This study introduces a kinetic model that significantly improves the interpretation of the oxygen radical absorbance capacity (ORAC) assay. Our model accurately simulates and fits the bleaching kinetics of fluorescein in the presence of various antioxidants, achieving high correlation values (R2 > 0.99) with the experimental data. The fit to the experimental data is achieved by optimizing two rate constants, k5 and k6. The k5 value reflects the reactivity of antioxidants toward scavenging peroxyl radicals, whereas k6 measures the ability of antioxidants to regenerate oxidized fluorescein. These parameters (1) allow the detailed classification of cinnamic acids based on their structure-activity relationships, (2) provide insights into the interaction of alkoxyl radicals with fluorescein, and (3) account for the regeneration of fluorescein radicals by antioxidants. The application of the model to different antioxidants and fruit extracts reveals significant deviations from the results of traditional ORAC tests based on the area under the curve (AUC) approach. For example, lemon juice, rich in 'fast' antioxidants such as ascorbic acid, shows a high k5 value, in contrast to its low AUC values. This finding underscores the limitations of the AUC approach and highlights the advantages of our kinetic model in understanding antioxidative dynamics in food systems. This study presents a comprehensive, quantitative, mechanism-oriented approach to assessing antioxidant reactivity, demonstrating a significant improvement in ORAC assay applications.
Keywords: antioxidant activity; antioxidant mechanism; fluorescein decay; hydroxycinnamic acids; peroxyl and alkoxyl radicals; structure–activity relationship.