The Mechanism Of Active Components Of Two Chinese Traditional Medicines On Melanogenesis

The main reason of gray hair is the decrease of melanins, resulting from the deficiency of melanocytes, melanosomes and tyrosinase activity. There are many Chinese herbs used for hair graying treatment, of them Polygonum multiflorum Thunb and Semen Sesami Nigrum are the two most frequently used herbs, indicating their potential in stimulating melanogenesis. Although there are many reports about the two traditional herbs on gray hair, little is known about their effective components and related mechanism on melanogenesis. The present study is to clarify their effect, mechanism and to find out the effective component for melanogenesis. The results above indicated the relationship between the two traditional pigmentation herbs and melanogenensis. The major results are summarized as follows:1. The water extract of Polygonum multiflorum Thunb or Semen Sesami Nigrum was found to have a strong pigmentation effect by increasing mushroom tyrosinase activity and melanin content, suggesting their potential for effective components development. The mushroom tyrosinase activity and melanin synthesis were measured by dopaoxidase and NaOH methods respectively. The results showed that the water extract of Polygonum multiflorum Thunb or Semen Sesami Nigrum increased the mushroom tyrosinase activity and melanin content significantly in a dose-dependent manner, while their ethanol extract had no significant effect.2. The water extract of Polygonum multiflorum Thunb or Semen Sesami Nigrum induces melanogenesis by increasing cell viability, melanin content and tyrosinase activity, as well as the expression of melanogenesis key enzymes. The tyrosinase activity and melanin synthesis were measured in B16 melanoma cells by dopaoxidase and NaOH methods respectively. The protein and mRNA expression of melanogenesis key enzymes were investigated by western blot and RT-PCR. The results showed that the water extract of Polygonum multiflorum Thunb or Semen Sesami Nigrum could increase the tyrosinase activity and melanin content significantly in a dose-dependent manner in B16 cells. The increasing mRNA and protein expression of TYR and MITF (microphthalmia-associated transcription factor) were also observed in a dose-dependent manner.3. The 2, 3, 5, 4'-tetrahydroxystilbene-2-O-β-D-glucoside (THSG), a water-soluble component extracted from dried tuber root of Polygonum multiflorum Thunb, was found to induce pigmentation in B16 cells by MAP kinase activation and tyrosinase upregulation. L-Dopa zymography was used to study the tyrosinase activity and NaOH method was used for melanin content assay. We found that THSG could increase the melanin content, tyrosinase activity and tyrosinase expression in a concentration-dependent manner. We then investigated whether THSG influences the expression of microphthalmia-associated transcription factor (MITF), which is required for tyrosinase expression. THSG was found to induce sustained MITF up-regulation and cAMP response element (CRE) binding protein (CREB) activation, suggesting that THSG-mediated MITF activation may be cAMP dependent. Furthermore, Western blot analysis revealed that THSG could elevate the level of phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) significantly; a p38 MAPK inhibitor, SB203580, almost completely attenuated the THSG-mediated up-regulation of melanin synthesis and induction of MITF and tyrosinase expression.4. Sesamin, an active lignan of Semen Sesami Nigrum, was found to have a strong pigmentation effect in BI6 melanoma cells via activation of protein Kinase A and p38 MAP kinase. Treatment of B16 cells with sesamin increased the melanin content, tyrosinase activity and tyrosinase expression in a dose-dependent manner. Moreover, result of Western blot showed that sesamin induced sustained MITF (microphthalmia-associated transcription factor) activation, CRE-binding protein phosphorylation, p38 MAPK up-regulation but ERK down-regulation in a time-dependent manner. These results indicate that the protein Kinase A (PKA) and MAP kinase (MAPK) signaling pathways are both involved in sesamin-induced melanogenesis. Furthermore, intracellular cAMP concentration and CRE promoter activity were significantly increased by sesamin. Additionaly, we found that sesamin could induce protein kinase A (PKA) activation in B16 cells. All these results suggest the involvement of cAMP-PKA pathway in sesamin-induced melanogenesis. Consistent with this, inhibitors of PKA and p38 MAPK almost completely attenuated sesamin-inediated up-regulation of melanin synthesis and induction of MITF and tyrosinase. These data suggest that sesamin induces melanogenesis via the cAMP-PKA and MAPK signaling pathways.