Cupric Ferricyanide Reaction in Solution for Determination of Reducing Properties of Plant Antioxidants

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 potentially developed to an assay for estimating reducing activity in extracts from woody plants or green tea.