Novel Total Antioxidant Capacity Index for Dietary Polyphenols and Vitamins C and E, Using Their Cupric Ion Reducing Capability in the Presence of Neocuproine:  CUPRAC Method

Abstract

The chemical diversity of antioxidants makes it difficult to separate and quantify antioxidants from the vegetable matrix. Therefore, it is desirable to establish a method that can measure the total antioxidant activity level directly from vegetable extracts. The current literature clearly states that there is no "total antioxidant" as a nutritional index available for food labeling because of the lack of standard quantitation methods. Thus, this work reports the development of a simple, widely applicable antioxidant capacity index for dietary polyphenols and vitamins C and E, utilizing the copper(II)-neocuproine [Cu(II)-Nc] reagent as the chromogenic oxidizing agent. Because the copper(II) (or cupric) ion reducing ability of polyphenols is measured, the method is named by our research group "cupric reducing antioxidant capacity" abbreviated as the CUPRAC method. This method should be advantageous over the ferric reducing antioxidant power (FRAP) method because the redox chemistry of copper(II)-as opposed to that of ferric ion-involves faster kinetics. The method comprises mixing of the antioxidant solution (directly or after acid hydrolysis) with a copper(II) chloride solution, a neocuproine alcoholic solution, and an ammonium acetate aqueous buffer at pH 7 and subsequent measurement of the developed absorbance at 450 nm after 30 min. Because the color development is fast for compounds such as ascorbic acid, gallic acid, and quercetin but slow for naringin and naringenin, the latter compounds were assayed after incubation at 50 degrees C on a water bath for 20 min [after Cu(II)-Nc reagent addition] so as to force the oxidation reaction to reach completion. The flavonoid glycosides were hydrolyzed to their corresponding aglycons by refluxing in 1.2 M HCl-containing 50% MeOH so as to exert maximal reducing power toward Cu(II)-Nc. Certain compounds also needed incubation after acid hydrolysis to fully exhibit their reducing capability. The CUPRAC antioxidant capacities of synthetic mixtures of antioxidants were experimentally measured as Trolox equivalents and compared to those theoretically found by making use of the principle of additivity of absorbances assuming no chemical interaction between the mixture constituents. Because ascorbic acid is not resistant to elevated temperature incubation, it should be assayed initially by measuring the absorbance (at 450 nm) difference of original and ascorbate oxidase-added mixture solutions at the end of 1 min of Cu(II)-Nc reagent addition. Thus, the total CUPRAC antioxidant capacity of a mixture containing various antioxidants should be that finally measured after a suitable combination of hydrolysis and incubation procedures, added to the initially measured capacity due to ascorbate. The antioxidant polyphenolic compounds tested demonstrate that the highest capacities in the CUPRAC method were observed for epicatechin gallate, epigallocatechin gallate, quercetin, fisetin, epigallocatechin, catechin, and caffeic acid in this order, in accordance with theoretical expectati...

Antioxidant properties of fruits of raspberry and blackberry grown in central Europe

“…The CUPRAC (cupric ion reducing antioxidant capacity) assay in which utilisation of copper (II)-neocuproine as chromogenic oxidizing agent is measured was performed as described by Apak et al [35]. In the test tube 1 mL of 10 mM CuCl 2 , 1 mL of 7.5 mM neocuproine and 1 mL of 1 M NH 4 Ac buffer, pH 7.0, were mixed with 0.025-0.05 mL of plant extracts and 0.8 mL of distilled water.…”

Evaluation of biological activities of Physalis peruviana ethanol extracts and expression of Bcl-2 genes in HeLa cells

“…The Trolox (Sigma, 238813) equivalent antioxidant capacity (TEAC) values of the samples were determined using bis(neocuproine)copper (II) chloride, also known as cuprac reagent as described by Apak et al (2004). The plant extracts at certain concentrations were mixed with copper (II) chloride (Sigma, 222011), neocuproine (Sigma, N1501) and ammonium acetate (Sigma, A1542) buffer at pH 7.…”

“…These results suggest that further studies should be done for identifying these compounds from tissue cultures of Physalis peruviana. Apak et al (2004) developed CUPRAC method to measure antioxidant capacity index for polyphenols and plant materials. CUPRAC was expressed as mmol Trolox equivalents (TE)/g extract.…”

Determinação da atividade antioxidante e do teor total de polifenol em amostras de chá de ervas comercializadas em sachets

“…O método CUPRAC (cupric ion reducing antioxidant capacity) já foi anteriormente utilizado para a determinação da CAT em amostras de chás 11 , se baseia na variação da coloração dos complexos de Cu(II) formados com o ligante neocuproína 12 e utiliza a espectrofotometria, técnica bastante comum em laboratórios analíticos.…”