Inhibited Melanin Synthesis Research Articles

Effect of cultured medium of human umbilical cord blood-derived mesenchymal stem cells on melanogenic enzyme activity in mouse B16 melanoma cells

The cultured medium of mesenchymal stem cell is a valuable resource for a variety of stem cell-derived cytokines and growth factors with a potential therapeutic effect on skin diseases. Here, we investigated the effect of the cultured medium of human umbilical cord blood-derived mesenchymal stem cell (CM-hCBSC) on melanogenesis in mouse B16 melanoma cells stimulated by α-melanocyte stimulating hormone (α-MSH). Our results show that CM-hCBSC inhibits melanin synthesis due to the decrease in cellular tyrosinase activity in B16 melanoma cells without cytotoxicity. The activity of melanogenic enzymes such as tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2 were evaluated by western blot and semi-quantitative RT-PCR. The CM-hCBSC down-regulated the protein and mRNA expression levels of tyrosinase and TRP-1 in a dose-dependent manner. In addition, TRP-2 protein and mRNA expression levels were found to be down-regulated to a lesser extent. We also measured the level of cytokines in CM-hCBSC using the Luminex total system. It was found that the top enriched cytokine in the CMhCBSC was interukine-6 (IL-6), known as a potent regulator of melanin synthesis together with IL-1α and tumor necrosis factor α (TNF- α). These results indicate that secretary factors including IL-6 in the CM-hCBSC may inhibit melanin synthesis by down-regulating the expression of tyrosinase and TRP1. This study suggests that CMhCBSC has a therapeutic effect as an anti-melanogenic agent and may be effective against hyperpigmentation disorders.

B16F1세포에서 항산화 활성 및 멜라닌 합성에 대한 유백피 에탄올 추출물의 효능

Melanin plays a key role in the protection of skin from ultraviolet light that generates reactive oxygen species (ROS), such as superoxide, hydroxyl radical, singlet oxygen and hydrogen peroxide. However, the ROS leading to the oxidation of lipids, proteins and DNA are involved in the overproduction of melanin that is known to cause melasma, age spots and freckles. Among the herb medicines, Ulmus macrocarpa used in this study was reported to contain flavonoids as a main component. The aim of this study is to investigate the whitening and anti-oxidant effects of Ulmus macrocarpa ethanolic extracts (UMEE) in B16F1 cells. UMEE below did not show cytotoxicity. In an anti-oxidant experiment, UMEE showed not only high reducing power and scavenging activity on DPPH, but it was also observed that UMEE exhibit an inhibitory effect on lipid peroxidation. UMEE did not display an inhibitory effect on tyrosinase activity in vitro. However, UMEE inhibited melanin synthesis in B16F1 cells. In addition, UMEE reduced the expression levels of tyrosinase and tyrosinase-related protein-2 (TRP-2), which are key enzymes in melanogenesis. These results indicate that UMEE exert a whitening effect through the inhibition of both tyrosinase and TRP-2 expressions as well as anti-oxidant activity, suggesting that UMEE could have the functional potential for a whitening effect on the skin.

Mechanistic studies of anti-hyperpigmentary compounds: elucidating their inhibitory and regulatory actions.

Searching for depigmenting agents from natural sources has become a new direction in the cosmetic industry as natural products are generally perceived as relatively safer. In our previous study, selected Chinese medicines traditionally used to treat hyperpigmentation were tested for anti-hyperpigmentary effects using a melan-a cell culture model. Among the tested chemical compounds, 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were found to possess hypopigmentary effects. Western blot analysis, reverse transcriptase polymerase chain reaction (RT-PCR), cyclic adenosine monophosphate (cAMP) assay, protein kinase A (PKA) activity assay, tyrosinase inhibition assay and lipid peroxidation inhibition assay were performed to reveal the underlying cellular and molecular mechanisms of the hypopigmentary effects. 4-Ethylresorcinol and 4-ethylphenol attenuated mRNA and protein expression of tyrosinase-related protein (TRP)-2, and possessed antioxidative effect by inhibiting lipid peroxidation. 1-Tetradecanol was able to attenuate protein expression of tyrosinase. The hypopigmentary actions of 4-ethylresorcinol, 4-ethylphenol and 1-tetradecanol were associated with regulating downstream proteins along the PKA pathway. 4-Ethylresorcinol was more effective in inhibiting melanin synthesis when compared to 4-ethylphenol and 1-tetradecanol.

