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1.
Mol Genet Genomics ; 295(4): 911-922, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32367255

RESUMO

Tyrosinase (TYR) converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and L-DOPA into L-dopaquinone, which can produce melanin pigment. The abrogation of the functional activity of TYR can result in albino skin and eye diseases because of a deficiency in melanin pigment production. In this study, we developed and characterized an inducible knockout TYR platform comprising the heat-inducible heat-shock-promoter-70-driving CRISPR/Cas9 system and a zU6-promoter-driving tyr single guide RNA (sgRNA) system to investigate the temporal expression of TYR genes. To overcome the difficulty of identifying zebrafish germline integrations and facilitate the observation of Cas9 expression, heart-specific cmlc2:enhanced green fluorescent protein (EGFP; used to confirm tyr sgRNA expression) and two selectable markers (P2A-mCherry and internal ribosomal entry site-EGFP) were applied in our system. Heat shock treatment administered to Cas9 transgenic embryos induced mCherry or EGFP fluorescence expression throughout the embryos' bodies, and Cas9 protein was detected 1 h after heat shock treatment. Mutations were created by direct injection and line crossing, which led to mosaic and complete depigmentation phenotypes in approximately 50% and 100% of the embryos, respectively. Using our system, conditional TYR knockout in zebrafish was achieved efficiently and simply.


Assuntos
Sistemas CRISPR-Cas/genética , Resposta ao Choque Térmico/genética , Monofenol Mono-Oxigenase/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Proteína 9 Associada à CRISPR/genética , Embrião não Mamífero , Desenvolvimento Embrionário , Regulação Enzimológica da Expressão Gênica , Técnicas de Inativação de Genes , Mutação em Linhagem Germinativa , Proteínas de Fluorescência Verde/genética , Melaninas/biossíntese , Melaninas/genética , Cadeias Leves de Miosina/genética , Regiões Promotoras Genéticas/genética , RNA/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética
2.
Toxicon ; 183: 29-35, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32445842

RESUMO

The ant-like bethylid ectoparasitoid Scleroderma guani (Hymenoptera: Bethylidae) envenomates host to suppress immune response. Yet, the roles of its venom in inhibiting melanization of the host hemolymph have not been fully characterized. Here, we demonstrated that S. guani envenomation induced strong inhibition of melanization of the hemolymph from Tenebrio molitor (Coleoptera: Tenebrionidae), permitting the successful development of parasitoid offspring. To reveal venom component associated with such function, a serine proteinase homolog (SguaSPH) rich in the venom of S. guani was characterized. It was found that one of the catalytic triad residues for serine proteinase is absent in the amino acid sequence of SguaSPH. This venom component was abundantly expressed in venom apparatus and adult stages. By enzymatic assays, SguaSPH displayed low trypsin and no chymotrypsin activity, and was able to inhibit phenoloxidase activity in the hemolymph of Ostrinia furnacalis (Lepidoptera: Crambidae). The findings suggest that SguaSPH is essential for interfering with hemolymph melanization of S. guani envenomated host via phenoloxidase cascade disruption.


Assuntos
Monofenol Mono-Oxigenase/metabolismo , Serina Proteases/metabolismo , Animais , Hemolinfa/metabolismo , Proteínas de Insetos/metabolismo , Larva , Tenebrio/metabolismo
3.
Sheng Li Xue Bao ; 72(2): 139-147, 2020 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-32328607

RESUMO

Increasing evidence suggests that stress may induce changes in hair color, with the underlying mechanism incompletely understood. In this study, female C57BL/6 mice subjected to electric foot shock combined with restraint stress were used to build chronic stress mouse model. The melanin contents and tyrosinase activity were measured in mouse skin and B16F10 melanoma cells. The enzyme-linked immunosorbent assay (ELISA) was used to determine the content of tumor necrosis factor α (TNF-α), interleukin- 1ß (IL-1ß) and interleukin-6 (IL-6) in the mouse skin. The content of nuclear factor κB (NFκB)/p65 subunit in mouse skins was valued by immunofluorescence staining. The results demonstrated that under chronic stress, the fur color turned from dark to brown in C57BL/6 mice due to the decrease of follicle melanocytes and tyrosinase activity in C57BL/6 mouse skin. Simultaneously, inflammatory responses in skins were detected as shown by increased NFκB activity and TNF-α expression in stressed mouse skin. In cultured B16F10 melanoma cells, TNF-α reduced the melanogenesis and tyrosinase activity in a dose-dependent manner. These findings indicate that chronic stress induces fur color change by decreasing follicle melanocytes and tyrosinase activity in female C57BL/6 mice, and TNF-α may play an important role in stress-induced hair color change.


