Optimized enzymatic colorimetric assay for determination of hydrogen peroxide (H2O2) scavenging activity of plant extracts

The classical method to determine hydrogen peroxide (H2O2) scavenging activity of plant extracts is evaluated by measuring the disappearance of H2O2 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 H2O2, 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 H2O2 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 H2O2 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 H2O2 in the system (EC50 value) calculated for several plant extracts and standard antioxidants resulted in coefficient of variance (CV%) of the EC50 values less than 3.0% and correlation coefficient values (R2) > 0.95 for all dose response curves obtained.
• This method is convenient and very precise which is suitable for the rapid quantification of H2O2 scavenging ability of standard antioxidants and natural antioxidants present in plant extracts.

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