In vitro measurement of the anthocyanin antioxidant activity in a living epithelium

Aim: Anthocyanins are natural pigments belonging to the group of polyphenols. They are known for the strong antioxidant activity mainly due to the "scavenging" of free radicals in the cell. The aim of this work is to study the antioxidant activity of an anthocyanin extract on a living tissue in vitro by using the probe CM-H2DCFDA, whose fluorescence (at the excitation wave length of 488 nm) increases in the presence of Reactive Oxigen Species (ROS). The study was carried out on the eel intestine, which is a useful model system for functional studies of epithelia that perform near-isosmotic fluid absorption. For the first time CM-H2DCFDA was used for ROS visualization in a living tissue. Methods: The anthocyanin extract was obtained from the skins of black grapes. Small segments of the eel middle intestine were incubated with the anthocyanin extract, charged with the fluorescent probe, and then exposed to hydrogen peroxide as oxidant agent. Under confocal microscopy observation serial z-cross-sectional images of the epithelium were performed in order to visualize ROS production through the thickness of the tissue. Results: Hydrogen peroxide exposure significantly increased the fluorescence through the thickness of the epithelium. The fluorescence intensity was dependent on the time of peroxide incubation. On the other hand in tissues previously incubated with the anthocyanin extract the hydrogen peroxide induced fluorescence was dramatically reduced. Conclusions: The anthocyanins cointained in the skin of black grapes are able to strongly protect the intestinal epithelium from oxidative stress. This protective role is of particular importance for the intestinal epithelium since this tissue may be exposed to prooxidant substances ingested with the diet. In this work for the first time a method for the measurement of the antioxidant activity of a substance directly on a living epithelium was proposed.