Assuntos
Pelo Animal , Melanócitos/enzimologia , Monofenol Mono-Oxigenase/metabolismo , Pele/fisiopatologia , Estresse Fisiológico , Animais , Cor , Feminino , Melaninas , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Pigmentação
4.
J Cosmet Sci ; 71(1): 11-22, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32271705

RESUMO

Darkening of fruits is the result of the oxidative activation of polyphenol oxidase converting low-molecular weight phenols present in the fruit body into quinone intermediates. Then, through polymerization, these reactive quinones convert to light yellow and red low-molecular weight melanin and, given enough time, to darker, higher molecular weight brown and black melanin. The process that occurs in the flesh of cut fruit is very similar to the process that human skin cells use to make melanin: the oxidative activation of tyrosinase and conversion of tyrosine to dopaquinone and eventually to darker melanin. The conversion of the phenols by tyrosinase to quinones is the rate-limiting step in the biochemical manufacture of melanin. This article will discuss a new and cost effective way to screen skin-brightening ingredients by the use of apple slices as a model for skin using a chromameter to measure the change in color that occurs in apple slices over a short time course. Such measurements have been popularly used by food manufacturers to examine ingredients that inhibit fruit browning. Interestingly, as will be noted, many of the ingredients used commercially to inhibit food browning are also popular skin-brightening ingredients. We have found that a DermaLab (Cortex Technologies, Hadsund, Denmark) chromameter measuring the erythema index of apple slice darkening appears to be able to differentiate the benefit of a formulation containing azelaic acid, a known skin-lightening ingredient, to minimize the darkening effects that occur in sliced apples. We will discuss how different apples behave differently when cut and how to best use the chromameter to analyze the changes that occur that can potentially help rapidly screen ingredients for their skin-brightening benefits.


Assuntos
Malus , Frutas , Humanos , Reação de Maillard , Monofenol Mono-Oxigenase , Fenóis
5.
J Cosmet Sci ; 71(2): 65-76, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32271709

RESUMO

Five-aminolevulinic acid (5-ALA)-photodynamic therapy combined with infrared radiation is an effective and safe therapy for facial acne. Although there are various available agents for treating acne, therapies for resistant or severe strains have been limited. The aim of this study was to investigate the inhibitory efficacy of ALACELL synthesized by combining 5-ALA with Y-G-G-F-L peptide against Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Yersinia enterocolitica, as well as Cutibacterium acnes. Furthermore, other effects of ALACELL on human skin cells, melanin formation, intracellular tyrosinase activity, and Ultra Violet B (UVB)-irradiated cell death were measured by treatment of ALACELL in vitro. ALACELL particularly showed a growth inhibitory effect on C. acnes and no inhibitory effect on the four bacteria strains. ALACELL reduced melanin formation and intracellular tyrosinase activity by α-melanin cell-stimulating hormone (α-MSH) in B16F10 cells, with no cytotoxicity. ALACELL also improved cell viability in UVB-irradiated HaCaT cells. The results of the experiment show that ALACELL exhibits more efficacy than 5-ALA against antimicrobial activity, melanin formation, intracellular tyrosinase activity, and UVB-irradiated cell death. Therefore, ALACELL is recommended as a candidate for clinical application in the treatment of acne and skin aging and will be further investigated to study the mode of action and in actual situations.


Assuntos
Raios Ultravioleta , Ácido Aminolevulínico , Humanos , Melaninas , Monofenol Mono-Oxigenase
6.
Life Sci ; 250: 117602, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32240677

RESUMO

AIMS: Extrinsic ageing or photoageing relates to the onset of age-linked phenotypes such as skin hyperpigmentation due to UV exposure. UV induced upregulated production of tyrosinase enzyme, which catalyses the vital biochemical reactions of melanin synthesis is responsible for the inception of skin hyperpigmentation. We aimed to generate a validated QSAR model with a dataset consisting of 69 thio-semicarbazone derivatives to elucidate the physicochemical properties of compounds essential for tyrosinase inhibition and to identify novel lead molecules with enhanced tyrosinase inhibitory activity and bioavailability. MAIN METHODS: Lead optimization and insilico approaches were employed in this research work. QSAR model was generated and validated by exploiting Multiple Linear Regression method. Prioritization of lead-like compounds was accomplished by performing multi parameter optimization depleting molecular docking, bioavailability assessments and toxicity prediction for 69 compounds Derivatives of best lead compound were retrieved from chemical spaces. KEY FINDINGS: Molecular descriptors explicated the significance of chemical properties essential for chelation of copper ions present in the active site of tyrosinase protein target. Further, derivatives which comprise of electron donating groups in their chemical structure were predicted and analysed for tyrosinase inhibitory activity by employing insilico methodologies including chemical space exploration. SIGNIFICANCE: Our research work resulted in the generation of a validated QSAR model with higher degree of external predictive ability and significance to tyrosinase inhibitory activity. We propose 11 novel derivative compounds with enhanced tyrosinase inhibitory activity and bioavailability.


