Synthesis and Characterization of Metronidazole and Heterocycle Ester Dyads for Tyrosinase Inhibitory Activity

Tyrosinase is an important copper-based enzyme mainly involved in skin pigmentation. The inhibition of the tyrosinase enzyme attracts importance in cosmetic and medicinal chemistry industry for its applications in skin whitening and anti-browning agents for humans as well as in food, agriculture industries. Imidazole based Metronidazole and its derivatives are widely accepted drug for wide range of diseases. Therefore, the present report involves the synthesis of heterocyclic derivatives of metronidazole and investigation of its efficacy towards the tyrosinase inhibitory activity. A series of metronidazole esters were synthesized and their chemical structures were confirmed using spectral techniques like, infrared spectroscopy (IR), proton nuclear magnetic resonance (1H-NMR), and liquid chromatography-mass spectrometry (LC-MS). All the compounds were evaluated for its tyrosinase inhibitory activity by oxidation of 3,4-dihydroxyphenylalanine in the presence of the synthesized esters(I-VIII) with kojic acid as standard. Among the synthesized compounds, VII (isonicotinic ester) and VIII (quinoline ester) demonstrated significant activity IC50 values 92.5 and 91.8µM respectively. Further molecular docking experiments were carried out for the synthesized compounds with 2y9w protein exhibited greater number of physical interactions for compounds VII and VIII than the other compounds in the series confirming the mushroom tyrosinase activity.

Epimedin B exhibits pigmentation by increasing tyrosinase family proteins expression,activity,and stability / 药物分析学报

Epimedin B(EB)is one of the main flavonoid ingredients present in Epimedium brevicornum Maxim.,a traditional herb widely used in China.Our previous study showed that EB was a stronger inducer of melanogenesis and an activator of tyrosinase(TYR).However,the role of EB in melanogenesis and the mechanism underlying the regulation remain unclear.Herein,as an extension to our previous investi-gation,we provide comprehensive evidence of EB-induced pigmentation in vivo and in vitro and eluci-date the melanogenesis mechanism by assessing its effects on the TYR family of proteins(TYRs)in terms of expression,activity,and stability.The results showed that EB increased TYRs expression through microphthalmia-associated transcription factor-mediated p-Akt(referred to as protein kinase B(PKB))/glycogen synthase kinase 3β(GSK3β)/β-catenin,p-p70 S6 kinase cascades,and protein 38(p38)/mitogen-activated protein(MAP)kinase(MAPK)and extracellular regulated protein kinases(ERK)/MAPK pathways,after which EB increased the number of melanosomes and promoted their maturation for melanogenesis in melanoma cells and human primary melanocytes/skin tissues.Furthermore,EB exerted repigmentation by stimulating TYR activity in hydroquinone-and N-phenylthiourea-induced TYR inhibitive models,including melanoma cells,zebrafish,and mice.Finally,EB ameliorated monobenzone-induced depigmentation in vitro and in vivo through the enhancement of TYRs stability by inhibiting TYR misfolding,TYR-related protein 1 formation,and retention in the endoplasmic reticulum and then by downregulating the ubiquitination and proteolysis processes.These data conclude that EB can target TYRs and alter their expression,activity,and stability,thus stimulating their pigmentation function,which might provide a novel rational strategy for hypopigmentation treatment in the pharmaceutical and cosmetic industries.

Melanogenesis of quality markers in Vernonia anthelmintica Injection based on UPLC-Q-TOF-MS combined network pharmacology / 中国中药杂志

