Сrude Plant Extracts Mediated Polyphenol Oxidation Reactions in the Presence of 3-Methyl-2-Benzothiazolinone Hydrazone for the Determination of Total Polyphenol Content in Beverages

Abstract

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 the quality of plant-derived products. Methods: Phenol oxidase activity of green bean (Phaseolus vulgaris) crude extract (in the 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 the presence of green bean crude extract and hydrogen peroxide; all studied compounds except ferulic acid have been oxidized in the presence of banana pulp crude extract. Michaelis constants (Km) and maximum reaction rates (Vmax) have been determined for oxidation in the 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 oxidation in the 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 the 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 1.8×10-4 - 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 9.5×10-5 - 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.

Spectrophotometric and Smartphone-Assisted Determination of Phenolic Compounds Using Crude Eggplant Extract

“…The effect of MBTH on the EE enzymatic oxidation (decrease of K m values and increase of V max values) is similar to that observed for other crude plant extracts [4]. K m values decreased by a factor of 2 to 3 on the average for EE, which is a less pronounced effect than for banana extract (a factor of 12 on the average).…”

“…The polyphenol oxidase activity of crude eggplant extract (EE), prepared in this study, was determined by comparing the reaction speed of catechol oxidation in the presence of EE and purified mushroom tyrosinase. EE was characterized by 160 ± 6 U/mL (n = 3, P = 0.95) polyphenol oxidase activity which is 2.5 times higher than for banana extract that we studied before [4]. Total protein content of EE was also determined (1.1 ± 0.3 mg/mL, n = 3, P = 0.95).…”

“…3-Methyl-2-benzothiazolinone hydrazone (MBTH) is known to couple with the product of phenolic compounds oxidation (both enzymatic and non-enzymatic) and accelerate the reaction speed [4,25,26,27]. MBTH is widely used in optical biosensors based on laccase and tyrosinase enzymes [28,29,30,31,32].…”

“…Food antioxidant activity is linked to its total polyphenol content (TPC), which is often determined without analyzing individual antioxidant components. Different phenolic compounds can be chosen as a reference: caffeic [1,2], gallic [4,5,6,7], and chlorogenic acids [8], catechol [9], guaiacol [10], etc. Due to their role in human health the determination of phenolic antioxidants in plant-derived products and phenolic neurotransmitters in biological fluids requires simple, fast and cheap procedures, including screening tools.…”

“…All these advantages translate into a significant interest of creating electrochemical and spectrophotometric biosensor systems based on crude plant extracts. Many works have been published describing the analytical use of such extracts with PPO activity: banana [4,9,13], sapota [3], jurubeba [14], sweet potato [15], cara [16].…”