A kinetic-based stopped-flow DPPH• method

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 center dot. The method consists of a stopped-flow spectrophotometric system, which monitors the decay of DPPH center dot during its interaction with antioxidants. A kinetic-based reaction mechanism fits the experimental data. Kinetic parameters include a second order kinetics (k(1)) and, depending on the type of antioxidant, a side reaction (k(2)). Ascorbic acid was the fastest antioxidant (k(1) = 21,100 +/- 570 M-1 s(-1)) in comparison with other eleven phenols, showing k(1) 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 (k(2) 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 center dot 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.