Assuntos
Química Farmacêutica/métodos , Biologia Computacional/métodos , Indóis/antagonistas & inibidores , Monofenol Mono-Oxigenase/antagonistas & inibidores , Pele/efeitos dos fármacos , Agaricales/metabolismo , Domínio Catalítico , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Elétrons , Inibidores Enzimáticos/farmacologia , Humanos , Ligação de Hidrogênio , Ligantes , Simulação de Acoplamento Molecular , Estrutura Molecular , Monofenol Mono-Oxigenase/metabolismo , Relação Quantitativa Estrutura-Atividade , Pigmentação da Pele/efeitos dos fármacos , Tiossemicarbazonas/química , Raios Ultravioleta
7.
Aquat Toxicol ; 222: 105464, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32160575

RESUMO

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing allows for the disruption or modification of genes in a multitude of model organisms. In the present study, we describe and employ the method for use in the fathead minnow (Pimephales promelas), in part, to assist in the development and validation of adverse outcome pathways (AOPs). The gene coding for an enzyme responsible for melanin production, tyrosinase (tyr), was the initial target chosen for development and assessment of the method since its disruption results in abnormal pigmentation, a phenotype obvious within 3-4 d after injection of fathead minnow embryos. Three tyrosinase-targeting guide strands were generated using the fathead minnow sequence in tandem with the CRISPOR guide strand selection tool. The strands targeted two areas: one stretch of sequence in a conserved region that demonstrated homology to EGF-like or laminin-like domains as determined by Protein Basic Local Alignment Search Tool in concert with the Conserved Domain Database, and a second area in the N-terminal region of the tyrosinase domain. To generate one cell embryos, in vitro fertilization was performed, allowing for microinjection of hundreds of developmentally-synchronized embryos with Cas9 proteins complexed to each of the three guide strands. Altered retinal pigmentation was observed in a portion of the tyr guide strand injected population within 3 d post fertilization (dpf). By 14 dpf, fish without skin and swim bladder pigmentation were observed. Among the three guide strands injected, the guide targeting the EGF/laminin-like domain was most effective in generating mutants. CRISPR greatly advances our ability to directly investigate gene function in fathead minnow, allowing for advanced approaches to AOP validation and development.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Cyprinidae/genética , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário , Poluentes Químicos da Água/toxicidade , Animais , Cyprinidae/crescimento & desenvolvimento , Cyprinidae/metabolismo , Embrião não Mamífero/enzimologia , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Melaninas/genética , Monofenol Mono-Oxigenase/genética , Mutação , Fenótipo , Pigmentação/genética
8.
Food Chem ; 320: 126649, 2020 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-32217433

RESUMO

The effect of frozen storage on the chemical properties and ingredient functionalities of Lesser mealworms was investigated at -20 °C for 2 months. Major changes occurred in the first week of frozen storage. Proteins, among which heavy chain myosin, underwent denaturation and aggregation, as shown by a decrease in solubility, SDS-PAGE pattern, and Confocal Laser Scanning Microscopy. The ice melting point in larvae was -32.5 °C as determined by DSC: 25% of water is not frozen at -20 °C, possibly due to anti-freezing proteins preventing ice formation. The presence of unfrozen water favoured various enzymatic activities as shown by a pH decrease, indicating protein hydrolysis. The molecular changes during frozen storage increased the browning reactions due to phenoloxidase activity. Foaming ability, foam stability and gel network stability increased upon frozen storage due to protein denaturation. Results provide important information regarding the opportunity of frozen storage of insect larvae for both research and industrial purposes.


