1–1,000 μM). The absorbance value was monitored for 10 min. IC50 (at 375 μM substrate concentration) was determined using inhibition curves. Mark “–” means no inhibitory effect on amidolytic activity of thrombin Polyphenolic compounds effect on thrombin proteolytic activity selleck inhibitor Fibrin polymerization was monitored as the changes in the absorbance values over time at 595 nm. Thrombin
preincubation with cyanidin, quercetin and silybin resulted in the inhibition of thrombin ability to induce fibrinogen polymerization, depending on their concentration (Fig. 1a–c). When thrombin was preincubated with cyanin, (+)-catechin and (−)-epicatechin and then added to fg the inhibitory effect of polymerization of human fibrinogen was not observed (Fig. 1d–f). Contrary to cyanin, (+)-catechin and (−)-epicatechin cyanidin in a dose-dependent manner reduced the initial velocity of fibrin polymerization; and at a concentration of 5 μM, total inhibition of thrombin activity was observed (Fig. 1a). Similar results were obtained for quercetin (Fig. 2b), but the concentration caused the total inhibition of thrombin activity to be ten times higher (50 μM) than in the case of cyanidin. Silybin also decreased in a dose-dependent manner the initial velocity of fibrin polymerization; however
at the highest concentration (1,000 μM) used, complete inhibition of thrombin activity was not observed (Fig. 1c). Fig. 1 The effect of polyphenolic compounds [cyanidin, quercetin, silybin, why cyanin, (+)-catechin and (−)-epicatechin] on the rate of thrombin-induced fibrinogen polymerization.
see more Thrombin was preincubated with each if the polyphenolic compounds at the selected concentrations, at 37 °C for 10 min. Thrombin-catalyzed fibrinogen polymerization was monitored for 20 min, as the change of turbidity at 595 nm. The results are expressed as % of maximal velocity V max of fg polymerization of the control samples (thrombin without tested polyphenols). Data represent mean ± SD of 12 independent experiments done in duplicates Fig. 2 The effect of polyphenolic compounds [cyanidin, quercetin, silybin, cyanin, (+)-catechin and (−)-epicatechin] on thrombin-induced cross-linked fibrin formation, after treatment of fibrinogen (containing factor XIII). 100 μl of control thrombin or preincubated with polyphenols was mixed with 50 μl of fibrinogen (3 mg/ml), and, after the specified time, 150 μl of Laemmli sample buffer containing 8 M urea and 10 % β-mercaptoethanol was added to digest the mixture. Proteins were separated on 7.5 % SDS-PAGE gel and staining with Coomassie Blue R250. Positions of fibrinogen chains (Aα, Bβ and γ) and the cross-linked fibrin chains (α, β, γ–γ dimer and α-polymers) are indicated. a Control thrombin, b thrombin preincubated with cyanidin (0.25 and 2.5 μM), c thrombin preincubated with quercetin (1.