Subhashini Rajakumaran1*, Ruth Christiya C2
1Assistant Professor, Department of Biochemistry, D.G. Govt. Arts College for Women, Mayiladuthurai, 609001
2Assistant Professor, PG and Research Department of Biochemistry,
Rabiammal Ahamed Maideen College for Women, Tiruvarur - 610001, Tamil Nadu, India.
Medicinal plants maintain the health and vitality of individuals, and also have potential curative effect on various diseases, including cancer. Bone cancer is the foremost cause of deaths among women worldwide. In this present study, the antiproliferative effects of methanol extracts of Euphorbia hirta L leaves was investigated on MG 63 cell line. The methanolic extract of plant exhibited significant dose dependent antiproliferative activity against MG63 cell line which was ranged between 89.43% and 20.63% at concentrations of 50µg/ml and 350µg/ml correspondingly (24 hours). Moreover, the plant found to decrease the cell viability in dose dependent manner. The results of this study show that Euphorbia hirta L is a potential source of compounds that may serve as leads for anticancer property.
Cancer is one of the most dreadful diseases globally and it appears to be due to extreme free radical damage, which eventually causes damage to the DNA, lipids and protein. In cell cycle, growth and division of normal cells occur in a well-ordered manner, however in cancerous cells, defective caspase-mediated cell death (apoptosis) leads to increased cell proliferation1. The International Association of Cancer Registries (IACR – GLOBOCAN database) reported that 12.7 million new cases of cancer, and 7.6 million deaths occur due to cancer worldwide2. The ratio increased to 14.1 million new cases and 8.2 million deaths by 20123 The recently updated database has reported new cancer cases of 18.1 million, and deaths of 9.6 million, which proved the rising burden of cancer frequency as well as mortality4
Osteosarcoma, the primary bone malignancy, is one of the most common cancers worldwide. Chemotherapy for cancers using agents such as cisplatin, methotrexate, and cyclophosphamide is widely used for treating osteosarcoma. It may result in drug resistance, as well as several side effects including drug-cytotoxicity which also cause damage to normal tissues5. So, there is a necessitate to identify the new molecules for the treatment of cancer with low prices, high-efficient curing, less side effects. The plant kingdom contains a great source of new bioactive compounds which, due to their intrinsic biological properties, may be used in medicine as well as in other human health promoting areas6. But there is no scientific evidence on some medicinal plants of their biological activities. In this point of view, we aimed the present study to evaluate the in vitro anti proliferative activity of Euphorbia hirta L on the activity of cytotoxicity in bone cancer MG-63 cells.
MATERIALS AND METHODS:
Plant Collection and Extraction:
The fresh leaves of plant Euphorbia hirta L were collected from Thiruvarur district, Tamil Nadu in December 2019. The plant materials were shade dried, powdered and extracted with methanol using soxhlet extraction process.
Cell Culture and Maintenance:
Human MG-63 cell lines (Bone cancer) were procured from the cell repository of National Centre for Cell Sciences (NCCS), Pune, India. Dulbecco`s Modified Eagle Media (DMEM) was used for maintaining the cell line, which was supplemented with 10% Fetal Bovine Serum (FBS). Penicillin (100U/ml), and streptomycin (100μg/ml) were added to the medium to prevent bacterial contamination. The medium with cell lines was maintained in a humidified environment with 5% CO2 at 37°C7
Cell Viability assay:
MG-63 cell line was seeded in six-well plate and incubated at 37°C in a 5% CO2. The cells were treated with different concentrations of 50, 150, 250 and 350 µg/mL of Euphorbia hirta L methanolic extract for 24 hours. The doses induced 50% or more cell death with MTT assay and were selected for trypan blue staining. The culture medium was removed and replaced with trypan blue (0.4%), and kept for 3-5 minutes. The cells were washed three times with PBS and observed under a microscope. Blue color cells represent as dead cells8.Viability rate was measured according to the below formula:
Cell viability (%) =
(Live cells) / (Total number of cells) ×100
MG-63 viable cells were seeded in 96 well plates and then incubated at 37°C in a humidified 95% air and 5% CO2 incubator for 24 h. Once the cells placed in wells, the different concentrations of Euphorbia hirta L leaves extract 50 to 350μg/ml were added and kept in incubator 24 hours. Then the samples were removed from the well, and washed with phosphate-buffered saline (pH 7.4) then the samples were removed from the well, and washed with phosphate-buffered saline (Ph 7.4) or DMEM without serum. 0.5% 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl–tetrazolium bromide (MTT) was added to each well (100μl/well) and incubated for 4 hours at 37°C. The purple precipitated formazan crystals formed was dissolved in 100µl of concentrated dimethyl sulfoxide (DMSO). The absorbance was read at 570nm using a multi-well plate reader. The results were expressed at the percentage of stable cells with respect to the control. The percentage cell viability was calculated by determining the ratio between A570 of treated cells and A570 of control cells, multiplied by 1009. The half maximal inhibitory concentration (IC50) values were calculated.
