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1.
J Toxicol Sci ; 45(11): 695-700, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33132243

RESUMO

Coumarin is a dietary-derived substance that is extensively metabolized by human liver to excretable 7-hydroxycoumarin. Although coumarin under daily dietary consumption is generally regarded as nontoxic, the substance is of toxicological and clinical interest because of its potential association with hepatotoxicity, which is especially evident in rats. In this study, the pharmacokinetics of coumarin were modeled after virtual oral administration in humans. The adjusted monitoring equivalents of coumarin, along with the biotransformation of coumarin to o-hydroxyphenylacetic acid (via 3,4-epoxidation) based on reported plasma concentrations from rat studies, were scaled to human coumarin equivalents using known species allometric scaling factors. Using rat and human liver preparations, data on the rapid in vitro metabolic clearance for humans (~50-fold faster than in rats) were obtained for in vitro-in vivo extrapolation. For human physiologically based pharmacokinetic (PBPK) modeling, the metabolic ratios to o-hydroxyphenylacetic acid and 7-hydroxycoumarin were set at minor (0.1) and major (0.9) levels for the total disappearance of coumarin. The resulting modeled plasma concentration curves in humans generated by simple PBPK models were consistent with reported simulated coumarin maximum concentrations. These results provide basic information to simulate plasma levels of coumarin and its primary metabolite 7-hydroxycoumarin or its secondary activated metabolite o-hydroxyphenylacetic acid (via 3,4-epoxidation) resulting from dietary foodstuff consumption. Under the current assumptions, little toxicological impact of coumarin was evident in humans, thereby indicating the usefulness of forward dosimetry using PBPK modeling for human risk assessment.


Assuntos
Cumarínicos/sangue , Cumarínicos/toxicidade , Animais , Simulação por Computador , Cumarínicos/metabolismo , Cumarínicos/farmacocinética , Conjuntos de Dados como Assunto , Humanos , Técnicas In Vitro , Fígado/metabolismo , Masculino , Modelos Biológicos , Fenilacetatos/sangue , Ratos Sprague-Dawley , Medição de Risco , Umbeliferonas/sangue
2.
Ecotoxicol Environ Saf ; 205: 111348, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32979803

RESUMO

Transgenerational effects induced by environmental stressors are a threat to ecosystems and human health. However, there is still limited observation and understanding of the potential of chemicals to influence life outcomes over several generations. In the present study, we investigated the effects of two environmental contaminants, coumarin 47 and permethrin, on exposed zebrafish (F0) and their progeny (F1-F3). Coumarin 47 is commonly found in personal care products and dyes, whereas permethrin is used as a domestic and agricultural pyrethroid insecticide/insect repellent. Zebrafish (F0) were exposed during early development until 28 days post-fertilization and their progeny (F1-F3) were bred unexposed. On one hand, the effects induced by coumarin 47 suggest no multigenerational toxicity. On the other hand, we found that behavior of zebrafish larvae was significantly affected by exposure to permethrin in F1 to F3 generations with some differences depending on the concentration. This suggests persistent alteration of the neural or neuromuscular function. In addition, lipidomic analyses showed that permethrin treatment was partially correlated with lysophosphatidylcholine levels in zebrafish, an important lipid for neurodevelopment. Overall, these results stress out one of the most widely used pyrethroids can trigger long-term, multi- and possibly transgenerational changes in the nervous system of zebrafish. These neurobehavioral changes echo the effects observed under direct exposure to high concentrations of permethrin and therefore call for more research on mechanisms underlying effect inheritance.


Assuntos
Cumarínicos/toxicidade , Repelentes de Insetos/toxicidade , Permetrina/toxicidade , Animais , Comportamento Animal/efeitos dos fármacos , Cumarínicos/metabolismo , Ecossistema , Fertilidade/efeitos dos fármacos , Larva/efeitos dos fármacos , Metabolismo dos Lipídeos , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia
3.
Plant Mol Biol ; 104(3): 327-337, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32761540

