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1.
Talanta ; 206: 120195, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514831

RESUMO

The ability to separate bioactive compounds from herbal medicines, which contain abundant components, is crucial for drug discovery. Conventional Countercurrent chromatography (CCC) methods for separating bioactive compounds are labor intensive and show low efficiency. Here, we present a novel integrative CCC method for separating lysine-specific demethylase 1 (LSD1) inhibitors from the roots of Salvia miltiorrhiza (RSM). The methanol extracts of RSM were separated into hydrosoluble and liposoluble fractions, which were online stored in coils. Subsequently, the targeting LSD1 constituents were isolated using isocratic, gradient, or recycling elution mode. All separation processes could be accomplished using one CCC apparatus. Using our separation strategy, two phenylpropanoids and four tanshinones were isolated, which were determined to be new classes of natural LSD1 inhibitors. Salvianolic acid B, which showed the most potent inhibitory activity with an IC50 of 0.11 µM, exhibiting a considerable potential as an anticancer agent. Promisingly, the integrative CCC could be a crucial tool for the target separation of enzyme inhibitors from herbal medicines.


Assuntos
Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Raízes de Plantas/química , Salvia miltiorrhiza/química , Benzofuranos/isolamento & purificação , Benzofuranos/metabolismo , Benzofuranos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Cinamatos/isolamento & purificação , Cinamatos/metabolismo , Cinamatos/farmacologia , Distribuição Contracorrente/métodos , Depsídeos/isolamento & purificação , Depsídeos/metabolismo , Depsídeos/farmacologia , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/metabolismo , Histona Desmetilases/metabolismo , Humanos , Simulação de Acoplamento Molecular , Fenantrenos/isolamento & purificação , Fenantrenos/metabolismo , Fenantrenos/farmacologia , Ligação Proteica
2.
Zhongguo Zhong Yao Za Zhi ; 44(21): 4704-4712, 2019 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-31872668

RESUMO

Rosmarinic acid,a hydrosoluble polyphenolic hydroxyl compound,is the active ingredient in such traditional Chinese medicines as Menthae Haplocalycis Herba,Salviae Miltiorrhizae Radix et Rhizoma,Rosemary,Perillae Folium. Because of its good anti-inflammatory,anti-oxidant and anti-tumor effects,it is widely used in food,medicine and other fields. However,the metabolic process and metabolites of rosmarinic acid in vivo have not been completely defined. In this study,an efficient method of ultra-high performance liquid chromatography combined with linear ion trap-Orbitrap(UHPLC-LTQ-Orbitrap) mass spectrometer was used to analyze the metabolites in vivo of rosmarinic acid in rats. Plasma,urine and feces samples were collected after oral administration of rosmarinic acid. After biological samples were processed by solid phase extraction,Acquity UPLC  BEH C18 column(2. 1 mm × 100 mm,1. 7 µm) was used with 0. 1% formic acid(A)-acetonitrile(B) solution as the mobile phase at the speed of 0. 30 m L·min-1 and temperature of 35 ℃ under gradient conditions. The plasma,urine,feces and the blank samples were then analyzed by ESI-LTQ-Orbitrap under both negative and positive ion modes. Based on the accurate mass measurement(<5),MS/MS fragmentation patterns,standards and literatures,a total of 36 metabolites were screened out and identified in the biological samples collected from rats after intragastric administration. Three were identified 3 from rat plasma,31 from urine,and 7 from feces. The main metabolic pathways of rosmarinic acid in rats can be divided into five parts. Rosmarinic acid were first decomposed into small molecules,such as trans-caffeic acid,coumaric acid,m-hydroxybenzoic acid and Danshensu,which were followed by sulfation,methylation,glucuronic acid conjugation and glucose conjugation. The results showed that UHPLC-LTQ-Orbitrap mass spectrometer could be used to analyze the metabolism of rosmarinic acid in rats,and provide reference for further studies on toxicology,pharmacodynamics and secondary development of Chinese medicine.


Assuntos
Cinamatos/metabolismo , Depsídeos/metabolismo , Medicamentos de Ervas Chinesas/metabolismo , Espectrometria de Massas em Tandem , Animais , Cromatografia Líquida de Alta Pressão , Ratos
3.
Enzyme Microb Technol ; 131: 109392, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615678

RESUMO

Rosmarinic acid (RA), as a hydroxycinnamic acid ester of caffeic acid (CA) and 3,4-dihydroxyphenyllactic acid (3,4-DHPL), is a phenylpropanoid-derived plant natural product and has diverse biological activities. This work acts as a modular platform for microbial production using a two-cofactor (ATP and CoA) regeneration system to product RA based on a cell-free biosynthetic approach. Optimal activity of the reaction system was pH 8 and 30 °C. Total turnover number for ATP and CoA was 820.60 ±â€¯28.60 and 444.50 ±â€¯9.65, respectively. Based on the first hour data, the RA productivity reached 320.04 mg L-1 h-1 (0.889 mM L-1 h-1).


