New Class of Tyrosinase Inhibitors, Rotenoids, from Amorpha fruticosa

Abstract

A series of rotenoids including a new one from the seeds of Amorpha fruticosa were found to have significant potential as tyrosinase inhibitors. All of the isolated rotenoids (1-6) displayed inhibitory activity against tyrosinase, both as a monophenolase for the oxidation of l-tyrosine and as a diphenolase for the oxidation of l-DOPA. The three most active compounds (1, 5, and 6) showed significant monophenolase inhibition with IC₅₀ values of 2.1, 1.7, and 1.2 μM, respectively. They also inhibited diphenolase function with IC₅₀ values in the range of 9.5-21.5 μM. The inhibition kinetics established all compounds to be competitive inhibitors of both oxidation processes. All rotenoids formed the E_met·I complex effectively around their IC₅₀ values with long lag times. Tyrosinase inhibition of the new rotenoid 6 was additionally demonstrated using high-performance liquid chromatography (HPLC) analysis with N-acetyl-l-tyrosine. Molecular docking disclosed that the sugar moiety of 5 interacted with the bottom of the catalytic gorge.

Figure 1

Chemical structures of the rotenoids (1–6) isolated from the seeds of A. fruticosa.

Figure 2

HMBC correlation (H → C) of new compound 6.

Figure 3

(A) Dose-dependent behavior of rotenoids (1–6) and kojic acid (positive control) on the oxidation of tyrosinase. (B) Catalytic activity of monophenolase as a function of the enzyme at different concentrations of compound 6 on monophenolase. (C–F) Lineweaver–Burk plots for the inhibition of compounds 1, 3, 5, and 6 on the monophenolase activity of tyrosinase.

Figure 4

(A–C) Lineweaver–Burk plots for the inhibition of the diphenolase activity of tyrosinase by compounds 1, 5, and 6. (D–F) Dixon plots for the inhibition of the diphenolase activity of tyrosinase by compounds 1, 5, and 6.

Figure 5

(A) Time course of oxidation of l-tyrosine catalyzed by tyrosinase in the presence of compound 6 at different concentrations (0, 0.35, 0.7, 1.4, 2.8, 5.6, and 11.2 μM). (B) Steady-state rate (v_ss) of monophenolase activity and the lag period of monophenolase for the oxidation of l-tyrosine.

Figure 6

HPLC chromatogram of the change in N-acetyl-l-tyrosine (peak A, t_R = 3.6 min) as a substrate for monophenolase at different incubation times (0, 15, 30, 45, and 60 min) by compound 6 treated at (A) 0, (B) 10, and (C) 20 μM.

Figure 7

Predicted binding methods of compounds 1 and 5 at the catalytic site of Ab mushroom tyrosinase. Overview modes (A, B) and detailed binding networks (C, D) of the docked structures of compounds 1 and 5 in the catalytic site of Ab mushroom tyrosinase are shown. In panel (A, B), compounds 1 and 5 are in the electrostatic surface model to clearly show the binding pocket and overall binding shapes. (C, D) Detailed binding networks between compounds 1 and 5 and the protein. All interactions are shown as dashed lines. Conventional hydrogen bonds, carbon–hydrogen bonds, pi–pi T-shaped interactions, pi–sigma interactions, and pi–alkyl interactions are shown in green, bright green, hot pink, purple, and pink, respectively.