Parallel determination of human plasma serine proteases complexed with C1-inhibitor

The demand for treatment of hyperpigmentation disorders are on the rise. Artemisia capillaris is a traditional herbal plant widely used in skin protective remedies. In the present study, the inhibitory effect of Artemisia capillaris ethanol extract on the production of melanin is examined, and the active compound was isolated from the crude extract and identified.The structure of the purified active compound of A. capillaris ethanol extract (ACE) was elucidated by NMR spectroscopy. ACE at the concentration of 6.25, 12.5, 25, and 50 μg/ml and active compound at the concentration of 37.5, 75, and 150 μg/ml were treated alpha-melanocyte stimulating hormone (α-MSH) induced in B16F10 melanoma cells. Melanin contents, tyrosinase activity, and protein expression of melanogenesis-related proteins were analyzed in ACE or active compound treated or untreated control.ACE significantly inhibited melanogenesis induced by α-MSH and tyrosinase activity without cell cytotoxicity in a dose-dependent manner. Western blot demonstrated that ACE downregulated the expression of melanocyte-specific proteins such as tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2, which catalyzes the rate-limiting oxidation of tyrosine to melanin. The active compound was finally identified as leukodin. It inhibited melanin pigment synthesis and tyrosinase activity in B16F10 melanoma cells without cytotoxicity. In addition, the leukodin decreased TRPs expression in a dose-dependent manner.Bioactivity-guided fraction identified leukodin is the active compound in ACE extract. Leukodin suppressed melanin synthesis through inhibition of the expression of melanogenic enzymes.