TY - JOUR
T1 - Optimized enzymatic colorimetric assay for determination of hydrogen peroxide (H2O2) scavenging activity of plant extracts
AU - Fernando, Chamira Dilanka
AU - Soysa, Preethi
N1 - Funding Information:
Freeze-dried samples of S . parvifolia and M . cerviana were donated by Ms. D.D.P. Jayarathna and Dr. D.N. Jayasinghe. We acknowledge financial assistance by Department of Biochemistry & Molecular Biology, Faculty of Medicine, University of Colombo and National Science Foundation, Sri Lanka . Authors like to thank Ms. Sudeepa Sugathadasa and Ms. Pushpa Jeewandara, Department of Botany, Bandaranayake Memorial Ayurveda Research Institute, Nawinna, Colombo, Sri Lanka, for the identification of the plant material used in this study. The technical assistance offered by Mr. Thisira Andrahennadi, Mr. Jayantha Weerasinghe, Mr. Saman Kolombage and Ms. Nilusha Rajapakse, Department of Biochemistry & Molecular Biology, Faculty of Medicine, University of Colombo, is gratefully acknowledged. MethodsX thanks the reviewers of this article for taking the time to provide valuable feedback.
Publisher Copyright:
© 2015 The Authors. Published by Elsevier B.V.
PY - 2015/6/4
Y1 - 2015/6/4
N2 - 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.
AB - 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.
KW - Method name Enzymatic colorimetric assay for HO scavenging activity
UR - https://www.scopus.com/pages/publications/84931261765
U2 - 10.1016/j.mex.2015.05.001
DO - 10.1016/j.mex.2015.05.001
M3 - Article
AN - SCOPUS:84931261765
SN - 2215-0161
VL - 2
SP - 283
EP - 291
JO - MethodsX
JF - MethodsX
ER -