Abstract:
Phikud Navakot (PN), a Thai traditional remedy recommended for treatment of cardiovascular symtomps, comprises an equal weight proportion of nine herb species. This study aimed to investigate the effect of hydroethanolic extract of PN, and one of its constituents, β-sitosterol against H2O2-induced oxidative stress in HepG2 cells to assess it hepatoprotective effect. Effect of this extract on human cytochrome P450 (CYP) were also investigated in vitro for assessing its herb-drug interaction potential. For the first aimed, cells were treated with different concentrations of PN (0-1 mg/mL) or β-sitosterol (0-20 µM) prior to incubation with H2O2. Cell viability and ROS generation were assessed by MTT and DCFH-DA assays, respectively. Oxidative defense mechanisms were determined by measuring glutathione levels and the activities of antioxidant enzymes. Expression levles of Nrf2 and HO-1 mRNA and proteins were investigated by qRT-PCR and western blot analyses, respectively. The results demonstrated that PN extract (0.001-0.1 mg/mL) and β-sitosterol (1-20 µM) significantly improved cell viability and prevented ROS generation induced by H2O2 in HepG2 cells. PN and β-sitosterol also increased the activities of antioxidant enzymes (CAT, GPx, GR and SOD), total GSH, GSH and GSH/GSSG ratio, but decreased GSSG. In addition, pretreatment of PN reversed Nrf2 and HO-1 protein while β-sitosterol affected only HO-1 protein. It was concluded that PN extract possessed hepatoprotective action associated with the antioxidant effects which may be attributed from it constituents such as β-sitosterol. Regarding herb-drug interaction study, selective substrates of CYPs were used to investigate the inhibitory effects of PN using human liver microsomes. Primary human hepatocytes were used to assess the inductive effect of PN using P450 GloTM CYP assay and western blot analysis. The results showed that, PN inhibited the activities of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 with the maximal inhibitory concentration (IC50) values of 13, 62, 67 and 88 µg/mL, respectively. Inhibition of PN on these CYPs was not a time dependent type but a reversible inhibition with the Ki of 34, 80, 12 and 150 µg/mL for CYP1A2, CYP2C9, CYP2D6 and CYP3A4, respectively. Meanwhile, it had no effect on the activities of CYP2C19 and CYP2E1 (IC50 > 1 mg/mL). PN did not have an induction effect on CYP1A2, CYP2C9, CYP2C19 and CYP3A4 in primary human hepatocytes. Thus, PN may cause herb-drug interactions via inhibition of CYP1A2, CYP2C9, CYP2D6 and CYP3A4, and precautions should be taken when PN is coadministered with drugs that are metabolized by these CYP enzymes.
