Identification of tyrosinase specific inhibitors from Xanthium strumarium fruit extract using ultrafiltration-high performance liquid chromatography

• Two new tyrosinase specific inhibitors were identified through the proposed UF-HPLC screening strategy.
• The problems of false negatives and false positives were overcome.
• A relationship between experimental data and binding affinity was established.
• The proposed technique is simple and fast that could be used in high-throughput screening.

In this study, a strategy based on ultrafiltration-high performance liquid chromatography coupled with diode array detection (UF-HPLC-DAD) was proposed for screening tyrosinase specific inhibitors in Xanthii fructus. The false negatives were distinguished by optimizing the UF-HPLC-DAD parameters to reduce the background noise; the false positives were distinguished by introducing a blocked tyrosinase in the control group for comparison. To obtain the best blocker, the competitive experiments were performed using various known ligands. Using this strategy, three competitive inhibitors (protocatechuic acid; 3,5-di-O-caffeoylquinic acid; and 1,5-di-O-caffeoylquinic acid) and one mixed-type inhibitor (chlorogenic acid) were identified. These results were verified using tyrosinase inhibition assay, kinetic analysis, and structural simulation of the complex. Our experimental results suggest that the proposed strategy could be useful for high-throughput identification of tyrosinase specific inhibitors in natural products.