RESUMO

KEY MESSAGE: Psoralen synthase and angelicin synthase responsible for the formation of psoralen and angelicin in Peucedanum praeruptorum Dunn were identified and functionally characterized, respectively. Furanocoumarins were reported to possess several activities such as anticancer, anti-inflammatory and neuroprotective, and function as phytotoxin and allelochemical in plants. Furanocoumarins are the main bioactive ingredient in P. praeruptorum which is a commonly used traditional Chinese medicine. Phenylalanine ammonia lyase (PAL), 4-coumarate: CoA ligase (4CL), p-coumaroyl CoA 2'-hyfroxylase (C2'H) were cloned previously to elucidate the biosynthetic mechanism of coumarin lactone ring. However, the genes involved in complex coumarins in P. praeruptorum have not been explored. Herein, putative psoralen synthase CYP71AJ49 and angelicin synthase CYP71AJ51 were cloned from P. praeruptorum. In vivo and in vitro yeast assays were conducted to confirm their activities. Furthermore, the results of High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS) verified that CYP71AJ49 catalyzed the conversion of marmesin to psoralen, and CYP71AJ51 catalyzed columbianetin to angelicin. Subsequently, the expression profile showed that CYP71AJ49 and CYP71AJ51 were easily affected by environmental conditions, especially UV and temperature. The genes tissue-specific expression and compounds tissue-specific distribution pattern indicated the existence of substance transport in P. praeruptorum. Phylogenetic analysis was conducted with 27 CYP71AJs, CYP71AJ49 and CYP71AJ51 were classified in I-4 and I-2, respectively. These results provide further insight to understand the biosynthetic mechanism of complex coumarins.


Assuntos
Apiaceae/enzimologia , Apiaceae/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Furocumarinas/metabolismo , Proteínas de Plantas/metabolismo , Apiaceae/genética , China , Cromatografia Líquida de Alta Pressão/métodos , Coenzima A Ligases/genética , Cumarínicos/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Furocumarinas/química , Furocumarinas/genética , Regulação da Expressão Gênica de Plantas , Cinética , Medicina Tradicional Chinesa , Fenilalanina Amônia-Liase/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , Transcriptoma
4.
PLoS One ; 15(6): e0233963, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32530961

RESUMO

Eclipta alba L., also known as false daisy, is well known and commercially attractive plant with excellent hepatotoxic and antidiabetic activities. Light is considered a key modulator in plant morphogenesis and survival by regulating important physiological cascades. Current study was carried out to investigate growth and developmental aspects of E. alba under differential effect of multispectral lights. In vitro derived callus culture of E. alba was exposed to multispectral monochromatic lights under controlled aseptic conditions. Maximum dry weight was recorded in culture grown under red light (11.2 g/L) whereas negative effect was observed under exposure of yellow light on callus growth (4.87 g/L). Furthermore, red light significantly enhanced phenolics and flavonoids content (TPC: 57.8 mg/g, TFC: 11.1 mg/g) in callus cultures compared to rest of lights. HPLC analysis further confirmed highest accumulation of four major compounds i.e. coumarin (1.26 mg/g), eclalbatin (5.00 mg/g), wedelolactone (32.54 mg/g) and demethylwedelolactone (23.67 mg/g) and two minor compounds (ß-amyrin: 0.38 mg/g, luteolin: 0.39 mg/g) in red light treated culture whereas stigmasterol was found optimum (0.22 mg/g) under blue light. In vitro based biological activities including antioxidant, antidiabetic and lipase inhibitory assays showed optimum values in cultures exposed to red light, suggesting crucial role of these phytochemicals in the enhancement of the therapeutic potential of E. alba. These results clearly revealed that the use of multispectral lights in in vitro cultures could be an effective strategy for enhanced production of phytochemicals.


Assuntos
Antioxidantes/metabolismo , Eclipta/metabolismo , Eclipta/efeitos da radiação , Hipoglicemiantes/metabolismo , Compostos Fitoquímicos/metabolismo , Antioxidantes/química , Cumarínicos/metabolismo , Eclipta/crescimento & desenvolvimento , Flavonoides/metabolismo , Hipoglicemiantes/química , Luz , Fenóis/metabolismo , Compostos Fitoquímicos/química , Metabolismo Secundário/efeitos da radiação , Técnicas de Cultura de Tecidos
5.
Sci Rep ; 10(1): 7850, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398726

RESUMO

Environmental and genetic factors are associated with pandemic obesity since childhood. However, the association of overweight-obesity with these factors, acting as a consortium, has been scarcely studied in children. We aimed here to assess the probabilities of being overweighed-obese in a randomly recruited cohort of Spanish children and adolescents (n = 415, 5-17 years-old) by estimating the odds ratios for different predictor variables, and their relative importance in the prediction. The predictor variables were ethnicity, age, sex, adherence to the Mediterranean diet (KIDMED), physical activity, urolithin metabotypes (UM-A, UM-B and UM-0) as biomarkers of the gut microbiota, and 53 single-nucleotide polymorphisms (SNPs) from 43 genes mainly related to obesity and cardiometabolic diseases. A proportional-odds logistic ordinal regression, validated through bootstrap, was used to model the data. While every variable was not independently associated with overweight-obesity, however, the ordinal logistic model revealed that overweight-obesity prevalence was related to being a young boy with either UM-B or UM-0, low KIDMED score and high contribution of a consortium of 24 SNPs, being rs1801253-ADRB1, rs4343-ACE, rs8061518-FTO, rs1130864-CRP, rs659366-UCP2, rs6131-SELP, rs12535708-LEP, rs1501299-ADIPOQ, rs708272-CETP and rs2241766-ADIPOQ the top-ten contributing SNPs. Additional research should confirm and complete this model by including dietary interventions and the individuals' gut microbiota composition.


