Characterization of Phenethyl Cinnamamide Compounds from Hemp Seed and Determination of Their Melanogenesis Inhibitory Activity.
ABSTRACT: Hyperpigmentation is induced by the overactivation of tyrosinase, which is a rate-limiting enzyme in melanogenesis. The defatted extract of hemp (Cannabis sativa L.) seed is known to have inhibitory effects on melanogenesis; however, effective compounds in the extract have not been identified yet. In this study, three phenethyl cinnamamides present in hemp seed extract were prepared by purification and chemical synthesis and were assessed for their inhibitory effect on melanogenesis in B16F10 melanoma cells. A comparison of the anti-melanogenesis and anti-tyrosinase activity of hemp seed solvent fractions revealed that the ethyl acetate fraction possessed the greatest potential for suppressing melanogenesis in melanoma cells by decreasing tyrosinase activity. We tentatively identified 26 compounds in the ethyl acetate fraction by comparing spectroscopic data with the literature. Three phenethyl cinnamamides such as N-trans-caffeoyltyramine, N-trans-coumaroyltyramine, and N-trans-feruloyltyramine present abundantly in the ethyl acetate fraction were prepared and their anti-melanogenesis and anti-tyrosinase activities in melanoma cells were evaluated. We found that N-trans-caffeoyltyramine and N-trans-feruloyltyramine inhibited alpha melanocyte stimulating hormone (α-MSH)-induced melanogenesis without cytotoxicity, while N-trans-coumaroyltyramine inhibited melanogenesis with cytotoxicity. IC50 values of N-trans-caffeoyltyramine, N-trans-feruloyltyramine, and N-trans-coumaroyltyramine for inhibition of α-MSH-mediated tyrosinase activation were 0.8, 20.2, and 6.3 μM, respectively. Overall, N-trans-caffeoyltyramine possessed the strongest anti-melanogenesis activity among the three phenethyl cinnamamides evaluated. The inhibitory effect of N-trans-caffeoyltyramine was verified by determining the melanin content and tyrosinase activity in melanoma after treating the cells with synthetic compounds. Thus, N-trans-caffeoyltyramine isolated from hemp seed extract could be useful in cosmetics as a skin-whitening agent.
Project description:In therapeutic interventions associated with melanin hyperpigmentation, tyrosinase is regarded as a target enzyme as it catalyzes the rate-limiting steps in mammalian melanogenesis. Since many known agents have been proven to be toxic, there has been increasing impetus to identify alternative tyrosinase inhibitors, especially from natural sources. In this study, we investigated 900 extracts from Greek plants for potential tyrosinase inhibitive properties. Among the five most potent extracts, the methanol extract of Morus alba wood (MAM) demonstrated a significant reduction in intracellular tyrosinase and melanin content in B16F10 melanoma cells. Bioassay-guided isolation led to the acquisition of twelve compounds: oxyresveratrol (1), kuwanon C (2), mulberroside A (3), resorcinol (4), dihydrooxyresveratol (5), trans-dihydromorin (6), 2,4,3'-trihydroxydihydrostilbene (7), kuwanon H (8), 2,4-dihydroxybenzaldehyde (9), morusin (10), moracin M (11) and kuwanon G (12). Among these, 2,4,3'-trihydroxydihydrostilbene (7) is isolated for the first time from Morus alba and constitutes a novel potent tyrosinase inhibitor (IC50 0.8 ± 0.15). We report here for the first time dihydrooxyresveratrol (5) as a potent natural tyrosinase inhibitor (IC50 0.3 ± 0.05). Computational docking analysis indicated the binding modes of six tyrosinase inhibitors with the aminoacids of the active centre of tyrosinase. Finally, we found both MAM extract and compounds 1, 6 and 7 to significantly suppress in vivo melanogenesis during zebrafish embryogenesis.
Project description:Five phenolic compounds, namely N-trans-coumaroyltyramine (1), N-trans-feruloyltyramine (2), N-trans-feruloyloctopamine (3), 5,7-dihydroxy-8-methoxyflavone (4) and (3S)3,5,4'-trihydroxy-7-methoxy-6-methylhomoisoflavanone (5), were isolated from the fibrous roots of Liriope muscari (Liliaceae). Compounds 2-5 were isolated for the first time from the Liriope genus. Their in vitro antioxidant activities were assessed by the DPPH and ABTS scavenging methods with microplate assays. The structure-activity relationships of compounds 1-3 are discussed.
