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°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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Apak, R., Güçlü, K., Özyürek, M., and Karademir, S. E. (2004) A novel total antioxidant capacity index for dietary polyphenols, vitamins c and e, using their cupric ion reducing capability in the presence of neocuproine: CUPRAC method. J. Agric. Food Chem. 52, 7970–7981.
Apak, R., Güçlü, K., Özyürek, M., Karademir, S. E., and Altun, M. (2005) total antioxidant capacity assay of human serum using copper(II)-neocuproine as chromogenic oxidant: The CUPRAC method. Free Radic. Res. 39, 949–961.
Apak, R., Güçlü, K., Demirata, B., Özyürek, M., Çelik, S. E., Bektas¸og˘lu, B., Berker, K. I., and Özyurt, D. (2007) Comparative evaluation of total antioxidant capacity assays applied to phenolic compounds, and the CUPRAC assay. Molecules 12, 1496–1547.
Tütem, E., Apak, R., and Baykut, F. (1991) Spectrophotometric determination of trace amounts of copper(I) and reducing agents with neocuproine in the presence of copper(II). Analyst 116, 89–94.
Özyürek, M., Güçlü, K., Bektas¸og˘lu, B., and Apak, R. (2007) Spectrophotometric determination of ascorbic acid by the modified CUPRAC method with extractive separation of flavonoids-La(III) complexes. Anal. Chim. Acta 588, 88–95.
Association of Official Analytical Chemists (1990) K. Helrick, Official Methods of Analysis (15th edn.), AOAC, Food Composition, Additives, Natural Contaminants, Washington, DC, 2, p. 1059.
Lykkesfeldt, J. (2000) Determination of ascorbic acid and dehydroascorbic acid in biological samples by high-performance liquid chromatography using subtraction methods: reliable reduction with tris(2-carboxyethyl]phosphine hydrochloride. Anal. Biochem. 282, 89–93.
Güçlü, K., Altun, M., Özyürek, M., Karademir, S. E., and Apak, R. (2006) Antioxidant capacity of fresh, sun- and sulfited-dried Malatya apricot (Prunus armeniaca) assayed by CUPRAC, ABTS/TEAC and Folin methods. Int. J. Food Sci. Technol. 41, 76–85.
Garcia-Alonso, M., Pascual-Teresa, S., Santos-Buelga, C., and Rivas-Gonzalo, J. C. (2004) Evaluation of the antioxidant properties of fruits. Food Chem. 84, 13–18.
Apak, R., Güçlü, K., Özyürek, M., Karademir, S. E., and Erc¸ag˘, E. (2006) The cupric ion reducing antioxidant capacity (CUPRAC) and polyphenolic content of some herbal teas. Int. J. Food Sci. Nutr. 57, 292–304.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., and Rice-Evans, C. (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26, 1231–1237.
Singleton, V. L., Orthofer, R., and Lamuela-Raventos, R. M. (1999) Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Meth. Enzymol. 299, 152–178.
Singleton, V. L., and Rossi, J. A. (1965) Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagent. Am. J. Enol. Vitic. 16, 144–158.
Bors, W., Hellers, W., Michel, C., and Saran, M. (1990) Radical Chemistry of Flavonoid Antioxidants. In: I. Emerit, L. Packer and C. Auclair (Eds.), Antioxidants in Therapy and Preventive Medicine, Plenum Press, New York, 1, 165–170.
Rice-Evans, C. A., Miller, N. J., and Paganga, G. (1997) Antioxidant properties of phenolic compounds. Trends Plant Sci. 2, 152–159.
Castelluccio, C., Bolwell, G. P., Gerrish C., and Rice-Evans, C. A. (1996) Differential distribution of ferulic acid to the major plasma constituents in relation to its potential as an antioxidant. Biochem. J. 316, 691–694.
Kanski, J., Aksenova, M., Stoyanova, A., and Butterfield, D. A. (2002) Ferulic acid antioxidant protection against hydroxyl and peroxyl radical oxidation in synaptosomal and neuronal cell culture systems in vitro: structure-activity studies. J. Nutr. Biochem. 13, 273–281.
