Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.142
Filtrar
1.
Anal Bioanal Chem ; 411(28): 7431-7440, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31655858

RESUMO

Alkaline phosphatase (ALP) is an important enzyme that is associated with many human diseases, so the quantitative detection of ALP is vital from a clinical perspective. Nevertheless, most fluorescent assays for monitoring ALP depend on aggregation-induced quenching (ACQ), single-signal modulation, or a "signal off" mode, which suffer from poor sensitivity, a "false positive" problem, and low signal output. In this work, we utilized the electrostatically driven self-assembly of glutathione-capped gold nanoclusters (GSH-AuNCs, which show aggregation-induced emission, AIE) and amino-modified silicon nanoparticles (SiNPs) to create a hybrid probe (SiNPs@GSH-AuNCs). This nanohybrid probe showed emission from the SiNPs at around 470 nm as well as aggregation-induced emission enhancement (AIEE) of the GSH-AuNCs at 580 nm. The AIEE of the GSH-AuNCs was quenched in the presence of KMnO4, but the AIEE was recovered by adding ascorbic acid as an oxidation-reduction reaction occurred between KMnO4 and the ascorbic acid. The fluorescence of the SiNPs remained constant whether the AIEE was quenched or not, meaning that the fluorescence of the SiNPs could be used as an internal reference. In a typical enzymatic reaction, ascorbic acid 2-phosphate is hydrolyzed by ALP to produce ascorbic acid. Therefore, the hybrid probe was shown to allow the ratiometric detection of ALP, with a linear range of 0.5-10 U L-1 and a limit of detection (LOD) of 0.23 U L-1. Finally, the proposed analytical strategy was successfully applied to detect ALP in human serum samples and to determine the concentration of an ALP inhibitor. Graphical Abstract.


Assuntos
Fosfatase Alcalina/sangue , Ouro/química , Fosfatase Alcalina/análise , Ácido Ascórbico/análise , Inibidores Enzimáticos/análise , Glutationa/química , Humanos , Limite de Detecção , Nanopartículas Metálicas/química , Padrões de Referência , Silício/química , Espectrometria de Fluorescência
2.
Anal Bioanal Chem ; 411(27): 7327-7336, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31520170

RESUMO

Histone acetylase (HAT p300), which has aroused great concern in fundamental research and clinical applications, serves as one class of significant tumor markers. In our work, a sensitive electrochemical immunoassay for testing HAT p300 based on both graphene-assisted supported AuPd nanomaterial (AuPd@GO composite) and a typical amperometric i-t technique with fast response is developed favorably. The AuPd@GO-based sensing mechanisms are distributed as follows: the HAT p300 derived acetylation reaction occurs at the customized peptide-immobilized electrode; the AuPd@GO composite acts as carrier to immobilize acetyl antibody, thus constructing a sandwich-type electrochemical immunosensor via an antigen and antibody interaction; importantly, a distinct electrochemical signal could be caught due to the AuPd@GO nanomaterial with a favorable electrocatalytic property to the commercialized 3,3,5',5'-tetramethyl benzidine solution (TMB). Taking advantage of AuPd@GO composite, the established immunosensor displays a wide linear range from 1 pM to 1000 nM, and the detection limit is 0.5 pM (S/N = 3) for HAT p300. Next, the biosensor is also used to analyze the inhibitor of HAT p300 successfully, which is promising for promoting the development of electrochemical HAT-related biodetection and drug discovery. Graphical abstract A sensitive electrochemical immunoassay for testing HAT p300 based on both graphene-assisted supported AuPd nanomaterial (AuPd@GO composite) and a typical amperometric i-t technique with fast response is developed favorably.


