Inhibitory effect of miconazole on melanogenesis

Miconazole (MIC), a regional antifungal agent, has been used worldwide in the treatment of superficial mycosis. However, the effect of MIC on skin pigmentation is not known. In this study, we investigated the inhibitory effect of MIC on melanogenesis in B16 melanoma cells. Tyrosinase activity and melanin content were dose dependently decreased by MIC as compared with untreated cells. The level of tyrosinase protein expression was reduced with treatment MIC. A decrease in cell proliferation was observed in B16 cells treated with MIC 30 microM, indicating that the MIC-induced depigmenting effect was caused by inhibition of melanin synthesis and not by destruction of B16 cells. Furthermore, MIC markedly suppressed alpha-melanocyte stimulating hormone or forskolin-induced tyrosinase activity in B16 cells. Therefore the depigmenting effect of MIC might be due to the inhibition of tyrosinase activity and tyrosinase expression, which eventually slows melanin biosynthesis. These results indicate that MIC may be a useful inhibitor of melanogenesis in B16 cells and suggest that it may have beneficial effects in the treatment of hyperpigmentation disorders such as ephelis and melasma.     

Curcumin-like diarylpentanoid analogues as melanogenesis inhibitors

Anti-melanogenesis screening of 47 synthesized curcumin-like diarylpentanoid analogues was performed to show that some had a potent inhibitory effect on the melanogenesis in B16 melanoma cells. Their actions were considered to be mostly due to tyrosinase inhibition, tyrosinase expression inhibition, and melanin pigment degradation. The structure–activity relationships of those curcumin-like diarylpentanoid analogues which inhibited the melanogenesis and tyrosinase activity were also discussed. Of those compounds assayed, (2E,6E)-2,6-bis(2,5-dimethoxybenzylidene)cyclohexanone showed the most potent anti-melanogenesis effect, the mechanism of which is considered to be the degradation of the melanin pigment in B16 melanoma cells, affecting neither the tyrosinase activity nor tyrosinase expression.     

Piperlonguminine from Piper longum with inhibitory effects on alpha-melanocyte-stimulating hormone-induced melanogenesis in melanoma B16 cells

Skin hyperpigmentations such as melasma, freckles and senile lentigines can be subjectively treated by depigmenting agents. In our ongoing study to find melanogenesis inhibitors from natural sources, Piper longum L (fruits, Piperaceae) was discovered to have an inhibitory effect on alpha-melanocyte-stimulating hormone (alpha-MSH)-induced melanogenesis in melanoma B16 cells. Piperlonguminine has been identified as the melanogenesis inhibitor from P. longum by activity-guided extraction and isolation. The compound showed dose-dependent inhibitory effects with 85.1 +/- 4.9% inhibition at 25 microM, 62.1 +/- 6.1% at 12.5 microM, 36.4 +/- 4.6% at 6.3 microM and 18.4 +/- 5.1% at 3.1 microM on alpha-MSH-induced melanogenesis, showing an IC50 value of 9.6 microM. As a positive control, kojic acid exhibited an IC50 value of 44.6 microM on the melanogenesis. As to the mode of action, piperlonguminine showed an inhibitory effect on alpha-MSH-induced tyrosinase synthesis, documented by Western immunoblot analysis. However, piperlonguminine did not show an inhibitory effect on tyrosinase activity or a direct depigmenting effect of melanin.     

Inhibitory effects of Morinda citrifolia extract and its constituents on melanogenesis in murine B16 melanoma cells

The objective of this study was to examine the effects of Morinda citrifolia (noni) extract and its constituents on α-melanocyte stimulating hormone (α-MSH)-stimulated melanogenesis in cultured murine B16 melanoma cells (B16 cells). A 50% ethanolic extract of noni seeds (MCS-ext) showed significant inhibition of melanogenesis with no effect on cell proliferation. MCS-ext was more active than noni leaf and fruit flesh extracts. Activity guided fractionation of MCS-ext led to the isolation of two lignans, 3,3'-bisdemethylpinoresinol (1) and americanin A (2), as active constituents. To elucidate the mechanism of melanogenesis inhibition by the lignans, α-MSH-stimulated B16 cells were treated with 1 (5?μM) and 2 (200?μM). Time-dependent increases of intracellular melanin content and tyrosinase activity, during 24 to 72?h, were inhibited significantly by treatment with the lignans. The activity of 1 was greater than that of 2. Western blot analysis suggested that the lignans inhibited melanogenesis by down regulation of the levels of phosphorylation of p38 mitogen-activated protein kinase, resulting in suppression of tyrosinase expression.     