Tepe, B., Eminagaoglu, O., Akpulat, H. A., and Aydin, E. (2007) Antioxidant potentials of rosmarinic acid levels of methanolic extracts of Salyvia verticillata (L.) subsp. verticillata and S. verticillata (L.) subsp. amasiaca (Freyn & Bornm.) Bornm. Food Chem. 100, 985–989.
Chen, J. H., and Ho, C. -T. (1997) Antioxidant activities of caffeic acid and its related hydroxycinnamic acid compounds. J. Agric. Food Chem. 45, 2374–2378.
Cervellati, R., Renzulli, C., Guerra, M. C., and Speroni, E. (2002) Evaluation of antioxidant activity of some natural polyphenolic compounds using the briggs-rauscher reaction method. J. Agric. Food Chem. 50, 7504–7509.
Kim, D. -O., and Lee, C. Y. (2004) Comprehensive study on vitamin C equivalent antioxidant capacity (VCEAC) of various polyphenolics in scavenging a free radical and its structural relationship. Critic. Rev. Food Sci. Nutr. 44, 253–273.
Miliauskas, G., van Beek, T. A., Venskutonis, P. R., Linssen, J. P. H., and de Waard, P. (2004) Antioxidative activity of Geranium Macrorrhizum. Eur. Food Res. Technol. 218, 253–261.
Firuzi, O., Lacanna, A., Petrucci, R., Marrosu, G., and Saso, L. (2005) Evaluation of the antioxidant activity of flavonoids by “ferric reducing antioxidant power” assay and cyclic voltammetry. Biochim. Biophys. Acta. 1721, 174–184.
Santos-Buelga, C., and Scalbert, A. (2000) Proanthocyanidins and tannin-like compounds: nature, occurence, dietary intake and effects on nutrition and health (review). J. Sci. Food Agric. 80, 1094–1117.
Asma, B. M. (2000) Kayisi yetistiriciligi (Apricot Growing). Malatya, Turkey: Evin Publishers.
Halvorsen, B. L., Holte, K., Myhrstad, M. C. W., Barikmo, I., Hvattum, E., Remberg, S. V., et al. (2002). A systematic screening of total antioxidants in dietary plants. J. Nutr. 132, 461–471.
Tütem, E., and Apak, R. (1991) Simultaneous spectrophotometric determination of cystine and cysteine in amino acid mixtures using copper(II)-neocuproine reagent. Anal. Chim. Acta 255, 121–125.
Benzie, I. F. F., and Strain, J. J. (1996) The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal. Biochem. 239, 70–76.
Özçelik, B., Lee, J. H., and Min, D. B. (2003) Effects of light, oxygen, and pH on the absorbance of 2,2-diphenyl-1-picrylhydrazyl. J. Food Sci. 68, 487–490.
Özyürek, M., Çelik, S. E., Berker, K. I., Güçlü, K., Tor, I., and Apak, R. (2007) Sensitivity enhancement of CUPRAC and iron(III)-phenanthroline antioxidant assays by preconcentration of colored reaction products on a weakly acidic cation exchanger. React. Func. Polym. 67, 1478–1486.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Apak, R., Güçlü, K., Özyürek, M., Bektas¸oğlu, B., Bener, M. (2008). Cupric Ion Reducing Antioxidant Capacity Assay for Food Antioxidants: Vitamins, Polyphenolics, and Flavonoids in Food Extracts. In: Armstrong, D. (eds) Advanced Protocols in Oxidative Stress I. Methods In Molecular Biology, vol 477. Humana Press. https://doi.org/10.1007/978-1-60327-517-0_14
Download citation
DOI: https://doi.org/10.1007/978-1-60327-517-0_14
Publisher Name: Humana Press
Print ISBN: 978-1-60327-218-6
Online ISBN: 978-1-60327-517-0
eBook Packages: Springer Protocols