Assuntos
Técnicas Eletroquímicas/instrumentação , Inibidores Enzimáticos/análise , Ouro/química , Grafite/química , Histona Acetiltransferases/análise , Nanopartículas Metálicas/química , Paládio/química , Sequência de Aminoácidos , Técnicas Biossensoriais , Histona Acetiltransferases/antagonistas & inibidores , Limite de Detecção , Peptídeos/química , Reprodutibilidade dos Testes
3.
J Chromatogr Sci ; 57(9): 838-846, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31504273

RESUMO

There is an increasing interest in screening and developing natural tyrosinase inhibitors widely applied in medicinal and cosmetic products, as well as in the food industry. In this study, an approach by ultrafiltration LC-MS and molecular docking was used to screen and identify tyrosinase inhibitors from Semen Oroxyli extract. The samples were first incubated with the tyrosinase to select the optimal binding conditions including tyrosinase concentration, incubation time and the molecular weight of ultrafiltration membrane. By comparison of the chromatographic profiles of the extracts after ultrafiltration with activated and inactivated tyrosinase, the potential inhibitors were obtained and then identified by LC-MS. The relative binding affinities of the potential inhibitors were also calculated based on the decrease of peak areas of those. As a result, seven compounds were fished out as tyrosinase inhibitors by this assay. Among them, oroxin A and baicalein showed higher tyrosinase inhibitory than resveratrol as positive drug, and their binding mode with enzyme was further verified via the molecular docking analysis. The test results showed that the proposed method was a simple, rapid, effective, and reliable method for the discovery of natural bioactive compounds, and it can be extended to screen other bioactive compounds from traditional Chinese medicines.


Assuntos
Bignoniaceae , Descoberta de Drogas/métodos , Inibidores Enzimáticos , Monofenol Mono-Oxigenase/antagonistas & inibidores , Extratos Vegetais , Cromatografia Líquida de Alta Pressão/métodos , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Flavonas/análise , Flavonas/química , Flavonas/metabolismo , Glucosídeos/análise , Glucosídeos/química , Glucosídeos/metabolismo , Espectrometria de Massas/métodos , Simulação de Acoplamento Molecular/métodos , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/metabolismo , Extratos Vegetais/análise , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Ultrafiltração/métodos
4.
Appl Microbiol Biotechnol ; 103(19): 8021-8033, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31372707

RESUMO

8-oxoguanine (GO) is a major lesion found in DNA that arises from guanine oxidation. The hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans encodes an archaeal GO DNA glycosylase (Tg-AGOG). Here, we characterized biochemically Tg-AGOG and probed its GO removal mechanism by mutational studies. Tg-AGOG can remove GO from DNA at high temperature through a ß-elimination reaction. The enzyme displays an optimal temperature, ca.85-95 °C, and an optimal pH, ca.7.0-8.5. In addition, Tg-AGOG activity is independent on a divalent metal ion. However, both Co2+ and Cu2+ inhibit its activity. The enzyme activity is also inhibited by NaCl. Furthermore, Tg-AGOG specifically cleaves GO-containing dsDNA in the order: GO:C, GO:T, GO:A, and GO:G. Moreover, the temperature dependence of cleavage rates of the enzyme was determined, and from this, the activation energy for GO removal from DNA was first estimated to be 16.9 ± 0.9 kcal/mol. In comparison with the wild-type Tg-AGOG, the R197A mutant has a reduced cleavage activity for GO-containing DNA, whereas both the P193A and F167A mutants exhibit similar cleavage activities for GO-containing DNA. While the mutations of P193 and F167 to Ala lead to increased binding, the mutation of R197 to Ala had no significant effect on binding. These observations suggest that residue R197 is involved in catalysis, and residues P193 and F167 are flexible for conformational change.


Assuntos
DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Análise Mutacional de DNA , Guanina/análogos & derivados , Thermococcus/enzimologia , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Guanina/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Temperatura Ambiente
5.
Biotechnol Lett ; 41(10): 1187-1200, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31418101

RESUMO

OBJECTIVES: Bifunctional alginate lyase can efficiently saccharify alginate biomass and prepare functional oligosaccharides of alginate. RESULTS: A new BP-2 strain that produces alginate lyase was screened and identified from rotted Sargassum. A new alginate lyase, Alg17B, belonging to the polysaccharide lyase family 17, was isolated and purified from BP-2 fermentation broth by freeze-drying, dialysis, and ion exchange chromatography. The enzymatic properties of the purified lyase were investigated. The molecular weight of Alg17B was approximately 77 kDa, its optimum reaction temperature was 40-45 °C, and its optimum reaction pH was 7.5-8.0. The enzyme was relatively stable at pH 7.0-8.0, with a temperature range of 25-35 °C, and the specific activity of the purified enzyme reached 4036 U/mg. A low Na+ concentration stimulated Alg17B enzyme activity, but Ca2+, Zn2+, and other metal ions inhibited it. Substrate specificity analysis, thin-layer chromatography, and mass spectrometry showed that Alg17B is an alginate lyase that catalyses the hydrolysis of sodium alginate, polymannuronic acid (polyM) and polyguluronic acid to produce monosaccharides and low molecular weight oligosaccharides. Alg17B is also bifunctional, exhibiting both endolytic and exolytic activities toward alginate, and has a wide substrate utilization range with a preference for polyM. CONCLUSIONS: Alg17B can be used to saccharify the main carbohydrate, alginate, in the ethanolic production of brown algae fuel as well as in preparing and researching oligosaccharides.


