Kinetics of Tyrosinase Inhibition Catalyzed by Black Bean Skin Anthocyanins

In order to study the kinetic mechanism of black bean skin anthocyanins inhibiting tyrosinase catalytic reaction, we studied the inhibitory properties of black bean skin anthocyanins on tyrosinase monophenolase and diphenolase under different conditions, and determined the optimal inhibitory conditions, inhibitory types and the semi-inhibitory concentration(IC₅₀). The UV and fluorescence spectra were used to analyze the interaction mechanism. The orthogonal analysis showed that the optimal conditions of anthocyanins on tyrosinase monophenol were as follows, 0.6 mg·mL^-1 anthocyanins concentration, 6 mmol·L^-1 L-tyrosine concentration, 37 ℃, 15 min, and the inhibition rate was 50.37%. The optimal action conditions of diphenol enzyme were as follows, 0.8 mg·mL^-1 anthocyanin concentration, 6 mmol·L^-1 L-dopa concentration, 35 ℃, 10 min, the inhibition rate of diphenolase was 52.45%. Compared with VC, black bean skin anthocyanins had stronger inhibitory effect on tyrosinase monophenolase and diphenolase.The kinetic experiments showed that anthocyanins from black seed could inhibit tyrosinase mono phenolase and diphenolase activities with IC₅₀ of 1.03 and 0.82 mg·mL^-1, respectively. It was a reversible mixed inhibition of tyrosinase, and the inhibition constant K_i was 0.61 mg· mL^-1. The ultraviolet spectrum analysis showed that the anthocyanins in black bean skin caused the blue shift of tyrosinase, indicating that the conformation of tyrosinase was changed. The fluorescence spectra showed that the binding-site number was 0.76, 0.71 and 0.65 at 298, 303 and 308 K, and the binding constant K_A was 159.66, 94.23 and 54.89 L·mol^-1, respectively.The result shows that black bean skin anthocyanins can effectively inhibit the activity of tyrosinase as a natural plant origin tyrosinase inhibitor.