Assuntos
Cumarínicos/metabolismo , Dieta Mediterrânea , Microbioma Gastrointestinal , Obesidade/genética , Obesidade/microbiologia , Polimorfismo de Nucleotídeo Único , Adolescente , Criança , Estudos de Coortes , Exercício Físico , Feminino , Humanos , Masculino , Obesidade/metabolismo , Razão de Chances
6.
Biochim Biophys Acta Proteins Proteom ; 1868(9): 140445, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32405284

RESUMO

Coumarins represent well-established structures to introduce fluorescence into tool compounds for biochemical investigations. They are valued for their small size, chemical stability and accessibility as well as their tunable photochemical properties. As components of fluorophore/quencher pairs or FRET donor/acceptor pairs, coumarins have frequently been applied in substrate mapping approaches for serine and cysteine proteases. This review also focuses on the incorporation of coumarins into the side chain of amino acids and the exploitation of the resulting fluorescent amino acids for the positional profiling of protease substrates. The protease-inhibiting properties of certain coumarin derivatives and the utilization of coumarin moieties to assemble activity-based probes for serine and cysteine proteases are discussed as well.


Assuntos
Cumarínicos/química , Cumarínicos/metabolismo , Cisteína Proteases/metabolismo , Serina Proteases/metabolismo , Domínio Catalítico , Cumarínicos/farmacologia , Cisteína Proteases/efeitos dos fármacos , Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Serina/metabolismo , Serina Proteases/efeitos dos fármacos , Especificidade por Substrato
7.
J Med Chem ; 63(11): 5723-5733, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32374603

RESUMO

The serine protease kallikrein-related peptidase 7 (KLK7) is a member of the human tissue kallikreins. Its dysregulation leads to pathophysiological inflammatory processes in the skin. Furthermore, it plays a role in several types of cancer. For the treatment of KLK7-associated diseases, coumarinic esters have been developed as small-molecule enzyme inhibitors. To characterize the inhibition mode of these inhibitors, we analyzed structures of the inhibited protease by X-ray crystallography. Electron density shows the inhibitors covalently attached to His57 of the catalytic triad. This confirms the irreversible character of the inhibition process. Upon inhibitor binding, His57 undergoes an outward rotation; thus, the catalytic triad of the protease is disrupted. Besides, the halophenyl moiety of the inhibitor was absent in the final enzyme-inhibitor complex due to the hydrolysis of the ester linkage. With these results, we analyze the structural basis of KLK7 inhibition by the covalent attachment of aromatic coumarinic esters.


Assuntos
Cumarínicos/química , Calicreínas/antagonistas & inibidores , Inibidores de Proteases/química , Sítios de Ligação , Domínio Catalítico , Cumarínicos/metabolismo , Cristalografia por Raios X , Ésteres/química , Humanos , Calicreínas/genética , Calicreínas/metabolismo , Simulação de Dinâmica Molecular , Inibidores de Proteases/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas em Tandem
8.
Nature ; 579(7799): 421-426, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32188939

RESUMO

Bioorthogonal chemistry capable of operating in live animals is needed to investigate biological processes such as cell death and immunity. Recent studies have identified a gasdermin family of pore-forming proteins that executes inflammasome-dependent and -independent pyroptosis1-5. Pyroptosis is proinflammatory, but its effect on antitumour immunity is unknown. Here we establish a bioorthogonal chemical system, in which a cancer-imaging probe phenylalanine trifluoroborate (Phe-BF3) that can enter cells desilylates and 'cleaves' a designed linker that contains a silyl ether. This system enabled the controlled release of a drug from an antibody-drug conjugate in mice. When combined with nanoparticle-mediated delivery, desilylation catalysed by Phe-BF3 could release a client protein-including an active gasdermin-from a nanoparticle conjugate, selectively into tumour cells in mice. We applied this bioorthogonal system to gasdermin, which revealed that pyroptosis of less than 15% of tumour cells was sufficient to clear the entire 4T1 mammary tumour graft. The tumour regression was absent in immune-deficient mice or upon T cell depletion, and was correlated with augmented antitumour immune responses. The injection of a reduced, ineffective dose of nanoparticle-conjugated gasdermin along with Phe-BF3 sensitized 4T1 tumours to anti-PD1 therapy. Our bioorthogonal system based on Phe-BF3 desilylation is therefore a powerful tool for chemical biology; our application of this system suggests that pyroptosis-induced inflammation triggers robust antitumour immunity and can synergize with checkpoint blockade.


