Tyrosinase Inhibition and Kinetic Details of Puerol A Having But-2-Enolide Structure from Amorpha fruticosa

Abstract

Puerol A (1) from Amorpha fruticosa showed highly potent inhibition against both monophenolase (IC50 = 2.2 mu M) and diphenolase (IC50 = 3.8 mu M) of tyrosinase. We tried to obtain a full story of enzyme inhibitory behavior for inhibitor 1 because the butenolide skeleton has never been reported as a tyrosinase inhibitor. Puerol A was proved as a reversible, competitive, simple slow-binding inhibitor, according to the respective parameters; k(3) = 0.0279 mu M-1 min(-1) and k(4) = 0.003 min(-1). A longer lag-phase and a reduced static-state activity of the enzyme explained that puerol A had a tight formation of the complex with E-met. Dose-dependent inhibition was also confirmed by high-performance liquid chromatography (HPLC) analysis using N-acetyl-l-tyrosine as a substrate, which was completely inhibited at 20 mu M. A high binding affinity of 1 to tyrosinase was confirmed by fluorescence quenching analysis. Moreover, puerol A decreased melanin content in the B16 melanoma cell dose-dependently with an IC50 of 11.4 mu M.