Response to silybin (1,424 RU) was higher than to (+)-catechin and (−)-epicatechin, but lower than cyanidin and quercetin. Fig. 4 Overlay sensorgrams for SPR analysis of polyphenolic compounds [cyanidin, quercetin, silybin, cyanin, (+)-catechin and (−)-epicatechin] bound to human thrombin immobilized on CM5 sensor chip. Polyphenols were injected at a concentration of 1,000 μM to the channel with immobilized LY2228820 thrombin. Sensorgrams were collected using BIAcore

system and BIAevalution software 3.1 The kinetic parameters obtained from the sensorgram analyses of the interaction of immobilized thrombin with polyphenolic compounds received using BIAcore system and BIAevaluation 3.1 software, presented in Table 2, show that cyanidin and quercetin association to thrombin was kinetically promoted (k a for cyanidin is 85.6 M–1 s–1, and for quercetin is 43.2 M–1 s–1), whereas cyanin showed the lowest association rate PXD101 datasheet (k a = 0.95 M–1 s–1). Analyses

of equilibrium constants demonstrate that the highest affinity to thrombin has cyanidin (K A = 1.28 × 108 M–1, K D = 7.79 × 10−9 M) and quercetin (K A = 2.59 × 107 M–1, K D = 3.87 × 10−8 M). Cyanin and (−)-Hydroxylase inhibitor epicatechin show the lowest affinity to thrombin (cyanin K A = 115 M–1 and K D = 8.63 × 10−3 M, while (−)-epicatechin K A = 192 M–1, K D = 5.19 × 10−3 M). Table 2 Kinetic parameters of the thrombin interaction with polyphenolic compounds Compound RU k Succinyl-CoA a (1/M s) k d (1/s) K A (1/M) K D (M) Cyanidin 2,251 85.60 6.67 × 10−7 1.28 × 108 7.79 × 10−9 Quercetin 1,882 43.20 1.67 × 10−6 2.59 × 107 3.87 × 10−8 Silybin 1,424 7.11 1.32

× 10−4 5.39 × 104 1.86 × 10−5 Cyanin 827 0.95 8.24 × 10−3 1.15 × 102 8.63 × 10−3 (+)-Catechin 717 3.62 1.78 × 10−4 2.03 × 104 4.92 × 10−5 (−)-Epicatechin 431 4.37 2.27 × 10−2 1.92 × 102 5.19 × 10−3 The association rate (k a), the dissociation rate (k d), equilibrium association constants K A and equilibrium dissociation constants K D were obtained in BIAcore analysis (from 5 sensorgrams at the concentrations ranging from 50 to 1,000 μM) using BIAevaluation 3.1 software. Response (RU) was shown for maximum used concentration of the analyte (1,000 μM) Analysis of thrombin inhibition parameters The analysis of the kinetic parameters obtained from Lineweaver–Burk curves shows that cyanidin, quercetin, silybin, (+)-catechin and (−)-epicatechin (Fig. 5) act as competitive inhibitors. These compounds resulted in an increase in the Michaelis constant (K m) value, whereas the maximum speed (V max) of chromogenic substrate decomposition reaction by thrombin remained unchanged (Table 3). In the case of the Lineweaver–Burk curve (Fig.