Kaliziri extract upregulates tyrosinase, TRP-1, TRP-2 and MITF expression in murine B16 melanoma cells

Abstract

Background: Kaliziri extract (KZE) is a traditional Uyghur medicine (TUM), used by traditional hospitals in China as an injection for treatment of vitiligo for more than 30 years. Clinical application has shown that this medicine has obvious therapeutic effects. However, its phytochemical analysis and mechanism have not been examined.

Methods: KZE was extracted from seeds of Kaliziri [Vernonia anthelmintica (L.) Willd.] in ethanol-water (80:20, v/v), its components were identified by LC-MS/MS, and the signaling pathway of melanin synthesis in KZE treated murine B16 melanoma cells was examined by western blotting.

Results: Liquid chromatography-mass spectrometry analysis confirmed that the main components of KZE are flavonoids. KZE increased the tyrosinase activity and melanin content in a dose-dependent manner at concentrations of 5-40 μg/ml, and treatment with 20 μg/ml of KZE enhanced the expression of tyrosinase in B16 cells in a time-dependent manner.

Conclusions: KZE induced melanogenesis by increasing the expression of TYR, TRP-1, TRP-2 and MITF in B16 cells.

Figure 1

LC–MS/MS analyze of KZE. Compounds detected in KZE in negative and positive MRM mode. LC–MS/MS conditions as described in the text.

Figure 2

The effect of KZE on mushroom tyrosinase activity. Mushroom tyrosinase activity was determined by L-DOPA oxidation in a cell-free system. Stimulation of tyrosinase activity in vitro by KZE at 0.3125-5 mg/ml. MOP being positive controls at 500 μM. Results shown are means ± SEM and are representative of three independent experiments. Data were analyzed by One-Way Analysis of Variance (ANOVA) followed by post hoc Tukey test. ^**P < 0.01, compared with control.

Figure 3

Cytotoxicity of KZE in B16 melanoma cells. Effect of KZE on B16 cell viability. B16 cells were treated for 48 h with various concentrations of KZE (6.25-800 μg/ml) and cell viability was determined by the MTT reduction assay. Data are expressed as mean ± SD (n = 6).

Figure 4

Tyrosinase activity was determined by L-DOPA oxidation. A. Stimulation of tyrosinase activity of B16 cell by KZE at 5-40 μg /ml. B. Melanin content were performed as described in "Materials and methods", B16 cells, the same by KZE at 5-40 μg /ml.MOP being positive controls at 50 μM. Results shown are means ± SEM and are representative of three independent experiments. Data were analyzed by One-Way Analysis of Variance (ANOVA) followed by post hoc Tukey test. **P<0.01, compared with control.

Figure 5

Effect of KZE on the protein levels of MITF and tyrosinase in B16 cells. The cells were treated with 20 μg /ml of KZE for the indicated times. Western blot assays were performed to examine MITF and tyrosinase expression levels. Results were normalized against β-actin expression. Results shown are means ± SEM and are representative of three independent experiments. Data were analyzed by One-Way Analysis of Variance (ANOVA) followed by post hoc Tukey test ^**P < 0.01, compared with control.

Figure 6

Effect of KZE on the protein levels of TRP-1 and TRP-2 in B16 cells. The cells were treated with 20 μg/ml of KZE for the indicated times. Western blot assays were performed to examine TRP-1 and TRP-2 expression levels. Results were normalized against β-actin expression. Results shown are means ± SEM and are representative of three independent experiments. Data were analyzed by One-Way Analysis of Variance (ANOVA) followed by post hoc Tukey test ^**P < 0.01, compared with control.