Assuntos
Besouros/química , Insetos Comestíveis/química , Armazenamento de Alimentos/métodos , Animais , Congelamento , Concentração de Íons de Hidrogênio , Larva/química , Reação de Maillard , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/metabolismo , Desnaturação Proteica/efeitos dos fármacos , Solubilidade , Substâncias Viscoelásticas/química , Água/química
9.
Subcell Biochem ; 94: 219-231, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32189301

RESUMO

Hemocyanin (Hc), a copper-containing extracellular multimeric protein, is the major protein component of hemolymph in different arachnid groups. Hc possesses 7 or 8 very well-characterized types of monomers with molecular weights ranging from 70 to 85 kDa, organized in hexamers or multiple of hexamers. The present chapter compiles the existing data with relation to the function of this protein in the arachnids. Hc has as main function the reversible transport of O2, but it shows many secondary though not less important functions. With reference to this, it has been described that Hc can transport hydrophobic molecules (lipid-derived hormones and lipids) to the different organs, having a key role in the lipid transport system. In arachnids, like in other arthropods and invertebrates, Hc has phenoloxidase function which is related to different metabolic processes such as melanin formation and defense against pathogens. In addition, Hc has additional defensive functions since it can serve as precursor for the production of antimicrobial peptides. In short, the evolution of this protein has led to the development of multiple functions essential for organisms possessing this protein.


Assuntos
Aracnídeos , Hemocianinas , Animais , Aracnídeos/enzimologia , Aracnídeos/metabolismo , Hemocianinas/metabolismo , Monofenol Mono-Oxigenase/metabolismo
10.
Subcell Biochem ; 94: 233-250, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32189302

RESUMO

The copper-containing hemocyanins are proteins responsible for the binding, transportation and storage of dioxygen within the blood (hemolymph) of many invertebrates. Several additional functions have been attributed to both arthropod and molluscan hemocyanins, including (but not limited to) enzymatic activity (namely phenoloxidase), hormone transport, homeostasis (ecdysis) and hemostasis (clot formation). An important secondary function of hemocyanin involves aspects of innate immunity-such as acting as a precursor of broad-spectrum antimicrobial peptides and microbial/viral agglutination. In this chapter, we present the reader with an up-to-date synthesis of the known functions of hemocyanins and the structural features that facilitate such activities.


Assuntos
Artrópodes , Hemocianinas , Animais , Artrópodes/enzimologia , Artrópodes/imunologia , Artrópodes/metabolismo , Hemocianinas/imunologia , Hemocianinas/metabolismo , Hemolinfa/metabolismo , Imunidade Inata , Monofenol Mono-Oxigenase/metabolismo
11.
Food Chem ; 317: 126415, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32087518

RESUMO

This paper focused on improving antityrosinase ability of quercetin, cinnamic acid, and ferulic acid (named Q-CA-FA) from Asparagus by combining heating with ultrasound treatments. Fluorescence spectroscopy and UPLC-MS were used to evaluate inhibitory mechanisms. Results showed that the impacts of combining heating (150 °C for 30 min) with ultrasound (600 W for 30 min) treatments were similar to heating treatment (150 °C for 120 min) alone, and the inhibition rate could reach 98.2% in the addition of 5 mM Q-CA-FA. Fluorescence quenching indicated that treated Q-CA-FA-tyrosinase complex was more stable, but combining treatments did not change the major force between tyrosinase and polyphenols. Thermodynamic analysis illustrated that the randomness of compounds was also increased. Interestingly, 2-hydroxy-3-(3-hydroxy-4-methoxy-phenyl)-propionic acid 4-(2,3-dihydroxy-propyl)-phenyl ester was newly detected, which might be the major reason for enhancing antityrosinase ability. Taken together, these results provide a creative insight on increasing antityrosinase activity by combining heating with ultrasound treatments.


Assuntos
Monofenol Mono-Oxigenase/metabolismo , Polifenóis/metabolismo , Sonicação , Asparagus (Planta)/química , Asparagus (Planta)/metabolismo , Cromatografia Líquida de Alta Pressão , Cinamatos/análise , Cinamatos/metabolismo , Ácidos Cumáricos/análise , Ácidos Cumáricos/metabolismo , Temperatura Alta , Monofenol Mono-Oxigenase/antagonistas & inibidores , Polifenóis/análise , Quercetina/análise , Quercetina/metabolismo , Espectrometria de Fluorescência , Espectrometria de Massas em Tandem , Termodinâmica
12.
J Food Sci ; 85(3): 696-706, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32043592