Assuntos
Trifosfato de Adenosina/metabolismo , Anti-Inflamatórios não Esteroides/metabolismo , Cinamatos/metabolismo , Coenzima A/metabolismo , Depsídeos/metabolismo , Sistema Livre de Células , Concentração de Íons de Hidrogênio , Temperatura Ambiente
4.
Nat Commun ; 10(1): 4071, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492833

RESUMO

Biological production and application of a range of organic compounds is hindered by their limited solubility and toxicity. This work describes a process for functionalization of phenolic compounds that increases solubility and decreases toxicity. We achieve this by screening a wide range of sulfotransferases for their activity towards a range of compounds, including the antioxidant resveratrol. We demonstrate how to engineer cell factories for efficiently creating sulfate esters of phenolic compounds through the use of sulfotransferases and by optimization of sulfate uptake and sulfate nucleotide pathways leading to the 3'-phosphoadenosine 5'-phosphosulfate precursor (PAPS). As an example we produce the antifouling agent zosteric acid, which is the sulfate ester of p-coumaric acid, reaching a titer of 5 g L-1 in fed-batch fermentation. The described approach enables production of sulfate esters that are expected to provide new properties and functionalities to a wide range of application areas.


Assuntos
Fontes de Energia Bioelétrica , Cinamatos/metabolismo , Fenóis/metabolismo , Sulfatos/metabolismo , Ésteres do Ácido Sulfúrico/metabolismo , Reatores Biológicos , Cinamatos/química , Escherichia coli/metabolismo , Glucose/metabolismo , Filogenia , Saccharomyces cerevisiae/metabolismo , Sulfotransferases/metabolismo , Ésteres do Ácido Sulfúrico/química , Transcriptoma/genética , Tirosina/metabolismo
5.
J Biotechnol ; 303: 72-79, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31381941

RESUMO

Carboxylic acid reductases (CARs) catalyze ATP- and NADPH-dependent reduction of carboxylic acids to corresponding aldehydes. Although successful applications of these enzymes for the bioconversion of monocarboxylic acids have already been reported, their applicability for the reduction of dicarboxylic acids is not well understood. Here, we explored the possibility of engineering CARs for enhanced activity toward succinic acid for potential applications in 1,4-butanediol production. Structural models of the carboxylate-binding pocket of the CAR enzyme MAB4714 from Mycobacterium abscessus suggested that its reactivity toward succinic acid could be enhanced by reducing the pocket volume. Using site-directed mutagenesis, we introduced larger side chains into the MAB4714 carboxylate binding pocket and compared the activity of 16 mutant proteins against cinnamic and succinic acids. These experiments revealed that, although the reaction rates remain low, the replacement of Leu284 or Thr285 with Trp increased activity toward succinic acid more than two times. The T285E mutant protein also showed increased activity toward succinic acid, but it was lower than that of T285W. The mutated residues of MAB4714 are located on the flexible loop covering the carboxylate-binding pocket, which appears to contribute to substrate preference of CARs. Thus, reductase activity of CARs against succinic acid can be improved by introducing large side chains into the carboxylate-binding pocket. We also discovered that alanine replacement of the conserved Ser713 in the CAR phosphopantetheine attachment site resulted in complete degradation of the full-length protein into separate A and R domains, suggesting that CAR phosphopantetheinylation is important for its stability in solution.


Assuntos
Mycobacterium abscessus/enzimologia , Oxirredutases/genética , Oxirredutases/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Butileno Glicóis/metabolismo , Cinamatos/metabolismo , Clonagem Molecular , Mutagênese Sítio-Dirigida , Mycobacterium abscessus/genética , Proteólise , Ácido Succínico/metabolismo
6.
J Biotechnol ; 304: 44-51, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31419454

