M.A. Morosanova, A.S. Fedorov and E.I. Morosanova* Pages 11 - 20 ( 10 )
Background: The consumption of antioxidants, including phenolic compounds, is considered important for preventing the oxidative damage diseases and ageing. The total polyphenol content (TPC) is the parameter used to estimate of plant derived products quality.
Methods: Phenol oxidase activity of green bean (Phaseolus vulgaris) crude extract (in presence of hydrogen peroxide) and banana (Musa sp.) pulp crude extract has been studied spectrophotometrically using catechol, gallic acid, caffeic acid, ferulic acid, and quercetin as substrates. All studied compounds have been oxidized in presence of green bean crude extract and hydrogen peroxide; all studied compounds except ferulic acid have been oxidized in presence of banana pulp crude extract. Michaelis constants (Km) and maximum reaction rates (Vmax) have been determined for the oxidation in presence of green bean crude extract and hydrogen peroxide (Km are 3.8×10-4 M, 1.6×10-3 M, 2.2×10-4 M, 2.3×10-4 M, 1.4×10-4 M and Vmax are 0.046 min-1, 0.102 min-1, 0.185 min-1, 0.053 min-1, 0.041 min-1 for catechol, gallic acid, caffeic acid, ferulic acid, and quercetin, respectively) and for the oxidation in presence of banana pulp crude extract (Km are 1.6×10-3 M, 3.8×10-3 M, 2.2×10-3 M, 4.2×10-4 M and Vmax are 0.058 min-1, 0.025 min-1, 0.027 min-1, 0.015 min-1 for catechol, gallic acid, caffeic acid, and quercetin, respectively). The influence of 3-methyl-2-benzothiazolinone hydrazone (MBTH) on the oxidation reactions kinetics has been studied: Michaelis constants values decrease and maximum reaction rates increase, which contributes to the increase in sensitivity of the determination.
Results: Kinetic procedures of total polyphenol content (TPC) determination using crude plants extracts in presence of MBTH have been proposed (time of analysis is 1 min). For gallic acid (used as a standard for TPC determination) detection limit is 5.3×10-5 M, quantitation limit is 1.8×10-4 M, and linear range is 6.0×10-5 – 1.3×10-3 M for green bean crude extract; detection limit is 2.9×10-5 M, quantitation limit is 9.5×10-5 M, and linear range is 3.0×10-5 - 2.4×10-3 M for banana pulp crude extract. Proposed procedures are characterized by higher interference thresholds for sulfites, ascorbic acid, and citric acid compared to pure enzymes (horseradish peroxidase and mushroom tyrosinase) in the same conditions. Compared with standard Folin-Ciocalteu (FC) method the procedures described in this work are also characterized by less interference and more rapid determination.
Conclusion: The procedures have been applied to TPC determination in tea, coffee, and wine samples. The results agree with the FC method for tea and coffee samples, and are lower for wine samples, probably, due to sulfites interference.
Phenol oxidase enzymatic activity, green bean crude extract, banana crude extract, 3-methyl-2-benzothiazolinone hydrazone, total polyphenol content determination, tea polyphenols, wine polyphenols.
Analytical Chemistry Division, Chemistry Department, Lomonosov Moscow State University, Moscow, Analytical Chemistry Division, Chemistry Department, Lomonosov Moscow State University Moscow, Analytical Chemistry Division, Chemistry Department, Lomonosov Moscow State University, Moscow