S-(-)-10,11-dihydroxyfarnesoic acid methyl ester inhibits melanin synthesis in murine melanocyte cells.

The development of antimelanogenic agents is important for the prevention of serious aesthetic problems such as melasmas, freckles, age spots, and chloasmas. In the course of screening for melanin synthesis inhibitors, we found that the culture broth from an insect morphopathogenic fungus, Beauveria bassiana CS1029, exhibits potent antimelanogenic activity. We isolated and purified an active metabolite and identified it as S-(−)-10,11-dihydroxyfarnesoic acid methyl ester (dhFAME), an insect juvenile hormone. To address whether dhFAME inhibits melanin synthesis, we first measured the size of the melanin biosynthesis inhibition zone caused by dhFAME. dhFAME also showed inhibitory activity against mushroom tyrosinase in Melan-a cells. Intracellular, dose-dependent tyrosinase inhibition activity was also confirmed by zymography. In addition, we showed that dhFAME strongly inhibits melanin synthesis in Melan-a cells. Furthermore, we compared levels of TYR, TRP-1, TRP-2, MITF, and MC1R mRNA expression by reverse-transcription polymerase chain reaction and showed that treatment of Melan-a cells with 35 μM dhFAME led to an 11-fold decrease in TYR expression, a 6-fold decrease in TRP-2 expression, and a 5-fold decrease in MITF expression. Together, these results indicate that dhFAME is a potent inhibitor of melanin synthesis that can potentially be used for cosmetic biomaterial(s).

Inhibitory Effects of Saposhnikoviae Radix Extracts on the Melanin Production and Elastase Activity in B16F10 cells

Saposhmikoviae Radix can treat various skin disease by anti-pruitus and anti-inflammatory effects. This study was designed to investigate effects of Saposhmikoviae Radix Extracts(SRE) on skin elasticity and whitening using B16F10 cell lines. In this experiment, We observed effect of SRE on cell viability, inhibition of melanin synthesis and inhibitory effect on tyrosinase and elastase. In results, SRE treated group showed lowered proliferation rates significantly compared to non-treated group. More than SRE 125 ㎍/㎖ of treated groups were lower levels of melanin synthesis respectively. SRE did not show inhibitory effect on tyrosinase activities in vitro and in B16F10 cells. Finally, SRE suppressed elastse type Ⅰ and Ⅳ activities in dose-dependent manner in vitro. And SRE also slightly suppressed elastase activities in B16F10 cells. In conclusion, these results suggest that SRE can inhibit melanin synthesis and inhibt elastase activity. So, We suggest that SRE can be maintained skin elasticity or whitening.

쓴메밀 추출물의 미백 개선 효과

This study was performed to DPPH radical scavenging activity, ABTS radical scavenging activity, tyrosinase inhibition activity and intracellular melanin synthesis inhibition to verify the whitening effect of Fagopyrum tataricum (bitter buckwheat) extract as contrasted with Fagopyrum esculentum (sweet buckwheat) extract. F. tataricum extract in consequence showed higher antioxidant activities, tyrosinase inhibition activity and melanin synthesis inhibition compared with F. esculentum extract. We investigated skin bright value during 8 weeks after induction of pigmentation in human skin from UV irradiation. In result, we obtained statistically a significant skin whitening effect from visual and mechanical evaluation. Accordingly, F. tataricum extract is expected to be high availability as functional cosmetic material for skin whitening.

Rhododendrol, a depigmentation-inducing phenolic compound, exerts melanocyte cytotoxicity via a tyrosinase-dependent mechanism.