RESUMO

A novel type III fungal CAR was identified from the organism Thermothelomyces thermophila. High expression levels were observed in E. coli using the pETDuet-1 plasmid system in combination with an autoinduction protocol. A broad substrate scope ranging from aromatic to aliphatic carboxylic acids was tested and TtCAR showed activity for all substrates. High specific activities for aromatic substrates and short chain aliphatic substrates were observed, comparable to those of NcCAR, the first type III fungal CAR. TtCAR's pH and temperature optima were at 6.5 and 30 °C, respectively. Up to 20% (v/v) cosolvents did not show a decrease in specific activity of TtCAR using (E)-cinnamic acid as a substrate. Its half-life at 40 °C was determined to be 8.25 h and its melting temperature (Tm) was 56 °C. In vitro reactions with TtCAR reduced 95.2% of 10 mM vanillic acid, which correlated to a titer of 1.4 g L-1 of vanillin. The space time yield of 0.029 g L-1 h-1 indicates that further improvements would be necessary for an industrially relevant application. This would be especially important when competing against de novo synthesis of bio vanillin by microbial strains producing >30 g L-1. In de novo and in vivo biosynthesis systems, by-products are fairly common. By contrast, we were pleased to observe less than 0.7% of vanillyl alcohol formed, making the cell-free acid reduction in the envisaged sequential two-step bioconversion from eugenol to vanillin very attractive.


Assuntos
Benzaldeídos/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Saccharomycetales/enzimologia , Cinamatos/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Oxirredutases/química , Conformação Proteica , Engenharia de Proteínas , Saccharomycetales/genética , Especificidade por Substrato , Termodinâmica , Ácido Vanílico/metabolismo
7.
J Antibiot (Tokyo) ; 72(10): 775-778, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31327868

RESUMO

A new N-cinnamoyl tripeptide, designated cipralphelin (1), was isolated from a cultured broth of Penicillium brevicompactum FKJ-0123 by physicochemical (PC) screening. Compound 1 was purified by silica gel and ODS column chromatography followed by preparative HPLC. The structure of 1 was determined as N-cinnamoyl-prolyl-alanyl-phenylalanine methyl ester by nuclear magnetic resonance and mass spectrometry analyses. The absolute configurations of three amino acids were determined by an advanced Marfey's method applied to the hydrolysate of 1. Compound 1 was evaluated for its cytotoxicity, anti-microbial activity, and ability to scavenge or quench reactive oxygen species (ROS) such as superoxide anion radicals, hydroxy radicals, and singlet oxygen. Compound 1 exhibited potent scavenging activity against hydroxy radicals.


Assuntos
Antioxidantes/farmacologia , Produtos Biológicos/farmacologia , Cinamatos/farmacologia , Depuradores de Radicais Livres/farmacologia , Oligopeptídeos/farmacologia , Penicillium/metabolismo , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/metabolismo , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/metabolismo , Cromatografia Líquida , Cinamatos/química , Cinamatos/isolamento & purificação , Cinamatos/metabolismo , Depuradores de Radicais Livres/química , Depuradores de Radicais Livres/isolamento & purificação , Depuradores de Radicais Livres/metabolismo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , Oligopeptídeos/química , Oligopeptídeos/isolamento & purificação , Oligopeptídeos/metabolismo , Penicillium/crescimento & desenvolvimento
8.
Aquat Toxicol ; 214: 105241, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31301543

RESUMO

The purpose of this study was to investigate the bioaccumulation and biochemical responses exposed to one of the main organic ultraviolet (UV) pollutants in the environment, ethylhexyl methoxy cinnamate (EHMC), and its main transformation product, either alone or in combination in zebrafish (Danio rerio). Four-month-old zebrafish were exposed to EHMC (34.4, 344 nmol/L) solution for 14 days, the species and contents of EHMC transformation products in zebrafish were determined and 3,5-dichloro-2-hydroxyacetophenone (3,5DCl2HAcP) was the one with the highest concentration in transformation products. Then, zebrafish were exposed to EHMC, 3,5DCl2HAcP alone and mixed solution for 21 days. At 7, 14 and 21 d, the related indexes of antioxidant defense system were determined. Results showed that both EHMC and 3,5DCl2HAcP can lead to the increase of malondialdehyde (MDA) and glutathione (GSH) contents, superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) activities in visceral mass compared with the corresponding control group, thus produced oxidative stress effect in organism and 3,5DCl2HAcP even showed stronger oxidative stress than EHMC. The effects of the two lower concentration co-exposure groups were similar and more significant to that of single exposure groups, while excessive oxidative stress occurred at the highest co-exposure group indicated by the decrease of GSH content, SOD, CAT, GR activities and the continued increase of MDA content. At 21 d, estradiol (E2), vitellogenin (Vtg) and testosterone (T) contents, estrogen receptor (Esr), progesterone receptor (Pgr), androgen receptor (Ar), Vtg1, P450 aromatase (Cyp19a1) and 17ß-hydroxysteroid dehydrogenase (Hsd17b3) expression were all significantly increased when exposed to 3,5DCl2HAcP alone, showing complex estrogen and androgen effects. When exposed to EHMC alone, E2 and Vtg contents, Esr, Pgr, Vtg1, Cyp19a1 and Hsd17b1 gene expression levels decreased significantly, and T content and Ar and Hsd17b3 expression increased significantly, indicated that EHMC can produce anti-estrogen and androgen effect. Last, the decrease of estrogen effect and increase of androgen effect in co-exposure group suggested that 3,5DCl2HAcP might weaken the estrogen effect and promote the androgen effect of EHMC.


