|Year : 2015 | Volume
| Issue : 4 | Page : 210-217
In vitro anti-cancer activity of ethanolic extract of Momordica charantia on cervical and breast cancer cell lines
CR Shobha1, Prashant Vishwanath1, MN Suma1, Akila Prashant1, Chandini Rangaswamy1, Basavana H Gowdappa2
1 Center of Excellence in Molecular Biology and Regenerative Medicine, Department of Biochemistry, JSS Medical College, JSS University, Mysore, Karnataka, India
2 Department of Medicine, JSS Medical College, JSS University, Mysore, Karnataka, India
|Date of Web Publication||20-Oct-2015|
Center of Excellence in Molecular Biology and Regenerative Medicine, Departments of Biochemistry, JSS Medical College, JSS University, Mysore - 570 015, Karnataka
Source of Support: None, Conflict of Interest: None
Objectives: To estimate the total phenol content (TPC) of the ethanolic extract of Momordica charantia (EEMC) whole fruit and to study the cytotoxic activity of this extract against cell lines representing carcinomas of cervix and breast. Materials and Methods: Cervical and breast carcinoma cell lines (HeLa and MCF-7) were procured from National Center for Cell Sciences, Pune, and cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1 mM L-glutamine. EEMC was prepared by graded ethanol fractionation method and the TPC determined using Folin–Ciocalteu assay. For cytotoxicity studies, 5000 cells/well in 100 μl DMEM-10% FBS medium were seeded in a 96 well plate; and treated with increasing concentration of EEMC. Efficacy of EEMC was determined by measuring the cell number using sulforhodamine B assay. Percentage inhibition was calculated using dimethyl sulfoxide vehicle control. The IC (50) value was calculated from the plot of inhibition (%) in dose- and time-dependent manner using GraphPad PRISM software. Results: The total phenolic content of EEMC decreased with increasing ethanol concentration from 50% to 100%. Cytotoxicity studies identified 50% ethanolic extract as the most active fraction. A time- and dose-dependent increase in the efficacy of 50% ethanolic extract for inhibiting cervical and breast carcinoma cell growth was noticed. The IC (50) dose was 12.31 μg/ml and 0.769 μg/ml for 50% EEMC at 48 h incubation for HeLa and MCF-7 cell lines, respectively. Conclusion: The presence of high total phenolic acid content in 50% ethanolic extract indicates that the anti-cancer activity of Momordica charantia could be due to the secondary metabolites. Based on the IC (50) value we conclude that the 50% EEMC is more potent against breast cancer cell lines. Further studies are required to know the exact cause for the increase in cell inhibition at 48 h incubation than in 72 h.
Keywords: Anti-cancer activity, breast cancer, cervical cancer, Momordica charantia, total phenolic content
|How to cite this article:|
Shobha C R, Vishwanath P, Suma M N, Prashant A, Rangaswamy C, Gowdappa BH. In vitro anti-cancer activity of ethanolic extract of Momordica charantia on cervical and breast cancer cell lines. Int J Health Allied Sci 2015;4:210-7
|How to cite this URL:|
Shobha C R, Vishwanath P, Suma M N, Prashant A, Rangaswamy C, Gowdappa BH. In vitro anti-cancer activity of ethanolic extract of Momordica charantia on cervical and breast cancer cell lines. Int J Health Allied Sci [serial online] 2015 [cited 2020 Jun 5];4:210-7. Available from: http://www.ijhas.in/text.asp?2015/4/4/210/167649
| Introduction|| |
The search for a better therapeutic agent makes cancer the most sought after in research among all other chronic debilitating diseases. Since ancient times, many of the dietary components such as curcumin, pepper, and garlic have been known to prevent cancer and other 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. 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. 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. 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., Physiological benefits include hypoglycemia, hypolipidemia, anti-viral, anti-bacterial, immunomodulatory, and anti-carcinogenic effects. 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 , and increase apoptosis in leukemia and colon cancer cell lines. Oral administration of Momordica charantia extract (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). 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.
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.
The obtained different percentage solutions of EEMC were then concentrated using Rotovapor R-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% concentrated extracts completely at reduced pressure after being frozen at −80°C. Once completely dehydrated, the concentrated 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. 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).
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. 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.
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 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.
|Figure 1: Percentage of total phenol content in 50%, 70%, and 100% ethanolic extract of Momordica charantia|
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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 [Figure 2], [Figure 3], [Figure 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.
|Figure 2: Time- and dose-dependent percentage inhibition of HeLa cell lines during treatment with 50% ethanolic extract of Momordica charantia|
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|Figure 3: Time- and dose-dependent percentage inhibition of HeLa cell lines during treatment with 70% ethanolic extract of Momordica charantia|
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|Figure 4: Time- and dose-dependent percentage inhibition of HeLa cell lines during treatment with 100% ethanolic extract of Momordica charantia|
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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 [Figure 5], [Figure 6], [Figure7]. 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.
|Figure 5: Time- and dose-dependent percentage inhibition of MCF-7 cell lines during treatment with 50% ethanolic extract of Momordica charantia|
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|Figure 6: Time- and dose-dependent percentage inhibition of MCF-7 cell lines during treatment with 70% ethanolic extract of Momordica charantia|
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|Figure 7: Time- and dose-dependent percentage inhibition of MCF-7 cell lines during treatment with 100% ethanolic extract of Momordica charantia|
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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].
|Figure 8: IC (50) of ethanolic extract of Momordica charantia on HeLa cell lines|
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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].
|Figure 9: IC (50) of ethanolic extract of Momordica charantia on MCF-7 cell lines|
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| Discussion|| |
In MC, primary metabolites found are common sugars, proteins, and chlorophyll, while the secondary metabolites commonly seen are alkaloids, flavonoids, phenols, tannins, etc. MC is a good source of phenolic compounds mainly Gallic acid that may have the potential as antioxidant and antimutagen.
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.
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. 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. 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., 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., 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.
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.
Weng et al., determined that the bioactive constituent 3β,7β-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 γ activation provides a mechanistic basis to account for the antitumor activity of wild bitter gourd.
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.
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. 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%. 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 HeLa cells was 0%, 30%, and 70% at concentrations of 140, 160, and 180 μg/ml for SiHa cells.
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.
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.
We thank the Department of Science and technology-Fund for the improvement of science and technology infrastructure 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
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
[Table 1], [Table 2]