Project description:Hyperpigmentation resulting from the overactivation of tyrosinase leads to darker spots or patches on the human skin. Although these phenomena are harmless, there is still great demand for melanogenesis inhibitors to prevent hyperpigmentation by inhibiting the tyrosinase, a rate-limiting enzyme in melanogenesis. Although <i>Lepisorus thunbergianus</i> has been used in folk remedies as a diuretic and hemostatic agent, its effect on melanogenesis has not yet been reported. In this study, we prepared an <i>L. thunbergianus</i> extract and its solvent fractions and evaluated their biological activity against free radical and melanin synthesis. The extract of <i>L. thunbergianus</i> inhibited mushroom tyrosinase activity more efficiently than, and with similar antioxidant activity to, arbutin <i>in vitro</i>. Comparative evaluation of the anti-melanogenesis and anti-tyrosinase activity of <i>L. thunbergianus</i> solvent fractions demonstrated that, by inhibiting tyrosinase activity, the butanol fraction has the highest potential for the inhibition of melanogenesis in melanoma cells. We found by structural analysis using high-performance liquid chromatography (HPLC) and NMR spectroscopy that the major compounds in butanol fraction were three caffeoylquinic acid derivatives. The three derivatives had similar radical scavenging and anti-tyrosinase activities <i>in vitro</i>, while only 5-caffeoylquinic acid had an inhibitory effect on ?-MSH-induced melanogenesis. The inhibitory effect of 5-caffeoylquinic acid was verified by the determination of the melanin content and tyrosinase activity in melanoma after treating the cells with a commercial compound. Further, we revealed that 5-caffeoylquinic acid inhibited melanogenesis by chelating a copper cation from a copper-tyrosinase complex. Thus, 5-caffeoylquinic acid or butanol fraction isolated from <i>L. thunbergianus</i> might be useful in cosmetics as a skin-whitening agent.
Project description:The individual parts of Morus alba L. including root bark, branches, leaves, and fruits are used as a cosmetic ingredient in many Asian countries. This study identified several anti-melanogenesis constituents in a 70% ethanol extract of M. alba leaves. The ethyl acetate fraction of the initial ethanol extract decreased the activity of tyrosinase, a key enzyme in the synthetic pathway of melanin. Twelve compounds were isolated from this fraction and their structures were identified based on spectroscopic spectra. Then, the authors investigated the anti-melanogenesis effects of the isolated compounds in B16-F10 mouse melanoma cells. Compounds 3 and 8 significantly inhibited not only melanin production but also intracellular tyrosinase activity in alpha-melanocyte-stimulating-hormone (?-MSH)-induced B16-F10 cells in a dose-dependent manner. These same compounds also inhibited melanogenesis-related protein expression such as microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein-1 (TRP-1). Compound 3 modulated the cAMP-responsive element-binding protein (CREB) and p38 signaling pathways in ?-MSH-activated B16-F10 melanoma cells, which resulted in the anti-melanogenesis effects. These results suggest that compound 3, isolated from M. alba leaves, could be used to inhibit melanin production via the regulation of melanogenesis-related protein expression.
Project description:The methanol extract of Cassia auriculata seeds was found to inhibit melanogenesis in B16 melanoma 4A5 cells under conditions of theophylline stimulation. Two new phlegmacin-type anthracenone dimer glycosides, auriculataosides A and B, were isolated from the active methanol fraction, and their inhibitory effects were observed in the concentration range of 0.03 to 0.3 ?M. Inhibition of microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 protein expression was observed, suggesting that the inhibition of these factors is part of the mechanism of action underlying melanogenesis inhibition.
Project description:BACKGROUND AND PURPOSE:Kazinol U is a prenylated flavan isolated from an extract of Broussonetia kazinoki Sieb (Moraceae). Kazinol U has shown cytoprotective effects against cytokine-induced apoptotic cell death and induces AMP kinase (AMPK) activation through LKB1 activation. However, kazinol U has not been tested as a regulator of melanogenesis, although bark extract of B. kazinoki has been used as a cosmetic ingredient for skin conditioning. EXPERIMENTAL APPROACH:We cultured mouse, human melanoma cells and normal human melanocytes to demonstrate anti-melanogenic effects of kazinol U. A tyrosinase activity assay, Western blot, RT-qPCR and a luciferase reporter gene assay were performed to determine the anti-melanogenic mechanisms of kazinol U. We confirmed its effect on melanogenesis in vivo using zebrafish. KEY RESULTS:Kazinol U inhibited the expression and activity of tyrosinase, the rate-limiting enzyme in melanogenesis, and reduced tyrosinase expression and activity in response to cAMP-inducing agents. Kazinol U reduced the expression of other melanogenic enzymes, such as tyrosinase-related protein (Tyrp) 1 and Tyrp2, and down-regulated microphthalmia-associated transcription factor (MITF), the master regulator of the tyrosinase gene family. Moreover, kazinol U induced phosphorylation of AMPK and MAPK proteins, which are MITF inhibitors. It also exhibited anti-melanogenic effects in zebrafish, a recently developed in vivo model. CONCLUSIONS AND IMPLICATIONS:Our findings suggest that kazinol U reduces melanogenesis via its inhibitory effect on MITF and its downstream target genes, tyrosinase, Tyrp1 and Tyrp2. This work may provide a basis for the application of kazinol U for the treatment of hyperpigmentation skin disorders.