Assuntos
Cinamatos/metabolismo , Peixe-Zebra/metabolismo , Acetofenonas/metabolismo , Animais , Antioxidantes/metabolismo , Biotransformação/efeitos dos fármacos , Catalase/metabolismo , Cinamatos/toxicidade , Estradiol/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Malondialdeído/metabolismo , Músculos/efeitos dos fármacos , Músculos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Testosterona/metabolismo , Vitelogeninas/metabolismo , Poluentes Químicos da Água/toxicidade
9.
Food Chem ; 297: 124910, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253292

RESUMO

Polyphenols can inhibit the enzymatic browning in food, but their indistinct synergistic effect and conformational change have limited their applications. In this paper, the mixture of quercetin, cinnamic acid and ferulic acid (Group 11, KI = 0.239 mM) possessed a higher inhibition ability than quercetin (KI = 0.361 mM), which could promote the spontaneous binding process. The final Group 11-tyrosinase complex is more stable, and the hydrophobic effect is the major driving force during the binding process. Moreover, there is not a direct relationship between the destruction of secondary structures and catalytic activity of tyrosinase. The interaction between ferulic acid and tyrosinase could destroy the secondary structures of enzyme but it had little impact on the tyrosinase activity. Molecular docking suggested that three polyphenols from Group 11 have synergistic effect on tyrosinase. This study provides new perspectives about the development of tyrosinase inhibitors in food products.


Assuntos
Inibidores Enzimáticos/química , Monofenol Mono-Oxigenase/antagonistas & inibidores , Polifenóis/química , Sítios de Ligação , Cinamatos/química , Cinamatos/metabolismo , Ácidos Cumáricos/química , Ácidos Cumáricos/metabolismo , Inibidores Enzimáticos/metabolismo , Cinética , Simulação de Acoplamento Molecular , Monofenol Mono-Oxigenase/metabolismo , Estrutura Secundária de Proteína , Termodinâmica
10.
Sci Total Environ ; 686: 729-736, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31195281

RESUMO

Ethylhexyl methoxycinnamate (EHMC) is one of the most frequently used UV filters in sunscreens and other cosmetic products. Its ubiquitous presence in various environmental matrices and its endocrine disrupting properties have been widely reported. However, we know little about the effect of EHMC exposure on humans, mainly due to its fast metabolism. In this study, urine and plasma of EHMC-dosed rats were analysed to identify its major metabolites. Five metabolites were found, with four firstly reported. Two metabolites were putatively identified as 4-methoxycinnamic acid (4-MCA) and 4'-methoxyacetophenone (4'-MAP). Quantitative results revealed that their excretion concentrations were much higher than the parent compound. Because of these high concentrations, for the human biomonitoring study, EHMC and these two metabolites were detected simultaneously in urine samples from Chinese children and adolescents. The results indicated wide exposure to EHMC, 4-MCA and 4'-MAP. The correlation between urinary concentration of EHMC and 4-MCA as well as 4-MCA and 4'-MAP provided important clues as to the sources and metabolic pathways among these three compounds. Several demographic factors were also assessed with the exposure level. As the first human exposure study of EHMC in a Chinese population, this report would help to establish an exposure database facilitating health risk assessment of EHMC.


Assuntos
Cinamatos/metabolismo , Protetores Solares/metabolismo , Animais , Cinamatos/urina , Disruptores Endócrinos/metabolismo , Humanos , Ratos , Medição de Risco
11.
Biomed Chromatogr ; 33(9): e4599, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31108569