Rhododendrol, an inhibitor of melanin synthesis developed for lightening/whitening cosmetics, was recently reported to induce a depigmentary disorder principally at the sites of repeated chemical contact. Rhododendrol competitively inhibited mushroom tyrosinase and served as a good substrate, while it also showed cytotoxicity against cultured human melanocytes at high concentrations sufficient for inhibiting tyrosinase. The cytotoxicity was abolished by phenylthiourea, a chelator of the copper ions at the active site, and by specific knockdown of tyrosinase with siRNA. Hence, the cytotoxicity appeared to be triggered by the enzymatic conversion of rhododendrol to active product(s). No reactive oxygen species were detected in the treated melanocytes, but up-regulation of the CCAAT-enhancer-binding protein homologous protein gene responsible for apoptosis and/or autophagy and caspase-3 activation were found to be tyrosinase dependent. These results suggest that a tyrosinase-dependent accumulation of ER stress and/or activation of the apoptotic pathway may contribute to the melanocyte cytotoxicity.

Involvement of Transglutaminase-2 in α-MSH-Induced Melanogenesis in SK-MEL-2 Human Melanoma Cells.

Skin hyperpigmentation is one of the most common skin disorders caused by abnormal melanogenesis. The mechanism and key factors at play are not fully understood. Previous reports have indicated that cystamine (CTM) inhibits melanin synthesis, though its molecular mechanism in melanogenesis remains unclear. In the present study, we investigated the effect of CTM on melanin production using ELISA reader and the expression of proteins involved in melanogenesis by Western blotting, and examined the involvement of transglutaminase-2 (Tgase-2) in SK-MEL-2 human melanoma cells by gene silencing. In the results, CTM dose-dependently suppressed melanin production and dendrite extension in α-MSH-induced melanogenesis of SK-MEL-2 human melanoma cells. CTM also suppressed α-MSH-induced chemotactic migration as well as the expressions of melanogenesis factors TRP-1, TRP-2 and MITF in α-MSH-treated SK-MEL-2 cells. Meanwhile, gene silencing of Tgase-2 suppressed dendrite extension and the expressions of TRP-1 and TRP-2 in α-MSH-treated SK-MEL-2 cells. Overall, these findings suggested that CTM suppresses α-MSH-induced melanogenesis via Tgase-2 inhibition and that therefore, Tgase-2 might be a new target in hyperpigmentation disorder therapy.

백합뿌리 추출물의 멜라닌 생성 억제효과

In order to develop a skin-whitening agent, melanin contents and intracellular tyrosinase activity were determined by western blotting. Ethyl acetate fractions of 80% ethanol extracts from lily (Lilium Oriental Hybrid ‘Siberia’) bulbs (R-EA) inhibited melanin synthesis in a dose-dependent manner in α-melanocyte stimulating hormone (α-MSH)-treated B16/F10 murine melanoma cells. Intracellular tyrosinase activity and melanin contents were suppressed by 45% and 74%, respectively, in response to treatment with 100 μg/mL of R-EA. Examination of protein expression associated with α-MSH-induced melanogenesis showed that tyrosinase related protein (TRP)-1 was inhibited more strongly than tyrosinase, and these results were correlated with stronger inhibition of melanin synthesis than intracellular tyrosinase activity. Taken together, R-EA containing p-coumaric acid and resveratrol could be used as a hypopigmentation agent through suppression of sustained extracellular signal-regulated kinase (ERK) activation via melanogenic induction.

The inhibitory effect of a Platycodon root extract on ultraviolet B-induced pigmentation due to a decrease in Kit expression.