This study aimed to evaluate the biological effect and mechanism of Vernonia anthelmintica Injection(VAI) on melanin accumulation. The in vivo depigmentation model was induced by propylthiouracil(PTU) in zebrafish, and the effect of VAI on melanin accumulation was evaluated based on the in vitro B16F10 cell model. The chemical composition of VAI was identified according to the high-performance liquid chromatography quadrupole-time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS). Network pharmaco-logy was applied to predict potential targets and pathways of VAI. A "VAI component-target-pathway" network was established, and the pharmacodynamic molecules were screened out based on the topological characteristics of the network. The binding of active molecules to key targets was verified by molecular docking. The results showed that VAI promoted tyrosinase activity and melanin production in B16F10 cells in a dose-and time-dependent manner and could restore the melanin in the body of the zebrafish model. Fifty-six compounds were identified from VAI, including flavonoids(15/56), terpenoids(10/56), phenolic acids(9/56), fatty acids(9/56), steroids(6/56), and others(7/56). Network pharmacological analysis screened four potential quality markers, including apigenin, chrysoeriol, syringaresinol, and butein, involving 61 targets and 65 pathways, and molecular docking verified their binding to TYR, NFE2L2, CASP3, MAPK1, MAPK8, and MAPK14. It was found that the mRNA expression of MITF, TYR, TYRP1, and DCT in B16F10 cells was promoted. By UPLC-Q-TOF-MS and network pharmacology, this study determined the material basis of VAI against vitiligo, screened apigenin, chrysoeriol, syringaresinol, and butein as the quality markers of VAI, and verified the efficacy and internal mechanism of melanogenesis, providing a basis for quality control and further clinical research.

Effect of miRNA-193b-5p-mediated decreased expression of transcriptional regulator CITED2 on melanogenesis / 中华皮肤科杂志

Objective:To investigate the effect of miRNA (miR) -193b-5p on melanogenesis and its possible mechanisms.Methods:Human primary melanocytes were isolated from discarded normal foreskin tissues of healthy males after circumcision, and cultured in vitro. miR-NC mimics (miR-NC mimic group) and miR-193b-5p mimics (miR-193b-5p mimic group) were transfected into human primary melanocytes and human MNT1 melanoma cells, separately. After transfection, real-time quantitative PCR (RT-qPCR) was performed to determine the overexpression efficiency of miR-193b-5p at 48 hours, Western blot analysis to determine the expression of melanogenesis-related proteins tyrosinase (TYR) and microphthalmia-associated transcription factor (MITF) in human primary melanocytes and human MNT1 melanoma cells at 72 hours, and the melanin content in the above cells was determined by a sodium hydroxide solubilization method at 1 week. The target gene of miR-193b-5p was predicted by using Targetscan algorithms and verified by dual-luciferase reporter assay, and RT-qPCR and Western blot analysis were performed to analyze changes in mRNA and protein expression of the target gene respectively after the overexpression of miR-193b-5p. Two-independent-samples t test was used for comparisons between two groups. Results:In human primary melanocytes and human MNT1 melanoma cells, the miR-193b-5p expression levels were significantly higher in the miR-193b-5p mimic groups than in the miR-NC mimic groups ( t = 65.57, 22.49, respectively, both P < 0.001) , and the melanin content was significantly lower in the miR-193b-5p mimic groups (0.091 ± 0.007, 0.130 ± 0.004, respectively) than in the miR-NC mimic groups (0.117 ± 0.002, 0.188 ± 0.032, t = 5.98, 3.24, P < 0.01, < 0.05, respectively) . Western blot analysis showed that the expression of melanogenesis-related proteins TYR and MITF in both human primary melanocytes and human MNT1 melanoma cells was significantly lower in the miR-193b-5p mimic groups than in the miR-NC mimic groups (all P < 0.01) . TargetScan analysis and dual-luciferase reporter assay revealed a binding site for miR-193b-5p in the 3′ untranslated region of the transcriptional regulator CITED2. After up-regulation of miR-193b-5p expression in human primary melanocytes and human MNT1 melanoma cells, the CITED2 mRNA and protein expression levels significantly decreased compared with the miR-NC mimic groups (all P < 0.05) . Conclusion:miR-193b-5p overexpression can down-regulate the expression of melanogenesis-related proteins TYR and MITF, and then inhibit melanogenesis, which may be related to the targeted inhibition of CITED2 expression.