RESUMO

Rosmarinic acid (RA) is a caffeic acid derivative and one of the most abundant and bioactive constituents in Java tea (Orthosiphon stamineus), which has significant biological activities. However, relatively few studies have been conducted to describe this compound's metabolites in vivo. Therefore, an ultra-high-performance liquid chromatography coupled to quadrupole-time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) analysis with a three-step data mining strategy was established for the metabolic profile of RA. Firstly, the exogenously sourced ions were filtered out by the MarkerView software and incorporated with Microsoft Office Excel software. Secondly, a novel modified mass detects filter strategy based on the predicted metabolites was developed for screening the target ions with narrow, well-defined mass detection ranges. Thirdly, the diagnostic product ions and neutral loss filtering strategy were applied for the rapid identification of the metabolites. Finally, a total of 16 metabolites were reasonably identified in urine, bile and feces, while metabolites were barely found in plasma. The metabolites of RA could also be distributed rapidly in liver and kidney. Glucuronidation, methylation and sulfation were the primary metabolic pathways of RA. The present findings might provide the theoretical basis for evaluating the biological activities of RA and its future application.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cinamatos/análise , Cinamatos/metabolismo , Mineração de Dados/métodos , Depsídeos/análise , Depsídeos/metabolismo , Orthosiphon/química , Espectrometria de Massas em Tandem/métodos , Animais , Cinamatos/química , Cinamatos/farmacocinética , Depsídeos/química , Depsídeos/farmacocinética , Masculino , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual
12.
Phytochemistry ; 164: 50-59, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31078779

RESUMO

Methyl (E)-cinnamate is a specialized metabolite that occurs in a variety of land plants. In flowering plants, it is synthesized by cinnamic acid methyltransferase (CAMT) that belongs to the SABATH family. While rarely reported in bryophytes, methyl (E)-cinnamate is produced by some liverworts of the Conocephalum conicum complex, including C. salebrosum. In axenically grown thalli of C. salebrosum, methyl (E)-cinnamate was detected as the dominant compound. To characterize its biosynthesis, six full-length SABATH genes, which were designated CsSABATH1-6, were cloned from C. salebrosum. These six genes showed different levels of expression in the thalli of C. salebrosum. Next, CsSABATH1-6 were expressed in Escherichia coli to produce recombinant proteins, which were tested for methyltransferase activity with cinnamic acid and a few related compounds as substrates. Among the six SABATH proteins, CsSABATH6 exhibited the highest level of activity with cinnamic acid. It was renamed CsCAMT. The apparent Km value of CsCAMT using (E)-cinnamic acid as substrate was determined to be 50.5 µM. In contrast, CsSABATH4 was demonstrated to function as salicylic acid methyltransferase and was renamed CsSAMT. Interestingly, the CsCAMT gene from a sabinene-dominant chemotype of C. salebrosum is identical to that of the methyl (E)-cinnamate-dominant chemotype. Structure models for CsCAMT, CsSAMT and one flowering plant CAMT (ObCCMT1) in complex with (E)-cinnamic acid and salicylic acid were built, which provided structural explanations to substrate specificity of these three enzymes. In phylogenetic analysis, CsCAMT and ObCCMT1 were in different clades, implying that methyl (E)-cinnamate biosynthesis in bryophytes and flowering plants originated through convergent evolution.


Assuntos
Cinamatos/metabolismo , Hepatófitas/metabolismo , Metiltransferases/metabolismo , Cinamatos/química , Relação Dose-Resposta a Droga , Hepatófitas/química , Metiltransferases/química , Estrutura Molecular , Relação Estrutura-Atividade
13.
Int J Mol Sci ; 20(10)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096565

RESUMO

Many medicinal plant species are currently threatened in their natural habitats because of the growing demand for phytochemicals worldwide. A sustainable alternative for the production of bioactive plant compounds are plant biofactories based on cell cultures and organs. In addition, plant extracts from biofactories have significant advantages over those obtained from plants, since they are free of contamination by microorganisms, herbicides and pesticides, and they provide more stable levels of active ingredients. In this context, we report the establishment of Satureja khuzistanica cell cultures able to produce high amounts of rosmarinic acid (RA). The production of this phytopharmaceutical was increased when the cultures were elicited with coronatine and scaled up to a benchtop bioreactor. S. khuzistanica extracts enriched in RA were found to reduce the viability of cancer cell lines, increasing the sub-G0/G1 cell population and the activity of caspase-8 in MCF-7 cells, which suggest that S. khuzistanica extracts can induce apoptosis of MCF-7 cells through activation of the extrinsic pathway. In addition, our findings indicate that other compounds in S. khuzistanica extracts may act synergistically to potentiate the anticancer activity of RA.