The signaling of stem cell factor (SCF) through its receptor Kit is known to play an important role in regulating cutaneous melanogenesis. In the course of UVB-induced pigmentation, the expression of membrane-bound SCF by epidermal keratinocytes is upregulated at an early phase and subsequently activates neighboring melanocytes via their Kit receptors. In order to identify effective skin-lightening materials, we screened botanical extracts to determine their abilities to diminish Kit expression in melanocytes. A Platycodon root extract was consequently found to have a remarkable inhibitory activity on Kit expression. When the extract was applied to three-dimensional human skin substitutes in vitro and to human skin in vivo after UVB irradiation, their pigmentation was significantly reduced, confirming the substantial contribution of the suppression of SCF/Kit signaling to preventing or inhibiting melanin synthesis. These data demonstrate that a Platycodon root extract is a promising material for a skin-lightening product to improve pigmentation-related diseases.

A derivative of imidazobenzimidazole, ML106, inhibits melanin synthesis via p38 MAPK activation.

We investigated the effects of ML106 on melanogenesis in B16F10 melanoma cells. Our results showed that ML106 decreased melanin content and tyrosinase activity in a dose-dependent manner. Interestingly, ML106 did not inhibit microphthalmia-associated transcription factor (MITF) expression, but did decrease tyrosinase expression. Thus, we further investigated the expression and degradation of tyrosinase and related signal transduction pathways. Although ML106 increased glycogen synthase kinase 3beta (GSK3beta) activation, the level of beta-catenin level was not affected. Thus, we excluded the involvement of GSK3beta and beta-catenin in ML106-induced hypopigmentation. However, ML106 induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK), causing down-regulation of tyrosinase. Thus, we next investigated whether tyrosinase down-regulation was due to proteasomal degradation by p38 MAPK activation. We found that ML106-induced tyrosinase down-regulation was restored by MG132, a proteasome inhibitor. Thus, we propose that ML106 has hypopigmentary activity through tyrosinase degradation via p38 MAPK phosphorylation.

Assessment of Cuscuta chinensis seeds׳ effect on melanogenesis: Comparison of water and ethanol fractions in vitro and in vivo

Ethnopharmacological relevanceCuscuta chinensis seeds have traditionally been used to treat freckles and melasma in Asia, although recent reports have revealed that Semen cuscutae is a promoter of melanogenesis. The present study aims to investigate the mechanism of this opposite effect of Semen cuscutae on melanogenesis. Materials and methodsIn accordance with traditional usage, the water fraction and the ethanol fraction from Semen cuscutae (WFSC/EFSC) were extracted to determine the herbal effects by examining the activity of mushroom tyrosinase, cellular melanin contents, tyrosinase activity assay, quantitative-reverse transcription polymerase chain reaction (qRT-PCR), and Western blot analysis for tyrosinase in B16F10 mouse melanoma cells. The melanocyte phenotypes of zebrafish larvae were observed while the in vivo melanin contents and tyrosinase activity were determined. ResultsThe activity of mushroom tyrosinase assay shown that WFSC was an uncompetitive inhibitor of mushroom tyrosinase, while EFSC indicated dose-dependent activation of the mushroom tyrosinase activity. The WFSC markedly inhibited 3-isobutyl-1-methylxanthine (IBMX)-stimulated melanin synthesis and tyrosinase activity in vitro. Howeveran accelerant role in melanin synthesis and tyosinase activity. Neither fraction had any effect on the IBMX-induced expression of tyrosinase protein or mRNA. The WFSC strongly inhibited melanin synthesis and cellular tyrosinase activity in vivo. Furthermore, with the function of WFSC at a higher concentration, a punctate melanocyte pattern appeared that was similar to the pattern induced by arbutin or Mequinol (MQ). The EFSC had no effect on the melanocytes of zebrafish larvae. It was discovered that WFSC did not show a stable inhibitory effect until it was extracted 1 month later. ConclusionsThese results suggest that the opposite effects of Cuscuta chinensis seeds were caused by the extraction methods and that time has an important role on the effect of WFSC. Both WFSC and EFSC significantly influence melanogenesis by regulating enzymatic activity of tyrosinase. In addition, the data indicate that wildtype (WT) zebrafish may be an ideal model for testing inhibitors of melanogenesis from clinically active herbs.