RESUMO

The aim of this study was to extract and purify anthocyanins from Lycium ruthenicum Murr. and evaluate their tyrosinase inhibitory activity. Response surface methodology was devoted to optimize enzyme-assisted extraction of anthocyanins from L. ruthenicum dried fruits. Extraction at 38 °C for 37 min using water-containing pectinase (52.04 mg/100 g dried fruit) rendered an anthocyanin extraction yield of 19.51 ± 0.21 mg/g. The purified anthocyanins were separated from the extract by macroporous resin XDA-6. Antioxidant tests in vitro suggested that the extract and the purified anthocyanins exhibited a potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity, 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging capacity, hydroxyl radical scavenging capacity, superoxide radical scavenging capacity, and total reducing power. Thirteen anthocyanins from L. ruthenicum dried fruits were analyzed by HPLC-MS. Moreover, the purified anthocyanins had inhibitory effect on tyrosinase monophenolase (IC50 = 1.483 ± 0.058 mg/mL), and the type of inhibition was competitive inhibition (Ki = 39.83 ± 1.4 mg/mL). The maximum inhibitory activity of the purified anthocyanins (3.00 mg/mL) on tyrosinase diphenolase was 42.16 ± 0.77%, and the type of inhibition was anticompetitive inhibition (Kis = 2.387 ± 0.10 mg/mL). PRACTICAL APPLICATION: The anthocyanins from L. ruthenicum dried fruits can be used as tyrosinase inhibitors in medicine, cosmetics, and food preservation industries.


Assuntos
Antocianinas/química , Inibidores Enzimáticos/química , Lycium/química , Monofenol Mono-Oxigenase/antagonistas & inibidores , Extratos Vegetais/química , Antocianinas/isolamento & purificação , Antioxidantes/química , Antioxidantes/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/isolamento & purificação , Frutas/química , Espectrometria de Massas , Monofenol Mono-Oxigenase/química , Oxirredução , Extratos Vegetais/isolamento & purificação
13.
J Biosci Bioeng ; 129(5): 638-645, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31926815

RESUMO

Fermented extracts have evolved to be a potential alternative to synthetic chemicals, owing to their anti-inflammatory and anti-bacterial properties. This study intends to assess the potential of fermented Zanthoxylum schinifolium extract for use in biomedical applications. Probiotic bacteria, Lactobacillus rhamnosus A6-5, were deployed as a seed culture for fermentation. The fermented extract showed greater tyrosinase inhibitory activity and reduced melanin production (58.3%) compared with the raw extract. Cytotoxicity assay inferred that 500 µg/mL is the ideal non-toxic concentration with maximum cell viability. In addition, DAPI staining did not show any damage to the chromatin structure of the cells. The anti-aging property of the fermented extract was confirmed by a decrease in IL-6 content. The fermented extract showed lower MIC (40 mg/mL) and MBC (60 mg/mL), indicating greater anti-bacterial activity than the raw extract. The results confirmed that the fermented Z. schinifolium extract has high biomedical properties compared with the raw extract and can be used as an ideal skin whitening agent.


Assuntos
Antibacterianos/farmacologia , Anti-Inflamatórios/farmacologia , Lactobacillus rhamnosus/metabolismo , Melaninas/química , Extratos Vegetais/farmacologia , Zanthoxylum/química , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Reatores Biológicos , Linhagem Celular , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Fermentação , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Lactobacillus rhamnosus/genética , Lactobacillus rhamnosus/isolamento & purificação , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Melaninas/metabolismo , Camundongos , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/química , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Células RAW 264.7 , Staphylococcus epidermidis/efeitos dos fármacos , Staphylococcus epidermidis/crescimento & desenvolvimento , Estrelas-do-Mar/microbiologia , Zanthoxylum/microbiologia
14.
Food Chem ; 312: 126042, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31911351

RESUMO

Although mango leaves are the main ingredients in some traditional Chinese medicine preparations and folk tea, they with considerable quantities are usually discarded as agricultural waste. Thus, to extend their potential, reverse ultrafiltration-HPLC-DAD-QTOF-MS/MS combining with key ion filtering strategy was proposed to efficiently fish and systematically identify tyrosinase inhibitors in ethyl acetate fraction of mango leaves, which has the highest total phenolic content (40.00 ± 0.84 mg GAE/g DW) and tyrosinase inhibition activity (IC50, 17.62 ± 1.26 µg/mL). Finally, 36 polyphenolic tyrosinase inhibitors were unambiguously characterized or tentatively identified, and three of them were found in mango leaves for the first time. Results suggested that the proposed strategy was powerful for effective identification of bioactive compounds in complex mixtures (e.g. food, agricultural and sideline products), and the findings would lay a foundation for potential applications of mango leaves in pharmaceutical, cosmetic, and food industrial fields.