Project description:Melanin is a natural pigment produced by cells to prevent damage caused by ultraviolet radiation. Previously, resveratrol was shown to reduce melanin synthesis. As a natural polyphenol with various biological activities, resveratrol occurs in a variety of beverages and plant foods, such as grapes. Therefore, we investigated whether grape extracts containing resveratrol also had the ability to regulate melanin synthesis. In this study, we used mouse B16F10 melanoma cells as a model for melanin synthesis with the melanogenesis-inducing α-melanocyte-stimulating hormone (α-MSH) as a positive control. Our results confirmed previous reports that resveratrol reduces melanin synthesis by reducing the activity of the rate-limiting enzyme tyrosinase. In contrast, the grape extract could not reduce melanin synthesis, and in fact promoted melanogenesis in the presence of α-MSH. The expression of genes related to melanin synthesis, such as tyrosinase, tyrosinase-related protein-1, tyrosinase-related protein-2, and microphthalmia-associated transcription factor, also supports these phenomena, which means that even in the presence of resveratrol, grape extract will strengthen the function of α-MSH in promoting melanin synthesis. Therefore, these results also provide a point of view for research on cosmetics.
Project description:Despite the critical role of melanin in the protection of skin against UV radiation, excess production of melanin can lead to hyperpigmentation and skin cancer. Pear fruits are often used in traditional medicine for the treatment of melasma; therefore, we investigated the effects of pear extract (PE) and its component, protocatechuic acid (PCA), on melanogenesis in mouse melanoma cells. We found that PE and PCA significantly suppressed melanin content and cellular tyrosinase activity through a decrease in the expression of melanogenic enzymes and microphthalmia-associated transcription factor (<i>Mitf</i>) in ?-melanocyte stimulating hormone-stimulated mouse melanoma cells. Moreover, PCA decreased cyclic adenosine monophosphate (cAMP) levels and cAMP-responsive element-binding protein phosphorylation, which downregulated <i>Mitf</i> promoter activation and subsequently mediated the inhibition of melanogenesis. These results suggested that pear may be an effective skin lightening agent that targets either a tyrosinase activity or a melanogenic pathway.
Project description:To evaluate possibility as a skin whitening agent of Sorghum bicolor (S. bicolor), its antioxidant activity and anti-melanogenic effect on 3-isobutyl-1-methylxanthine (IBMX)-induced melanogenesis in B16/F10 melanoma cells were investigated. The result of total phenolic contents (TPC) indicated that 60% ethanol extract of S. bicolor (ESB) has the highest contents than other ethanol extracts. Antioxidant activity was evaluated using the 2,2'-azino-bis-(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt (ABTS)/1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activities and malondialdehyde (MDA) inhibitory effect. These results showed ESB has significant antioxidant activities. Inhibitory effect against tyrosinase was also assessed using L-tyrosine (IC50 value = 89.25 ?g/mL) and 3,4-dihydroxy-L-phenylalanine (L-DOPA) as substrates. In addition, ESB treatment effectively inhibited melanin production in IBMX-induced B16/F10 melanoma cells. To confirm the mechanism on anti-melanogenic effect of ESB, we examined melanogenesis-related proteins. ESB downregulated melanogenesis by decreasing expression of microphthalmia-associated transcription factor (MITF), tyrosinase and tyrosinase-related protein (TRP)-1. Finally, 9-hydroxyoctadecadienoic acid (9-HODE), 1,3-O-dicaffeoylglycerol and tricin as the main compounds of ESB were analyzed using the ultra-performance liquid chromatography-ion mobility separation-quadrupole time of flight/tandem mass spectrometry (UPLC-IMS-QTOF/MS2). These findings suggest that ESB may have physiological potential to be used skin whitening material.
Project description:Two compounds termed 1 and 2 were isolated from the leaves of Capsicum chinense using column chromatography. Their structures were identified as amide scaffolds by analyzing spectroscopic signals. Compounds 1 and 2 have been confirmed to be competitive soluble epoxide hydrolase (sEH) inhibitors that suppress the catalytic reaction of sEH in a dose-dependent manner in vitro. Molecular docking was used for analyzing two binding clusters of ligand and receptor. The results confirmed that the key amino acids interacting with the ligand were Asp335, Tyr383, and Gln384. On the basis of molecular dynamics, inhibitors 1 and 2 were noted to interact at a distance of 3.5?Å from Asp335, Tyr383, Leu408 and Tyr466, and Asp335, Tyr383, and Tyr466, respectively. These results highlight the potential of N-trans-coumaroyltyramine (1) and N-trans-feruloyltyramine (2) as sEH inhibitors.