An aqueous extract of Limoniastrum guyonianum gall induces anti-tumor effects in melanoma-injected mice via modulation of the immune response

The objectives of this study were to evaluate the in vitro and in vivo anti-tumor potential of the aqueous gall extract (G extract) from Limoniastrum guyonianum and to elucidate its immunological mechanisms, in part, by assessing its effects on the growth of transplanted tumors and the immune response in these tumor-bearing mice. Here, mice were inoculated with B16F10 mouse tumor cells and then treated intraperitoneally with G extract at 25 or 50mg extract/kgBW for 7, 14, or 21days. At each timepoint, effects of the extract on the tumor growth, splenocytes proliferation, NK cell activity, and CTL activity among splenocytes isolated from the mice were measured. G extract-induced tumor growth inhibition was associated with characteristic apoptotic changes in the tumor cells, like nuclear condensation. In addition, the extract inhibited melanin synthesis and tyrosinase activity among melanoma cells in a concentration-related manner. G extract did not only significantly inhibit the growth of the transplantable tumor, but also remarkably increased splenocytes proliferation and both NK and CTL activities in tumor-bearing mice. The extract was also seen to have promoted lysosomal activity of host macrophages and gave rise to enhanced cellular anti-oxidant activity in several cell types in mice.

8-hydroxyarctigenin isolated from safflower sprouts inhibits melanogenesis of melan-a cells and light quality during the sprout growth determines the compound yield

Safflower sprouts were grown under different light frequencies and conditions, including fluorescent white light, red LED or blue LED light and darkness. The sprout extracts were tested for their inhibitory effect on melanin synthesis. Two compounds from safflower sprout extract were isolated and tested for their activities. Safflower seeds were sown in growth chambers set to lights and harvested at 9 days after sowing. Methanolic extracts of safflower sprouts were subjected to melanin synthesis inhibitory effect on MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetraxolium Bromide] and melanin bioassay methods with a dose dependent manner. Two compounds were identified by using open column chromatography and LC-MS (liquid chromatography-mass spectrometry) analysis. The MTT and melanin bioassays with the identified compounds were conducted. Darkness or fluorescent white light irradiation induced extracts with inhibitory effects on melanin synthesis (in a dose dependent manner). In contrast, blue light induced extracts to slightly increase melanin synthesis. It was found that 8-hydroxyarctigenin, a compound isolated from the safflower sprouts grown under fluorescent white light for 9 days after germination, induced inhibitory effect of melanin synthesis. The compound was contained more in the darkness or fluorescent white light treatment than others. Safflower sprouts grown under dark or fluorescent white light conditions were more effective on inhibiting melanin synthesis than conditions under red or blue light. An active compound, 8-hydroxyarctigenin, may be a useful whitening agent in cosmetic resources.

The Effects of Salacia reticulata on Anti-Cellular Oxidants and Melanogenesis Inhibition in α-MSH-stimulated and UV Irradiated B16 Melanoma Cells

The purposes of this study were to investigate the inhibitory effects of Salacia reticulata Tul. root extract on cellular oxidants and melanogenesis in B16 melanoma cells. Cells treated with non-toxic doses of S. reticulata root extract were investigated for their effects on melanogenesis, cellular tyrosinase activity and cellular oxidant scavenging activity. The results indicated that S. reticulata extract inhibited melanin synthesis and tyrosinase activity in alpha-MSH-induced or UV-irradiated B16 melanoma cells in a dose dependent manner. Additionally, the extract also exhibited anti-cellular oxidants in UV-induced radical melanoma cells. Altogether, these results suggested that S. reticulata root extract has roles in suppression of melanogenesis and oxidant inhibition. S. reticulata root extract may be a potential source for the development of pharmaceutical products for treatment of skin hyperpigmentation disorders.

