Determination of Free Radical Scavenging Activity of Plant Extracts Through DPPH Assay: An EPR and UV–Vis Study

Abstract

Several methods have been developed to assess the radical scavenging activity. Among them, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) spectrophotometric method is one of the most widely applied and is appreciated for its reliability. In this study, a comparison of two spectroscopic methods (electron paramagnetic resonance (EPR) and ultraviolet–visible (UV–Vis) spectroscopy) was performed analysing the spectroscopic features of DPPH in mixed ethanol/water solution and the free radical scavenging properties of myrtle leaves extracts and citrus juices. When DPPH was dissolved in mixed solvents, EPR enabled to identify the aggregation phenomena that occur when high amounts of water were employed. On the contrary, UV–Vis revealed only small differences in the absorption maximum among solutions with increasing water contents, without detecting aggregation. EC50 values of myrtle leaf extracts and citrus juices calculated from UV–Vis data were lower than those calculated with EPR. In myrtle extracts, the DPPH depletion measured by UV–Vis was not concentration dependent, revealing the interference in the analysis of the decomposition products of the antioxidants, which absorb at 517 nm. EPR spectroscopy was proven to be most reliable with all types of matrix since it is not dependent on the chemical composition of the extract.