Spectrofluorimetric determination of hydrogen peroxide scavenging activity

Homovanillic acid (HVA) is widely used for the detection and imaging of oxidative enzymes—peroxidase, glucose oxidase and xanthine oxidase, but antioxidant activity has not been determined so far with the use of HVA. We have developed a simple, sensitive and in-field spectrofluorimetric method for the determination of hydrogen peroxide (H₂O₂) scavenging activity. The assay is based on the oxidation of HVA to its fluorescent biphenyl dimer in the presence of H₂O₂ and peroxidase. The presence of substances with H₂O₂ scavenging activity prevents the oxidation of HVA by removing H₂O₂. The decrease in fluorescence intensity is proportional to the antioxidative (H₂O₂ scavenging) activity. The method was evaluated using Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), BHA (3-t-butyl-4-hydroxyanisole) and ferulic, vanillic, caffeic, chlorogenic, protocatechuic and oxalic acids. Additionally, tea and herb infusions known for their antioxidant properties were evaluated.