DKK3 Regulates Melanogenesis in Melanocytes of Alpaca / 中国生物化学与分子生物学报

Dickkopf-3 (DKK3) , as a critical inhibitor of the Wnt/p-catenin signaling pathway, may he involved in melanogenesis.In the current study, we investigated the effects of DKK3 on melanogenesis in melanocytes of alpaca.Overexpression of DKK3 in alpaca melanocytes, the expression of Wntl, Lefl , Myc and the major target genes termed microphthalmia-associated transcription factor (M1TF) and its downstream genes, including tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1) and tyrosinase- related protein 2 (TYRP2) were significantly decreased at both mRNA and protein levels (P<0.05); total alkali melanin, pheomelanin and eumelanin were decreased by 80.30%, 72.17% and 64.60% (P <0.05), respectively.In contrast, in the melanocytes transfected with siRNA-DKK3 (a small interference RNA targeting DKK3) , the expression of Wntl, Lefl, Myc, MITF, TYR, TYRPl and TYRP2 were significantly increased at both mRNA and protein levels (P<0.05) ; total alkali melanin, pheomelanin and eumelanin were significantly increased by 1.65 folds, 1.25 folds and 1.21 folds (P< 0.05) , respectively.These results indicate that DKK3 regulates melanogenesis in alpaca melanocytes via the Wnt/p-catenin signaling pathway and down-regulates MITF.

MicroRNA-100-5p Regulates Melanogenesis in Alpaca Melanocytes by Targeting FGF21 / 中国生物化学与分子生物学报

Although many microRNAs (miRNAs) are known to function as regulators of coat color and melanogenesis, the underlying molecular mechanisms of miR-100-5p governing melanogenesis were not completely known.The goal of this study was to determine the effect of miR-l()()-5p on melanogenesis in alpaca melanocytes.Fibroblast growth factor 21 (FGF21) is a predicted target gene of miR-100-5p and the luciferase reporter assay demonstrated that miR-100-5p regulates FGF21 by binding to its 3' untranslated region (3'UTR).In this study, alpaca melanocytes were transfected with miR-100-5p, inhibitor and negative control plasmid.Results showed that miR-100-5p overexpression significantly decreased mRNA and protein expression of FGF2\.Meanwhile, the ERK signal pathway was inhibited, with subsequent up-regulation of microphthalmia-associated transcription factor (MITF) , tyrosinase (TYR) and tyrosinase-related protein 2 (TYRP2), which increased melanin production.The results suggest that miR-100-5p may regulate melanogenesis by targeting FGF21 via extracellular regulated MAP kinase (ERK) signaling pathway.

Antioxidant and anti-melanogenic activities of ultrasonic extract from Stichopus japonicus

Objective: To investigate the antioxidant and anti-melanogenesis activities of an ultrasonic extract of red sea cucumber, Stichopus japonicus, collected from Jeju Island. Methods: Antioxidant activity experiments were assessed by an electron spin resonance system and a cellular model of immortalized human keratinocytes (HaCaT) to determine its radical scavenging activity and protective effects against 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress. Anti-melanogenic activity of the ultrasonic extract of red sea cucumber was also examined using the melanoma cell model B16F10 and mushroom tyrosinase. Following the induction by α-melanocyte-stimulating hormone, the effects of the ultrasonic extract of red sea cucumber on intracellular tyrosinase activity, melanin content and the melanogenic protein expression of microphthalmia-associated transcription factor, tyrosinase, and tyrosinase-related proteins (TRP-1, and TRP-2) were examined. Results: The ultrasonic extract of red sea cucumber significantly scavenged 2,2-diphenyl-1-picrylhydrazyl and alkyl radicals [IC50:(0.924±0.035) and (0.327±0.006) mg/mL, respectively], as well as showed a protective effect against oxidative stress and attenuated generation of intracellular reactive oxygen species on AAPH-induced HaCaT cells, with no cytotoxicity (12.5-400 μg/mL). The ultrasonic extract of red sea cucumber also exhibited a tyrosinase inhibitory effect [IC50: (2.750±0.006) mg/mL]. On α-melanocyte-stimulating hormone-stimulated B16F10 melanoma cells, the ultrasonic extract of red sea cucumber (25-200 μg/mL) significantly inhibited not only melanin synthesis and tyrosinase activity, but also protein expressions of microphthalmia-associated transcriptional factor, tyrosinase, TRP-1, and TRP-2. Conclusions: The ultrasonic extract of red sea cucumber shows antioxidant and anti-melanogenic potential and may be a natural candidate for anti-aging as well as a whitening agent in the cosmeceuticals industry.