Inhibitory effects of 5‐chloroacetyl‐2‐piperidino‐1,3‐selenazole,a novel selenium‐containing compound,on skin melanin biosynthesis

Objectives Increased production and accumulation of melanin leads to many hyperpigmentation disorders such as melasma, freckles and geriatric pigment spots. Thus, there is a need for the development of depigmenting agents. Based on our previous reports, selenium derivatives as anti‐melanogenic lead compounds could be very important. The aim of this study was to investigate the depigmenting effect of novel selenium‐containing compounds. Methods The inhibitory effects of 5‐chloroacetyl‐2‐piperidino‐1,3‐selenazole (CS1), a novel selenium‐containing compound, on melanogenesis were investigated in B16F10 melanoma cells and cultured brownish guinea pig skin tissue with α‐melanocyte‐stimulating hormone stimulation. Key findings We found that CS1 inhibited melanin production in B16F10 cells by suppressing tyrosinase activity and its protein expression. In addition, Western blotting analysis revealed that CS1 suppressed the expression of tyrosinase‐related protein (TRP)‐1 and TRP‐2. Therefore, the depigmenting effect of CS1 might have been due to inhibition of tyrosinase activity and expression of melanogenic enzymes. Furthermore, CS1 had inhibitory effects on melanin biosynthesis of primary cultured skin of brownish guinea pig. Conclusions The results suggested that CS1 could be a useful candidate for the treatment of skin hyperpigmentation.     

Inhibitory effect of p-hydroxybenzyl alcohol on tyrosinase activity and melanogenesis

Tyrosinase is a key enzyme catalyzing the rate-limiting step for the biosynthesis pathway of melanin pigment, which is the most important determinant of the color of skin. Inhibiting tyrosinase and repressing melanocyte metabolism can reduce melanin production. Among the possible melanin reducing compounds, tyrosinase inhibitors are most promising for treating pigmentation and are used as skin-whitening agents in the cosmetic industry. In our investigation, some new tyrosinase inhibitors from plants have been identified to have high tyrosinase inhibitory activity. Specifically, p-hydroxybenzyl alcohol (4HBA) was found to inhibit the monophenolase activity of mushroom tyrosinase. When 4HBA binds with the enzyme, conformation of the enzyme is altered and its activity decreases. The inhibitory effect of 4HBA on melanogenesis has been studied using cultured mouse melanoma cells. Melanin synthesis in cell culture with 4HBA at 1.0 mM was decreased to 45% of control and below 1.0 mM there was no effect on cell growth. The inhibitory effects of 4HBA on melanogenesis are due to the direct inhibition of melanosomal tyrosinase activity, rather than to the suppression of tyrosinase gene. These results showed that 4HBA is a promising and safe agent for skin whitening.     

Inhibitory effects of 1,3‐thiazine derivatives on melanogenesis

Objectives The aim of this study was to identify a novel skin‐depigmenting agent from synthetic 1,3‐thiazine derivatives. Methods We investigated the inhibitory effects of six kinds of 1,3‐thiazine derivative on melanogenesis by examining their effects on tyrosinase activity and melanin biosynthesis in melan‐a cells and the zebrafish model. Key findings Of the six compounds, 4‐hydroxy‐2,6‐dimethyl‐5,6‐dihydro‐4H‐1,3‐thiazine (TZ‐6) had the strongest anti‐melanogenic effects in cultured melan‐a cells (30.4% inhibition at 100 μM). In addition, TZ‐6 exhibited an inhibitory effect on mushroom and cellular tyrosinase. Based on the results of Western blotting, TZ‐6 reduced the expression of tyrosinase at 100 mM. Additionally, TZ‐6 reduced body pigmentation and inhibited tyrosinase activity in the zebrafish model. Conclusions The results have provided useful information for the development of a skin whitening agent.     

