The hepatoprotective capacity of selected natural products from South Africa

Abstract

Drug-induced liver injury (DILI) is recognized as a significant clinical problem, which may account for up to 50% of all cases of acute liver failure. DILI is initiated by the bioactivation of parent drug molecules to produce chemically reactive metabolites. These reactive intermediates induce mitochondrial dysfunction and oxidative stress leading to glutathione (GSH) depletion and damage to cellular proteins, lipids and nucleic acids, which eventually culminates in necrotic cell death. The principal objective of this study is to establish an in vitro screening platform to identify potential hepatoprotective natural products (plants and mushrooms) from South Africa (SA). Aqueous plant extracts (Cyclopia intermedia, Opuntia ficus indica and Kigelia africana), and aqueous and ethanolic macrofungal extracts (Ganoderma lucidum, Russula capensis, Pleurotus ostreatus and Lenzites elegans) were prepared and screened against HepG2 and VERO cells to assess their safety using Hoechst 33342-PI dual labelling. A drug-induced hepatotoxic model was established, using the dietary supplement menadione (vitamin K3). Hoechst 33342- PI, -CellROX® Orange and -TMRE dual labelling was used for necrosis, oxidative stress and mitochondrial membrane potential depolarization (ΔΨm) detection, respectively. The accuracy of the hepatoprotection model was confirmed through HepG2 cellbased assays (Hoechst 33342- PI, -CellROX® Orange and -TMRE dual labelling) that measured the protective effects of natural products against the menadione-induced toxicity, anti-oxidant assays (DPPH, NO, ORAC, CAPe and FRAP) that measured their anti-oxidant potential and enzyme assays (βglucuronidase, carboxylesterase and CYP450 isoform 3A4) that measured their effects on drug metabolism. Silymarin was used as a positive control for each assay. Menadione displayed significant cell death, increased oxidative stress and decreased ΔΨm at an elevated concentration of 100 μM; confirming the hepatotoxicity model, where necroptosis was suspected to be menadione’s cell death mode. Only ethanolic G. lucidum was cytotoxic. All three aqueous plant extracts demonstrated strong anti-oxidant capacities out of all the tested extracts; where C. intermedia displayed the most promising DPPH, NO, ORAC, CAPe and FRAP activity, followed by aqueous G. lucidum. Aqueous plant and ethanolic macrofungal extracts (C. intermedia, O. ficus indica, K. africana, and ethanolic P. ostreatus, R. capensis) displayed decreased menadione-induced ROS production and protected against menadione-induced ΔΨm depolarization, posing them and aqueous G. lucidum potential therapeutic interventions for DILI. Ethanolic L. elegans demonstrated the highest enzyme inhibition for each assay and presented genotoxicity, ruling it out as a therapeutic strategy against DILI. Together these assays addressed several aspects relating to DILI and hepatoprotection, and served as a good starting point in evaluating the therapeutic value of natural products from South Africa