An Ultrafiltration High-performance Liquid Chromatography Coupled with Diode Array Detector and Mass Spectrometry Approach for Screening and Characterising Tyrosinase Inhibitors from Mulberry Leaves

Tyrosinase is a key enzyme in melanin synthesis. Its inhibitor may be used to efficiently treat hyperpigmentation and widely applied in cosmetic products and food supplements. In the present study, a new assay based on ultrafiltration high-performance liquid chromatography coupled with diode array detector and mass spectrometry (HPLC-DAD-MS) was developed for the rapid screening and identification of ligands for tyrosinase. Experiments were carried out to select the optimal binding conditions, tyrosinase concentration, and incubation time. Non-specific binding to the denatured tyrosinase was also investigated. Twelve compounds with tyrosinase binding activity were found in mulberry leaf extracts. The identities of these compounds were characterised by HPLC-DAD-MS(n). Particularly, two compounds, namely, quercetin-3-O-(6-O-malonyl)-β-D-glucopyranoside and kaempferol-3-O-(6-O-malonyl)-β-D-glucopyranoside, were identified as new tyrosinase inhibitors. The screening results were verified by tyrosinase inhibition assays. Experimental results proved that the proposed method could rapidly screen tyrosinase inhibitors in complex mixtures.