Antioxidant and anti-melanogenic activities of ultrasonic extract from Stichopus japonicus

To investigate the antioxidant and anti-melanogenesis activities of an ultrasonic extract of red sea cucumber, Stichopus japonicus, collected from Jeju Island. Methods: Antioxidant activity experiments were assessed by an electron spin resonance system and a cellular model of immortalized human keratinocytes (HaCaT) to determine its radical scavenging activity and protective effects against 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress. Antimelanogenic activity of the ultrasonic extract of red sea cucumber was also examined using the melanoma cell model B16F10 and mushroom tyrosinase. Following the induction by ?-melanocytestimulating hormone, the effects of the ultrasonic extract of red sea cucumber on intracellular tyrosinase activity, melanin content and the melanogenic protein expression of microphthalmia-associated transcription factor, tyrosinase, and tyrosinase-related proteins (TRP-1, and TRP-2) were examined. Results: The ultrasonic extract of red sea cucumber significantly scavenged 2,2-diphenyl-1-picrylhydrazyl and alkyl radicals [IC50: (0.9240.035) and (0.3270.006) mg/mL, respectively], as well as showed a protective effect against oxidative stress and attenuated generation of intracellular reactive oxygen species on AAPHinduced HaCaT cells, with no cytotoxicity (12.5-400 ug/mL). The ultrasonic extract of red sea cucumber also exhibited a tyrosinase inhibitory effect [IC50: (2.7500.006) mg/mL]. On ?-melanocytestimulating hormone-stimulated B16F10 melanoma cells, the ultrasonic extract of red sea cucumber (25-200 ug/mL) significantly inhibited not only melanin synthesis and tyrosinase activity, but also protein expressions of microphthalmia-associated transcriptional factor, tyrosinase, TRP-1, and TRP-2. Conclusions: The ultrasonic extract of red sea cucumber shows antioxidant and anti-melanogenic potential and may be a natural candidate for anti-aging as well as a whitening agent in the cosmeceuticals industry.

Critical role of metabotropic glutamate receptor 4 in bone marrow-derived dendritic cells in the Th17 cell differentiation and the melanogenesis of B16 cells

Vitiligo is an acquired pigmentary disorder resulting from selective destruction of melanocytes. Emerging studies have suggested that T helper cell 17 (Th17) is potentially implicated in vitiligo development and progression. It was recently discovered that metabotropic glutamate receptor 4 (mGluR4) can modulate Th17-mediated adaptive immunity. However, the influence of mGluR4 on melanogenesis of melanocytes has yet to be elucidated. In the present study, we primarily cultured mouse bone marrow-derived dendritic cells (BMDC) and then knocked down and over-expressed mGluR4 using transfection. Transduced BMDC were co-cultured with CD4+ T cells and the expression of Th17-related cytokines were measured. The morphology and melanogenesis of B16 cells were observed after being treated with co-culture medium of CD4+ T cells and transduced BMDC. We found that mGluR4 knockdown did not affect the co-stimulatory CD80 and CD86 upregulation after lipopolysaccharide stimulation but did increase the expression of Th17-related cytokines, and further down-regulated the expression of microphthalmia-associated transcription factor (MITF) and the downstream genes, decreased melanin production, and destroyed the morphology of B16 cells. Conversely, over-expression of mGluR4 reduced the expression of CD80 and CD86, suppressed the production of Th17-related cytokines, increased the expression of MITF, and did not destroy the morphology of B16 cells. Our study confirmed that mGluR4 modulated the Th17 cell polarization and resulted in the alteration of melanogenesis and morphology of B16 cells. Collectively, these findings suggest mGluR4 might be a potent target involved in the immune pathogenesis of vitiligo.

