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.
HPLC Determination of Polyphenols from Calendula officinalis L. Flowers
