| Summary: | Puerol A (<b>1</b>) from <i>Amorpha fruticosa</i> showed highly potent inhibition against both monophenolase (IC<sub>50</sub> = 2.2 μM) and diphenolase (IC<sub>50</sub> = 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><sub>3</sub> = 0.0279 μM<sup>−1</sup> min<sup>−1</sup> and <i>k</i><sub>4</sub> = 0.003 min<sup>−1</sup>. 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<sub>met</sub>. Dose-dependent inhibition was also confirmed by high-performance liquid chromatography (HPLC) analysis using <i>N</i>-acetyl-<span style="font-variant: small-caps;">l</span>-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 IC<sub>50</sub> of 11.4 μM.
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