Inhibitory and Acceleratory Effects of Inonotus obliquus on Tyrosinase Activity and Melanin Formation in B16 Melanoma Cells

Abstract

The aim of the present study is to preliminarily investigate the antimelanogenesis effect of Inonotus obliquus extracts by cell-free mushroom tyrosinase assay. It was found that petroleum ether and n-butanol extracts might contain unknown potential tyrosinase inhibitors, while its ethyl acetate extract might contain some unknown accelerators. Six compounds were isolated and their structures were identified by interpretation of NMR data and nicotinic acid was first discovered in Inonotus obliquus. In cells testing, betulin and trametenolic acid decreased tyrosinase activity and melanin content, while inotodiol and lanosterol significantly increased tyrosinase activity and melanin content, showing an AC⁡50 of 9.74 and 8.43 μM, respectively. Nicotinie acid, 3β,22,25-trihydroxy-lanosta-8-ene, had a little or no effect on tyrosinase. Betulin exhibited a mode of noncompetitive inhibition with a K I = K IS of 0.4 μM on tyrosinase activity showing an IC50 of 5.13 μM and being more effective than kojic acid (6.43 μM), and trametenolic acid exhibited a mode of mixed inhibition with a K I of 0.9 μM, K IS of 0.5 μM, and an IC50 of 7.25 μM. We proposed betulin and trametenolic acid as a new candidate of potent tyrosinase inhibitors and inotodiol and lanosterol as accelerators that could be used as therapeutic agent.

Figure 1

Screening of tyrosinase inhibitors with using Tyr as the substrate, concentrations of fraction were 10 μg/mL (a); inhibition (acceleration) 50% of enzyme activity (IC₅₀, AC₅₀) was determinated by tyrosinase activity versus the concentrations of every fraction (0, 4, 6, 8, 10, 12 μg/mL) (b).

Figure 2

Chemical structure of betulin (Fa-a), trametenolic acid (Fa-b), inotodiol (Fb-a), lanoserol (Fb-b), nicotinie acid (Fc-a), and 3β,22,25-trihydroxy-lanosta-8-ene (Fd-a).

Figure 3

Effects of test compounds and kojic acid on cellular tyrosinase activity in B16 melanoma cells. Data are expressed as a percentage of control which was set at 100%. Each column represents the mean ± SD of three independent experiments. *P < 0.05 and **P < 0.01 compared with the control.

Figure 4

Effects of test compounds and kojic acid on cellular tyrosinase activity in a-MSH-stimulated B16 melanoma cells compared with kojic acid. The cells were incubated with 100 μM a-MSH alone or together with increasing doses of tested compounds or kojic acid for 72 h following which cellular tyrosinase activity was measured. Data are expressed as a percentage of control which was set at 100%. Each column represents the mean ± SD of three independent experiments. ****P < 0.001 versus control group (without a-MSH). ***P < 0.005, **P < 0.01, and *P < 0.05 versus a-MSH-treated group.

Figure 5

Effects of test compounds and kojic acid on cellular melanin content in B16 melanoma cells. The control readings were set as 100%. Data are expressed as a percentage of control which was set at 100%. Each column represents the mean ± SD of three independent experiments. **P < 0.01, *P < 0.05 compared with the control.

Figure 6

Effects of test compounds and kojic acid on cellular melanin content in a-MSH-stimulated B16 melanoma cells compared with kojic acid. The cells were incubated with 100 μM alone or together with increasing doses of tested compounds or kojic acid for 72 h following which total cellular melanin activity was measured. Baseline melanin content in control wells not exposed to a-MSH and any test compounds or kojic acid was set at 100%. Data from experimental wells were expressed as a percentage of control. Each column represents the mean ± SD of three independent experiments. ****P < 0.0005 versus control group (without a-MSH). ***P < 0.001, **P < 0.01, and *P < 0.05 versus a-MSH-treated group.

Figure 7

(a) Inhibitory effects of Fa-a on tyrosinase activity in B16 melanoma cells. Lineweaver-Burk plots in the absence (control) or in the presence of Fa-a with L-DOPA as the substrate are shown. Concentrations of Fa-a for the curve 1–5 at 0, 25, 50, 100, and 180 μM, respectively. (b) represents the secondary plot of the slope of the straight lines versus concentration of Fa-a.

Figure 8

(a) Inhibitory effects of Fa-b on tyrosinase activity in B16 melanoma cells. Lineweaver-Burk plots in the absence (control) or in the presence of Fa-b with L-DOPA as the substrate are shown. Concentrations of Fa-b for the curve 1–5 at 0, 25, 50, 100, and 180 μM, respectively. (b) The plot of slope versus the concentration of Fa-b for determining the inhibition constants K _I. (c) The plot of intercept versus the concentration of Fa-b for determining the inhibition constants K _IS.