Assuntos
Preparações de Ação Retardada/administração & dosagem , Neoplasias Mamárias Experimentais/imunologia , Piroptose/imunologia , Animais , Cumarínicos/administração & dosagem , Cumarínicos/química , Cumarínicos/metabolismo , Cumarínicos/farmacocinética , Preparações de Ação Retardada/química , Preparações de Ação Retardada/metabolismo , Preparações de Ação Retardada/farmacocinética , Feminino , Proteínas de Fluorescência Verde/administração & dosagem , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/farmacocinética , Células HeLa , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/química , Imunoconjugados/metabolismo , Imunoconjugados/farmacocinética , Inflamassomos/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/administração & dosagem , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacocinética , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteínas/administração & dosagem , Proteínas/química , Proteínas/metabolismo , Proteínas/farmacocinética , Silanos/administração & dosagem , Silanos/química , Silanos/metabolismo , Silanos/farmacocinética , Linfócitos T/imunologia , Trastuzumab/administração & dosagem , Trastuzumab/química , Trastuzumab/metabolismo , Trastuzumab/farmacocinética , Ensaios Antitumorais Modelo de Xenoenxerto
9.
J Agric Food Chem ; 68(14): 4205-4214, 2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32141744

RESUMO

Polyphenol extracts derived from gastrointestinal digestates of buckwheat (Fagopyrum Mill) were studied for their intestinal transport and lipid-lowering effects in Caco-2/HepG2 coculture models. The relative amounts of all phenolic compounds throughout the digestion and intestinal absorption process were determined by UHPLC-Q-Orbitrap mass spectrometry. The digestible and easily transported phenolic compounds in buckwheat extract were identified. Herein, four main phenolic compounds and their metabolites were found on both the apical and basolateral sides of the Caco-2 cell transwell model. The transepithelial transport rates in the Caco-2 cell monolayer were scoparone (0.97) > hydroxycinnamic acid (0.40) > rutin (0.23) > quercetin (0.20). The main metabolism of hydroxycinnamic acid, quercetin, and scoparone in transepithelial transport was found to be methylation. Furthermore, results indicated that triglyceride, low-density lipoprotein cholesterol, total cholesterol, aspartate aminotransferase, and alanine aminotransferase levels in HepG2 cells on the basolateral side of coculture models can be suppressed by 53.64, 23.44, 36.49, 27.98, and 77.42% compared to the oleic acid-induced group (p < 0.05). In addition, the mRNA expression of Fabp4 relative to the control was found to be significantly upregulated (85.82 ± 10.64 to 355.18 ± 65.83%) by the easily transported buckwheat polyphenol components in HepG2 cells (p < 0.01).


Assuntos
Digestão , Fagopyrum/metabolismo , Extratos Vegetais/metabolismo , Polifenóis/metabolismo , Alanina Transaminase/metabolismo , Transporte Biológico , Células CACO-2 , Colesterol/metabolismo , LDL-Colesterol/metabolismo , Técnicas de Cocultura , Ácidos Cumáricos/metabolismo , Cumarínicos/metabolismo , Fagopyrum/química , Células Hep G2 , Humanos , Absorção Intestinal , Extratos Vegetais/química , Polifenóis/química , Quercetina/metabolismo , Rutina/metabolismo , Transaminases/metabolismo , Triglicerídeos/metabolismo
10.
Chem Biol Interact ; 322: 109053, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32198085

RESUMO

Notopterol (NOT) is a major bioactive ingredient extracted from the rhizomes of either Notopterygium incisum Ting ex H. T. Chang or N. forbesii Boiss (Qianghuo in Chinese), a botanical drug that was adopted as a traditional Chinese medicine. NOT is suggested to show analgesic and anti-inflammatory effects in clinical practice. The inhibitory effects of NOT on human cytochrome P450 enzymes were investigated in the present study. Our results indicate that NOT inhibited the activity of CYP2D6 in a time-, concentration- and NADPH-dependent manner. The values of KI and kinact were 10.8 µM and 0.62 min-1, respectively. The calculated kobs at 10 µM was 0.29 min-1, above the 0.02 min-1 risk level. After incubation with NOT at 10 µM for 9 min, approximately 92% of CYP2D6 activity was inhibited. Such loss of enzyme activity was not restored through dialysis, which indicates that the observed enzyme inhibition was irreversible. Partition ratio of the inactivation was approximately 29. Quinidine, a competitive CYP2D6 inhibitor, demonstrated protection on enzymes against the NOT-induced inactivation, but such protection was not found in incubation systems fortified with glutathione or catalase/superoxide dismutase. Additionally, CYP3A4 was observed to function as an enzyme mainly involved in the biotransformation of NOT. Taken together, these findings indicate that NOT served as a mechanism-based inactivator of CYP2D6, meanwhile, those observed effects may induce the latent drug-drug interactions. The metabolic activation of NOT may be the key to trigger the inactivation of the enzyme.


