Cellular Antioxidant Activity (CAA) Assay for Assessing Antioxidants, Foods, and Dietary Supplements

TLDR

The CAA assay uses dichlorofluorescin, which becomes fluorescent dichlorofluorescein (DCF) upon oxidation, and measures antioxidant capacity by the reduction of DCF fluorescence, providing a biologically relevant assessment that incorporates cellular uptake, metabolism, and localization. In HepG2 cells, the assay evaluates compounds by inhibiting ABAP‑generated peroxyl radical–induced DCF formation, reporting activity as quercetin equivalents per 100 µmol phytochemical or per 100 g fruit. The assay identified quercetin as the most potent pure antioxidant, with blueberry, cranberry, apple, red grape, and green grape ranking in decreasing order of activity.

Abstract

A cellular antioxidant activity (CAA) assay for quantifying the antioxidant activity of phytochemicals, food extracts, and dietary supplements has been developed. Dichlorofluorescin is a probe that is trapped within cells and is easily oxidized to fluorescent dichlorofluorescein (DCF). The method measures the ability of compounds to prevent the formation of DCF by 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-generated peroxyl radicals in human hepatocarcinoma HepG2 cells. The decrease in cellular fluorescence when compared to the control cells indicates the antioxidant capacity of the compounds. The antioxidant activities of selected phytochemicals and fruit extracts were evaluated using the CAA assay, and the results were expressed in micromoles of quercetin equivalents per 100 micromol of phytochemical or micromoles of quercetin equivalents per 100 g of fresh fruit. Quercetin had the highest CAA value, followed by kaempferol, epigallocatechin gallate (EGCG), myricetin, and luteolin among the pure compounds tested. Among the selected fruits tested, blueberry had the highest CAA value, followed by cranberry > apple = red grape > green grape. The CAA assay is a more biologically relevant method than the popular chemistry antioxidant activity assays because it accounts for some aspects of uptake, metabolism, and location of antioxidant compounds within cells.