Protective effect of detoxified Rhus verniciflua stokes on human keratinocytes and dermal fibroblasts against oxidative stress and identification of the bioactive phenolics.

Oxidative stress due to the over-production of reactive oxygen species (ROS) is associated with human skin aging. This study was designed to identify the bioactive phenolics in detoxified Rhus verniciflua Stokes (DRVS) that may protect human skin against oxidative stress. Under oxidative stress caused by H2O2, the 40% (v/v) aqueous methanol extract of DRVS protected human keratinocytes in a dose-dependent manner. The expression of matrix metalloproteinase-1 (MMP-1) was also inhibited by the DRVS extract in human dermal fibroblasts-neonatal cells exposed to ultraviolet A. The major bioactive phenolics of DRVS were tentatively identified by LC/Q-TOF-ESI-MS/MS, and included gallic acid, 2-(ethoxymethoxy)-3-hydroxyphenol, fustin, a fustin isomer, tetragalloyl glucose, pentagalloyl glucose, fisetin, sulfuretin, a sulfuretin isomer, and butein. The results suggest that a DRVS extract may be effective in slowing skin aging through its antioxidative properties and by down-regulating MMP-1 expression. Further studies are needed to examine whether this effect would be mediated by the phenolics identified in this study.

Statistically designed enzymatic hydrolysis for optimized production of icariside II as a novel melanogenesis inhibitor.

Three kinds of prenylated flavonols, icariside I, icariside II, and icaritin, were isolated from an icariin hydrolysate and their effects on melanogenesis evaluated based on mushroom tyrosinase inhibition and quantifying the melanin contents in melanocytes. Although none of the compounds had an effect on tyrosinase activity, icariside II and icaritin both effectively inhibited the melanin contents with an IC50 of 10.53 and 11.13 MM, respectively. Whereas icariside II was obtained from a reaction with beta-glucosidase and cellulase, the icariin was not completely converted into icariside II. Thus, for the high-purity production of icariside II, the reaction was optimized using the response surface methodology, where an enzyme concentration of 5.0 mg/ml, pH 7, 37.5 degrees C;, and 8 h reaction time were selected as the central conditions for the central composite design (CCD) for the enzymatic hydrolysis of icariin into icariside II using cellulase. Empirical models were developed to describe the relationships between the operating factors and the response (icariside II yield). A statistical analysis indicated that all four factors had a significant effect (p<0.01) on the icariside II production. The coefficient of determination (R2) was good for the model (0.9853), and the optimum production conditions for icariside II was an enzyme concentration of 7.5 mg/ml, pH 5, 50 degrees C, and 12 h reaction time. A good agreement between the predicted and experimental data under the designed optimal conditions confirmed the usefulness of the model. A laboratory pilot scale was also successful.

Fructose 1,6-diphosphate alleviates UV-induced oxidative skin damage in hairless mice.

Reactive oxygen species (ROS) are involved in the deleterious effects of UV light on skin. The antioxidant defense system is considered to be crucial for protecting skin from ROS. Recently, we showed that fructose 1,6-diphosphate (FDP), a glycolytic metabolite, reduced oxidative stress in UVB-irradiated keratinocytes. This study set out to determine whether topically applied FDP could exert protective effects against UV-induced skin damage in hairless mice. An in vitro skin permeation study using Franz-type diffusion cells showed that the amount of [14C]-FDP that diffused through the skin increased in a time-dependent manner, and about 3.5% of the applied FDP penetrated the skin after 24 h. Topical application of FDP (1%) preserved the endogenous antioxidant capacity of skin such as catalase and glutathione, which were significantly reduced after UVB irradiation without FDP. FDP also reversed the loss of catalase protein and prevented the accumulation of carbonylated proteins induced by UVB irradiation. These results provide evidence that topically administered FDP could penetrate into the skin and attenuate UVB-induced oxidative skin damage in hairless mice.

Zebrafish as a new model for phenotype-based screening of melanogenic regulatory compounds.