Assuntos
Aziridinas/farmacologia , Cinamatos/metabolismo , Cinamatos/farmacologia , Cicloexenos/farmacologia , Depsídeos/metabolismo , Depsídeos/farmacologia , Espécies em Perigo de Extinção , Extratos Vegetais/farmacologia , Satureja/metabolismo , Adenocarcinoma/tratamento farmacológico , Reatores Biológicos , Caspase 8/metabolismo , Caspases/metabolismo , Técnicas de Cultura de Células , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Hep G2/efeitos dos fármacos , Humanos , Células MCF-7 , Compostos Fitoquímicos/farmacologia , Plantas Medicinais/química , Satureja/crescimento & desenvolvimento
14.
J Pharm Biomed Anal ; 172: 126-138, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31035094

RESUMO

Salvia miltiorrhiza Bunge is a traditional Chinese medicine, and its water-soluble phenolic acid active compounds have very important medicinal value; however, the synthesis pathways of the main active ingredients remain unknown. Here, we employed nuclear magnetic resonance (NMR)-based metabolomics and transcriptomics techniques to study the biosynthesis mechanism of salvianolic acids. High-performance liquid chromatography (HPLC) combined with NMR showed an improvement over traditional techniques, and 54 metabolites were detected. The results of the multivariate statistical analysis showed that salvianolic acid B (SAB), rosmarinic acid (RA), caffeic acid, succinate, and citrate were among the multiple compounds that were increased in the methyl jasmonate (MeJA)-elicited group; the levels of sucrose, fructose, glutamine, and tyrosine were decreased. Combined with the differentially expressed genes (DEGs) found by transcriptome sequencing, we speculate that the synthesis of RA after MeJA treatment mostly occurred through caffeic acid and bypassed 4-hydroxyphenyllactic acid. This provides useful information for the study of salvianolic acids synthesis.


Assuntos
Benzofuranos/metabolismo , Cinamatos/metabolismo , Depsídeos/metabolismo , Espectroscopia de Prótons por Ressonância Magnética/métodos , Salvia miltiorrhiza/metabolismo , Acetatos/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Células Cultivadas , Cromatografia Líquida de Alta Pressão/métodos , Ciclopentanos/farmacologia , Perfilação da Expressão Gênica/métodos , Metabolômica/métodos , Oxilipinas/farmacologia , Fenilpropionatos/metabolismo , Raízes de Plantas/citologia
15.
Acta Pharmacol Sin ; 40(8): 1106-1118, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30792487

RESUMO

Cinnamic acid and its analogues (pyragrel and ozagrel) undergo chain-shortened (ß-oxidative) and reductive metabolism on acyl side chain. In this study, we characterized the ß-oxidative and reductive metabolism on acyl side chain of cinnamic acid and its analogues using primary rat hepatocytes, hepatic mitochondrial, and microsomal systems. A compartmental model including parent compounds and metabolites was developed to characterize in vivo ß-oxidative and reductive metabolism following an intravenous dose of parent compounds to rats. The fitted total in vivo clearance values were further compared with the in vitro values predicted by the well-stirred model. We showed that hepatic microsomal CYP450s did not catalyze ß-oxidative or reductive metabolism of the three compounds. Similar to ß-oxidation of fatty acids, ß-oxidative metabolism on their acyl side chain occurred mainly in mitochondria, which was highly dependent on ATP, CoA and NAD+. Fatty acids and NADH inhibited the ß-oxidative metabolism. Reductive metabolism occurred in both mitochondria and microsomes. Reduction in mitochondria was ATP-, CoA-, and NAD(P)H-dependent and reversible, which was suppressed by enoyl reductase inhibitor triclosan. Reduction in microsomes was ATP-, CoA-, and NADPH-dependent but little affected by triclosan. Both plasma concentrations of ß-oxidative metabolites and reductive metabolites were successfully fitted using the compartmental model. The estimated total in vivo clearance values were consistent with those predicted from hepatocytes and organelles, implicating significance of in vitro kinetics. These findings demonstrate the roles of hepatic mitochondria and microsomes in ß-oxidative and reductive metabolism on acyl side chain of cinnamic acid and its analogues along with their metabolic characteristics.


Assuntos
Cinamatos/metabolismo , Metacrilatos/metabolismo , Pirazinas/metabolismo , Animais , Cinamatos/química , Cinamatos/farmacocinética , Ácidos Graxos/metabolismo , Hepatócitos/metabolismo , Masculino , Metacrilatos/química , Metacrilatos/farmacocinética , Microssomos Hepáticos/metabolismo , Mitocôndrias Hepáticas/metabolismo , Estrutura Molecular , NAD/metabolismo , Oxirredução/efeitos dos fármacos , Pirazinas/química , Pirazinas/farmacocinética , Ratos Sprague-Dawley , Triclosan/farmacologia
16.
Chem Biol Drug Des ; 93(2): 139-146, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30216691

RESUMO

This study describes the activity of five natural hydroxycinnamic acids and derived compound: caffeic (1), rosmarinic (2), chlorogenic (3), and cryptochlorogenic (4), acids and isoverbascoside (5). All compounds inhibited Leishmania amazonensis arginase with IC50 -in range of 1.5-11 µM. Compounds 2 and 5 also showed activity against promastigotes of L. amazonensis with IC50  = 61 (28-133) µM and IC50  = 14 (9-24) µM, respectively. Further computational studies applying molecular docking simulations were performed on the competitive inhibitors to gain insight into the molecular basis for arginase inhibition and could be exploited to the development of new antileishmanials drug targeting parasite arginase.


