Formation of hydrogen peroxide in cell culture media by apple polyphenols and its effect on antioxidant biomarkers in the colon cell line HT‐29

Beneficial health effects of diets containing fruits have partly been attributed to polyphenols which display a spectrum of bioactive effects, including antioxidant activity. However, polyphenols can also exert prooxidative effects in vitro . In this study, polyphenol‐mediated hydrogen peroxide (H 2 O 2 ) formation was determined after incubation of apple juice extracts (AEs) and polyphenols in cell culture media. Effects of extracellular H 2 O 2 on total glutathione (tGSH; =GSH + GSSG) and cellular reactive oxygen species (ROS) level of HT‐29 cells were studied by coincubation ± catalase (CAT). AEs (⪈30 μg/mL) significantly generated H 2 O 2 in DMEM, depending on their composition. Similarly, H 2 O 2 was measured for individual apple polyphenols/degradation products (phenolic acids > epicatechin, flavonols > dihydrochalcones). Highest concentrations were generated by compounds bearing the o ‐catechol moiety. H 2 O 2 formation was found to be pH dependent; addition of CAT caused a complete decomposition of H 2 O 2 whereas superoxide dismutase was less/not effective. At incubation of HT‐29 cells with quercetin (1–100 μM), generated H 2 O 2 slightly contributed to antioxidant cell protection by modulation of tGSH‐ and ROS‐level. In conclusion, H 2 O 2 generation in vitro by polyphenols has to be taken into consideration when interpreting results of such cell culture experiments. Unphysiologically high polyphenol concentrations, favoring substantial H 2 O 2 formation, are not expected to be met in vivo , even under conditions of high end nutritional uptake.