Assuntos
Cumarínicos/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Ativação Metabólica , Apiaceae/química , Apiaceae/metabolismo , Cumarínicos/química , Citocromo P-450 CYP2D6/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Glutationa/antagonistas & inibidores , Glutationa/metabolismo , Humanos , Cinética , NADP/química , NADP/metabolismo , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/metabolismo
11.
Xenobiotica ; 50(9): 1076-1089, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32174209

RESUMO

Coumarins have aroused high interests due to their diverse bioactivities. Understanding of its metabolism contributes to determine the druggability of coumarin in vivo.A sensitive and efficient strategy based on ultra-performance liquid chromatography-mass spectrometer (UPLC-MS) analysis combined with various data-processing techniques including metabolomics and multiple mass defect filter (MMDF) was established for the comprehensive screening and elucidation of potential coumarin metabolites.Total 20 metabolites of scoparone were identified in this study, including 14 undescribed metabolites. The metabolism of two other similar coumarins scopoletin and esculetin also could be determined using this strategy.By the established strategy, this study gives the insights about the major metabolic pathways of scoparone in vivo and in vitro metabolism, including demethylation, hydroxylation, hydration, cysteine conjugation, glucuronide conjugation and sulfate conjugation. Additionally, the metabolic pathways of scopoletin and esculetin were determined as hydroxylation, glucuronidation and sulfation. These results contribute to the understanding of metabolic characterization of coumarins, and demonstrate that the combination of UPLC-MS-based metabolomics and MMDF is a powerful approach to determine the metabolic pathways of coumarin compounds.


Assuntos
Cumarínicos/metabolismo , Metabolômica , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Hidroxilação , Redes e Vias Metabólicas , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem
12.
PLoS One ; 15(2): e0228675, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32049975

RESUMO

Aspergillus tubingensis is an important pathogen of economically important crops. Different biotic stresses strongly influence the balance of metabolites in plants. The aim of this study was to understand the function and response of resistance associated metabolites which, in turn are involved in many secondary metabolomics pathways to influence defense mechanism of cotton plant. Analysis of non-targeted metabolomics using ultra high performance liquid chromatography-mass spectrometry (UPLC-MS) revealed abundant accumulation of key metabolites including flavonoids, phenylpropanoids, terpenoids, fatty acids and carbohydrates, in response to leaf spot of cotton. The principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and partial least squares discriminant analysis (PLS-DA) score plots illustrated the evidences of variation between two varieties of cotton under mock and pathogen inoculated treatments. Primary metabolism was affected by the up regulation of pyruvate and malate and by the accumulation of carbohydrates like cellobiose and inulobiose. Among 241 resistance related (RR) metabolites, 18 were identified as resistance related constitutive (RRC) and 223 as resistance related induced (RRI) metabolites. Several RRI metabolites, identified in the present study were the precursors for many secondary metabolic pathways. These included phenylpropanoids (stilbenes and furanocoumarin), flavonoids (phlorizin and kaempferol), alkaloids (indolizine and acetylcorynoline) and terpenoids (azelaic acid and oleanolic acid). Our results demonstrated that secondary metabolism, primary metabolism and energy metabolism were more active in resistant cultivar, as compared to sensitive cultivar. Differential protein and fatty acid metabolism was also depicted in both cultivars. Accumulation of these defense related metabolites in resistant cotton cultivar and their suppression in susceptible cotton cultivar revealed the reason of their respective tolerance and susceptibility against A. tubingensis.


Assuntos
Aspergillus/patogenicidade , Resistência à Doença , Gossypium/metabolismo , Metaboloma , Folhas de Planta/metabolismo , Cumarínicos/metabolismo , Ácidos Graxos/metabolismo , Flavonoides/metabolismo , Gossypium/microbiologia , Folhas de Planta/microbiologia , Estilbenos/metabolismo , Terpenos/metabolismo
13.
Spectrochim Acta A Mol Biomol Spectrosc ; 230: 118063, 2020 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-32000060

RESUMO

In the present study the binding of diversin (DIV), a prenylated coumarin isolated from Ferula diversivittata, to bovine serum albumin (BSA) was investigated using surface plasmon resonance (SPR), spectrofluorimetry, and molecular docking approaches. Following the activation of carboxylic groups, via NHS/EDC, BSA was immobilized on the carboxymethyl dextran (CMD) hydrogel coated Au sensor, and was used for real-time monitoring of the interactions between DIV and BSA. KD value of DIV binding to BSA increased with increasing temperature, confirmed that the affinity between BSA and DIV decreases with rising temperature. In addition, the fluorescence and synchronous fluorescence spectroscopic data revealed that the intrinsic emission intensity of BSA was quenched via a dynamic mechanism. In addition, the micro-region around BSA tyrosine residue was changed upon interaction with DIV. The thermodynamic parameter findings suggested that the hydrophobic interactions were dominant in the binding and formation of the BSA and DIV complex. The molecular docking outputs indicated that there is only one binding site on BSA for DIV, in agreement with experimental data, and DIV bind BSA in subdomain IB.


