A Novel Stoichio-Kinetic Model for the DPPH• Assay: The Importance of the Side Reaction and Application to Complex Mixtures

The 2,2-diphenyl-1-picrylhydrazyl (DPPH center dot) assay is widely used to determine the antioxidant activity of food products and extracts. However, the common DPPH center dot protocol uses a two-point measurement and does not give information about the kinetics of the reaction. A novel stoichio-kinetic model applied in this study monitors the consumption of DPPH center dot by common antioxidants following the second order reaction. The fitting of such decay yields the rate constant k(1), which describes the main reaction between antioxidants and DPPH center dot, and the rate constant k(2), which is attributed to a slower side reaction considering the products generated between the transient radicals (AO center dot) and another molecule of DPPH center dot. The model was first applied to antioxidant standards. Sinapic acid, Trolox and ascorbic and chlorogenic acids did not show any side reaction. Instead gallic, ferulic and caffeic acids achieved the best fitting with k(2). The products of the side reaction for these compounds were confirmed and identified with high-resolution mass spectrometry. Finally, the kinetic model was applied to evaluate the antioxidant activity of eight herbal extracts. This study suggests a new kinetic approach to standardize the common DPPH center dot assay for the determination of antioxidant activity.

Automated summary

A novel stoichio-kinetic model was applied in this study to monitor the consumption of DPPH center dot by common antioxidants, yielding rate constants k(1) and k(2) to describe the main and side reactions. This approach was used to evaluate the antioxidant activity of various compounds and herbal extracts, providing insights into the kinetics of the reactions. The study suggests a new kinetic approach to standardize the common DPPH center dot assay for the determination of antioxidant activity.