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

Abstract

Puerol A (<b>1</b>) from <i>Amorpha fruticosa</i> showed highly potent inhibition against both monophenolase (IC₅₀ = 2.2 μM) and diphenolase (IC₅₀ = 3.8 μM) of tyrosinase. We tried to obtain a full story of enzyme inhibitory behavior for inhibitor <b>1</b> 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; <i>k</i>₃ = 0.0279 μM^-1 min^-1 and <i>k</i>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 <i>N</i>-acetyl-l-tyrosine as a substrate, which was completely inhibited at 20 μM. A high binding affinity of <b>1</b> 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 μM.