Anti-Melanogenic Effect from Submerged Mycelial Cultures of Ganoderma weberianum

Compounds from Lingzhi has been demonstrated the ability for inhibiting tyrosinase (a key enzyme in melanogenesis) activity. In this study, we investigated the anti-melanogenic activity from the submerged mycelial culture of Ganoderma weberianum and elucidated the skin lightening mechanism by B16-F10 murine melanoma cells. From the cellular context, several fractionated mycelium samples exhibited anti-melanogenic activity by reducing more than 40% extracellular melanin content of B16-F10 melanoma cells. In particular, the fractionated chloroform extract (CF-F3) inhibited both secreted and intracellular melanin with the lowest dosage (25 ppm). Further analysis demonstrated that CF-F3 inhibited cellular tyrosinase activity without altering its protein expression. Taken together, our study has demonstrated that the chemical extracts from submerged mycelial culture of G. weberianum have the potential to serve as an alternative anti-melanogenic agent.

Research progress in melanogenesis signaling pathway / 生物工程学报

Melanogenesis is a biosynthetic pathway to produce melanin pigment in melanocyte, involving a series of intricate enzymatic and chemical catalyzed reactions. Melanogenesis involves five signaling pathways that converge on microphthalmia-associated transcription factor. In addition, many cytokines, involved in the regulation of melanogenesis, play an important role in the development, proliferation, differentiation and migration of melanocytes. Polyoxometalate can be used as a potential inhibitor of melanin production. Hence, this paper reviews the signaling pathways of melanogenesis and their regulatory mechanism, to apply polyoxometalates in the melanin production pathway, and briefly introduces the regulatory factors of related pathways.

Effect of latanoprost on cell proliferation of and melanogenesis in human epidermal melanocytes and its mechanism / 中华皮肤科杂志

Objective To evaluate the effect of latanoprost on cell proliferation of and melanogenesis in human epidermal melanocytes,and to explore its mechanism.Methods Latanoprost was added into the 254 medium to prepare latanoprost solutions at different concentrations of 10-5,10-6 and 10-7 mol/L.In vitro cultured human epidermal melanocytes were divided into 4 groups to be cultured with media containing no latanoprost (control group) or 10-5,10-6 and 10-7 mol/L latanoprost for 48 hours.Cell counting kit-8 (CCK8) assay was performed to evaluate the proliferative activity of melanocytes,dopa oxidation assay to estimate the activity of tyrosinase.Sodium hydroxide (NaOH)-lysis method was used to determine the content of melanin,and Masson-Fontana staining to observe the number and distribution of melanin granules.Westernblot analysis and real-time fluorescence-based quantitative PCR were performed to determine the protein and mRNA expression of melanogenesis-related genes including microphthalmia-associated transcription factor (MITF),tyrosinase (TYR) and tyrosinase-related protein 1 (TYRP1).Comparison among the 4 groups and multiple comparisons were done by using one-way analysis of variance and least significant difference (LSD)-t test.Results Compared with the control group,the 10-6-,10-5-mol/L latanoprost groups showed significantly increased proliferative activity of melanocytes (1.064 ± 0.172 and 1.078 ± 0.080 vs.0.784 ± 0.015;t =3.289,3.454 respectively,both P ＜ 0.05),increased activity of tyrosinase (0.510 ± 0.017 and 0.454 ± 0.009 vs.0.355 ± 0.041;t =6.139,3.939 respectively,P ＜ 0.01 or 0.05),and increased content of melanin (t =7.232,5.967,both P ＜ 0.01).However,there were no significant differences in the proliferative activity of melanocytes,activity of tyrosinase or content of melanin between the 10-7-mol/L latanoprost group and control group (all P ＞ 0.05).Masson-Fontana staining showed more and darker melanin granules on melanocyte dendrites in the 10-5-,10-6-,10-7-mol/L latanoprost groups than in the control group,and the color of melanin granules changed from light brown to black brown along with the increase in the concentration of latanoprost.The mRNA expression of MITF increased along with the increase in the concentration of latanoprost (P ＜ 0.01),and the protein expression of MITF wassignificantly higher in the 10-6,10-5-mol/L latanoprost groups than in the control group and 10-7-mol/L latanoprost group (all P ＜ 0.01).The 10-6-mol/L latanoprost group showed significantly increased mRNA and protein expression of TYR and TYRP1 compared with the control group,10-7-,10-5-mol/L latanoprost groups (all P ＜ 0.01).Conclusion Latanoprost can increase the proliferation of human epidermal melanocytes,and promote tyrosinase activity and melanogenesis likely by enhancing the mRNA and protein expression of MITF,TYR,TYRP1.

