Articles
Brahmi saponins inhibit proliferation of Hep G2 cells by blocking cell cycle progression and inducing apoptosis
Article number
1125_21
Pages
173 – 180
Language
English
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.
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.
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
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