The values are expressed as mean ± SD. The statistical comparisons were performed by one-way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT), using SPSS version 12.0 for windows (SPSS Inc. Chicago; http://www.spss.com). The values are considered statistically significant if the p value was less than 0.05.
RESULTS AND DISCUSSION:
The MG-63cells were treated with various concentration of methanolic leaf extract of Euphorbia hirta L (50-350 µg/ml) for 24 h and the results are expressed as a percentage of the control value in presenting as a cell cytotoxicity ratio for MG-63 cells using MTT assay. The cytotoxicity effects of methanolic extract of Euphorbita hirta were shown in figure 1. The cell viability of MG63 cells ranged between 89.43% and 20.63% at extract concentrations of 50µg/ml and 350µg/ml correspondingly in an incubation period of 24 hours. The IC50 value was found to be 187μg/ml. The methanolic extract of Euphorbita hirta showed cytotoxicity against MG63 cells in concentration dependent manner. Exposure to increased concentration of the extract has grossly reduced the number of viable osteosarcoma cells, and their architecture was found to be disrupted. The assay detects the reduction of dimethylthiazole diphenyl tetrazolium bromide (MTT) salt to a coloured formazan product by mitochondrial enzyme succinate dehydrogenase, the intensity of the colour was measured using spectrophotometer, which measures the quantity of viable cells10. The cell viability of MG63 cells decreased with increasing dose of the extract confirming its anti-proliferative property
Figure 1: Anti-proliferative effect of Euphorbia hirta L on MG63 cell line
Photomicrograph represents morphological changes in MG-63 cells such as shrinkage, detachment, membrane blebbing and distorted shape induced by MG-63 treatment (200µg/ml for 24 h) as compared with control. Control showed normal intact cell morphology and their images were captured by light microscope. The anti proliferative property of the plant extract may be attributed due to their phytoconstituents particularly phenolic and flavanoid compounds.
Figure 2: Morphological Changes in MG63 cell lines
Figure 3 results showed that 50, 150, 250, and 350 µg/mL of methanolic extract E.hirta significantly reduced the survival rate of MG63 cells after 24 hours (P < 0.05). The lowest cell survival rate occurred after administration of 350µg/mL (P < 0.05). The more cell death was observed in higher concentration of plant extract. The findings obtained with trypan blue confirmed our previous MTT results. The antiproliferative properties of the extract might explain its bioactive components, including flavonoids, alkaloids etc. Several flavonoids regulate the genes, which are critical for the control of proliferation, cell cycle and apoptosis pathway in cancer cells11. It has been found that methanolic extract of E.hirta can decrease the cell viability of MG63 cell lines in a concentration-dependent manner
Figure 3: MG63 cell viability after treatment with various concentration of Euphorbia hirta L methanolic extract after 24 hours incubation
With the results of the present findings, it may be inferred that methanolic extract of Euphorbita hirta possess therapeutic activity against bone cancers. Further studies should be focused on Fractionation of the active components in plant extract to take the research forward for further exploration.
The authors would like to thank the cell repository of National Centre for Cell Sciences (NCCS), Pune, India, for providing the cancer cell lines and their help in doing the anti-cancer activity successfully.
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Received on 10.04.2020 Modified on 05.05.2020
Accepted on 30.05.2020 © RJPT All right reserved