Fluvastatin increases tyrosinase synthesis induced by α-melanocyte-stimulating hormone in B16F10 melanoma cells

The aim of this study was to evaluate the effect of fluvastatin on the α-melanocyte-stimulating hormone-mediated increase in tyrosinase activity in the melanoma B16F10 cell line and to establish whether Akt and extracellular signal-regulated kinase (Erk) inhibition is involved in tyrosinase synthesis after fluvastatin administration. Fluvastatin modulates α-melanocyte-stimulating hormone induced melanogenesis by increasing tyrosinase mRNA production, as shown by real time PCR, or tyrosinase protein synthesis, as presented by western blot technique. The stimulatory effect of fluvastatin on melanogenesis was, in part, induced by modulation of cell proliferation (decreased melanoma cell proliferation in G2/M phase) and possibly decrease of Akt. These findings indicate that fluvastatin increases tyrosinase synthesis induced by α-melanocyte-stimulating hormone in B16F10 cells and reveal an unknown effect of statin use: their influence on melanin production.     

Inhibitory effect of gnetin C, a resveratrol dimer from melinjo (Gnetum gnemon), on tyrosinase activity and melanin biosynthesis

Tyrosinase is the key enzyme involved in melanogenesis. The aim of this study was to investigate the in vitro inhibitory effects of gnetin C, a resveratrol dimer isolated from melinjo (Gnetum gnemon) seeds, on tyrosinase activity and melanin biosynthesis in murine B16 cells. The inhibitory activities of gnetin C and resveratrol were shown to be almost equal against tyrosinase and melanin biosynthesis in the cells. The IC(50) values of gnetin C activity against tyrosinase and melanin biosynthesis were 7.0 and 7.6 μM, respectively, whereas resveratrol demonstrated IC(50) values of 7.2 and 7.3 μM, respectively. These results indicated that gnetin C inhibited melanogenesis, in a manner similar to that of resveratrol, by inhibiting tyrosinase and may therefore function as a new skin-whitening agent. However, the direct effects of gnetin C and resveratrol on murine tyrosinase activities were not equal. The IC(50) value of resveratrol was 10.1 μM, while gnetin C only exhibited a 25.2% enzyme inhibition at 16 μM. The IC(25) values for gnetin C and resveratrol were 15.5 and 4.0 μM, respectively. Therefore, it is suggested that the effects of gnetin C may be due to mechanisms other than the direct inhibition of tyrosinase activity.     

Inhibitors of mammalian melanocyte tyrosinase: in vitro comparisons of alkyl esters of gentisic acid with other putative inhibitors

To discover safe and effective topical skin-lightening agents, we have evaluated alkyl esters of the natural product gentisic acid (GA), which is related to our lead compound methyl gentisate (MG), and four putative tyrosinase inhibitors, utilizing mammalian melanocyte cell cultures and cell-free extracts. Desirable characteristics include the ability to inhibit melanogenesis in cells (IC50 < 100 microg/mL) without cytotoxicity, preferably due to tyrosinase inhibition. Of the six esters synthesized, the smaller esters (e.g. methyl and ethyl) were more effective enzyme inhibitors (IC50 approximately 11 and 20 microg/mL, respectively). For comparison, hydroquinone (HQ), a commercial skin "bleaching" agent, was a less effective enzyme inhibitor (IC50 approximately 72 microg/mL), and was highly cytotoxic to melanocytes in vitro at concentrations substantially lower than the IC50 for enzymatic inhibition. Kojic acid was a potent inhibitor of the mammalian enzyme (IC50 approximately 6 microg/mL), but did not reduce pigmentation in cells. Both arbutin and magnesium ascorbyl phosphate were ineffective in the cell-free and cell-based assays. MG at 100 microg/mL exhibited a minimal inhibitory effect on DHICA oxidase (TRP 1) and no effect on DOPAchrome tautomerase (TRP-2), suggesting that MG inhibits melanogenesis primarily via tyrosinase inhibition. MG and GA were non-mutagenic at the hprt locus in V79 Chinese hamster cells, whereas HQ was highly mutagenic and cytotoxic. The properties of MG in vitro, including (1) pigmentation inhibition in melanocytes, (2) tyrosinase inhibition and selectivity, (3) reduced cytotoxicity relative to HQ, and (4) lack of mutagenic potential in mammalian cells, establish MG as a superior candidate skin-lightening agent.     