The Anti-melanogenesis Activities of Some Selected Brown Macroalgae from Northern Coasts of the Persian Gulf

Abstract Melanogenesis is a biological process which led to the synthesis of melanin pigment. Abnormal melanin production results in melasma, solar lentigo, post inflammatory melanoderma, etc. In this study, we examined the potential inhibitory effects of 17 brown macroalgae from Persian Gulf on melanogenesis. The effects of various concentrations (100, 250 and 500 µg/mL) of methanolic extracts of macroalgae belonging to four genera (including: Padina, Colpomonia, Cystoseira and Sargassum) were studied on oxidation of L-Dopa by mushroom tyrosinase. Subsequently, the activity of macroalgae with high inhibitory effect on monophenolase activity of mushroom tyrosinase and zebrafish was investigated using L-tyrosine as a substrate. Anti-melanogenesis effects of algae extracts were studied on zebrafish as an alternative in vivo model. Kojic acid was used as a positive control. All the tested macroalgae showed inhibitory effect on activities of diphenolase and monophenolase (of mushroom tyrosinase). P. boergesinii exhibited the most in vivo anti-tyrosinase activity compared with other samples. P. boergesenii inhibited zebrafish tyrosinase more potent than kojic acid (83% vs 50% inhibition for kojic acid). Moreover, it reduced melanin synthesis in zebrafish 42% (kojic acid: 50%).

Chemical constituents from the leaves of Anisopus mannii and the melanogenesis inhibitory activities / 中国药科大学学报

@#The phytochemical studies on the leaves of Anisopus mannii led to the isolation of seven compounds by silica gel, ODS, DIAION HP-20, Sephadex LH-20 colunmn chromatographer, their structures were elucidated on the basis of the spectroscopic analyses(NMR, HRMS)and the comparisons with the literatures as 3β-acetoxylup-20(29)-ene(1), 1-acetoxy-2-isopropyl-1-tridecene(2), rutin(3), 3, 6′-diferuloylsucrose(4), 3-O-β-D-glucopyranosyl-3-β-hydroxyolean-12-en-28-oic acid 28-O-［α-L-rhamnopyra- nosyl-(1→2)-β-D-glucopyranosyl］ ester(5), conduritol A(6), hoyacarnoside I(7). Meanwhile, the isolated compounds were evaluated for their inhibitory activities against melanogensis in B16 melanoma cells, as the results, all compounds exhibited melanogenesis-inhibitory activity and compound 5 showed a strongest activity(Melanin content: (27. 4±3. 5)%, Cell Viability: (54. 9±5. 6)% with a concentration of 30 μmol/L)which could be further developed.

Effects of six compounds with different chemical structures on melanogenesis / 中国天然药物

Several chemical compounds can restore pigmentation in vitiligo through mechanisms that vary according to disease etiology. In the present study, we investigated the melanogenic activity of six structurally distinct compounds, namely, scopoletin, kaempferol, chrysin, vitamin D, piperine, and 6-benzylaminopurine. We determined their effectiveness, toxicity, and mechanism of action for stimulating pigmentation in B16F10 melanoma cells and in a zebrafish model. The melanogenic activity of 6-benzylaminopurine, the compound identified as the most potent, was further verified by measuring green fluorescent protein concentration in tyrp1 a: eGFP (tyrosinase-related protein 1) zebrafish and mitfa: eGFP (microphthalmia associated transcription factor) zebrafish and antioxidative activity. All the tested compounds were found to enhance melanogenesis responses both in vivo and in vitro at their respective optimal concentration by increasing melanin content and expression of TYR and MITF. 6-Benzyamino-purine showed the strongest re-pigmentation action at a concentration of 20 μmol·Lin vivo and 100 μmol·Lin vitro, and up-regulated the strong fluorescence expression of green fluorescent protein in tyrp1a: eGFP and mitfa: eGFP zebrafish in vitro. However, its relative anti-oxidative activity was found to be very low. Overall, our results indicated that 6-benzylaminopurine stimulated pigmentation through a direct mechanism, by increasing melanin content via positive regulation of tyrosinase activity in vitro, as well as up-regulating the expression of the green fluorescent protein in transgenic zebrafish in vivo.

