Evaluation Study on Intracellular Antioxidant Activity

Abstract

Reactive oxygen species (ROS), generated by ultraviolet radiation or diverse oxidative stress, are believed to promote aging. Various methods for evaluation of antioxidant activity have been proposed. In particular, the methods using cultured cells are thought to be capable of evaluating antioxidant activity in a way similar to in vivo conditions. In this study, we investigated a method which reflected the intracellular antioxidant activity more precisely than the conventional method, by introducing a novel ROS detection reagent, 5-(and-6)-chloromethyl-2', 7-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA), which permeates cell membranes better and is retained longer within the cells, normal human dermal fibroblast (NHDF), than conventional reagents. Cellular antioxidant activity (CAA) values for representative polyphenols, caffeic acid, quercetin and EGCg were measured by the investigated method. As a result, CAA values for caffeic acid (100μM), quercetin (50μM) and EGCg (50μM) were 82, 48, and 68% respectively, and it was supposed that the intracellular antioxidant activity, including the penetration amount of samples into the cells, was evaluated more precisely.