仙藿胶囊对酪氨酸酶活性作用的实验研究

目的探讨仙藿胶囊对酪氨酸酶活性可能的作用机制,为临床治疗白癜风提供实验依据。方法离体实验采用蘑菇酪氨酸酶多巴速率氧化法,检测仙藿胶囊乙醇提取物对酪氨酸酶活性及黑素生成的影响。结果仙藿胶囊可上调酪氨酸酶的活性（P〈0．05）及黑素生成量。结论仙藿胶囊对酪氨酸酶有显著激活作用;提示仙藿胶囊治疗白癜风可能是通过调节酪氨酸酶活性促进黑素合成而发挥作用。     

Antimelanogenic and antioxidant properties of gallic acid

To find novel skin-whitening agents, the melanogenesis inhibitory action of gallic acid (GA) was investigated. In this current study, the effects of GA on mushroom tyrosinase, tyrosinase inhibitory activity, and melanin content were assessed in B16 melanoma cells (B16 cells). Results indicated that GA has a strong antityrosinase activity (IC50=3.59x10(-6) M). Furthermore, data on murine tyrosinase activity and melanin biosynthesis revealed that GA effectively suppressed murine tyrosinase action and the amount of melanin. To investigate the relation between GA's inhibition of melanogenesis and antioxidant activity, the effect of GA on reactive species (RS) generation and the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio in were determined in B16 cells. Results indicated that GA effectively down-regulated the RS generation and enhanced the GSH/GSSG ratio. Based on these results, I propose that GA exerts antimelanogenic activity coupled with antioxidant properties by suppressing RS generation and maintaining a higher GSH/GSSG ratio.     

Inhibitory effect of p-coumaric acid by Rhodiola sachalinensis on melanin synthesis in B16F10 cells

Rhodiola has been widely used in traditional Asian medicine. In this study, we tested the hypopigmentation effects of R. sachalinensis and its active compounds including catechin, chlorogenic acid, p-coumaric acid, and p-tyrosol. Results have shown that only p-coumaric acid inhibits melanin synthesis in B16F10 cells. However, p-coumaric acid did not inhibit tyrosinase activity when L-DOPA was used as a substrate. Instead, p-coumaric acid inhibited tyrosinase activity when L-tyrosine was used as a substrate. We further analyzed the changes of cAMP responsive element binding protein (CREB) phosphorylation and tyrosinase gene expression. The results indicate that p-coumaric acid does not affect CREB phosphorylation or tyrosinase protein production. In turn, these findings demonstrate that p-coumaric acid has no effect on the upstream regulation of tyrosinase gene expression, although p-coumaric acid showed a significant inhibitory effect on melanogenesis. Because p-coumaric acid showed different effects on tyrosinase activity according to different substrates, we tested whether tyrosinase can utilize p-coumaric acid as a substrate. Our findings revealed that competitive inhibition occurs between p-coumaric acid and tyrosine. Consequently, this finding could be a primary mechanism for the hypopigmenting action of p-coumaric acid.     

Inhibitory effects of 6-(3-hydroxyphenyl)-2-naphthol on tyrosinase activity and melanin synthesis

As a part of an ongoing project searching for new skin-lightening agents, the inhibitory property of 6-(3-Hydroxyphenyl)-2-naphthol (HPN) on melanogenesis was investigated. The inhibitory action of HPN (IC₅₀=15.2 μM) on mushroom tyrosinase was revealed. To further explore the action of HPN on melanogenesis, the inhibition of tyrosinase and melanin levels were measured in B16 melanoma cells (B16 cells). Results show that HPN inhibited tyrosinase activity and reduced melanin in B16 cells. Therefore, our data indicate HPN as a new candidate for depigmentation reagents. Contributed equally to this work.