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Antioxidative efficiency of an anthocyanin rich bilberry extract in the human colon tumor cell lines Caco-2 and HT-29


Bilberries (Vaccinium myrtillus L.) and its major polyphenolic constituents, the anthocyanins, are discussed to be preventive against diseases, such as colon cancer or inflammatory bowel diseases (e.g. Crohn’s disease, ulcerative colitis), are associated with oxidative stress. Therefore the gastrointestinal tract (GIT) might be a target for prevention of these diseases. In this study antioxidative efficiency of a commercially available anthocyanin rich bilberry extract (BE) was investigated in vitro in the human colon tumor cell lines Caco-2 and HT-29. The cell cytotoxicity of the BE was measured by alamar blue assay. Modulation of intracellular generated reactive oxygen species (ROS) levels was investigated by dichlorfluorescein assay (DCF). Oxidative DNA damage was monitored by single-cell gel electrophoresis (comet assay) with additional treatment of the DNA with formamido-pyrimidinglycosylase (FPG) to enhance sensitivity towards ROS induced DNA lesions. Modulation of the total glutathione (tGSH) level was assayed in a photometric kinetic assay. In a two step protocol cells were first treated with the protective extract (5–500 μg/ml; 1 and 24 h) and then with the redox-cycler menadione (Md) (HT-29: 20 μM and Caco-2: 6 μM) or the oxidant TBH (tert-butyl hydroperoxide) (250 μM, 40 min). Under all conditions tested BE was not cytotoxic in Caco-2 and HT-29 cells. The data achieved revealed that BE significantly reduce ROS level in HT-29 (250 μg/ml; 24 h, p < 0.05) and Caco-2 (50 μg/ml; 1 h, p < 0.05) cells. Significant decrease of induced DNA damage was detected in Caco-2 cells after BE treatment (5 μg/ml; 24 h; FPG, p < 0.05). Trend towards increase of tGSH was observed at concentrations of 50–500 μg/ml BE in Caco-2 cells after 24 h incubation. In total, the BE was shown to possess antioxidative activity under the used assay conditions towards prevention of oxidative DNA damage, reduction of intracellular ROS and cellular tGSH.