Aqueous pomegranate seed extract protects human hepatoma Hepg2 against oxidative stress induced by tert-butyl hydroperoxide

Abstract

The search for Advanced glycation end-products (AGEs) formation inhibitors has recently received much attention and certain by-products of plant extracts have been evaluated for their antiglycation potential. AGEs are a group of complex and heterogeneous products formed in the advanced stage of the Maillard reaction in foods and glycation in living bodies. AGEs are involved in the development of several health disorders such as diabetes and its complications [1], atherosclerosis [2], Alzheimer¿s disease and normal aging [3]. Recently, our group has identified a significant antiglycative activity in aqueous extracts of pomegranate seeds (Punica granatum). The aim of this study is to investigate the protective effect of aqueous pomegranate seed extract (PSE) on toxicity and oxidative stress induced by tert-butyl hydroperoxide (t-BOOH) in HepG2 cell lines before to be used as ingredient in foods and pharmacological preparations. Human hepatoma HepG2 cells initially isolated from a liver biopsy in a 15-year old Caucasian male. This cell line was grown in a humidified incubator containing 5% CO2 and 95% air at 37°C. They were grown in DMEM F-12 medium, supplemented with 2.5% Biowhitaker fetal bovine serum [4]. Cell viability was determined by using the Crystal Violet assay. Cytotoxicity was determined by the lactate dehydrogenase (LDH) leakage from cells. Cellular reactive oxygen species (ROS) were quantified by the dichlorofluorescein (DCFH) assay [4]. Pretreatment of HepG2 in the range of 0.01 to 0.1 mg/mL of PSE completely prevented LDH leakage from the cells. Reactive oxygen species generation induced by t-BOOH was significantly reduced when cells were pretreated for 20 h with 0.001 to 0.1 mg/mL PSE. It is concluded that treatment of HepG2 cells in culture with the aqueous extracts of pomegranate seeds strongly protects the cells against an oxidative insult, increasing cell viability as well.