Effect and molecular mechanism of selective natural compounds on melanin synthesis and degradation

Abstract

Melanin is the primary determinant of skin, hair, and eye color, and also has a critical role in photoprotection against ultraviolet radiation. The skin has epidermal units that are responsible for melanin production and distribution through a process known as melanogenesis. As facial hyperpigmentation is a common complaint in adult population and has profound impact on people’s quality of life, pharmaceutical and cosmetic industries have been continuously seeking new, safe and effective solutions. Natural compounds are increasingly used as melanogenic inhibitors or inducer of melanin degradation for skin whitening. Earlier research work from our lab has discovered that some natural chemicals showed strong mushroom tyrosinase inhibitory activity, which could be utilized as potential skin whitening agents. At the meanwhile, several chemicals isolated from Chinese herbs were selected for screening based on their ability of inducing autophagy. Through our preliminary screening, we found that 6-C-(E-phenylethenyl) naringenin (6-CEPN), 8-C-(E-phenylethenyl) qucercetin (8-CEPQ), 8-C-(E-phenylethenyl) norartocarpetin (8-CEPNar), isoartocarpesin, saikosaponin D, berberine hydrochloride, dihydroartemisinin and alisol B 23-acetate potently decreased melanin content and cellular tyrosinase activity in melan-a cells in a dose-dependent manner. However, few studies have examined their whitening mechanism in vitro and in vivo. In this study, we further investigated of isoartocarpesin and 6-CEPN for their action mechanisms against hyperpigmentation including the inhibition of melanin synthesis and induction of autophagy of melanosome using Melan-A cell line. MatTek's MelanoDerm™ artificial skin system and zebrafish model were also used to assess the depigmenting effectiveness of isoartocarpesin and 6CEPN, which proved that both compounds significantly reduced melanin content and tyrosinase activity in vivo. For the mechanism of inhibiting melanogenesis, the analysis of relative mRNA and protein expression indicated that isoartocarpesin and 6-CEPN inhibited the melanin production through the down-regulation of tyrosinase and tyrosinase related proteins (TRP1/2) at both mRNA and protein level. Isoartocarpesin and 6-CEPN also decreased the protein expression levels of the microphthalmia-associated transcriptional factor (MITF) in a transit time through the phosphorylation of extracellular responsive kinase (ERK) and phosphatidylinositol-3-kinase (PI3K/Akt). For the induction of autophagy of melanosome, autophagy process in melan-A cells treated with isoartocarpesin or 6-CEPN was detected by Fluorescence Microscopy. Western blot analysis was carried out by using autophagy pathway related antibody (ATG7, LC3 I/II) and siRNA of ATG7. With the presence of isoartocarpesin or 6-CEPN, the conversion of LC3 I to LC3 II in melan-a cells was found to be increased. While knocked down ATG7 by using specific siRNA, the conversion of LC3 I to LC3 II were reduced and the melanin content increased significantly, which further proved that isoartocarpesin and 6-CEPN could reduce melanin content through inducing autophagy process. Taken together, our data reveal that isoartocarpesin and 6CEPN can reduce melanin content through dual reactions, one is to inhibit the melanin synthesis and the other one is to induce the autophagy process to reduce the synthesized melanin.