Sensitive cell-based assay using DCFH oxidation for the determination of pro- and antioxidant properties of compounds and mixtures: Analysis of fruit and vegetable juices

Reactive oxygen species play a critical role in cardiovascular diseases, inflammatory diseases, neurodegenerative disorders, cancer and aging. Diets rich in foods containing antioxidants, such as fruits and vegetables, could help prevent these pathologies. It is therefore important to properly assay the antioxidant potentials of these antioxidant foods in order to have a guideline for their proper use. Actual in vitro methodologies are often very specific for one mode of action and do not necessarily reflect the normal biological context in which they are used. In this work, we have developed a cell-based assay using 2′,7′-dichlorofluorescin-diacetate (DCFH-DA), a useful indicator of reactive oxygen species (ROS), in order to determine the antioxidant properties of foods, extracts and molecules. Results show a dose-dependent antioxidant activity for pure compounds (in decreasing order of activity: quercetin > caffeic acid > gallic acid > α-tocopherol) and fruit juices (in decreasing order of activity: strawberries > highbush blueberries > kiwis > peaches). These results are in good agreement with results obtained using the ORACFL assay. However, the cell-based assay detected a pro-oxidant effect with broccoli and carrot juices which was not observed using the ORACFL assay. Mixed isomers of β,α-carotene isolated from carrots were also found to oxidize DCFH about 212% above control-level. Interestingly, the boiling of broccoli and carrot juices inhibits this pro-oxidant effect and restores the antioxidant properties of the juices. Moreover, the boiling of the β,α-carotene mixed isomers causes their partial degradation and significantly inhibits DCFH oxidation about 68%, suggesting that carotenoids present in broccoli and carrot juices are, in part, responsible for their pro-oxidant effects.