Ashwagandha (Withania somnifera) supercritical CO2 extract derived withanolides mitigates Bisphenol A induced mitochondrial toxicity in HepG2 cells

Abstract

Bisphenol A (BPA) safety aspects on human health are debated extensively for long time. In the present study, we have studied the toxicity induced by BPA at no observed adverse effect level (NOAEL) using HepG2 cells. We report that BPA at 100 nM induced cytotoxicity to HepG2 cells as determined by MTT assay at 0-72 h. The toxicity was result of reduced oxygen consumption and reduced mitochondrial membrane potential associated with decreased ATP production. The BPA treatment resulted in increase of malondialdehyde (MDA) content with decreased glutathione and other antioxidant enzymes. BPA derived toxicity is a concern to human health and alternative non-toxic natural products/derivatives or adjuvants that serve as antidote will be relevant. In this context, Ashwagandha (Withania somnifera) a widely used herb to treat arthritis, rheumatism and to improve longevity for time immemorial is investigated for its antidote effect. Ashwagandha supercritical CO₂ extract derived Withanolides (ADW) at 100 μg/ml protect HepG2 cells from BPA induced toxicity by suppressing mitochondrial damage and increased ATP production. Further, cellular MDA content was significantly suppressed with increased non-enzymic and antioxidant enzyme activities. These findings derived from the present study suggest the beneficial effect of ADW in mitigating BPA induced mitochondrial toxicity in HepG2 cells.

Keywords: ADW, Ashwagandha supercritical CO2 extract derived Withanolides; Antioxidant enzymes; Ashwagandha; Bisphenol A; CAT, catalase; GPx, glutathione peroxidase; GSH, reduced glutathione; Glutathione; HepG2 cells; LPO, lipid peroxidation; MDA, malondialdehyde; Mitochondrial toxicity; SOD, superoxide dismutase; ΔΨM, mitochondrial membrane potential.

Fig. 1

Mass chromatogram showing compounds of Ashwagandha supercritical fluid (CO₂) extract (ADW). The mass spectrometric conditions are as described in the text.

Fig. 2

Effect of various concentrations of Bisphenol A on HepG2 cell cytotoxicity at 0–72 h. The cells were incubated with incremental concentrations of BPA and incubated for 24, 48 and 72 h and the cytotoxicity was determined as described in Section 2. Values are Mean ± SEM of three independent experiments carried out in triplicates.

Fig. 3

Effect of incremental concentrations of Ashwagandha derived Withanolides (ADW) on HepG2 cell cytotoxicity at 0–72 h. The cells were incubated with various concentrations of ADW and incubated for 24, 48 and 72 h and the cytotoxicity was determined as described in Section 2. Values are Mean ± SEM of three independent experiments carried out in triplicates.

Fig. 4

Effect ADW on cell viability during BPA induced toxicity at 0–72 h. HepG2 cells were co-incubated with BPA (100 nM) in the presence or absence of ADW (100 μg/ml) for 0–72 h and the cytotoxicity was determined at 24, 48 and 72 h as described in Section 2. Antimycin A (10 μM) was used as positive control. Values are Mean ± SEM of three independent experiments carried out in triplicates. **Statistically significant at P < 0.05 compared to control. *Statistically significant at P < 0.05 compared to BPA.

Fig. 5

Effect ADW on mitochondrial function during BPA induced toxicity. HepG2 cells were incubated with BPA (100 nM) in the presence or absence of ADW (100 μg/ml) for 72 h and the (a) oxygen consumption rate, (b) ATP content and (c) mitochondrial membrane potential was determined as described in Section 2. Antimycin A (10 μM) was used as positive control. Values are Mean ± SEM of three independent experiments carried out in triplicates. **Statistically significant at P < 0.05 compared to control. *Statistically significant at P < 0.05 compared to BPA.

Fig. 6

Effect of ADW on lipid peroxidation during BPA induced toxicity in HepG2 cells. HepG2 cells were co-incubated with BPA (100 nM) in presence or absence of ADW (100 μg/ml), vitamin E (25 μM) or BHA (10 μM) and the lipid peroxidation was determined at 24 h as described in Section 2. Vitamin E (25 μM) and BHA (10 μM) was used as negative controls. Values are Mean ± SEM of three independent experiments carried out in triplicates. **Statistically significant at P < 0.05 compared to control. *Statistically significant at P < 0.05 compared to BPA.