Assuntos
Organismos Aquáticos/enzimologia , Gammaproteobacteria/enzimologia , Polissacarídeo-Liase/isolamento & purificação , Polissacarídeo-Liase/metabolismo , Sargassum/microbiologia , Alginatos/metabolismo , Ácido Algínico/metabolismo , Organismos Aquáticos/classificação , Organismos Aquáticos/genética , Organismos Aquáticos/isolamento & purificação , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Gammaproteobacteria/classificação , Gammaproteobacteria/genética , Gammaproteobacteria/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Peso Molecular , Monossacarídeos/metabolismo , Oligossacarídeos/metabolismo , Polissacarídeo-Liase/química , Polissacarídeo-Liase/genética , Polissacarídeos Bacterianos/metabolismo , Especificidade por Substrato , Temperatura Ambiente
6.
World J Microbiol Biotechnol ; 35(9): 135, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432264

RESUMO

The feather-degrading strain Thermoactinomyces sp. YT06 secretes an extracellular keratinolytic protease (KERTYT); however, the gene encoding this protease remains unknown. The kerT1 gene (1170 bp) encoding keratinase was cloned and expressed in Escherichia coli BL21(DE3). Purified recombinant keratinase (rKERTYT) was achieved at a yield of 39.16% and 65.27-fold purification with a specific activity of 1325 U/mg. It was shown that rKERTYT has many similarities to the native enzyme (KERTYT) by characterization of rKERTYT. The molecular weight of rKERTYT secreted by recombinant E. coli was approximately 28 kDa. The optimal temperature and the pH values of rKERTYT were 65 °C and 8.5, respectively, and the protein remained stable from 50 to 60 °C and pH 6-11. The keratinase was strongly inhibited by phenyl methane sulfonyl fluoride (PMSF), suggesting that it belongs to the serine protease family. It was significantly activated by Mn2+ and ß-mercaptoethanol (ß-Me). rKERTYT showed stability and retained over 80% activity with the existence of organic solvents such as acetone, methylbenzene and dimethyl sulfoxide. These findings indicated that rKERTYT will be a promising candidate for the enzymatic processing of keratinous wastes.


Assuntos
Clonagem Molecular , Escherichia coli/metabolismo , Expressão Gênica , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Thermoactinomyces/enzimologia , Ativadores de Enzimas/análise , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Thermoactinomyces/genética
7.
Analyst ; 144(16): 4871-4879, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31298663

RESUMO

Bacterial, fungal and viral infections in plant systems are on the rise, most of which tend to spread quickly amongst crops. These pathogens are also gaining resistance to known treatments, which makes their early detection a priority to avoid extensive loss of crops and the spreading of disease to animal systems. In this work, we propose a microfluidic platform coupled with integrated thin-film silicon photosensors for the detection of pathogen infections in grapes. This detection was achieved by monitoring the concentration of Azelaic Acid (AzA). This small organic acid plays a significant role in the defense mechanism in plant systems. In this platform, the enzyme tyrosinase was immobilized on microbeads inside a microfluidic system. By colorimetric monitoring of the inhibitory effect of AzA on the enzyme tyrosinase in real time, it was possible, in under 10 minutes, to detect different concentrations of AzA in both buffer and spiked solutions of grape juice, in both cases with limits of detection in the 5-10 nM range. In addition, with this microfluidic device, it was possible to clearly distinguish infected from healthy grape samples at three different grape maturation points. Healthy grape samples showed AzA concentrations in the range of 10-20 nM (post-dilution) while infected samples have an estimated increase of AzA of 10-30×, results which were confirmed using HPLC. In both juice and grape samples an integrated sample preparation stage that decreases the phenol content of the solutions was required to achieve fit-for-purpose sensitivities to AzA.


