Limonoids and unsaturated fatty acids present in Melia toosendan increase ceramide production in keratinocytes.

The skin barrier prevents moisture evaporation and the entry of foreign substances such as allergens. Ceramides are one of the most important factors for maintaining skin barrier function. Melia toosendan is a plant of the Meliaceae family, and its fruit extracts have been used in Traditional Chinese Medicine as analgesics and anthelmintics; however, its ability to increase ceramide levels has not been reported. In this study, we screened for compounds present in M. toosendan fruit extracts that increase ceramide levels in the skin. We fractionated the extracts based on their activity to identify the active components. Nimbolinins, limonoids such as toosendanin, and hydroxylated unsaturated fatty acids were found to be the major active components. The structure-activity relationship of toosendanin derivatives indicated that the sites around R4 and R5 contributed to the activity. To the best of our knowledge, this is the first report showing that limonoids promote ceramide production in skin cells. Therefore, M. toosendan fruit extracts may be used to develop products for improving the skin barrier function.

Structural insights from an in silico molecular docking simulation of complement component 3a receptor 1 with an antagonist.

Complement component 3a receptor 1 (C3aR) is an anaphylatoxin receptor that mediates inflammatory processes. Although considerable effort has gone into discovering the antagonists and agonists of C3aR, structural insights are required to search for effective ligands and to elucidate their binding modes and the mechanism of activation and inactivation. No experimental structural data of C3aR have yet been reported. We investigated the binding mode of an antagonist of C3aR using a combination of homology modeling, ligand docking, molecular dynamics simulations, and binding free energy calculations. We produced a plausible binding model consistent with the reported experimental data. We believe that this model is appropriate for the identification of new C3aR antagonists, as it can distinguish between antagonists and decoy compounds.

The Surprising Effect of Phenformin on Cutaneous Darkening and Characterization of Its Underlying Mechanism by a Forward Chemical Genetics Approach.

Melanin in the epidermis is known to ultimately regulate human skin pigmentation. Recently, we exploited a phenotypic-based screening system composed of ex vivo human skin cultures to search for effective materials to regulate skin pigmentation. Since a previous study reported the potent inhibitory effect of metformin on melanogenesis, we evaluated several biguanide compounds. The unexpected effect of phenformin, once used as an oral anti-diabetic drug, on cutaneous darkening motivated us to investigate its underlying mechanism utilizing a chemical genetics approach, and especially to identify alternatives to phenformin because of its risk of severe lactic acidosis. Chemical pull-down assays with phenformin-immobilized beads were performed on lysates of human epidermal keratinocytes, and subsequent mass spectrometry identified 7-dehydrocholesterol reductase (DHCR7). Consistent with this, AY9944, an inhibitor of DHCR7, was found to decrease autophagic melanosome degradation in keratinocytes and to intensely darken skin in ex vivo cultures, suggesting the involvement of cholesterol biosynthesis in the metabolism of melanosomes. Thus, our results validated the combined utilization of the phenotypic screening system and chemical genetics as a new approach to develop promising materials for brightening/lightening and/or tanning technologies.

Comprehensive quantification of ceramide species in human stratum corneum.

One of the key challenges in lipidomics is to quantify lipidomes of interest, as it is practically impossible to collect all authentic materials covering the targeted lipidomes. For diverse ceramides (CER) in human stratum corneum (SC) that play important physicochemical roles in the skin, we developed a novel method for quantification of the overall CER species by improving our previously reported profiling technique using normal-phase liquid chromatography-electrospray ionization-mass spectrometry (NPLC-ESI-MS). The use of simultaneous selected ion monitoring measurement of as many as 182 kinds of molecular-related ions enables the highly sensitive detection of the overall CER species, as they can be analyzed in only one SC-stripped tape as small as 5 mm x 10 mm. To comprehensively quantify CERs, including those not available as authentic species, we designed a procedure to estimate their levels using relative responses of representative authentic species covering the species targeted, considering the systematic error based on intra-/inter-day analyses. The CER levels obtained by this method were comparable to those determined by conventional thin-layer chromatography (TLC), which guarantees the validity of this method. This method opens lipidomics approaches for CERs in the SC.

Characterization of overall ceramide species in human stratum corneum.

Ceramides (CERs) in human stratum corneum (SC) play physicochemical roles in determining barrier and water-holding functions of the skin, and specific species might be closely related to the regulation of keratinization, together with other CER-related lipids. Structures of those diverse CER species, however, have not been comprehensively revealed. The aim of this study was to characterize overall CER species in the SC. First, we constructed 3D multi-mass chromatograms of the overall CER species, based on normal-phase liquid chromatography (NPLC) connected to electrospray ionization-mass spectrometry (ESI-MS) using a gradient elution system and a postcolumn addition of a volatile salt-containing polar solvent. The CERs targeted from the 3D chromatograms were structurally analyzed using NPLC-ESI-tandem mass spectrometry (MS/MS), which resulted in the identification of 342 CER species in the inner forearm SC. This led to the discovery of a new CER class consisting of alpha-hydroxy fatty acid and dihydrosphingosine moieties, in addition to the 10 classes generally known. The results also revealed that those CERs contain long-chain (more than C(18))-containing sphingoids and a great number of isobaric species. These novel results will contribute not only to physiochemical research on CERs in the SC but also to lipidomics approaches to CERs in the skin.