In situ detection of the intracellular antioxidant activity of grape skin polyphenolic extracts by confocal fluorescence microscopy: a study on rat superficial colonocytes

Colon epithelium is exposed to oxidative stress conditions via exogenous and endogenous routes and a number of colon diseases are associated with the imbalance of the cellular redox system. The aim of the present work was to study the intracellular antioxidant activity of red grape polyphenolic extracts on superficial enterocytes experimentally exposed to prooxidant conditions. In situ confocal microscopy observation on CM-H2DCFDA charged explants exposed to H2O2 (5, 10, and 15min) was performed. First, the ability of the experimental set up to detect intracellular ROS scavenging activity by intracellularly absorbed antioxidants was demonstrated by a dose-response inhibition of the H2O2 induced intracellular probe fluorescence following pre-incubation with Trolox, synthetic analog of vitamin E. Then, the explants were incubated with grape skin polyphenolic extracts for 1 h producing a significant decrease of the H2O2 induced fluorescence. This effect was more pronounced following 15 min H2O2 exposure with respect to 5 min and it was also more evident for extracts obtained from mature grapes, which showed an increased ORAC value and qualitative peculiarities in the polyphenolic composition. Results demonstrated that red grape polyphenolic extracts are able to exert an intracellular antioxidant activity on superficial colonocytes, inducing a protection action against pro-oxidant condition. Extracts obtained from mature grapes showed an increased protective activity