Anthocyanins inhibit peroxyl radical-induced apoptosis in Caco-2 cells

The antioxidant activity of anthocyanins has been well characterized in vitro; many cases has been postulated to provide an important exogenous mediator of oxidative stress in the gastrointestinal tract. The objective of this study was to evaluate the efficacy of anthocyanin protection against peroxyl radical (AAPH)-induced oxidative damage and associated cytotoxicity in Caco-2 colon cancer cells. Crude blackberry extracts were purified by gel filtration column to yield purified anthocyanin extracts that were composed of 371 mg/g total anthocyanin, 90.1% cyanidin-3-glucoside, and 4.9 mmol Trolox equivalent/g (ORAC) value. There were no other detectable phenolic compounds in the purified anthocyanin extract. The anthocyanin extract suppressed AAPH-initiated Caco-2 intracellular oxidation in a concentration-dependent manner, with an IC50 value of 6.5+/-0.3 microg/ml. Anthocyanins were not toxic to Caco-2 cells, but provided significant (P<0.05) protection against AAPH-induced cytotoxicity, when assessed using the CellTiter-Glo assay. AAPH-induced cytoxicity in Caco-2 cells was attributed to a significant (P<0.05) reduction in the G1 phase and increased proportion of cells in the sub G1 phase, indicating apoptosis. Prior exposure of Caco-2 cells to anthocyanins suppressed (P<0.05) the AAPH-induced apoptosis by decreasing the proportion of cells in the sub-G1 phase, normalized the proportion of cells in other cell cycle phases. Our results show that the antioxidant activity of anthocyanins principally attributed to cyanidin-3-O-glucoside and common to blackberry, are effective at inhibiting peroxyl radical induced apoptosis in cultured Caco-2 cells.