Assuntos
Antiprotozoários/química , Arginase/metabolismo , Cinamatos/química , Leishmania/enzimologia , Proteínas de Protozoários/metabolismo , Antiprotozoários/metabolismo , Antiprotozoários/farmacologia , Arginase/antagonistas & inibidores , Sítios de Ligação , Cinamatos/metabolismo , Concentração Inibidora 50 , Cinética , Leishmania/efeitos dos fármacos , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Proteínas de Protozoários/antagonistas & inibidores
17.
ACS Chem Neurosci ; 10(1): 424-437, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30226747

RESUMO

The discovery of extended catalytic versatilities is of great importance in both the chemistry and biotechnology fields. Fatty acid amide hydrolase (FAAH) belongs to the amidase signature superfamily and is a major endocannabinoid inactivating enzyme using an atypical catalytic mechanism involving hydrolysis of amide and occasionally ester bonds. FAAH inhibitors are efficacious in experimental models of neuropathic pain, inflammation, and anxiety, among others. We report a new multitarget drug, AGN220653, containing a carboxyamide-4-oxazole moiety and endowed with efficacious analgesic and anti-inflammatory activities, which are partly due to its capability of achieving inhibition of FAAH, and subsequently increasing the tissue concentrations of the endocannabinoid anandamide. This inhibitor behaves as a noncompetitive, slowly reversible inhibitor. Autoradiography of purified FAAH incubated with AGN220653, opportunely radiolabeled, indicated covalent binding followed by fragmentation of the molecule. Molecular docking suggested a possible nucleophilic attack by FAAH-Ser241 on the carbonyl group of the carboxyamide-4-oxazole moiety, resulting in the cleavage of the C-C bond between the oxazole and the carboxyamide moieties, instead of either of the two available amide bonds. MRM-MS analyses only detected the Ser241-assisted formation of the carbamate intermediate, thus confirming the cleavage of the aforementioned C-C bond. Quantum mechanics calculations were fully consistent with this mechanism. The study exemplifies how FAAH structural features and mechanism of action may override the binding and reactivity propensities of substrates. This unpredicted mechanism could pave the way to the future development of a completely new class of amidase inhibitors, of potential use against pain, inflammation, and mood disorders.


Assuntos
Amidoidrolases/metabolismo , Analgésicos/química , Analgésicos/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Cinamatos/química , Cinamatos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Analgésicos/administração & dosagem , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Carbono/química , Carbono/metabolismo , Catálise , Cinamatos/administração & dosagem , Camundongos , Simulação de Acoplamento Molecular/métodos , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Ratos
18.
Biomed Pharmacother ; 110: 111-117, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30466000

RESUMO

In light of the widespread use of herbal medicines containing rosmarinic acid (RA) and limited literature available thereon, we investigated the metabolic interactions of RA with human cytochrome P450 monooxygenases (CYPs) and uridine diphosphate glucuronosyltransferases (UGTs). The involvement of selected enzymes (CYP1A2, CYP2C19, CYP2E1, CYP3 A4, UGT1A1, UGT1A6, and UGT2B7) in the metabolism of RA and the inhibitory effect of RA on the enzyme activity were comprehensively evaluated using human recombinant isozyme system. Additionally, concentration-dependent RA metabolism mediated by phase I enzymes (including CYPs) or UGT was investigated in human liver microsome (HLM) system. A significant disappearance of RA was observed in the seven CYP and UGT isoforms studied, indicating their possible involvement in the metabolism of RA. Based on Michaelis-Menten kinetics, the metabolism study using HLM suggests that the UGT system may have a higher capacity and lower affinity for the metabolism of RA than phase I enzyme (including CYP) systems. Moreover, RA weakly inhibited CYP2C9 and 2E1 activities with IC50 values of 39.6 and 61.0 µM, respectively, while moderately inhibiting UGT1A1, 1A6, and 2B7 with IC50 values of 9.24, 19.1, and 23.4 µM, respectively. By constructing Line weaver-Burk plots, the type of inhibition exhibited by RA on CYP and UGT activities was determined as follows: CYP2C19, mixed inhibition; CYP2E1, UGT1A1, UGT1A6, and UGT2B7, competitive inhibition. Based on the comparison of the IC50 and Ki values obtained in the current study with the previously reported plasma concentrations of RA after oral dosing in humans, it is suggested that RA may significantly inhibit the activities of the tested UGTs, rather than CYPs, in clinical settings. Thus, the present study could provide a basis for further studies on clinically significant interactions between UGT substrate drugs and herbal medicines containing RA.