Assuntos
Cumarínicos/química , Cumarínicos/metabolismo , Ferula/metabolismo , Fluorescência , Simulação de Acoplamento Molecular , Monoterpenos/química , Monoterpenos/metabolismo , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Ressonância de Plasmônio de Superfície , Animais , Sítios de Ligação , Bovinos , Interações Hidrofóbicas e Hidrofílicas , Ligação Proteica , Espectrometria de Fluorescência , Termodinâmica
14.
Food Chem Toxicol ; 136: 111027, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31870919

RESUMO

The incubation system of CYP2E1 and CYP3A4 enzymes in rat liver microsomes was established to investigate the effects of psoralidin, isobavachalcone, neobavaisoflavone and daidzein from Fructus Psoraleae in vitro. The relevant metabolites were measured by the method of high performance liquid chromatography (HPLC), after probe substrates of 4-nitrophenol, testosterone and the drugs at different concentrations were added to the incubation systems. In addition, real-time RT-PCR was performed to determine the effect of psoralidin, neobavaisoflavone and daidzein on the mRNA expression of CYP3A4 in rat liver. The results suggested that psoralidin, isobavachalcone and neobavaisoflavone were Medium-intensity inhibitors of CYP2E1 with Ki values of 2.58, 1.28 and 19.07 µM, respectively, which could inhibit the increase of CYP2E1 and reduce diseases caused by lipid peroxidation. Isobavachalcone (Ki = 37.52 µM) showed a weak competitive inhibition on CYP3A4. Psoralidin and neobavaisoflavone showed obvious induction effects on CYP3A4 in the expression level of mRNA, which could accelerate the effects of drug metabolism and lead to the risk of inducing DDIs and serious adverse reactions. The results could be used for guideline of Fructus Psoraleae in clinic, which aimed to calculate the drug toxicity by studying the drug-drug interactions caused by the induction and inhibition of CYP450.


Assuntos
Benzofuranos/toxicidade , Chalconas/toxicidade , Cumarínicos/toxicidade , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP3A/metabolismo , Isoflavonas/toxicidade , Microssomos Hepáticos/metabolismo , Animais , Benzofuranos/metabolismo , Chalconas/metabolismo , Cumarínicos/metabolismo , Inibidores do Citocromo P-450 CYP2E1/metabolismo , Inibidores do Citocromo P-450 CYP2E1/toxicidade , Inibidores do Citocromo P-450 CYP3A/metabolismo , Inibidores do Citocromo P-450 CYP3A/toxicidade , Interações Medicamentosas , Isoflavonas/metabolismo , Ratos Sprague-Dawley
15.
Life Sci ; 239: 117044, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31715187

RESUMO

BACKGROUND: Galbanic acid (GBA), which is known as a sesquiterpene coumarin, has been reported to have various anti-tumor activities in different cells. Our study intended to investigate whether curcumin potentiates GBA-induced anti-tumor effect in non-small cell lung cancer cells. MATERIALS AND METHODS: The combined effect of GBA and curcumin on cell viability was examined by MTT analysis. Cellular apoptosis was evaluated by flow cytometry analysis. Autophagy was defined by autophagosome observed by confocal microscopy after infected with GFP-LC3 adenovirus. In addition, the expression of marker proteins involved in cell apoptosis, autophagy, and Akt/mTOR signaling pathway were estimated by qRT-PCR and Western Blotting assay. RESULTS: 15 µM curcumin combined with 40 µM GBA could obtain better synergistic repressive efficacy on cell viability and notably induced cell apoptosis in A549 cells. Besides, curcumin in alliance with GBA could significantly inhibit cell migration and invasion. GFP-LC3 infection experiments elaborated that curcumin could potentiate GBA induced cell autophagy and restrain the phosphorylation of Akt/mTOR/P70s6k signaling pathway. What's more, the reaction of migration, apoptosis, and autophagy induced by curcumin and GBA treatment could be reversed by mTOR inhibitor rapamycin and AKT activator insulin.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Cumarínicos/farmacologia , Curcumina/farmacologia , Células A549 , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cumarínicos/metabolismo , Curcumina/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
16.
Pestic Biochem Physiol ; 161: 68-76, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31685199