Assuntos
Inibidores Enzimáticos/farmacologia , Mangifera/química , Monofenol Mono-Oxigenase/antagonistas & inibidores , Cromatografia Líquida de Alta Pressão , Fenóis/análise , Folhas de Planta/química , Espectrometria de Massas em Tandem
15.
Int J Mol Sci ; 21(1)2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31906440

RESUMO

Fisetin is found in many fruits and plants such as grapes and onions, and exerts anti-inflammatory, anti-proliferative, and anticancer activity. However, whether fisetin regulates melanogenesis has been rarely studied. Therefore, we evaluated the effects of fisetin on melanogenesis in B16F10 melanoma cell and zebrafish larvae. The current study revealed that fisetin slightly suppressed in vitro mushroom tyrosinase activity; however, molecular docking data showed that fisetin did not directly bind to mushroom tyrosinase. Unexpectedly, fisetin significantly increased intracellular and extracellular melanin production in B16F10 melanoma cells regardless of the presence or absence of α-melanocyte stimulating hormone (α-MSH). We also found that the expression of melanogenesis-related genes such as tyrosinase and microphthalmia-associated transcription factor (MITF), were highly increased 48 h after fisetin treatment. Pigmentation of zebrafish larvae by fisetin treatment also increased at the concentrations up to 200 µM and then slightly decreased at 400 µM, with no alteration in the heart rates. Molecular docking data also revealed that fisetin binds to glycogen synthase kinase-3ß (GSK-3ß). Therefore, we evaluated whether fisetin negatively regulated GSK-3ß, which subsequently activates ß-catenin, resulting in melanogenesis. As expected, fisetin increased the expression of ß-catenin, which was subsequently translocated into the nucleus. In the functional assay, FH535, a Wnt/ß-catenin inhibitor, significantly inhibited fisetin-mediated melanogenesis in zebrafish larvae. Our data suggested that fisetin inhibits GSK-3ß, which activates ß-catenin, resulting in melanogenesis through the revitalization of MITF and tyrosinase.


Assuntos
Flavonoides/farmacologia , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Melaninas/biossíntese , beta Catenina/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Flavonoides/química , Flavonoides/toxicidade , Glicogênio Sintase Quinase 3 beta/química , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Larva/efeitos dos fármacos , Larva/metabolismo , Melanoma Experimental , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Simulação de Acoplamento Molecular , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/metabolismo , Pigmentação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , alfa-MSH/farmacologia , beta Catenina/antagonistas & inibidores , beta Catenina/genética
16.
Int J Mol Sci ; 21(1)2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31906476

RESUMO

Bioactive collagen/chitosan complexes were prepared by an ion crosslinking method using fish skin collagen and chitosan solution as raw materials. Scanning electron microscopy observation confirmed that the collagen/chitosan complexes were of a uniform spherical shape and uniform particle size. The complexes were stable at different pH values for a certain period of time through swelling experiments. Differential scanning calorimetry (DSC) showed the collagen/ chitosan complexes were more stable than collagen. X-ray diffraction (XRD) showed that the complexes had a strong crystal structure, and Fourier transform infrared spectroscopy (FTIR) data revealed the changes in the secondary structure of the protein due to chitosan and TPP crosslinking. The content of malondialdehyde (MDA) in the complex treatment group was considerably lower, but the content of SOD was significantly higher than that of the collagen group or chitosan group. In addition, the collagen/chitosan complexes could considerably reduce melanin content, inhibit tyrosinase activity, and down-regulate tyrosinase mRNA expression. In conclusion, the collagen/chitosan complexes were potential oral protein preparation for antioxidant enhancement and inhibiting melanin synthesis.


