$$ Analysis of thrombin inhibition parameters Thrombin was incubated with polyphenol compounds at Compound C IC50 concentration at 37 °C. After 10 min, 280 μl of thrombin control (without tested compounds) or thrombin ARN-509 chemical structure preincubated with polyphenol compounds was added to reaction well containing, respectively, 40 μl of 1.5, 3, 4.5 and 6 mM chromogenic substrate (final concentrations of chromogenic substrate was 187.5, 375, 562.5 and 750 μM respectively). Absorbance was monitored every 12 s for 10 min

in a 96-well microplate reader. The velocity of reaction was expressed as the increase in product (pNA) over time (∆ μmol/min) using a computer program Mikcroplate Manager® 8 and the extinction coefficient of p-nitroaniline. (ε = 8,270/M/cm). Then, the Lineweaver–Burk (1934) curves for thrombin in the presence and in the absence of polyphenol compounds were plotted. The Lineweaver–Burk equation, which is a transformation of the Michaelis–Menten model, looks as follows: $$\frac1V

= \fracK_\textm V_\hboxmax \cdot \frac1[S] + \frac1V_\hboxmax $$ Statistical analysis The statistical analysis was performed using StatSoft Inc. “Statistica” v. 6.0. All the values in this study were expressed as mean ± SD. Results were analyzed under the account of normality with Shapiro–Wilk test and equality of variance with Levene test. The significance CRT0066101 of differences between the values Resveratrol was analyzed depending on the Levene test by ANOVA followed by Tukey multiple comparisons test or Kruskal–Wallis test. A level p < 0.05 was accepted as statistically significant. Results Polyphenolic compounds effect on thrombin amidolytic activity Only six compounds: cyanidin, quercetin, silybin, cyanin, (+)-catechin and (−)-epicatechin, of all examined polyphenols, caused the inhibition of thrombin amidolytic activity (Table 1). It was observed that these six compounds in a dose-dependent manner

decreased the initial velocity of chromogenic substrate hydrolysis. The thrombin inhibition by the polyphenolic compound was expressed as IC50 value—the concentration of a polyphenol needed to inhibit 50 % of thrombin amidolytic activity. The strongest inhibitory effect was demonstrated by cyanidin and quercetin (IC50 for cyanidin at 0.25 μM and for quercetin 1.5 μM at 375 μM of substrate concentration). The six polyphenols manifesting inhibitory effect on thrombin amidolytic activity were selected for the next steps of the study. Table 1 The effect of polyphenolic compounds on the amidolytic activity of human thrombin Compound IC50 (Μm) Cyanidin 0.25 Quercetin 1.