A rapid and simplified DPPH assay for analysis of antioxidant interactions in binary combinations

The study of synergistic and antagonistic antioxidant interactions is particularly important for pharmaceutical and food industries. The application of methods like 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay is highly suited for the quantification of the antioxidant activity of single compounds or plant extracts or foods. However, for studying antioxidant interactions, these assays can become laborious and time-consuming, since each pair of combinations demands the preparation of several binary mixtures endowed with different combining concentrations. Consequently, these assays generate large amounts of data that need to be mathematically and statistically treated with specific and commercial software. In our work, we propose a novel strategy to shorten simultaneously the number of steps related to the preparation of binary mixtures and data treatment in the study of antioxidant interactions. To achieve this goal, binary mixtures were prepared using an adapted version of the checkerboard assay (two-dimensional microdilution) and the antioxidant interactions were quantified, analyzed, and presented using a free-web application (SynergyFinder Plus). Particularly, we studied the antioxidant interaction of binary combinations of trehalose with ascorbic acid, catechin, gallic acid, or quercetin. Our results show that trehalose presents a significant antagonistic activity in a binary combination with all four antioxidant compounds. In conclusion, this is the first time an adapted version of the checkerboard assay and SynergyFinder Plus has been used to study antioxidant interactions. Hence, a straightforward, efficient, and accessible method was developed to assess the antioxidant interactions of compounds in binary mixtures.