Ethyl linoleate inhibits α-MSH-induced melanogenesis through Akt/GSK3β/β-catenin signal pathway

Ethyl linoleate is an unsaturated fatty acid used in many cosmetics for its various attributes, such as antibacterial and anti-inflammatory properties and clinically proven to be an effective anti-acne agent. In this study, we investigated the effect of ethyl linoleate on the melanogenesis and the mechanism underlying its action on melanogenesis in B16F10 murine melanoma cells. Our results revealed that ethyl linoleate significantly inhibited melanin content and intracellular tyrosinase activity in α-MSH-induced B16F10 cells, but it did not directly inhibit activity of mushroom tyrosinase. Ethyl linoleate inhibited the expression of microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase related protein 1 (TRP1) in governing melanin pigment synthesis. We observed that ethyl linoleate inhibited phosphorylation of Akt and glycogen synthase kinase 3β (GSK3β) and reduced the level of β-catenin, suggesting that ethyl linoleate inhibits melanogenesis through Akt/GSK3β/β-catenin signal pathway. Therefore, we propose that ethyl linoleate may be useful as a safe whitening agent in cosmetic and a potential therapeutic agent for reducing skin hyperpigmentation in clinics.

Effects of six compounds with different chemical structures on melanogenesis / 中国天然药物

Several chemical compounds can restore pigmentation in vitiligo through mechanisms that vary according to disease etiology. In the present study, we investigated the melanogenic activity of six structurally distinct compounds, namely, scopoletin, kaempferol, chrysin, vitamin D, piperine, and 6-benzylaminopurine. We determined their effectiveness, toxicity, and mechanism of action for stimulating pigmentation in B16F10 melanoma cells and in a zebrafish model. The melanogenic activity of 6-benzylaminopurine, the compound identified as the most potent, was further verified by measuring green fluorescent protein concentration in tyrp1 a: eGFP (tyrosinase-related protein 1) zebrafish and mitfa: eGFP (microphthalmia associated transcription factor) zebrafish and antioxidative activity. All the tested compounds were found to enhance melanogenesis responses both in vivo and in vitro at their respective optimal concentration by increasing melanin content and expression of TYR and MITF. 6-Benzyamino-purine showed the strongest re-pigmentation action at a concentration of 20 μmol·Lin vivo and 100 μmol·Lin vitro, and up-regulated the strong fluorescence expression of green fluorescent protein in tyrp1a: eGFP and mitfa: eGFP zebrafish in vitro. However, its relative anti-oxidative activity was found to be very low. Overall, our results indicated that 6-benzylaminopurine stimulated pigmentation through a direct mechanism, by increasing melanin content via positive regulation of tyrosinase activity in vitro, as well as up-regulating the expression of the green fluorescent protein in transgenic zebrafish in vivo.

Effect of urea-extracted sericin on melanogenesis: potential applications in post-inflammatory hyperpigmentation

BACKGROUND: Hyperpigmentation disorders such as post-inflammatory hyperpigmentation are major concerns not only in light-skinned people but also in Asian populations with darker skin. The anti-tyrosinase and immunomodulatory effects of sericin have been known for decades. However, the therapeutic effects of sericin on hyperpigmentation disorders have not been well documented. METHODS: In this study, we used an in vitro model to study the anti-tyrosinase, tolerogenic, and anti-melanogenic effects of sericin on Staphylococcus aureus peptidoglycan (PEG)-stimulated melanocytes, dendritic cells (DCs), and artificial skin (MelanoDerm™). Enzyme-linked immunosorbent assay, conventional and immunolabeled electron microscopy, and histopathological studies were performed. RESULTS: The results revealed that urea-extracted sericin has strong anti-tyrosinase properties as shown by a reduction of tyrosinase activity in melanin pigments both 48 h and 10 days after allergic induction with PEG. Anti-inflammatory cytokines including interleukin (IL)-4, IL-10, and transforming growth factor-p were upregulated upon sericin treatment (10, 20, and 50 µg/mL), whereas production of allergic chemokines, CCL8 and CCL18, by DCs was diminished 48 h after allergic induction with PEG. Moreover, sericin lowered the expression of micropthalmia-associated transcription factor (MITF), a marker of melanogenesis regulation, in melanocytes and keratinocytes, which contributed to the reduction of melanin size and the magnitude of melanin deposition. However, sericin had no effect on melanin transport between melanocytes and keratinocytes, as demonstrated by a high retention of cytoskeletal components. CONCLUSION: In summary, sericin suppresses melanogenesis by inhibition of tyrosinase activity, reduction of inflammation and allergy, and modulation of MITF function.

