Tyrosinase inhibition mechanism of kojic acid-phenylpiperazine derivatives and their anti-browning effect on fresh-cut apples.

Tyrosinase, a rate-limiting enzyme in the enzymatic browning reaction, is a key target for fresh food preservation. Fifteen novel kojic acid-phenylpiperazine derivatives (4a-4o) were synthesized and structurally characterized by 1H NMR, 13C NMR and HRMS. In vitro assays showed most derivatives had stronger tyrosinase inhibitory activity than kojic acid (IC50 = 21.85 ± 0.23 μmol/L), with 4c performing best (IC50 = 8.19 ± 0.22 μmol/L). Kinetic analysis identified 4c as a mixed-type inhibitor, and spectral assays plus molecular docking revealed its precise binding to tyrosinase via static quenching, conformational induction and copper chelation. Fresh-cut apple assays confirmed 4c's excellent anti-browning efficacy, while cytotoxicity and acute oral toxicity tests proved its low toxicity to HEK-293 cells and no visceral damage to mice at effective concentrations. In conclusion, 4c exhibited promising anti-browning potential. Available toxicological data suggest that it possessed low toxicity, and it was worthy of further investigation as a lead compound of tyrosinase inhibitors in food preservation.