RESUMO

Over the last 50 years numerous studies were published by insect toxicologists using native microsomal membrane preparations in order to investigate in vitro cytochrome P450-(P450) mediated oxidative metabolism of xenobiotics, including insecticides. Whereas the preparation of active microsomal membranes from many pest insect species is straightforward, their isolation from honey bees, Apis mellifera (Hymenoptera: Apidae) remained difficult, if not impossible, due to the presence of a yet unidentified endogenous inhibitory factor released during abdominal gut membrane isolation. Thus hampering in vitro toxicological studies on microsomal oxidative phase 1 metabolism of xenobiotics, including compounds of ecotoxicological concern. The use of microsomal membranes rather than individually expressed P450s offers advantages and allows to develop a better understanding of phase 1 driven metabolic fate of foreign compounds. Here we biochemically investigated the problems associated with the isolation of active honey bee microsomes and developed a method resulting in highly active native microsomal preparations from adult female worker abdomens. This was achieved by removal of the abdominal venom gland sting complex prior to microsomal membrane preparation. Molecular sieve chromatography of the venom sac content leads to the identification of phospholipase A2 as the enzyme responsible for the immediate inhibition of cytochrome P450 activity in microsomal preparations. The substrate specificity of functional honey bee microsomes was investigated with different fluorogenic substrates, and revealed a strong preference for coumarin over resorufin derivatives. Furthermore we were able to demonstrate the metabolism of insecticides by honey bee microsomes using an approach coupled to LC-MS/MS analysis of hydroxylated metabolites. Our work provides access to a new and simple in vitro tool to study honey bee phase 1 metabolism of xenobiotics utilising the entire range of microsomal cytochrome P450s.


Assuntos
Abelhas/metabolismo , Microssomos/metabolismo , Fosfolipases A2/isolamento & purificação , Abdome , Animais , Cumarínicos/metabolismo , Hidroxilação , Microssomos/enzimologia , Neonicotinoides/metabolismo , Inibidores de Fosfolipase A2/farmacologia , Fosfolipases A2/metabolismo , Especificidade por Substrato , Xenobióticos/metabolismo
17.
Dalton Trans ; 48(48): 17818-17830, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31774094

RESUMO

An unsymmetrical o-phenylenediamine derivative, L (7-hydroxy-4-methyl-8-(1-(phenyl- (pyridin-2-yl)methyl)-1H-benzo[d]imidazol-2-yl)-2H-chromen-2-one), has been synthesized from (E)-N1-(phenyl(pyridine-2-yl)methylene)benzene-1,2-diamine with 7-hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde and characterized by X-ray, IR, 1H NMR and ESI-MS spectral analyses. The X-ray structure shows L as a cyclic benzimidazole derivative, but it undergoes ring-opening upon reaction with transition metal ions. L is non-emissive in 9 : 1 (v/v) EtOH/H2O (HEPES buffer, pH 7.4) but becomes highly fluorescent upon Zn2+ coordination, and the emissive Zn(ii) complex undergoes transmetallation in the presence of Cu2+ ions specifically, followed by amine to imine oxidation, i.e. an oxidative dehydrogenation (OD) reaction -(2e + 2H+) occurs. The transmetallation of Zn2+ from the complex by Cu2+ (CuCl2) separated the non-emissive X-ray diffractable crystal of [Cu(L'')Cl] (L'' = amine oxidized form of L). A square pyramidal [Cu(L'')][ClO4] complex was also isolated from the reaction of L with Cu(CH3CN)4(ClO4) in the presence of air, and in this complex the amine is also oxidized to the imine. Here, copper ions in the presence of air play an important role in the OD reaction of L as determined by EPR and cyclic voltammetry studies. The ligand, L, is used for Zn2+ ion recovery from a municipally supplied water sample. A paper strip detection kit for Zn2+ and Cu2+ is designed using L. The ligand is also used for intracellular Zn2+ detection in a human liver cancer cell line (HepG2).


Assuntos
Cobre/química , Cumarínicos/metabolismo , Complexos de Coordenação/química , Cumarínicos/química , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Modelos Moleculares , Estrutura Molecular , Oxirredução
18.
Molecules ; 24(19)2019 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-31569547

RESUMO

Natural coumarins are present in remarkable amounts as secondary metabolites in edible and medicinal plants, where they display interesting bioactivities. Considering the wide enzymatic arsenal of filamentous fungi, studies on the biotransformation of coumarins using these microorganisms have great importance in green chemical derivatization. Several reports on the biotransformation of coumarins using fungi have highlighted the achievement of chemical analogs with high selectivity by using mild and ecofriendly conditions. Prompted by the enormous pharmacological, alimentary, and chemical interest in coumarin-like compounds, this study evaluated the biotransformation of nine coumarin scaffolds using Cunninghamella elegans ATCC 10028b and Aspergillus brasiliensis ATCC 16404. The chemical reactions which were catalyzed by the microorganisms were highly selective. Among the nine studied coumarins, only two of them were biotransformed. One of the coumarins, 7-hydroxy-2,3-dihydrocyclopenta[c]chromen-4(1H)-one, was biotransformed into the new 7,9-dihydroxy-2,3-dihydrocyclopenta[c]chromen-4(1H)-one, which was generated by selective hydroxylation in an unactivated carbon. Our results highlight some chemical features of coumarin cores that are important to biotransformation using filamentous fungi.


