To discover an active skin depigmenting agent, we isolated a novel inhibitor of melanin biosynthesis from the methanol extract of Erigeron breviscapus using a bioactivity-guided fractionation and identified it as (2Z,8Z)-matricaria acid methyl ester by means of spectroscopic analysis. The compound showed strong whitening activity in melan-a cell. Compared with arbutin (IC 50 ‫0.4؍‬ mM) as a positive control, the depigmentation IC 50 value for (2Z,8Z)-matricaria acid methyl ester was 25.4 m mM in B16F10 melanoma cell. Moreover, its inhibitory effect on tyrosinase, the key enzyme of melanogenesis, was examined by in vivo and in vitro tyrosinase assay and Western blot. The results indicate that (2Z,8Z)-matricaria acid methyl ester isolated from Erigeron breviscapus is a promising compound that could be useful for treating hyper-pigmentation as skin-whitening agents.Key words melanin; Erigeron breviscapus; (2Z,8Z)-matricaria acid methyl ester; skin whitening Biol.

A protein chip was constructed to detect the binding of microphthalmia-associated transcription factor (Mitf) and E-box DNA. Mitf, a key regulatory transcriptional factor of pigmentation-related genes such as tyrosinase, binds to specific sequence (CATGTG) in E-box DNA within the promoter of tyrosinase in the melanocytes. We produced Mitf as a maltose-binding protein (MBP) fusion protein in Escherichia coli, purified it using an affinity column, and immobilized it on beta-cyclodextrin-coated glass plate. Binding of Mitf to its target DNA, E-box oligomer, was monitored by surface plasmon resonance (SPR), SPR imaging (SPRi), and fluorescence-based system. Among these detection methods, fluorescence method was the most reliable. In this method, fluorescent intensity was proportional to the DNA concentration (up to 20 microM) and Mitf (up to 500 microg/ml). Kinetics of DNA binding with Mitf showed Langmuir isotherm, and its kinetic constants were determined. It is expected that Mitf-E-box DNA chip can be used as a screening tool for depigmenting agents in the cosmetic industry.

Abstract. Transfer of melanin-containing melanosomes from melanocytes to neighboring keratinocytes results in skin pigmentation. To provide a more practical method of visualizing melanosomes in melanocytes as well as in keratinocytes, we attempted to use murine cell lines instead of human primary cells. We generated various fluorescent fusion proteins of tyrosinase, a melanin synthesis enzyme located in the melanosome, by using green fluorescent protein and red fluorescent protein. The intracellular localization of tyrosinase was then examined by fluorescence and confocal microscopy. Co-culture of murine melanocytes and keratinocytes was optimized and melanosome transfer was either stimulated with ·MSH or partially inhibited by niacinamide. To the best of our knowledge, this is the first study showing that a murine co-culture model, in addition to human primary cell co-culture, can be a good tool for depigmenting agent screening by monitoring melanosome transfer. IntroductionHyperpigmentation disorders, such as melasma, postinflammatory hyperpigmentation and lentigo senilis (LS), are associated with abnormal accumulation of melanin pigments, which can be improved by skin lightening reagents (1). Furthermore, Asian women prefer lighter skin color and there is a great demand to develop safer and more effective skin whitening agents (2). Although much effort has been made to develop novel therapeutic agents against pigmentation abnomalities, safer, more effective and less irritating lightening agents are still required.To date, research on the regulation of melanogenesis has focused on factors that can inhibit tyrosinase activity and decrease total melanin content (3). Considerable knowledge of basic pigment distribution and regulation processes has been accumulated to date and should be applicable to the discovery of new targets for developing depigmenting agents.As the present screening methods for depigmenting agents are time-consuming, rapid high-throughput screening (HTS) is required for depigmenting agent screening. Furthermore, HTS of diversified chemical libraries provide a major means for identification of lead candidates for drug or cosmetic ingredient development (4).Visible pigmentation in mammals requires the transfer of melanin granules from melanocytes to keratinocytes. For this intercellular transfer to be effective, melanosomes must first accumulate at the distal end of the melanocyte dendrites using cooperative transport mechanisms (5). These include long range bidirectional, microtubule-dependent melanosome movements along the dendrite length before coupling to myosin Va-dependent capture machinery within the distal actin-rich regions of the dendrite (6). Myosin Va is recruited onto the melanosome surface by a receptor complex containing Rab27a and melanophilin in a Guanosine triphosphate (GTP)-dependent fashion (7-9). The absence of any one of these three proteins collapses the myosin Va-dependent capture of melanosomes in the periphery, causing instead their accumulation in the perikaryon of th...

Phosphorylation of proteins by kinases plays an important role in regulating cellular processes including melanin production in the skin cells. Protein kinase C β (PKCβ) is known to be involved in phosphorylating tyrosinase, the key enzyme of melanin production, regulating the skin pigmentation process. In melanogenesis, PKCβ activates the tyrosinase by phosphorylation of its two serine residues. In this study, phosphorylation activity by PKCβ was monitored on a protein chip for the screening of depigmenting agents. As a tyrosinase mimic, 11 or 30 amino acids of the C-terminal of tyrosinase was fused with maltose-binding protein (MBP). After immobilizing the MBP-fused PKCβ substrate peptide on epoxy-treated slide surface, PKCβ reaction mix was applied over the immobilized MBP-fused PKCβ substrate peptide. Phosphorylation was detected with anti-phosphoSer/Thr antibodies, followed by fluorescence-labeled second antibodies. Phosphorylation of MBP-30aa was observed on a protein chip, and this phosphorylation was inhibited by the PKC inhibitor (GF109203X). These results indicate the potential of PKCβ protein chip as a high-throughput screening tool in the screening of depigmenting agents.

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