| Radix Polygoni Multiflori, the dried root of Polygonum multiflorum (PM), is well documented for its clinical effects in treating various diseases associated with aging and hair graying, but the evidence based-mechanisms remain largely unknown. In this study, PM was extracted with water and 70% ethanol and a major constituent, 2,3,5,4'-tetrahydroxystilbene-2- O-β-D-glucoside (THSG) of about 0.064% and 0.75%, respectively, were found in these extracts as analyzed by high-performance liquid chromatography (HPLC) coupled to mass spectrometry. The melanogenic properties and cytotoxicity of the two extracts and THSG were evaluated using murine and human melanocytes.;Both water and ethanol extracts of PM and THSG showed a dose-dependent anti-melanogenic activity in an in vitro murine melan-a melanocyte assay for reduction of L-DOPA conversion by tyrosinase. Of at least 5 melanoma and melanocyte cell lines tested, both water PM extract and THSG were relatively safe, which at doses <100 µg/ml did not demonstrate any significant cytotoxic effects. On the other hand, ethanol PM extract was about 3-4 folds more cytotoxic.;Tyrosinase is the rate-limiting enzyme for melanogenesis. In a cell-free kinetic analysis, THSG inhibited tyrosinase activity in a reversible and non-competitive manner. At the cellular level, this inhibition is mediated through a PKA-dependent melanogenic pathway, as well as in a dose-dependent manner, with IC50 = 123.0 µM and 61.5 µM for murine and human melanocytes, respectively. Tyrosinase was much more sensitive to the inhibitory effect of THSG in the melanocyte/keratinocyte co-culture system than in the melanocyte mono-culture system.;Functional tyrosinase is regulated at both transcriptional and post-translational modification levels. At the transcription level, THSG reduced expression of microphthalmia-associated transcription factor (MITF) resulted in a down-regulation of tyrosinase expression. At the post-translational modification level, THSG inhibited expression of PKC-β which is responsible for tyrosinase phosphorylation, and enhanced tyrosinase/TRP-1 complex formation. On the hand, THSG did not affect glycosylation of tyrosinase nor its trafficking from ER/ Golgi to endosomal/ lysosomal compartments.;Taken our results together, the anti-melanogenic property of PM extracts and THSG were firstly demonstrated in both mono- and co-culture system using murine or human melanocytes and keratinocytes. THSG is a reversible and competitive inhibitor, which lowered the tyrosinase activity at both transcription and post-translational modification levels via PKA-mediated melanogenesis. |