추출 온도에 따른 금불초 꽃 추출물의 항노화 효능

본 연구에서는 추출 온도에 따른 금불초 꽃(I. britannica var. chinensis) 추출물의 항노화 효능을 알아 보았다. 추출 온도에 따른 세포독성을 살펴본 결과, 상온, <TEX>$45^{\circ}C$</TEX>와 <TEX>$65^{\circ}C$</TEX> 추출물 모두 0.1% 이하의 농도에서 세포독성이 관찰되지 않았다. 0.1% 이하의 농도에서 항노화 효능평가를 수행한 결과, 0.1% 농도의 상온 추출물 경우에 멜라닌 생성 억제율은 24.5%로 <TEX>$45^{\circ}C$</TEX>와 <TEX>$65^{\circ}C$</TEX> 추출물과 비교하여 멜라닌 생성 억제율이 큰 것으로 나타났다. 0.1% 농도의 상온 추출물을 처리한 세포의 히알루론산 합성 관련 유전자 HAS-1, HAS-2 및 HAS-3의 발현율은 각각 123.3%, 137.8% 및 133.2%를 나타냈으며, 이는 비교물질인 L-ascorbic acid (115.6%, 121.0% 및 131.4%) 경우 보다 약간 크게 나타났다. 금불초 꽃 45 <TEX>$^{\circ}C$</TEX> 추출물의 경우, 0.1%의 농도에서 염증유발 유전자인 TNF-<TEX>${\alpha}$</TEX>, COX-2 및 IL-1<TEX>${\alpha}$</TEX>의 발현율이 대조군 대비 각각 30.3%, 12.8%, 25.7%로 감소하였으며, 이는 상온, <TEX>$65^{\circ}C$</TEX> 추출물과 비교하여 염증유발 유전자 발현 억제 효과가 더 큰 것으로 나타났다. 이상의 결과들로 볼 때, 금불초 꽃 추출물은 추출 온도에 따라 멜라닌 생성 억제, 히알루론산 합성 관련 유전자 발현 증가 효과, 염증유발 유전자 발현 억제 효과를 나타내며, 그로 인하여 피부에서 미백, 보습, 항염 효능을 통해 피부 항노화 효능을 가질 것으로 사료된다. 이는 금불초 추출물이 항노화 화장품 원료로서 응용 가능성이 있음을 시사한다. In this study, the extracts of Inula britannica var. chinensis (I. britannica) flower were extracted at three different temperatures (room temperature, <TEX>$45^{\circ}C$</TEX>, and <TEX>$65^{\circ}C$</TEX>) and their anti-aging effects were studied. Before investigating anti-aging effects of the extracts, their cytotoxicity was tested on B16F10, Hs683, and HaCaT cells. All extracts showed no cytotoxicity at the concentration less than 0.1% (v/v). Melanin synthesis inhibitory activities in B16F10 cells and reverse transcriptase polymerase chain reaction (RT-PCR) in Hs683 and HaCaT cells were used to see their anti-aging effects. The room temperature extract at 0.1% showed 24.5% melanin synthesis inhibition, which was better than the <TEX>$45^{\circ}C$</TEX> and <TEX>$65^{\circ}C$</TEX> extracts. In addition, expression rates of the room temperature extract at 0.1% on HAS-1, HAS-2, and HAS-3 related to hyaluronan synthase genes were 123.3%, 137.8%, 133.2%, respectively. which were higher than reference material of L-ascorbic acid. Expression rates of the <TEX>$45^{\circ}C$</TEX> extract at 0.1% on TNF-<TEX>${\alpha}$</TEX>, COX-2, and IL-<TEX>$1{\alpha}$</TEX>, which are inflammatory related genes, was suppressed to 30.3%, 12.8%, 25.7%, respectively. It was better in anti-in flammatory effect than the room temperature and <TEX>$65^{\circ}C$</TEX> extracts. As results, we showed that I. britannica var. chinensis flower extarcts decreased melanin production and expression of inflammatory related genes and increased the expression rate of hyaluronan synthase genes. Thus, it is believed that the extracts affect anti-aging effects of skin through whitening, moisturizing, and anti-inflammatory processes and could be applicable to cosmetics as a functional cosmetic ingredient.