Although many hypo-pigmenting agents are currently available, the demand for novel whitening agents is increasing, in part due to the weak effectiveness and unwanted side effects of currently available compounds. To screen for novel hypo-pigmenting agents, many methodologies such as cell culture and enzymatic assays are routinely used. However, these models have disadvantages in terms of physiological and economic relevance. In this study, we validated zebrafish as a whole-animal model for phenotype-based screening of melanogenic inhibitors or stimulators. We used both the well-known melanogenic inhibitors (1-phenyl-2-thiourea, arbutin, kojic acid, 2-mercaptobenzothiazole) and newly developed small molecule compounds (haginin, YT16i). All the tested compounds produced inhibitory effects on the pigmentation of zebrafish, most likely due to their inhibitory potential on tyrosinase activity. In simultaneous in vivo toxicity tests, a newly developed melanogenic inhibitor YT16i showed massive abnormalities in terms of deformed morphologies and cardiac function. Together, these results provide a rationale in screening and evaluating the putative melanogenic regulatory compounds. We suggest that the zebrafish system is a novel alternative to mammalian models, with several advantages including the rapidity, cost-effectiveness, and physiological relevance.

Influence of N-glycan processing disruption on tyrosinase and melanin synthesis in HM3KO melanoma cells.

Tyrosinase, a type I membrane glycoprotein, is synthesized and glycosylated in the endoplasmic reticulum (ER) and Golgi. The enzyme is subsequently transported to melanosomes where it participates in melanogenesis. Previous studies showed that the disruption of early ER N-glycan processing by deoxynojirimycin (DNJ), an inhibitor of alpha-glucosidase, suppresses tyrosinase enzymatic activity and melanogenesis. However, the disruption of late glycan processing, mainly performed by ER and Golgi alpha-1,2-mannosidases, on tyrosinase enzymatic activity and melanogenesis remains to be investigated. Following treatment of HM3KO human melanoma cells with deoxymannojirimycin (DMJ), an inhibitor of alpha-1,2-mannosidase, transport of tyrosinase to the melanosome, enzymatic activity, and melanogenesis were reduced in a dose-dependent manner. However, DMJ did not directly inhibit tyrosinase enzymatic activity and expression. Interestingly, an extract of Streptomyces subrutilus culture medium (ESSCM) containing DMJ and DNJ as the main components inhibited glycosylation and transport of tyrosinase to the melanosome as well as melanin synthesis, but with no negative effects on cell viability. These inhibitory effects of ESSCM were stronger than those of DMJ or DNJ alone. Tyrosinase glycosylation and melanogenesis in HM3KO melanoma cells were more effectively inhibited by DMJ and DNJ combined than DMJ or DNJ alone. Accordingly, we propose that ESSCM is a potential candidate for treating undesirable hyperpigmentation conditions, such as melasma, postinflammatory melanoderma, and solar lentigo.

Inhibition of skin pigmentation by an extract of Lepidium apetalum and its possible implication in IL-6 mediated signaling.

The development of effective skin-lightening agents is an increasingly important area of research aimed at the treatment of hyperpigmentation induced by UV irradiation or by medical conditions such as melasma, postinflammatory melanoderma and solar lentigo. Although some inhibit tyrosinase, identifying and understanding the mechanisms of action of other agents is an important goal if more effective pigmentation inhibitors are to be developed. We present here that an extract of Lepidium apetalum (ELA) decreased UV-induced skin pigmentation in brown guinea pigs and melanogenesis of HM3KO human melanoma cells. Interestingly, ELA did not reduce melanogenesis in HM3KO cells unless they were co-cultivated in keratinocyte-conditioned medium prepared by culturing keratinocytes with ELA. Under these conditions, ELA decreased tyrosinase mRNA and protein expression as well as melanin content via an ELA-mediated increase in keratinocyte IL-6 production which in turn was shown to decrease in the expression Mitf, a transcription factor implicated in tyrosinase gene expression and melanocyte differentiation. The results reveal that ELA may be an effective inhibitor of hyperpigmentation caused by UV irradiation or by pigmented skin disorders through a mechanism involving IL-6-mediated downregulation of Mitf rather than a direct inhibition of tyrosinase activity.