Abstract
BackgroundSsanghwa-tang (SHT) is a widely used medication for the treatment of fatigue, pain, inflammation, hypothermia, erectile dysfunction, cancer, and osteoporosis in Asia, however, role of SHT on the melanin synthesis has not been checked previously. Thus, the present study was designed to determine the effect of SHT on α-melanocyte stimulating hormone (α-MSH)-induced melanogensis and its mechanisms of action in murine B16F10 melanoma cells.MethodCellular melanin content and tyrosinase activity in murine B16F10 melanoma cells were determined after α-MSH stimulation with or without pre-treatment of SHT at the concentration of 250 and 500 μg/ml. Expression level of tyrosinase, tyrosinase-related protein 1 (TRP-1), TRP-2, microphthalmia-associated transcription factor (MITF), and activation of c-AMP-dependent protein kinase (PKA), c-AMP-related element binding protein (CREB), and mitogen-activated protein kinases (MAPKs) were examined by Western blot analysis.ResultsSHT significantly inhibited α-MSH-induced melanin synthesis and tyrosinase activity, and also decreased α-MSH-induced expression of MITF, tyrosinase, and TRP-1. In addition, SHT remarkably suppressed tyrosinase, CRE, and MITF luciferase reporter activity in a resting state as well as in α-MSH-stimulating condition. Phosphorylation of p38 MAPK by α-MSH stimulation was efficiently blocked by SHT pre-treatment. Moreover, SHT as an herbal cocktail showed synergistic anti-melanogenic effect compared with that of each single constituent herb.ConclusionSHT efficiently inhibited c-AMP-induced melanin synthesis in B16F10 cells via suppression of PKA and p38 MAPK signaling pathways and subsequently decreased the level of CREB phosphorylation, MITF, and melanogenic enzymes. These results indicate that SHT may be useful as herbal medicine for treating hyperpigmentation and cosmetics as a skin-whitening agent.
Highlights
Ssanghwa-tang (SHT) is a widely used medication for the treatment of fatigue, pain, inflammation, hypothermia, erectile dysfunction, cancer, and osteoporosis in Asia, role of SHT on the melanin synthesis has not been checked previously
SHT at non-cytotoxic concentrations inhibits melanin synthesis in B16F10 cells To exclude the possibility that the inhibitory effect of SHT on melanin synthesis was due to cytotoxicity, we determined whether SHT is toxic to B16F10 cells using a MTT assay
Treatment with α-melanocyte stimulating hormone (MSH), which stimulates cAMP production, caused a 280% accumulation of melanin in cells, resulting were incubated with indicated concentrations of SHT for 48 h, and the cell viability was determined by MTT assay. (C) B16F10 cells were pre-incubated with or without 250 and 500 μg/ml SHT for 12 h, stimulated with 1 μM of α-MSH for additional 36 h, and observed for the accumulation of melanin in the microscopic image or pigmentation of cell pellets (2 × 106 cells)
Summary
Ssanghwa-tang (SHT) is a widely used medication for the treatment of fatigue, pain, inflammation, hypothermia, erectile dysfunction, cancer, and osteoporosis in Asia, role of SHT on the melanin synthesis has not been checked previously. Since genes encoding tyrosinase and TRP-1 are under transcriptional control of the MITF, substances capable of inhibiting MITF expression and activity could substantially down-regulate melanogenesis. Depigmentation can be achieved by downregulation of the expression and activity of tyrosinase, TRP-1, and TRP-2, by regulation of the uptake and distribution of melanosomes in keratinocytes, and by degradation of melanin and melanosome. Due to the pivotal role of tyrosinase in melanogenesis, identification of tyrosinase inhibitors is the most potent approach for the development of cosmetic products and medicinal drug treating abnormal skin pigmentation [8]. Available natural melanin synthesis inhibitors including arbutin, kojic acid, and stilbene often cause undesirable side-effects. Novel skin-whitening agents with more potent efficacy but less adverse effect are necessary for cosmetic and medicinal purposes
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