Cupric Ferricyanide Reaction in Solution for Determination of Reducing Properties of Plant Antioxidants
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Cupric Ferricyanide Reaction in Solution for Determination of Reducing Properties of Plant Antioxidants Jaan Klõšeiko 1
Received: 16 January 2015 / Accepted: 7 April 2015 # Springer Science+Business Media New York 2015
Abstract To develop an antioxidant assay, the oxidation of phenolics and ascorbic acid by cupric ferricyanide in solution was estimated. Solutions (pH 6.6) were kept clear with citrate and gelatin. Reaction kinetics of the color formation at 480 nm indicated further slow increase after initial fast reaction with a sample. With the active phenolics (tannic acid, quercetin, rutin, chlorogenic acid), sufficiently stable endpoint was reached at 35 °C by 90 min. Calibration showed deviation of 5–10 % from the linearity. Antioxidant mixtures of tannic, chlorogenic, and ascorbic acids indicated small positive interaction between tannic and chlorogenic acids. Extracts from forest trees and green tea responded in the same way as individual phenolics to the oxidation. Common plant osmolytes in excess did not interfere with the analysis of tannic acid for its reducing activity. Due to a poor endpoint with ascorbic acid, a method for stabilizing the endpoint was looked further. Borate, molybdate, and lower pH greatly inhibited the reaction with ascorbic acid during the slow phase. By the presence of copper ions, it was possible that the autoxidation of ascorbic acid produced hydrogen peroxide during the slow phase. However, hydrogen peroxide gave substantial color response. Solvent N-methylpyrrolidone at low level inhibited or decreased the color formation for several analytes, suggesting that an analyte-dependent activation of oxygen could cause decolorization of cupric ferrocyanide. Current findings indicated that the cupric ferricyanide antioxidant reaction can be
* Jaan Klõšeiko [email protected] 1
Department of Ecophysiology, Institute of Forestry and Rural Engineering, Estonian University of Life Sciences, Mustamäe tee 4, 10621 Tallinn, Estonia
potentially developed to an assay for estimating reducing activity in extracts from woody plants or green tea. Keywords Antioxidant assay . Ascorbic acid . Colloid . Copper . Phenolics
Introduction Antioxidant substances are important in forest trees and plantderived food (Riedl and Hagerman 2001; Sakihama et al. 2002; Lavola et al. 2003; Boyer and Liu 2004). Phenolics tend to accumulate during stress in woody plants. Beneficial health effects of plant and food polyphenols are ascribed to their ease of oxidizability (low reduction-oxidation potential) and quick scavenging of free radicals. In this way, polyphenols protect vital cellular components from the free-radical generated oxidative stress. Poplar and tea leaves accumulate phenolic compounds by 15 % or more of the leaf biomass (Donaldson et al. 2006; Kerio et al. 2013). Among various polyphenols, hydrolyzable tannins are abundant in the biomass of tree leaves (Salminen et al. 2004). Quercetin and rutin accumulate in small concentrations, but they have high antioxidant activity and are poorly soluble i
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