A Cell-Based Assay for Antioxidant Behaviour of Phytochemicals: Influence of Exposure Time and Presence of Serum

Abstract

There is considerable interest in the ability of plant-derived antioxidants to protect against oxidative damage associated with disease or exposure to toxic agents. In this study, the cytoprotection effect of the direct antioxidants Quercetin (Q) and Epigallocatechin-3-Gallate (EGCG) and the indirect antioxidants, Sulforaphane (SFN) and Indole-3-Carbinol (I3C) was assessed in a cellular protection assay. This assay involved two cytoprotection patterns: (a) exposure to phytochemical for 20 hours followed by 5-hour exposure to t-Butylhydroperoxide (t-BHP); (b) simultaneous exposure to phytochemicals and t-BHP for 5 hours. HepG2 cells were cultured to a confluent monolayer and exposed to phytochemical +/- t-BHP in serum-free medium or serum-containing medium at high [10%(v/v)] or low [2%(v/v)] levels of foetal bovine serum, after which cell damage mediated by oxidant stress was assessed by uptake of neutral red. Results showed that Q, EGCG and, I3C were effective while SFN was inactive and toxic to the cells by itself at high concentration during long incubation. On the other hand, a short time of incubation with SFN displayed identical results to prolonged exposure. However, I3C was devoid of protection activity. Moreover, results showed that serum has a major impact on antioxidant activity.