Assuntos
Ácidos Dicarboxílicos/análise , Dispositivos Lab-On-A-Chip , Doenças das Plantas/microbiologia , Vitis/microbiologia , Biomarcadores/análise , Biomarcadores/química , Colorimetria/métodos , Ácidos Dicarboxílicos/química , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/química , Enzimas Imobilizadas/química , Sucos de Frutas e Vegetais/análise , Limite de Detecção , Técnicas Analíticas Microfluídicas/métodos , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/química
8.
Molecules ; 24(13)2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269660

RESUMO

Pesticides vary in the level of poisonousness, while a conventional rapid test card only provides a general "absence or not" solution, which cannot identify the various genera of pesticides. In order to solve this problem, we proposed a seven-layer paper-based microfluidic chip, integrating the enzyme acetylcholinesterase (AChE) and chromogenic reaction. It enables on-chip pesticide identification via a reflected light intensity spectrum in time-sequence according to the different reaction efficiencies of pesticide molecules and assures the optimum temperature for enzyme activity. After pretreatment of figures of reflected light intensity during the 15 min period, the figures mainly focused on the reflected light variations aroused by the enzyme inhibition assay, and thus, the linear discriminant analysis showed satisfying discrimination of imidacloprid (Y = -1.6525X - 139.7500), phorate (Y = -3.9689X - 483.0526), and avermectin (Y = -2.3617X - 28.3082). The correlation coefficients for these linearity curves were 0.9635, 0.8093, and 0.9094, respectively, with a 95% limit of agreement. Then, the avermectin class chemicals and real-world samples (i.e., lettuce and rice) were tested, which all showed feasible graphic results to distinguish all the chemicals. Therefore, it is feasible to distinguish the three tested kinds of pesticides by the changes in the reflected light spectrum in each min (15 min) via the proposed chip with a high level of automation and integration.


Assuntos
Inibidores Enzimáticos/análise , Dispositivos Lab-On-A-Chip , Óptica e Fotônica/métodos , Papel , Resíduos de Praguicidas/análise , Análise por Conglomerados , Ivermectina/análogos & derivados , Ivermectina/análise , Ivermectina/química , Neonicotinoides/análise , Neonicotinoides/química , Nitrocompostos/análise , Nitrocompostos/química , Forato/análise , Forato/química , Fatores de Tempo
9.
Appl Microbiol Biotechnol ; 103(16): 6571-6580, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31240367

RESUMO

Pyruvate carboxylase of Corynebacterium glutamicum serves as anaplerotic enzyme when cells are growing on carbohydrates and plays an important role in the industrial production of metabolites derived from the tricarboxylic acid cycle, such as L-glutamate or L-lysine. Previous studies suggested that the enzyme from C. glutamicum is very labile, as activity could only be measured in permeabilized cells, but not in cell-free extracts. In this study, we established conditions allowing activity measurements in cell-free extracts of C. glutamicum and purification of the enzyme by avidin affinity chromatography and gel filtration. Using a coupled enzymatic assay with malate dehydrogenase, Vmax values between 20 and 25 µmol min-1 mg-1 were measured for purified pyruvate carboxylase corresponding to turnover numbers of 160 - 200 s-1 for the tetrameric enzyme. The concentration dependency for pyruvate and ATP followed Michaelis-Menten kinetics with Km values of 3.76 ± 0.72 mM and 0.61 ± 0.13 mM, respectively. For bicarbonate, concentrations ≥5 mM were required to obtain activity and half-maximal rates were found at 13.25 ± 4.88 mM. ADP and aspartate inhibited PCx activity with apparent Ki values of 1.5 mM and 9.3 mM, respectively. Acetyl-CoA had a weak inhibitory effect, but only at low concentrations up to 50 µM. The results presented here enable further detailed biochemical and structural studies of this enzyme.


