Inhibitory effect of Gastrodia elata Blume extract on alpha-melanocyte stimulating hormone-induced melanogenesis in murine B16F10 melanoma

Abstract

Background/objectives: Gastrodia elata Blume (GEB), a traditional herbal medicine, has been used to treat a wide range of neurological disorders (e.g., paralysis and stroke) and skin problems (e.g., atopic dermatitis and eczema) in oriental medicine. This study was designed to investigate whether GEB extract inhibits melanogenesis activity in murine B16F10 melanoma.

Materials/method: Murine B16F10 cells were treated with 0-5 mg/mL of GEB extract or 400 µg/mL arbutin (a positive control) for 72 h after treatment with/without 200 nM alpha-melanocyte stimulating hormone (α-MSH) for 24 h. Melanin concentration, tyrosinase activity, mRNA levels, and protein expression of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein (Trp)1, and Trp2 were analyzed in α-MSH-untreated and α-MSH-treated B16F10 cells.

Results: Treatment with 200 nM α-MSH induced almost 2-fold melanin synthesis and tyrosinase activity along with increased mRNA levels and protein expression of MITF, tyrosinase, Trp1 and Trp2. Irrespective of α-MSH stimulation, GEB extract at doses of 0.5-5 mg/mL inhibited all these markers for skin whitening in a dose-dependent manner. While lower doses (0.5-1 mg/mL) of GEB extract generally had a tendency to decrease melanogenesis, tyrosinase activity, and mRNA levels and protein expression of MITF, tyrosinase, Trp1, and Trp2, higher doses (2-5 mg/mL) significantly inhibited all these markers in α-MSH-treated B16F10 cells in a dose-dependent manner. These inhibitory effects of the GEB extract at higher concentrations were similar to those of 400 µg/mL arbutin, a well-known depigmenting agent.

Conclusions: These results suggest that GEB displays dose-dependent inhibition of melanin synthesis through the suppression of tyrosinase activity as well as molecular levels of MITF, tyrosinase, Trp1, and Trp2 in murine B16F10 melanoma. Therefore, GEB may be an effective and natural skin-whitening agent for application in the cosmetic industry.

Keywords: Gastrodia elata; melanogenesis; tyrosinase.

Fig. 1. Effect of Gastrodia elata Blume (GEB) extract on cell viability in B16F10 cells.

The cells were plated at 4 × 10⁴ cells/well and incubated in media containing 0-25 mg/mL concentrations of GEB for 24 h after treatment with (panel B)/without (panel A) 200 nM alpha-melanocyte stimulating hormone (α-MSH) for 24 h. Each bar represents the mean ± SD (n = 5). Different letters indicate a significant difference according to the ANOVA (P < 0.05).

Fig. 2. Effect of Gastrodia elata Blume (GEB) extract on melanin content in B16F10 cells.

The cells were treated with 0-5 mg/mL of GEB extract or 400 µg/mL arbutin (Arb, a positive control) for 72 h after treatment with/without 200 nM α-MSH for 24 h. The melanin concentration in the samples was derived from a synthetic melanin standard curve and each sample was normalized to a total cellular protein. Each bar represents the mean ± SD (n = 4). Different letters indicate a significant difference according to the ANOVA (P < 0.05).

Fig. 3. Effect of Gastrodia elata Blume (GEB) extract on tyrosinase activity in B16F10 cells.

The cells were treated with 0-5 mg/mL of GEB extract or 400 µg/mL arbutin (Arb, a positive control) for 72 h after treatment with/without 200 nM α-MSH for 24 h. The supernatant lysed in RIPA buffer was incubated with 5 mM L-DOPA for 2 h and each sample was normalized to total cellular protein. Each bar represents the mean ± SD (n = 4). Different letters indicate a significant difference according to the ANOVA (P < 0.05).

Fig. 4. Effects of Gastrodia elata Blume (GEB) extract on mRNA levels of (A) MITF (B) tyrosinase (Tyr), (C) Trp1, and (D) Trp2 in B16F10 cells.

The cells were treated with 0-5 mg/mL of GEB extract or 400 µg/mL arbutin (Arb, a positive control) for 72 h after treatment with/without 200 nM α-MSH for 24 h. Real time-PCR was performed using gene specific primers for MITF, tyrosinase, Trp1, and Trp2. The expression levels were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Each bar represents the mean ± SD (n = 3). Different letters indicate a significant difference according to the ANOVA (P < 0.05).

Fig. 5. Effects of Gastrodia elata Blume (GEB) extract on protein expression of (A) MITF (B) tyrosinase (Tyr), (C) Trp1, and (D) Trp2 in B16F10 cells.

The cells were treated with 0-5 mg/mL of GEB extract or 400 µg/mL arbutin (Arb, a positive control) for 72 h after treatment with/without 200 nM α-MSH for 24 h. Immunoblotting was performed with specific antibodies for MITF, tyrosinase, Trp1, and Trp2. The expression levels were normalized to that of β-actin. Each bar represents the mean ± SD (n = 3). Different letters indicate a significant difference according to the ANOVA (P < 0.05).