Assuntos
Biotransformação , Cumarínicos/metabolismo , Fungos/metabolismo , Cromatografia Líquida de Alta Pressão , Cumarínicos/química , Cumarínicos/isolamento & purificação , Estrutura Molecular
19.
Med Sci Monit ; 25: 8025-8033, 2019 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-31654522

RESUMO

BACKGROUND This study aimed to investigate the effects of xanthoxyletin, a plant-derived coumarin, on human oral squamous cancer cells in vitro and in mouse xenografts in vivo. MATERIAL AND METHODS The study included SCC-1 human oral cancer cells and EBTr normal embryonic bovine tracheal epithelial cells, which were treated with 0 µM, 5 µM, 10 µM, and 20 µM of xanthoxyletin for 24 hours. The MTT assay assessed cell viability, and autophagy was detected by electron microscopy. Cell apoptosis was investigated using 4',6-diamidino-2-phenylindole (DAPI), annexin V, and propidium iodide (PI) fluorescence flow cytometry, which was also used to investigate the cell cycle. Protein expression was measured by Western blot. Mouse xenografts were used for the in vivo evaluation of the effects of xanthoxyletin. RESULTS Xanthoxyletin significantly inhibited the proliferation of oral cancer cells (IC50, 10-30 µM) with lower cytotoxicity for normal cells. Xanthoxyletin treatment was associated with G2/M arrest of the cell cycle and with increased apoptosis and autophagy of SCC-1 cells. Apoptosis and autophagy induced by xanthoxyletin were also associated with changes in expression of the apoptosis-associated proteins, Bax and Bcl-2, and the autophagy-associated proteins, LC3I, LC3II, Beclin 1, p62, and VSp34. Xanthoxyletin inhibited the expression of components of the signaling cascade of the MEK/ERK pathway in the SCC-1 oral cancer cells. The in vivo effects of xanthoxyletin showed inhibition of growth of mouse xenografts. CONCLUSIONS Xanthoxyletin inhibited the proliferation of human oral squamous carcinoma cells and induced apoptosis, autophagy, and cell cycle arrest by modulation of the MEK/ERK signaling pathway.


Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Cumarínicos/farmacologia , Neoplasias Bucais/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Bovinos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cumarínicos/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Nus , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
20.
BMC Plant Biol ; 19(1): 450, 2019 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-31655554

RESUMO

BACKGROUND: Secondary metabolites play an important role in the plant defensive response. They are produced as a defence mechanism against biotic stress by providing plants with antimicrobial and antioxidant weapons. In higher plants, the majority of secondary metabolites accumulate as glycoconjugates. Glycosylation is one of the commonest modifications of secondary metabolites, and is carried out by enzymes called glycosyltransferases. RESULTS: Here we provide evidence that the previously described tomato wound and pathogen-induced glycosyltransferase Twi1 displays in vitro activity toward the coumarins scopoletin, umbelliferone and esculetin, and the flavonoids quercetin and kaempferol, by uncovering a new role of this gene in plant glycosylation. To test its activity in vivo, Twi1-silenced transgenic tomato plants were generated and infected with Tomato spotted wilt virus. The Twi1-silenced plants showed a differential accumulation of Twi1 substrates and enhanced susceptibility to the virus. CONCLUSIONS: Biochemical in vitro assays and transgenic plants generation proved to be useful strategies to assign a role of tomato Twi1 in the plant defence response. Twi1 glycosyltransferase showed to regulate quercetin and kaempferol levels in tomato plants, affecting plant resistance to viral infection.


Assuntos
Cumarínicos/metabolismo , Flavonoides/metabolismo , Glicosiltransferases/metabolismo , Lycopersicon esculentum/enzimologia , Doenças das Plantas/imunologia , Tospovirus/patogenicidade , Regulação da Expressão Gênica de Plantas , Glicosilação , Glicosiltransferases/genética , Lycopersicon esculentum/genética , Lycopersicon esculentum/imunologia , Lycopersicon esculentum/virologia , Doenças das Plantas/virologia , Imunidade Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
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