Optimized enzymatic colorimetric assay for determination of hydrogen peroxide (H(2)O(2)) scavenging activity of plant extracts

The classical method to determine hydrogen peroxide (H(2)O(2)) scavenging activity of plant extracts is evaluated by measuring the disappearance of H(2)O(2) at a wavelength of 230 nm. Since this method suffers from the interference of phenolics having strong absorption in the UV region, a simple and rapid colorimetric assay was developed where plant extracts are introduced to H(2)O(2), phenol and 4-aminoantipyrine reaction system in the presence of horseradish peroxidase (HRP). This reaction yields a quinoneimine chromogen which can be measured at 504 nm. Decrease in the colour intensity reflects the H(2)O(2) scavenged by the plant material. • Optimum conditions determined for this assay were 30 min reaction time, 37 °C, pH 7, enzyme concentration of 1 U/ml and H(2)O(2) concentration of 0.7 mM. The limit of detection (LOD) and limit of quantitation (LOQ) were 136 μM and 411 μM, respectively. • Half maximal effective concentration required to scavenge 50% of H(2)O(2) in the system (EC(50) value) calculated for several plant extracts and standard antioxidants resulted in coefficient of variance (CV%) of the EC(50) values less than 3.0% and correlation coefficient values (R(2)) > 0.95 for all dose response curves obtained. • This method is convenient and very precise which is suitable for the rapid quantification of H(2)O(2) scavenging ability of standard antioxidants and natural antioxidants present in plant extracts.