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Cupric ion reducing antioxidant capacity assay for food antioxidants: vitamins, polyphenolics, and flavonoids in food extracts.
Methods Mol Biol. 2008; 477:163-93.MM

Abstract

Antioxidants are health beneficial compounds through their combat with reactive oxygen and nitrogen species and free radicals that may cause tissue damage leading to various diseases. This work reports the development of a simple and widely applicable antioxidant capacity index for dietary polyphenols, vitamins C and E, and plasma antioxidants utilizing the copper(II)-neocuproine (Cu(II)-Nc) reagent as the chromogenic oxidizing agent. This novel method based on an electron-transfer mechanism was named by our research group as 'cupric reducing antioxidant capacity', abbreviated as the CUPRAC method. The method is comprised of mixing the antioxidant solution with aqueous copper(II) chloride, alcoholic neocuproine, and ammonium acetate aqueous buffer at pH 7, and subsequently measuring the developed absorbance at 450 nm after 30 min. Since the color development is fast for compounds like ascorbic acid, gallic acid, and quercetin but slow for naringin and naringenin, the latter compounds are assayed after incubation at 50 degrees C on a water bath for 20 min. The flavonoid glycosides are hydrolyzed to their corresponding aglycones by refluxing in 1.2 M: HCl-containing 50% MeOH so as to exert maximal reducing power towards Cu(II)-Nc. The CUPRAC antioxidant capacities of synthetic mixtures are equal to the sum of individual capacities of antioxidant constituents, indicating lack of chemical deviations from Beer's law. Tests on antioxidant polyphenols demonstrate that the highest CUPRAC capacities are observed for epicatechin gallate, epigallocatechin gallate, quercetin, fisetin, epigallocatechin, catechin, and caffeic acid in this order, in accord with the number and position of the -OH groups as well the conjugation level of the molecule. The parallelism of the linear calibration curves of pure antioxidants in water and in a given complex matrix (plant extract) demonstrates that there are no chemical interactions of interferent nature among the solution constituents, and that the antioxidant capacities of the tested antioxidants are additive, in conformity to the Beer's law. For individual determination of ascorbic acid in fruit juices with a modified CUPRAC procedure, flavonoids are pre-extracted as their La(III) complexes prior to assay. For apricot extracts, a modified version of the CUPRAC assay based on anion exchange separation at pH 3 is applied, since sulfited-dried sample extracts contain the hydrosulfite anion interfering with the determination. For herbal tea infusions, the standard CUPRAC protocol is applied. The CUPRAC reagent is stable, easily accessible, low-cost, and is sensitive toward thiol-type antioxidants unlike FRAP. The reaction is carried out at nearly physiological pH as opposed to the acidic pH of FRAP or to the alkaline pH of Folin methods, constituting a basic advantage for the realistic assay of biological fluids.

Authors+Show Affiliations

Department of Chemistry, Faculty of Engineering, Istanbul University, Istanbul, Turkey.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19082947

Citation

Apak, Reşat, et al. "Cupric Ion Reducing Antioxidant Capacity Assay for Food Antioxidants: Vitamins, Polyphenolics, and Flavonoids in Food Extracts." Methods in Molecular Biology (Clifton, N.J.), vol. 477, 2008, pp. 163-93.
Apak R, Güçlü K, Ozyürek M, et al. Cupric ion reducing antioxidant capacity assay for food antioxidants: vitamins, polyphenolics, and flavonoids in food extracts. Methods Mol Biol. 2008;477:163-93.
Apak, R., Güçlü, K., Ozyürek, M., Bektas Oğlu, B., & Bener, M. (2008). Cupric ion reducing antioxidant capacity assay for food antioxidants: vitamins, polyphenolics, and flavonoids in food extracts. Methods in Molecular Biology (Clifton, N.J.), 477, 163-93. https://doi.org/10.1007/978-1-60327-517-0_14
Apak R, et al. Cupric Ion Reducing Antioxidant Capacity Assay for Food Antioxidants: Vitamins, Polyphenolics, and Flavonoids in Food Extracts. Methods Mol Biol. 2008;477:163-93. PubMed PMID: 19082947.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Cupric ion reducing antioxidant capacity assay for food antioxidants: vitamins, polyphenolics, and flavonoids in food extracts. AU - Apak,Reşat, AU - Güçlü,Kubilay, AU - Ozyürek,Mustafa, AU - Bektas Oğlu,Burcu, AU - Bener,Mustafa, PY - 2008/12/17/entrez PY - 2008/12/17/pubmed PY - 2009/1/8/medline SP - 163 EP - 93 JF - Methods in molecular biology (Clifton, N.J.) JO - Methods Mol. Biol. VL - 477 N2 - Antioxidants are health beneficial compounds through their combat with reactive oxygen and nitrogen species and free radicals that may cause tissue damage leading to various diseases. This work reports the development of a simple and widely applicable antioxidant capacity index for dietary polyphenols, vitamins C and E, and plasma antioxidants utilizing the copper(II)-neocuproine (Cu(II)-Nc) reagent as the chromogenic oxidizing agent. This novel method based on an electron-transfer mechanism was named by our research group as 'cupric reducing antioxidant capacity', abbreviated as the CUPRAC method. The method is comprised of mixing the antioxidant solution with aqueous copper(II) chloride, alcoholic neocuproine, and ammonium acetate aqueous buffer at pH 7, and subsequently measuring the developed absorbance at 450 nm after 30 min. Since the color development is fast for compounds like ascorbic acid, gallic acid, and quercetin but slow for naringin and naringenin, the latter compounds are assayed after incubation at 50 degrees C on a water bath for 20 min. The flavonoid glycosides are hydrolyzed to their corresponding aglycones by refluxing in 1.2 M: HCl-containing 50% MeOH so as to exert maximal reducing power towards Cu(II)-Nc. The CUPRAC antioxidant capacities of synthetic mixtures are equal to the sum of individual capacities of antioxidant constituents, indicating lack of chemical deviations from Beer's law. Tests on antioxidant polyphenols demonstrate that the highest CUPRAC capacities are observed for epicatechin gallate, epigallocatechin gallate, quercetin, fisetin, epigallocatechin, catechin, and caffeic acid in this order, in accord with the number and position of the -OH groups as well the conjugation level of the molecule. The parallelism of the linear calibration curves of pure antioxidants in water and in a given complex matrix (plant extract) demonstrates that there are no chemical interactions of interferent nature among the solution constituents, and that the antioxidant capacities of the tested antioxidants are additive, in conformity to the Beer's law. For individual determination of ascorbic acid in fruit juices with a modified CUPRAC procedure, flavonoids are pre-extracted as their La(III) complexes prior to assay. For apricot extracts, a modified version of the CUPRAC assay based on anion exchange separation at pH 3 is applied, since sulfited-dried sample extracts contain the hydrosulfite anion interfering with the determination. For herbal tea infusions, the standard CUPRAC protocol is applied. The CUPRAC reagent is stable, easily accessible, low-cost, and is sensitive toward thiol-type antioxidants unlike FRAP. The reaction is carried out at nearly physiological pH as opposed to the acidic pH of FRAP or to the alkaline pH of Folin methods, constituting a basic advantage for the realistic assay of biological fluids. SN - 1064-3745 UR - https://www.unboundmedicine.com/medline/citation/19082947/Cupric_ion_reducing_antioxidant_capacity_assay_for_food_antioxidants:_vitamins_polyphenolics_and_flavonoids_in_food_extracts_ L2 - https://dx.doi.org/10.1007/978-1-60327-517-0_14 DB - PRIME DP - Unbound Medicine ER -