Invitro Studies on the potential protective effects of Dietes bicolor plant against carbon tetrachloride-induced hepatotoxicity

Abstract

The present study aimed to investigate the potential hepatoprotective activity of Dietes bicolor DB leaf extract and (n-hexane, dichloromethane and n-butanol) fractions in vitro against CCl4-induced acute cytotoxicity in human hepatocellular carcinoma (HepG2) cells. This is in addition to the isolation and structural elucidation of the major compound of the biologically active fraction using UV, ESI-MS, 1H and 13C NMR. Moreover, this study was also extended to characterize the underlying mechanisms of these protective effects.

The main findings of this study can be summarized as follows: 1. Pretreatment with three different concentrations of DB leaf extract and fractions significantly reduced CCl4-induced increase in hepatocyte membrane integrity and leakage markers such as Alanine transaminase (ALT) and Aspartate transaminase (AST) in a comparable manner to that of silymarin (positive standard group). 2. Butanol fraction (Fr-C) showed the maximum protection as evidenced by a reduction in the leakage of alanine transaminase (ALT) -reaching the control levels- and aspartate transaminase (AST) in the culture medium by 72% and 64%, compared to CCl4. 3. The extract and fractions showed a potent antioxidant effect confirmed by an increase in reduced glutathione (GSH) levels and superoxide dismutase (SOD) activity and by a decrease in lipid peroxidation product, malondialdehyde (MDA), versus CCl4. 4. Fr-C exhibited the most potent antioxidant effect by normalizing GSH and SOD by 2 folds and 2.4 folds and by lowering MDA levels by about 65%, relative to CCl4. There was no significant difference compared to the effects produced by silymarin with the highest concentration, nearly normalizing MDA levels. 5. The same fraction showed a potent anti-inflammatory activity as evidenced by a52% reduction of CCl4-induced increase in prostaglandin E2 (PGE2) levels, versus CCl4,reaching to a comparable level to the control group. 6. Fr-C protected against CCl4-induced apoptosis, as indicated by elevated Bcl-2/Bax ratio and reduced caspase-3 activity, compared to CCl4. 7. Phytochemical analysis revealed that the main constituent of Fr-C is the flavone C-glycoside, vitexin. 8. Vitexin was shown to contribute to the observed protective effects by lowering leakage markers, boosting cellular antioxidant defenses, decreasing lipid peroxidation and inhibiting apoptosis.

In conclusion, pretreatment with DB leaf extract and fractions exhibited a promising protective activity against CCl4-induced hepatocyte damage. The flavone C glycoside, vitexin was found to be the main constituent of the most potent fraction (Fr-C). This protective effect can be, at least partly, attributed to their potent antioxidant, anti-inflammatory and anti-apoptotic effects. This study sets the stage for future experimentation in vivo and for clinical trials to explore the potential utility for such a promising plant extract in the protection against oxidative stress-induced liver diseases.