Abstract: To investigate the combined inhibitory effects of sophoroside (Sop), puerarin (Pue), mangiferin (Man), and kojic acid (KA) on tyrosinase (TYR), we determined the combination index (CI). Competitive binding experiments were done to evaluate the effect of the order of addition of TYR inhibitors on their binding to TYR, and the mechanism of the combined action of TYR inhibitors was explored by various spectroscopies. The results showed that the combinations of Pue + Sop, Sop + KA, and KA + Man exhibited a synergistic inhibitory effect on TYR, with addition orders of TYR-Pue-Sop, TYR-Sop-KA, and TYR-KA-Man. All inhibitor combinations statically quenched the fluorescence of TYR, forming a ternary complex. The binding constant of Pue + Sop to TYR was not significantly different from that of Sop, and the number of binding sites was approximately to 1, indicating that the binding sites of the two inhibitors were different. As evidenced by increased binding constant and decreased particle size, Sop and KA promoted the binding of KA and Man to TYR, respectively, which consequently became tighter. The interaction type of one inhibitor was not changed by the presence of another. Electrostatic interaction was dominant in TYR-Pue-Sop, hydrogen bond and van der Waals force in TYR-Sop-KA and hydrophobic interaction in TYR-KA-Man. Infrared spectroscopy showed that compared with TYR, TYR-Sop-KA and TYR-Pue-Sop tended to transit towards disorder, and the structure of TYR-KA-Man was more unstable. The results of differential scanning calorimetry (DSC) showed that the melting temperature (Tm) of the TYR-inhibitor ternary complexes was lower than that of the binary complexes, indicating decreased thermal stability. In conclusion, an increase in the binding constant of one TYR inhibitor and a decrease in the thermal stability of complexes in the presence of another may be reasons for the synergistic action of the two TYR inhibitors.

Key words: combined inhibition; sophoricoside; puerarin; mangiferin; kojic acid