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ISHS Acta Horticulturae 1125: XXIX International Horticultural Congress on Horticulture: Sustaining Lives, Livelihoods and Landscapes (IHC2014): V World Congress on Medicinal and Aromatic Plants and International Symposium on Plants, as Factories of Natural Substances, Edible and Essential Oils

Brahmi saponins inhibit proliferation of Hep G2 cells by blocking cell cycle progression and inducing apoptosis

Authors:   M. Kalachaveedu, D. Adapala, A.M. Punnoose, S. Kuruvilla
Keywords:   Bacopa monnieri, hepatocellular carcinoma, bacosides A & B, cytotoxicity, cell cycle analysis, p53, anti-cancer
DOI:   10.17660/ActaHortic.2016.1125.21
Abstract:
Bacopa monnieri Linn (Plantaginaceae) or 'Brahmi' is a well-researched aquatic herb used in Ayurveda for centuries as a neuro tonic and memory enhancing drug. The plant and its saponins are being investigated for multiple pharmacological activities. Hepatoprotective and anti-cancer activity of the plant extracts have been reported. The current study isolated the saponins NDASH bacosides A & B from the methanol extract and established their purity by melting point, TLC, IR, 1H NMR and 13C NMR studies. They were evaluated for anti-proliferative effect on human hepato cellular carcinoma cell line, Hep G2. In the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay, at the doses tested (5, 10, 12.5, 15, 25 and 50 g mL-1) bacosides A & B showed a dose dependent decrease in cell viability and their IC50 values were: bacoside A - 0.625 g mL-1 and bacoside B - 9.8 g mL-1. Laddering assay confirmed DNA fragmentation in the treated cells, which showed morphological changes indicating initiation of apoptosis. Fluorescence microscopy of acridine orange/ethidium Bromide (Et Br) stained control and treated cells demonstrated condensed fragmented nuclei that incorporated Et Br in the treated cells. Cell cycle analysis by flow cytometry indicated G2/M arrest by bacoside A and an S phase arrest by bacoside B. Qualitative reverse transcriptase-PCR analysis for mRNA expression of the tumour suppressor gene p53 indicated its upregulation by both the saponins with bacoside A having a greater effect. Immune blot analysis, showed that the pro apoptotic proteins, p16, p21 and Bax were over expressed relative to controls, while anti apoptotic Bcl 2 levels decreased. Our study suggests anti-proliferative activity of bacosides A & B on Hep G2 cells by blocking cell cycle progression and induction of apoptosis. These saponins may be further explored for their anti-cancer potential.

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