated the
phenolic compounds of MC in water and obtained
averages of 0.516 mg of GAE/ml and 0.202 mg GAE/ml,
respectively.[20,21]
Our study shows the presence of phenolic
activity in the whole fruit EEMC.
In our study, the highest percentage of phenolic activity
was seen in 50% (0.029) when compared to 70% (0.0098)
and 100% (0.0022) EEMC may be due to the fact that
biochemical components present in MC is water soluble.
Phenolic acid profiling using HPLC will be done in order
to specify the key compound present in the EEMC. Weng
and Yen (2012) have shown that the daily consumption
of natural dietary components that are rich in phenols is
beneficial for the prevention of cancer metastasis.[22]
To evaluate in vitro cytotoxic activity of cancer cell
lines, several methods have been employed based
on the principle of colorimetry or fluorometry.
The more commonly used are SRB assay and
3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium
bromide‑assay. In our study, we have evaluated the cytotoxic
activity using SRB‑assay.
Kwatra et al., determined that MCE enhanced the effect of
doxorubicin (DOX) on colon cancer cell lines, HT‑29 cells
and MDCK cell proliferation and sensitized the cells
toward DOX upon pretreatment and reduction in the
expression of multidrug resistance conferring proteins
P‑glycoprotein (P‑gp), multidrug resistance‑associated
protein 2 and breast cancer resistance protein. MCE
suppressed pregnane X receptor promoter activity thereby
suppressing its expression, and also MCE use different
pathways other than 5’
AMP‑activated protein kinase
pathway for the anti‑cancer and MDR modulating activities.
All this suggests that MCE can enhance the bioavailability
and efficacy of conventional chemotherapy.[23]
Weng et al., determined that the bioactive constituent 3,
7‑dihydroxy‑25‑methoxycucurbita‑5,23‑diene‑19‑al, a
cucurbitane‑type triterpene isolated from a crude extract of
wild bitter gourd, induced apoptotic death in breast cancer
cells, MCF‑7 and MDA‑MB‑231, through peroxisome
proliferator‑activated receptor activation provides a
mechanistic basis to account for the antitumor activity of
wild bitter gourd.[24]
Konishi et al., demonstrated that the MCE containing
1‑monopalmitin is most potent than soybean, dokudami,
and welsh onion in inhibiting the P‑gp activity in Caco‑2
intestinal cells.[25]
Ray et al., showed that MCE treatment on human breast
cancer cells, MCF‑7 and MDA‑MB‑231 and primary human
mammary epithelial cells resulted in a significant decrease
in cell proliferation and induced apoptotic cell death
accompanied by increased PARP cleavage and caspase
activation. The study also showed that MCE treatment of
breast cancer cells inhibited survivin and claspin expression
and enhanced p53, p21, and pChk1/2 and inhibited cyclin
B1 and cyclin D1 expression, suggesting an additional
mechanism involving cell cycle regulation.[26]
Deshmukh
et al., showed that the treatment of crude fruit and
endophytic extracts of MC on HeLa cell lines were shown
the highest antiproliferative activity which were ranged from
70% to 96%.[27]
Fongmoon et al., showed that cytotoxicity
effect of MCE on cervical cancer cell line, that is, 0%, 51%,
and 98% at concentrations of 80, 100, and 120 μg/ml for
50
70
100
0
5
10
15
20
25
50
70
100
Different percentage of EEMC
IC(50)valueinµg/ml
Figure 9: IC (50) of ethanolic extract of Momordica charantia
on MCF‑7 cell lines
8. Shobha, et al.: Anti‑cancer activity of Momordica charantia
International Journal of Health & Allied Sciences • Vol. 4 • Issue 4 • Oct-Dec 2015216
HeLa cells was 0%, 30%, and 70% at concentrations of 140,
160, and 180 μg/ml for SiHa cells.[28]
Even though in all this studies, they have shown that MC is
potent as anti‑cancer they have either used a crude extract or
a single percentage of extract from a solvent. In our study,
we have used a different percentage of ethanol for extraction.
As we know, there is an uncontrolled proliferation of cells
in cancer, our data showed EEMC inhibit cell growth and
hence possess anti‑cancer activity. The data presented in
this paper demonstrates that EEMC is potent in inhibiting
growth of cervical and breast cancer cell lines at a dose of
25 µg/ml and 50% ethanolic extract has the highest cytotoxic
effect when compared to 70% and 100%. This indicates
that the biochemical components present in MC are more
water soluble. The lowest IC (50) dose was 12.31 µg/ml,
16.72 µg/ml and 15.67 µ/ml for 50%, 70%, and 100% of
EEMC, respectively, at 48 h incubation for HeLa cell lines
and 0.769 µg/ml, 1.029 µg/ml and 0.846 µg/ml for 50%,
70%, and 100% of EEMC, respectively, at 48 h incubation
for MCF‑7 cell lines. Even though IC (50) value of 50%
EEMC is lower when compared to 100% EEMC, it is
not statistically significant in MCF‑7 cell lines. However,
in HeLa cell lines the IC (50) value of 50% EEMC is
significantly lower when compared to 70% and 100% EEMC
and taking into consideration the high phenol content in 50%
EEMC we would plan our further studies with 50% EEMC.
When compared to HeLa cell lines, 50% EEMC is showing
increased percentage of cell inhibition for MCF‑7 cell lines
with lower IC (50) value. Thus, we conclude that the 50%
EEMC is more potent against breast cancer cell lines when
compared to cervical cancer cell lines.
Future directions
Further studies are required to know the exact cause for the
increase in cell inhibition at 48 h incubation than in 72 h. The
bioactive compound present in the extract will be identified
by using high‑performance liquid chromatography and
mass spectrophotometer. The active compound identified
will be re‑tested on the breast cancer cell lines to confirm its
anti‑cancer activity and IC (50) value will be derived for the
purified active compound. Studies will be done to know the
exact mechanism of action of EEMC using this IC(50) values
also its effect on invasion and migration of cancer will be
tested. Animal experiments will be carried out to establish the
therapeutic index of the purified active compound and also to
identify the toxic symptoms that occur with the tested dose.
CONCLUSION
The presence of high total phenolic acid content in 50%
ethanolic extract indicates that the anti‑cancer activity of
MC could be due to the secondary metabolites which are
more soluble in water. The 50% EEMC with lowest IC(50)
value 0.769 µg/ml is more potent against breast cancer cell
lines. Hence, it may represent a novel therapeutic fruit for
the treatment of breast cancer.
Acknowledgment
We thank the Department of Science and technology‑Fund
fortheimprovementof scienceandtechnologyinfrastructure
for funding the Center of Excellence in Molecular Biology
and Regenerative Medicine (CEMR) laboratory and
facilitating the project. Dr. MVSST SubbaRao, Associate
Professor and Dr. Devananda D, Lecturer in the Department
of Biochemistry for their kind guidance.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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