COMPARISON OF TWO KINETIC METHOD OF DETERMINATION OF ANTIRADICAL ACTIVITY USING 2,2-DIPHENYL-1-PICRYLHYDRAZYL

Considerable work has been directed towards devising relevant assay method for evaluation of activity of free radical scavengers. A large number of methods using different approaches has been reported in the literature, but the simple spectrophotometric test based on scavenging of stable coloured 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical belongs to the most commonly used. In DPPH test, the scavenging ability of antioxidants is usually expressed as the antioxidant concentration (EC50) at which half the DPPH radicals is quenched in a predetermined time or by the number of DPPH radicals quenched by one molecule of antioxidant. However both these parameters are rather static, neither the EC50 value nor the stoichiometry have not reflect the reaction rate of antioxidants with DPPH. The aim of this work was developing of kinetic method of determination of antiradical activity by virtue of determination of rate constants of scavenging of DPPH. The rate constants and order of reaction between DPPH and antioxidant in methanol were estimated by order degeneration method. If the concentrations of antioxidants were much more higher than concentration of DPPH, the rate constant of reaction can not be determined due to nonlinear relationship between the rate constant of pseudo-first order and the concentration of antioxidants. However measurement at the excess of DPPH enables the assessment of the rate constants. Surprisingly, the decay of DPPH corresponds always to first-order reaction. For majority of antioxidants, the rate of the decrease of DPPH depends only on antioxidant concentration. However, for minority of antioxidants above all esters of caffeic acid, the rate of reaction depends only on DPPH concentration. We determined the rate constants of 25 antioxidants. The most efficient scavenger of DPPH was ascorbic acid, which reacts with DPPH approximately million times quickly than para-benzoic acid, the most weekly antioxidant. The relationship between the structure and antiradical activity is reported. The antiradical activity of antioxidants depends not so much on the number of phenolic hydroxyl, as is widely reported in literature, as on the reactivity of the most activated hydroxyl. The first order rate constant, can be used for simple and correct statement of antiradical activity.