A kinetic-based stopped-flow DPPH• method

Abstract

The reaction kinetics of antioxidants with free radicals is crucial to screen their functionality. However, studying antioxidant-radical interactions is very challenging for fast electron-donor substances, such as ascorbic acid, because the reaction ends in a few seconds. Accordingly, this work proposes a rapid and sensitive method for the determination of the absolute rate constant of the reaction between fast antioxidants and DPPH•. The method consists of a stopped-flow spectrophotometric system, which monitors the decay of DPPH• during its interaction with antioxidants. A kinetic-based reaction mechanism fits the experimental data. Kinetic parameters include a second order kinetics (k1) and, depending on the type of antioxidant, a side reaction (k2). Ascorbic acid was the fastest antioxidant (k1 = 21,100 ± 570 M−1 s−1) in comparison with other eleven phenols, showing k1 values from 45 to 3070 M−1 s−1. Compounds like catechin, epicatechin, quercetin, rutin, and tannic, ellagic and syringic acids presented a side reaction (k2 from 15 to 60 M−1 s−1). Among seven fruit juices, strawberry was the fastest, while red plum the slowest. Overall, the proposed kinetic-based DPPH• method is simple, rapid, and suitable for studying the activity and capacity of different molecules, and food samples rich in fast antioxidants, like fruit juices.