Novel pro-oxidant activity assay for polyphenols, vitamins C and E using a modified CUPRAC method.
In this study, a direct assay, a modified CUPRAC (Cupric Ion Reducing Antioxidant Capacity) method, is developed to determine transition metal ion (Cu(II))-catalyzed pro-oxidant activity of polyphenolic compounds, vitamins C and E, and herbal samples in the presence of proteins containing thiol groups. Since transition metal ion-catalyzed pro-oxidant activity of phenolics is usually initiated with the reduction of the metal to lower oxidation states (as a prerequisite of Fenton-type reactions), this method involves the reduction of copper(II) ions to copper(I) by polyphenolic compounds (simultaneously giving rise to reactive species), binding of the formed Cu(I) to egg white protein -SH groups, and liberation of copper(I)-neocuproine (Cu(I)-Nc) chelate (showing maximum absorbance at 450 nm) by treating the incubation product with a neocuproine-ammonium acetate mixture. The proposed method is validated against atomic absorption spectrometric (AAS) determination of protein-bound copper and protein carbonyl assay of oxidative stress. The proposed assay is faster and more specific than the carbonyl assay, and uses low-cost reagents and equipment. Pro-oxidant activity (i.e. proportional to absorbance) varies linearly over a relatively wide range with concentration, as opposed to the reciprocal correlations (i.e. linear regression of 1/(pro-oxidant activity) versus 1/concentration) of other similar assays. The pro-oxidant activity order of the tested antioxidant compounds in terms of 'Quercetin Equivalent Pro-oxidant Activity' (QREPA) coefficients is: gallic acid > epicatechin > quercetin ≈ catechin > α-tocopherol > rosmarinic acid > trolox > caffeic acid > ascorbic acid.
Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar 34320, Istanbul, Turkey.
Esin
E
Kondakçı
Mustafa
M
Özyürek
Kubilay
K
Güçlü
Reşat
R
Apak
D000085:Acetates / Q000737:chemistry; D000975:Antioxidants / Q000032:analysis*; D001205:Ascorbic Acid / Q000032:analysis*; D001681:Biological Assay / Q000379:methods* / Q000592:standards; D002138:Calibration; D002413:Cations, Divalent; D002414:Cations, Monovalent; D003300:Copper / Q000737:chemistry*; D004527:Egg Proteins / Q000737:chemistry; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000737:chemistry; D010936:Plant Extracts / Q000737:chemistry; D059808:Polyphenols / Q000032:analysis*; D050050:Protein Carbonylation; D012680:Sensitivity and Specificity; D012996:Solutions; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014810:Vitamin E / Q000032:analysis*
D000085:Acetates; D000975:Antioxidants; D002413:Cations, Divalent; D002414:Cations, Monovalent; D004527:Egg Proteins; D010618:Phenanthrolines; D010936:Plant Extracts; D059808:Polyphenols; D012996:Solutions; D013438:Sulfhydryl Compounds; D014810:Vitamin E; C028559:bathocuproine sulfonate; D003300:Copper; C002701:neocuproine; D001205:Ascorbic Acid; C018824:ammonium acetate
Modified CUPRAC method; Polyphenolic compounds; Pro-oxidant activity; Protein oxidation