The transdermal inhibition of melanogenesis by a cell-membrane-permeable peptide delivery system based on poly-arginine

Topical therapy is the most favored form of treatment for whitening against hyper-pigmentation and sunburn because it lends itself to self-administration, patient compliance and an absence of systemic adverse effects. However, high-molecular-weight, hydrophilic chemicals are difficult to use as transdermal delivery drugs and the use of topical drugs has been highly limited. There are now many potent tyrosinase inhibitors, for example, sulfite or kojic acid, but the efficacy of their skin transduction remains a big problem. Furthermore, melanogenesis inhibitors from natural sources have great potential, as they are considered to be safe and largely free from adverse side effects. We applied 11-arginine (11R), a cell-membrane-permeable peptide, as a transdermal delivery system with a skin delivery enhancer, pyrenbutyrate. We performed intracellular screening for melanogenesis inhibitors with 11R fused with several kinds of tyrosinase inhibitory peptides from natural sources. Of 28 tyrosinase peptides, 13 melanin synthesis inhibitory peptides were selected. Peptide No. 10 found in gliadin protein, a wheat component, most strongly inhibited melanin production. This No. 10 peptide, of only 8 amino acids, fused to 11R showed no cytotoxicity and inhibited melanin synthesis as determined through melanin content measured using an absorption spectrometer and observation with a transmission electron microscope. Next, we transduced this 11R-No. 10 into skin with an 11R transdermal delivery system after previous treatment with pyrenbutyrate and performed daily repetitive topical application for two weeks against a UV-induced sun-tanning guinea pig model. We observed a whitening effect in a model skin sample by Masson-Fontana staining and the 11R-No. 10 peptide-applied area showed significant melanogenesis inhibition. These results show that 11R using a transdermal drug delivery system with melanogenesis inhibitory peptide is a very safe and promising method for applications from cosmetics to the pharmaceutical industry.

Docosahexaenoic acid inhibits melanin synthesis in murine melanoma cells in vitro through increasing tyrosinase degradation

To investigate the effects of docosahexaenoic acid (DHA) on melanin synthesis and related regulatory mechanisms. B16F10 mouse melanoma cells were exposed to DHA for 3 d, and melanin content and tyrosinase activity were measured. Western blot analysis was used to analyze the protein levels in DHA-mediated signal transduction pathways. DHA (1-25 μmol/L) did not affect the viability of B16F10 cells, but decreased α-MSH-induced melanin synthesis in a concentration-dependent manner. DHA concentration-dependently reduced tyrosinase activity in the cells, but did not affect mushroom tyrosinase activity in a cell-free system. Furthermore, DHA treatment significantly reduced tyrosinase level without affecting microphthalmia-associated transcription factor (MITF) in the cells. DHA did not activate ERK and Akt in the cells. Pretreatment with the proteasome inhibitor MG132 (80 nmol/L) abolished DHA-induced tyrosinase reduction. DHA inhibits melanogenesis in B16F10 cells in vitro through increasing tyrosinase degradation. The results suggest that DHA may be a potential agent for treatment of hyperpigmentary disorders of skin.

Synthesis and Bioevaluation of 4,5,6,7-Tetrahydrobenzo[d]isoxazole Derivatives as Melanogenesis Inhibitors

