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

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 µg mL-1) bacosides A & B showed a dose dependent decrease in cell viability and their IC50 values were: bacoside A - 0.625 µg mL-1 and bacoside B - 9.8 µg 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.