Development, structure-activity relationships, and application of the cellular antioxidant activity assay

Reactive oxygen species (ROS) are involved in all stages of carcinogenesis. Reduction of damaging ROS can be achieved by increasing dietary antioxidants found in fruits and vegetables. Intake of fruits and vegetables and their antioxidant flavonoid constituents have been associated with reduced risk of cancer in many epidemiological studies and their effects on cell proliferation, survival, and apoptosis in vitro are numerous. Due to the potential of dietary antioxidants to prevent or delay cancer, it is important to be able measure their antioxidant activity. It is currently popular to measure the antioxidant activity using chemistry assays, but those methods do not take into account the complexity of biological systems. There is a need for a more biologically relevant assay. Therefore, the objectives of the study were (1) to develop a cell-based assay to measure antioxidant activity; (2) to determine the structure-activity relationships of flavonoids in the assay; and (3) to measure the cellular antioxidant activity of commonly consumed fruits.;A cellular antioxidant activity (CAA) assay for quantifying the antioxidant activity of phytochemicals, food extracts, and dietary supplements was developed. Dichlorofluorescin is a probe that is trapped within cells and is easily oxidized to the fluorescent dichlorofluorescein (DCF). The method measures the ability of compounds to prevent the formation of DCF by 2,2'-azobis(2-arnidinopropane) dihydrochloride (ABAP)-generated peroxyl radicals in human hepatocarcinoma HepG2 cells. The decrease in cellular fluorescence when compared to the control cells indicates the antioxidant efficacy of the compounds.;The structures that conferred flavonoids with the most antioxidant activity in the CAA assay were determined. They were a 3',4'-o-dihydroxyl group in the B-ring, a 2,3-double bond combined with a 4-keto group in the C-ring, and a 3-hydroxyl group. Isoflavones had no cellular antioxidant activity. Flavanols with a galloyl moiety had higher antioxidant activity than those without, and a B-ring 3',4',5'-trihydroxyl group further improved their efficacy. ORAC values for flavonoids were not related to their CAA values.;The cellular antioxidant activities of 25 fruits commonly consumed in the United States were measured. Pomegranate and the berries (wild blueberry, blackberry, raspberry, blueberry) had the highest CAA values, whereas banana and the melons (watermelon, cantaloupe, honeydew) had the lowest. Apples were found to be the largest contributors of fruit phenolics to the American diet, and apple and strawberries were the biggest suppliers of cellular antioxidant activity. Pomegranate had the highest cellular antioxidant quality of the fruits tested.;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 species within cells. Increasing phytochemical intake through fruit consumption is a logical strategy to increase antioxidant intake and decrease oxidative stress, and may lead to reduced risk of cancer.