Assuntos
Antioxidantes/farmacologia , Quitosana/química , Colágeno/química , Colágeno/farmacologia , Melaninas/biossíntese , Monofenol Mono-Oxigenase/antagonistas & inibidores , Animais , Varredura Diferencial de Calorimetria , Quitosana/farmacologia , Colágeno/ultraestrutura , Feminino , Concentração de Íons de Hidrogênio , Masculino , Malondialdeído/análise , Malondialdeído/metabolismo , Melaninas/análise , Melaninas/metabolismo , Melanoma Experimental , Camundongos , Microscopia Eletrônica de Varredura , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Tamanho da Partícula , Conformação Proteica , Espectroscopia de Infravermelho com Transformada de Fourier , Superóxido Dismutase/análise , Superóxido Dismutase/metabolismo , Difração de Raios X
17.
J Enzyme Inhib Med Chem ; 35(1): 424-431, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31899985

RESUMO

A series of 16 novel benzenesulfonamides incorporating 1,3,5-triazine moieties substituted with aromatic amines, dimethylamine, morpholine and piperidine were investigated. These compounds were assayed for antioxidant properties by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, 2,2`-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical decolarisation assay and metal chelating methods. They were also investigated as inhibitors of acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and tyrosinase, which are associated with several diseases such as Alzheimer, Parkinson and pigmentation disorders. These benzenesulfonamides showed moderate DPPH radical scavenging and metal chelating activity, and low ABTS cation radical scavenging activity. Compounds 2 b, 3d and 3 h showed inhibitory potency against AChE with % inhibition values of >90. BChE was also effectively inhibited by most of the synthesised compounds with >90% inhibition potency. Tyrosinase was less inhibited by these compounds.


Assuntos
Acetilcolinesterase/efeitos dos fármacos , Antioxidantes/farmacologia , Butirilcolinesterase/efeitos dos fármacos , Inibidores da Colinesterase/farmacologia , Inibidores Enzimáticos/farmacologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Sulfonamidas/química , Sulfonamidas/farmacologia , Triazinas/química , Benzotiazóis/química , Compostos de Bifenilo/química , Picratos/química , Ácidos Sulfônicos/química
18.
BMC Plant Biol ; 20(1): 39, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992195

RESUMO

BACKGROUND: Plants have been used as an important source of indispensable bioactive compounds in various cosmetics, foods, and medicines. However, the subsequent functional annotation of these compounds seems arduous because of the largely uncharacterized, vast metabolic repertoire of plant species with known biological phenotypes. Hence, a rapid multi-parallel screening and characterization approach is needed for plant functional metabolites. RESULTS: Fifty-one species representing three plant families, namely Asteraceae, Fabaceae, and Rosaceae, were subjected to metabolite profiling using gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) and ultrahigh-performance liquid chromatography quadrupole orbitrap ion trap tandem mass spectrometry (UHPLC-Q-orbitrap-MS/MS) as well as multivariate analyses. Partial least squares discriminant analysis (PLS-DA) of the metabolite profiling datasets indicated a distinct clustered pattern for 51 species depending on plant parts (leaves and stems) and relative phylogeny. Examination of their relative metabolite contents showed that the extracts from Fabaceae plants were abundant in amino acids, fatty acids, and genistein compounds. However, the extracts from Rosaceae had higher levels of catechin and ellagic acid derivatives, whereas those from Asteraceae were higher in kaempferol derivatives and organic acids. Regardless of the different families, aromatic amino acids, branch chain amino acids, chlorogenic acid, flavonoids, and phenylpropanoids related to the shikimate pathway were abundant in leaves. Alternatively, certain amino acids (proline, lysine, and arginine) as well as fatty acids levels were higher in stem extracts. Further, we investigated the associated phenotypes, i.e., antioxidant activities, affected by the observed spatial (leaves and stem) and intra-family metabolomic disparity in the plant extracts. Pearson's correlation analysis indicated that ellagic acid, mannitol, catechin, epicatechin, and quercetin derivatives were positively correlated with antioxidant phenotypes, whereas eriodictyol was positively correlated with tyrosinase inhibition activity. CONCLUSIONS: This work suggests that metabolite profiling, including multi-parallel approaches and integrated bioassays, may help the expeditious characterization of plant-derived metabolites while simultaneously unraveling their chemodiversity.


Assuntos
Metaboloma , Extratos Vegetais/química , Folhas de Planta/química , Caules de Planta/química , Aminoácidos/metabolismo , Antioxidantes/química , Antioxidantes/metabolismo , Asteraceae/química , Asteraceae/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Fabaceae/química , Fabaceae/metabolismo , Ácidos Graxos/metabolismo , Flavonoides/química , Flavonoides/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Redes e Vias Metabólicas , Metabolômica/métodos , Monofenol Mono-Oxigenase/metabolismo , Extratos Vegetais/metabolismo , Folhas de Planta/metabolismo , Caules de Planta/metabolismo , Rosaceae/química , Rosaceae/metabolismo , Espectrometria de Massas em Tandem
19.
PLoS One ; 15(1): e0227561, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935259