Assuntos
Cinamatos/metabolismo , Inibidores das Enzimas do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Depsídeos/metabolismo , Metabolismo Energético/fisiologia , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/metabolismo , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Cinamatos/farmacologia , Inibidores das Enzimas do Citocromo P-450/farmacologia , Depsídeos/farmacologia , Relação Dose-Resposta a Droga , Metabolismo Energético/efeitos dos fármacos , Humanos
19.
Lett Appl Microbiol ; 68(2): 165-172, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30480824

RESUMO

Dithiolopyrrolone antibiotics, produced by several micro-organisms, are known for their strong antimicrobial and antitumor activities. Among of this micro-organisms, Saccharothrix algeriensis NRRL B-24137, a rare actinobacterium, has the ability to produce several dithiolopyrrolones derivatives depending on precursors added in the culture medium. After 10 days of strain fermentation on semi-synthetic medium supplemented with cinnamic acid and HPLC purification, biosynthesis of benzoyl-pyrrothine dithiolopyrrolone was evidenced through complete spectroscopic (UV-visible and 1H and 13C NMR) and spectrometric (electron impact mass spectrum) analyses. The pure molecule showed appreciable minimum inhibitory concentration values against several Gram-positive bacteria and filamentous fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: Dithiolopyrrolone antibiotics, known for their strong antimicrobial activities, gained greater interest after the discovery of their antitumor properties. Depending on precursors added, Saccharothrix algeriensis NRRL B-24137 has the ability to produce several dithiolopyrrolones derivatives. Since biological activities of dithiolopyrrolones are related to their variable structure, discover of new natural analogues to be therapeutically explored remains a significant framework of research. In this study, a new dithiolopyrrolone derivative was purified from the fermentation broth of S. algeriensis NRRL B-24137. This new antibiotic, characterized as benzoyl-pyrrothine dithiolopyrrolone, was induced by adding cinnamic acid, as precursor, to a semi-synthetic medium.


Assuntos
Actinomycetales/metabolismo , Antibacterianos/farmacologia , Fungos/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Pirrolidinonas/farmacologia , Actinomycetales/classificação , Cinamatos/metabolismo , Meios de Cultura/farmacologia , Fermentação , Testes de Sensibilidade Microbiana , Pirrolidinonas/metabolismo
20.
Environ Toxicol Chem ; 38(3): 548-560, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30569498

RESUMO

In vitro biotransformation studies were performed to support the bioaccumulation assessment of 3 hydrophobic organic ultraviolet filters (UVFs), 4-methylbenzylidene camphor (4-MBC), 2-ethylhexyl-4-methoxycinnamate (EHMC), and octocrylene. In vitro depletion rate constants (kdep ) were determined for each UVF using rainbow trout liver S9 fractions. Incubations performed with and without added cofactors showed complete (4-MBC) or partial (EHMC and octocrylene) dependence of kdep on addition of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), suggesting that hydrolysis of EHMC and octocrylene by NADPH-independent enzymes (e.g., carboxylesterases) is an important metabolic route. The concentration dependence of kdep was then evaluated to estimate Michaelis-Menten parameters (KM and Vmax ) for each UVF. Measured kdep values were then extrapolated to apparent whole-body biotransformation rate constants using an in vitro-in vivo extrapolation (IVIVE) model. Bioconcentration factors (BCFs) calculated from kdep values measured at concentrations well below KM were closer to empirical BCFs than those calculated from kdep measured at higher test concentrations. Modeled BCFs were sensitive to in vitro binding assumptions employed in the IVIVE model, highlighting the need for further characterization of chemical binding effects on hepatic clearance. The results suggest that the tested UVFs are unlikely to accumulate to levels exceeding the European Union Registration, Evaluation, Authorisation, and Restriction regulation criterion for bioaccumulative substances (BCF > 2000 L kg-1 ). However, consideration of appropriate in vitro test concentrations and binding correction factors are important when IVIVE methods are used to refine modeled BCFs. Environ Toxicol Chem 2019;38:548-560. © 2018 SETAC.


Assuntos
Oncorhynchus mykiss/metabolismo , Protetores Solares/metabolismo , Acrilatos/química , Acrilatos/metabolismo , Animais , Biotransformação , Cânfora/análogos & derivados , Cânfora/química , Cânfora/metabolismo , Cinamatos/química , Cinamatos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Fígado/metabolismo , Protetores Solares/química
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