A new Fenton assay for Hydroxyl radical scavengers by monitoring Catechol oxidation

A new, simple assay has been optimized to generate and detect hydroxyl radical by Fenton reaction. The assay is based on oxidation of catechol, monitored at 360 nm following incubation at room temperature for ca. 30 minutes, with linearity over 0.25 through 2.5 μmole H2O2 using Fe (II) (0.5 μmole) and acidified catechol (0.5 mg). The Fenton reaction has been characterized by registering: (i) about 13 folds increase in response to H2O2 in presence of iron (II), (ii) inhibition by agents chelating iron and/or neutralizing H2O2 and, (iii) antagonism by established hydroxyl radical scavengers. Hydroxyl radical scavenging potential of the test agents, expressed as μmole scavenged μmole test agent, has been found in the order: quercetin > curcumin> morin > rutin> daflon> diosmin > ascorbic acid > thiourea > DMSO > mannitol > benzoic acid. Citric acid, oxalic acid, tartaric acid, acetic acid and their salts, disodium EDTA, sodium metabislphite, ethanol, methanol and acetone have been evaluated for their influence on the assay when present during or following color development. A comparable Fenton reaction has been also demonstrated while using 1 mg catechol and 10 μmole Cu (II) with linearity over 0.25 through 5.0 μmole H2O2. Key-words: Fenton reaction, Hydroxyl radical scavengers, Inhibitors of Fenton reaction, Dihydroxybenzenes.