RESUMO

Host-parasite interactions may be modulated by host- or parasite-associated microbes, but the role of these are often overlooked. Particularly for parasites with intestinal stages (either larval or adult), the host gut microbiome may play a key role for parasite establishment; moreover, the microbiome may change in response to invading parasites. Hypothesis testing at the organismal level may be hampered, particularly in mammalian definitive hosts, by ethical, logistical, and economical restrictions. Thus, invertebrates naturally serving as intermediate hosts to parasites with complex life cycles may inform the development of mammalian models as an early-stage host-parasite model. In addition, several important pathogens are vectored by insects, and insect gut microbiome-pathogen interactions may provide essential base-line knowledge, which may be used to control vectorborne pathogens. Here, we used the grain beetle, Tenebrio molitor, a host of the tapeworm Hymenolepis diminuta, to explore interactions between infection status and resident gut microbiota at two pre-determined time points (day two and seven) post infection. Using 16S/18S microbial profiling, we measured key parameters of the composition, relative abundance, and diversity of the host gut bacteriome and mycobiome. In addition, we quantified the systemic beetle immune response to infection by Phenoloxidase activity and hemocyte abundance. We found significant changes in the gut bacteriome and mycobiome in relation to infection status and beetle age. Thus, the relative abundance of Proteobacteria was significantly higher in the gut of infected beetles and driven mostly by an increased abundance of Acinetobacter. In addition, the mycobiome was less abundant in infected beetles but maintained higher Shannon diversity in infected compared with non-infected beetles. Beetles treated with a broad-spectrum antibiotic (Tetracycline) exhibited significantly reduced parasite establishment compared with the untreated control group, indicating that the host microbiome may greatly influence hatching of eggs and subsequent establishment of H. diminuta larvae. Our results suggest that experimental work using invertebrates may provide a platform for explorative studies of host-parasite-microbe interactions and their underlying mechanisms.


Assuntos
Besouros/parasitologia , Microbioma Gastrointestinal , Hymenolepis diminuta/fisiologia , Animais , Antibacterianos/farmacologia , Besouros/imunologia , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Microbioma Gastrointestinal/efeitos dos fármacos , Hemolinfa/metabolismo , Interações Hospedeiro-Parasita , Monofenol Mono-Oxigenase/metabolismo , Micobioma/efeitos dos fármacos , Análise de Componente Principal , Proteobactérias/genética , Proteobactérias/isolamento & purificação , Tetraciclina/farmacologia
20.
Phytochem Anal ; 31(3): 314-321, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31997462

RESUMO

INTRODUCTION: Tyrosinase is a multifunctional copper-containing oxidase enzyme that catalyses the first steps in the formation of melanin pigments. Identification of tyrosinase inhibitors is of value for applications in cosmetics, medicine and agriculture. OBJECTIVE: To develop an analytical method that allows identification of drug-like natural products that can be further developed as tyrosinase inhibitors. Results of in vitro and in silico studies will be compared in order to gain a deeper insight into the mechanism of action of enzyme inhibition. METHOD: Using an in vitro assay we tested tyrosinase inhibitor effects of five structurally related flavones, i.e. luteolin (1), eupafolin (2), genkwanin (3), nobiletin (4), and chrysosplenetin (5). The strongest inhibitors were further investigated in silico, using enzyme docking simulations. RESULTS: All compounds tested showed modest tyrosinase inhibitory effect compared to the positive control, kojic acid. The polymethoxy flavones 4 and 5 exhibited the strongest tyrosinase inhibitory effect with the half maximal inhibitory concentration (IC50 ) values of 131.92 ± 1.75 µM and 99.87 ± 2.38 µM respectively. According to kinetic analysis 2, 4 and 5 were competitive inhibitors, whereas 1 and 3 were non-competitive inhibitors of tyrosinase. Docking studies indicated that methoxy groups on 4 and 5 caused steric hindrance which prevented alternative binding modes in the tyrosinase; the methoxy groups on the B-ring of these flavones faced the catalytic site in the enzyme. CONCLUSIONS: The docking simulations nicely complemented the in vitro kinetic studies, opening the way for the development of predictive models for use in drug design.


Assuntos
Agaricales , Flavonas , Inibidores Enzimáticos , Cinética , Simulação de Acoplamento Molecular , Estrutura Molecular , Monofenol Mono-Oxigenase , Relação Estrutura-Atividade
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