Melanin, the main component of skin color, is synthesized in the melanosomes of melanocytes through a physiological process called melanogenesis and then transferred to neighboring keratinocytes. Melanin absorbs high-energy UV radiation and transforms it into the thermal energy. In this way, melanin plays a protective role against UV-induced DNA damage. However, abnormal skin pigmentation is a major dermatological concern. Abnormal production of melanin causes hypopigmentation or hyperpigmentation such as vitiligo, melasma, age spot, and freckle. In addition, it has been reported that alteration in melanin production is observed in melanoma and Parkinson’s disease. Although key factors including melanocortin 1 receptor (MC1R), Microphthalmia associated transcription factor (MITF) and tyrosinase have been found, the molecular mechanism of melanogenesis is not yet fully understood. Potent chemical regulators for melanogenesis could be a useful tool to elucidate the complex mechanisms. In particular, the regulation of dyschromias is of growing interest, and further options for the treatment of hyperpigmentation are in great demand across Asia. Such a demand has led to the discovery of a variety of natural or synthetic chemicals to regulate melanin biosynthesis (Figure 1). Most of the reported melanogenesis inhibitors decrease tyrosinase activity, and a few small molecules such as arbutin and kojic acid are available on the market as skin lightening agents. However, arbutin has shown insufficient activity and kojic acid has toxicity issue. We have reported cyclohepta[d]isoxazole derivatives as novel melanogenesis inhibitors that decrease the expression of tyrosinase. Investigation of the Structure-activity relationship (SAR) has been explored through modification of regions B and C, which led to the identification of potent melanogensis inhibitors in our previous study (Figure 1). In order to explore and further extend the SAR of this series of melanogenesis inhibitors, a variety of the ring contracted derivatives were synthesized. Herein, we describe the synthesis and melanogenesis activity of 4,5,6,7-tetrahydrobenzo[d]isoxazole derivatives. Compounds 4a-j were synthesized by amide coupling of arylpiperazines with 4,5,6,7-tetrahydrobenzo[d]isoxazole-3carboxylic acid chloride in the presence of triethylamine in dry THF. The acid chlorides 3a-b were obtained by reaction of commercially available 4,5,6,7-tetrahydrobenzo[d]isoxazole-3-carboxylic acid with oxalyl chloride and a catalytic amount of DMF in dichloromethane (Scheme 1). O-alkylation of 4f with iodoethane gave compound 5 (Scheme 2). Melanogenesis activity of melanin synthesis inhibition is shown in Table 1. The para-methoxy derivative 4b exhibited slightly greater potency than the phenol 4a. However, cytotoxicity was observed at 10 M. A shift of the methoxy group on the aryl group from the para to ortho position (4c) greatly reduced activity. The introduction of trifluoromethyl at para position (4d) resulted in almost complete loss of potency at even 10 μM. The para-fluoro substituent (4j) did not improve activity. Next, we synthesized compounds removing tert-butyl group in region A. Phenol 4f retained activity at 1 μM but showed cytotoxicity at 10 μM. Figure 1. Representative melanin biosynthesis inhibitors and a validated hit compound (MMS1001) as a down-regulator of tyrosinase expression. Scheme 1. (a) oxalyl chloride, DMF (cat.), CH2Cl2, rt, 2 h; (b) arylpiperazines, Et3N, THF, rt, 1 h, 60-79% for 2 steps.

Inhibitory Effects of Resveratrol on Melanin Synthesis in Ultraviolet B-Induced Pigmentation in Guinea Pig Skin

Resveratrol is a polyphenolic compound found in various natural products such as grapes and berries and possesses anti-cancer, anti-hyperlipidemia, and anti-aging properties. Recently, it has been reported that resveratrol inhibits α-melanocyte-stimulating hormone signaling, viability, and migration in melanoma cells. However, these effects have not been confirmed in vivo, specifically brownish guinea pigs. To evaluate the potential of resveratrol as a regulator of melanin for hyperpigmentation therapy, the influence of resveratrol on pigmentation was investigated by ultraviolet B-induced hyperpigmentation in brownish guinea pig skin. We found that resveratrol reduced the expression of melanogenesis-related proteins tyrosinase, tyrosinase-related proteins 1 and 2, and microphthalmia-associated transcription factor in melanoma cells. Furthermore, topical application of resveratrol was demonstrated to significantly decrease hyperpigmentation on ultraviolet B-stimulated guinea pig skin in vivo. Based on our histological data, resveratrol inhibits melanin synthesis via a reduction in tyrosinase-related protein 2 among the melanogenic enzymes. This study is the first to provide evidence supporting resveratrol as a depigmentation agent, along with further clinical investigation of resveratrol in ultraviolet B-induced skin disorders such as hyperpigmentation and skin photoaging.