Essential Oils of Alpinia nantoensis Retard Forskolin-Induced Melanogenesis via ERK1/2-Mediated Proteasomal Degradation of MITF

Abstract

The anti-melanogenic activity of essential oils of Alpinia nantoensis and their bioactive ingredients were investigated in vitro. Treatment with leaf (LEO) and rhizome (REO) essential oils of A. nantoensis, significantly reduced forskolin-induced melanin production followed by down-regulation of tyrosinase (TYR) and tyrosinase related protein-1 (TRP-1) expression at both transcriptional and translational levels. Further studies revealed that down-regulation TYR and TRP-1 were caused by LEO/REO-mediated suppression of Microphthalmia-associated transcription factor (MITF), as evidenced by reduced nuclear translocation of MITF. Also, we found that LEO/REO induce the sustained activation of ERK1/2, which facilitate subsequent proteasomal degradation of MITF, as confirmed by that LEO/REO failed to inhibits MITF activity in ERK1/2 inhibitor treated cells. In addition, a significant increase of ubiquitinated MITF was observed after treatment with LEO and REO. Furthermore, the chemical composition of LEO and REO were characterized by gas chromatography-mass spectrometry (GC-MS) resulted that camphor, camphene, α-pinene, β-pinene, isoborneol and _D-limonene were the major compounds in both LEO and REO. Further studies revealed that α-pinene and _D-limonene were the active components responsible for the anti-melanogenic properties of LEO and REO. Based on the results, this study provided a strong evidence that LEO and REO could be promising natural sources for the development of novel skin-whitening agents for the cosmetic purposes.

Keywords: Alpinia nantoensis; MITF; Zingiberaceae; anti-melanogenesis; essential oil; forskolin.

Figure 1

Inhibitory effect of leaf (LEO) and rhizome (REO) essential oils on melanin content and tyrosinase enzyme activity in forskolin (FRK)-induced B16-F10 melanoma cells. (A) Cells were treated with indicated concentrations of LEO, REO, arbutin (AB) and kojic acid (KA) and stimulated with FRK for 48 h. Melanin content was assessed by with an absorbance at 405 nm. Cellular melanin content was calculated by comparison with a melanin standard curve. (B) The relative activity of mushroom tyrosinase on L-DOPA in the presence of increasing concentrations of LEO, REO, KA and ascorbic acid (AA), compared to the control (100%). (C) Effect of cellular tyrosinase activity was determined using whole cell lysates. After treatment with indicated concentration of LEO, REO, AB and KA for 48 h. Cell lysates were used as enzyme source and L-DOPA as substrate. The effects on L-DOPA oxidation velocity was measured at 492 nm. (D–F) Relative mRNA expression levels of tyrosinase, TRP-1 and dopachrome tautomerase (DCT) were determined by Q-PCR analysis. (G–I) Protein expression levels of tyrosinase, TRP-1 and DCT were determined by Western blot analysis and the histogram showed relative protein expression, which were normalized with loading control GAPDH. Data represent the mean ± SD of three experiments. Statistical significance was set at # p < 0.05 compared to control vs. FRK and * p < 0.05, ** p < 0.01, *** p < 0.001 compared with FRK + sample treatment groups vs. control group.

Figure 2

Effect of LEO and REO on Microphthalmia-associated transcription factor (MITF) transcriptional activity. (A) The mRNA expression level of MITF was determined by Q-PCR after treatment with 6 h. The protein expression level of MITF was determined by western blot analysis. (B) Protein expression level of MITF was determined by Western blot analysis and the histogram showed relative protein expression, which was normalized with loading control GAPDH. (C) Cellular localization of MITF was determined by immunofluorescence with fluorescein isothiocyanate (FITC)-conjugated secondary antibody. (D) The effect of LEO and REO on melanin content was determined in cells transiently transfected with MITF siRNA or control siRNA. (E) The protein expression levels of CREB and Phos-CREB were determined by immunoblotting. (F) Cellular localization of Phos-CREB was measured by immunofluorescence after treatment with FRK in the presence or absence of LEO, REO and gallic acid (GA). Data represent the mean ± SD of three experiments. Statistical significance was set at # p < 0.05 compared to control vs. FRK and *** p < 0.001 compared with FRK + sample treatment groups vs. control group.

Figure 3

Effect of LEO and REO on ERK1/2 and AKT activation. (A) B16-F10 cells were treated with LEO or REO for 15 min to 6 h. The phosphorylation of ERK1/2 and AKT were determined by western blot analysis using specific antibodies. Histogram shows the relative protein expression of phos-ERK1/2 and phos-AKT levels, which are normalized with total levels of corresponding proteins. (B) The effect of LEO and REO on MITF protein expression under pharmacological inhibition of ERK1/2 and AKT were determined by western blot analysis. (C) Effect of LEO and REO on FRK-induced melanin content was determined after pre-treated with p38 MAPK, ERK1/2, JNK/SAPK and AKT inhibitors for 2 h. (D) Ubiquitination of MITF was determined in B16-F10 cells treated with LEO or REO with ERK1/2 and proteasome inhibitors for 6 h. The cells were immunoprecipitated using an anti-MITF antibody and immunoblotted using an anti-ubiquitin antibody. Data represent the mean ± SD of three experiments. Statistical significance was set at # p < 0.05 compared to control vs. FRK, Δ p < 0.05 compared to FRK vs. ERK1/2 inhibitor (PD98059) and * p < 0.05, ** p < 0.01, *** p < 0.001 compared with FRK + sample treatment groups vs. control group.

Figure 4

Effect of major compounds of LEO and REO on melanin production and cellular tyrosinase activity in FRK-stimulated cells. (A) Cells were treated with indicated concentration of major compounds and stimulated with FRK for 48 h. The melanin content was determined spectrometrically. (B,C) The effect of α-pinene and D-limonene on FRK-induced melanin production was determined in a dose-dependent manner. (D) The effect of cellular tyrosinase activity by major compounds of LEO and REO were determined. (E,F) Dose-dependent inhibitory effect of α-pinene and D-limonene were quantified. Data represent the mean ± SD of three experiments. Statistical significance was set at # p < 0.05 compared to control vs. FRK and *** p < 0.001 compared with FRK + sample treatment groups vs. control group.