Pimecrolimus increases the melanogenesis and migration of melanocytes in vitro

Vitiligo is an intriguing depigmentary disorder and is notoriously difficult to be treated. The ultimate goal of vitiligo treatment is to replenish the lost melanocytes by immigration from hair follicle and to restore the normal function of melanogenesis by residual melanocytes. There are two types of topical calcineurin inhibitors called tacrolimus and pimecrolimus, and are recommended as the first-line treatments in vitiligo. Although pimecrolimus is efficacious for the repigmentation of vitiligo, its intrinsic mechanisms have never been investigated in vitro. This research aimed to study the ability of pimecrolimus on stimulating melanogenesis, melanocyte migration and MITF (microphthalmia associated transcription factor) protein expression. Results showed that pimecrolimus at the dosages of 1, 10, 10² nM were neither mitogenic nor cytotoxic to melanocytes. The addition of pimecrolimus at 10, 10² and 10³ nM significantly increased intracellular tyrosinase activity, which was consistent with the elevated content of melanin content at the same concentrations. The peak effect was seen at 72 h in response to 10² nM pimecrolimus. Results of the wound scratch assay and Transwell assays indicate that pimecrolimus is effective in facilitating melanocyte migration on a collagen IV-coated surface. In addition, MITF protein yield reached the highest by pimecrolimus at 10² nM. In brief, pimecrolimus enhances melanin synthesis as well as promotes migration of melanocytes directly, possibly via their effects on MITF protein expression.

Inhibitory effect of Gastrodia elata Blume extract on alpha-melanocyte stimulating hormone-induced melanogenesis in murine B16F10 melanoma

BACKGROUND/OBJECTIVES: Gastrodia elata Blume (GEB), a traditional herbal medicine, has been used to treat a wide range of neurological disorders (e.g., paralysis and stroke) and skin problems (e.g., atopic dermatitis and eczema) in oriental medicine. This study was designed to investigate whether GEB extract inhibits melanogenesis activity in murine B16F10 melanoma. MATERIALS/METHOD: Murine B16F10 cells were treated with 0-5 mg/mL of GEB extract or 400 µg/mL arbutin (a positive control) for 72 h after treatment with/without 200 nM alpha-melanocyte stimulating hormone (α-MSH) for 24 h. Melanin concentration, tyrosinase activity, mRNA levels, and protein expression of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein (Trp)1, and Trp2 were analyzed in α-MSH-untreated and α-MSH-treated B16F10 cells. RESULTS: Treatment with 200 nM α-MSH induced almost 2-fold melanin synthesis and tyrosinase activity along with increased mRNA levels and protein expression of MITF, tyrosinase, Trp1 and Trp2. Irrespective of α-MSH stimulation, GEB extract at doses of 0.5-5 mg/mL inhibited all these markers for skin whitening in a dose-dependent manner. While lower doses (0.5-1 mg/mL) of GEB extract generally had a tendency to decrease melanogenesis, tyrosinase activity, and mRNA levels and protein expression of MITF, tyrosinase, Trp1, and Trp2, higher doses (2-5 mg/mL) significantly inhibited all these markers in α-MSH-treated B16F10 cells in a dose-dependent manner. These inhibitory effects of the GEB extract at higher concentrations were similar to those of 400 µg/mL arbutin, a well-known depigmenting agent. CONCLUSIONS: These results suggest that GEB displays dose-dependent inhibition of melanin synthesis through the suppression of tyrosinase activity as well as molecular levels of MITF, tyrosinase, Trp1, and Trp2 in murine B16F10 melanoma. Therefore, GEB may be an effective and natural skin-whitening agent for application in the cosmetic industry.