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20211
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Subject: Veterinary Pharmacology and Toxicology × Author: DHANUSHA G × End date: 2023 × Start date: 2020 ×
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    ThesisItemOpen Access
    MODULATION OF OESTROGENIC ACTIVITY BY ALCOHOLIC EXTRACTS OF Asparagus racemosus AND Boerhavia diffusa IN MCF-7 CELL LINE
    (COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2021-07-30) DHANUSHA G; Sujith S.
    The present study was undertaken to evaluate the modulatory effect of methanolic extracts of tubers of Asparagus racemosus (Shathavari) and leaves of Boerhaevia diffusa (Thazhuthama) on oestrogen levels in Michigan Cancer Foundation-7 (MCF -7) cell line and to compare that with the effect of Naringenin. Components of the active extract were identified and the expression of Steroidogenic Acute Regulatory protein (StAR), Cytochrome P45011A1 (CYP11A1) and Aromatase (CYP19) in extract treated cells were studied. The tubers of A. racemosus and whole plant of B. diffusa were collected locally, shade dried, extracted using methanol, concentrated and stored under refrigerated conditions till further use. The phytochemical constituents were assessed by using qualitative tests, GC-MS and FTIR analysis. Both the plant extracts and Naringenin were screened for their cytotoxic activity in MCF-7 breast carcinoma cell lines by MTT assay at the dose rates of 10, 20, 40, 80 and 160 µg/mL. Based on the results of MTT Assay, per cent cell viability, per cent inhibition and IC50 were calculated. On the basis of IC50 values obtained, the dose for further studies was calculated as IC50, half of the dose of IC50 and double the dose of IC50. The cells were exposed to these concentrations of the extract for 96 hours in 6 well plates and media was collected every 48 hours for analysis of oestrogen by ELISA. The cells were collected after 96 hours for the study of expression of CYP19, CYP11A1 and StAR keeping GAPDH as reference gene. The phytochemical analysis revealed the presence of alkaloids, steroids, tannins, saponins, phytosterols, flavonoids, diterpenes and glycosides in A. racemosus and alkaloids, steroids, tannins, flavonoids, glycosides and saponins in B. diffusa. GC-MS and FTIR analysis revealed the presence of short chain carboxylic acids, 5-Hydroxymethylfurfural, 1,2,3-Propanetriol, Linoleic acid methyl ester, and guanosine in extract of A. racemosus. 5-Hydroxymethylfurfural, 4-O-methyl-d arabinopyranoside, D-Allose, Hexadecanoic acid methyl ester were identified in the extract of B. diffusa. A dose dependent cytotoxicity was shown by both the extracts and Naringenin in MCF-7 cells. Based on the per cent inhibition, the IC50 of the 112 methanolic extracts of A. racemosus and B. diffusa were found to be 267 µg/mL and 170 µg/mL respectively whereas the IC50 of Naringenin was 395 µg/mL. The methanolic extracts of A. racemosusincreased the concentration of oestrogen secretion from MCF-7 cells, whereas of B. diffusa and Naringenin significantly reduced the concentration of oestrogen in a dose dependent manner. Maximum oestrogen concentration was noticed when cells were treated with 540 µg/mL of A. racemosus, whereas the lowest concentration was noticed when cells were treated with 340 µg/mL of B. diffusa. The oestrogen concentration were 19.69+0.26, 6.73+0.00 and 7.86+0.29 ng/mL, when cells were treated with (IC50) 270, 170, 395 µg/mL doses of A. racemosus, B. diffusa and Naringenin respectively. B. diffusa and Naringenin significantly downregulated the expression of CYP19 gene compared to control groups. There was a 0.43+0.039, 0.65+0.052 and 0.55+0.043 fold decrease in gene expression when MCF-7 cells were treated with different doses of B.diffusa and 0.37+0.03, 0.37+0.03and 0.22+0.02 fold decreases with different doses of Naringenin. Treatment with A. racemosus upregulated CYP19 gene expression by 1.49+0.09and 1.35+0.12 fold when treated with 135 and 540 µg/mL concentrations of the extract. The extract of B. diffusa and Naringenin significantly downregulated the expression of StAR gene. There was a 0.26+0.025, 0.22+0.018 and 0.24+0.022 fold decrease in gene expression when MCF-7 cells were treated with different doses of B. diffusa and 0.31+0.03, 0.27+0.02 and 0.18+0.06 fold decreases with different doses of Naringenin. The activity of CYP11A1 was upregulated by methanolic extract of A. racemosus and B. diffusa whereas Naringenin significantly downregulated the gene. There was a 2.14+0.14, 2.03+0.16and 2.00+0.16b fold increase in gene expression when MCF-7 cells were treated with different doses of A. racemosus and 3.83+0.31, 4.04+0.275 and 4.19+0.30 fold increase with different doses of B. diffusa. Treatment with Naringenin caused an downregulation of CYP19 gene expression by 0.32+0.03, 0.67+0.02 and 0.45+0.06fold when treated with 192, 395 and 790 µg/mL concentrations of the extract. There was a significant downregulation of StAR and CYP19 genes by B. diffusa and 113 Naringenin which might have decreased the secretion of oestrogen, whereas upregulation of CYP19 and CYP11A1 by A. racemosus which might have increased the oestrogen secretion by MCF-7 cells. From the study it could be concluded that A. racemosus produced a positive effect on oestrogen secretion whereas B. diffusa and Naringenin acted as antioestrogens.