Assuntos
Corynebacterium glutamicum/enzimologia , Piruvato Carboxilase/isolamento & purificação , Piruvato Carboxilase/metabolismo , Trifosfato de Adenosina/metabolismo , Cromatografia de Afinidade , Cromatografia em Gel , Inibidores Enzimáticos/análise , Concentração de Íons de Hidrogênio , Cinética , Multimerização Proteica , Ácido Pirúvico/metabolismo
10.
Appl Microbiol Biotechnol ; 103(16): 6825-6836, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31240368

RESUMO

Polychlorinated biphenyls (PCBs) are a group of persistent organic pollutants (POPs) widely existing in the environment. Arthrobacter sp. YC-RL1 is a biphenyl-degrading bacterium that shows metabolic versatility towards aromatic compounds. A 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoate (HOPDA) hydrolase (BphD) gene involved in the biodegradation of biphenyl was cloned from strain YC-RL1 and heterologously expressed in Escherichia coli BL21 (DE3). The recombinant BphDYC-RL1 was purified and characterized. BphDYC-RL1 showed the highest activity at 45 °C and pH 7. It was stable under a wide range of temperature (20-50 °C). The enzyme had a Km value of 0.14 mM, Kcat of 11.61 s-1, and Vmax of 0.027 U/mg. Temperature dependence catalysis exhibited a biphasic Arrhenius Plot with a transition at 20 °C. BphDYC-RL1 was inactivated by SDS, Tween 20, Tween 80, Trition X-100, DTT, CHAPS, NBS, PMSF, and DEPC, but insensitive to EDTA. Site-directed mutagenesis of the active-site residues revealed that the catalytic triad residues (Ser115, His275, and Asp247) of BphDYC-RL1 were necessary for its activity. The investigation of BphDYC-RL1 not only provides new potential enzyme resource for the biodegradation of biphenyl but also helps deepen our understanding on the catalytic process and mechanism.


Assuntos
Arthrobacter/enzimologia , Compostos de Bifenilo/metabolismo , Fungicidas Industriais/metabolismo , Hidrolases/metabolismo , Arthrobacter/genética , Biotransformação , Domínio Catalítico , Clonagem Molecular , Análise Mutacional de DNA , Inibidores Enzimáticos/análise , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Hidrolases/genética , Cinética , Mutagênese Sítio-Dirigida , Temperatura Ambiente
11.
Mikrochim Acta ; 186(5): 320, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31049712

RESUMO

The oxidase-like activity of nanoceria is low. This limits its practical applications. It is demonstrated here that pyrophosphate ion (PPi) can improve the oxidase-like activity of nanoceria. Specifically, nanoceria catalyzes the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to give a blue product (oxTMB) with an absorption peak at 645 nm in the presence of PPi. If, however, alkaline phosphatase (ALP) is present, it will hydrolyze PPi, and this results in a decreased oxidase-like activity of nanoceria. Hence, less blue oxTMB willl be formed. On the other hand, if the ALP inhibitor Na3VO4 is added to the system, the oxidase-like activity of nanoceria is gradually restored. On the basis of the above results, a spectrophotometric method was developed for determination of the activity of ALP. It works in the 0.5 to 10 mU.mL-1 activity range and has a 0.32 mU.mL-1 detection limit. Na3VO4 causes a 50% ALP inhibition if present in 71 µM concentration. The assay was successfully applied to the determination of ALP in spiked human serum and gave good recoveries. Graphical abstract Schematic presentation of pyrophosphate (PPi)-induced acceleration of the oxidase-like activity of nanoceria (CeO2) for determination of alkaline phosphatase enzyme (ALP) activity and its inhibitor NaVO3.


Assuntos
Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/sangue , Cério/química , Difosfatos/química , Inibidores Enzimáticos/análise , Benzidinas/química , Catálise , Humanos , Hidrólise , Limite de Detecção , Oxirredução , Oxirredutases/química , Espectrofotometria/métodos , Vanadatos/química
12.
Biomed Chromatogr ; 33(9): e4577, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31069821

RESUMO

A method based on enzyme blocking combined with ultrafiltration liquid chromatography-mass spectrometry (LC-MS) has been developed to identify xanthine oxidase (XOD) inhibitors in the roots of Lindera reflexa Hemsl (LR) and determine their binding positions. Allopurinol and febuxostat, known XOD inhibitors, which occupy different binding positions in XOD, were used as blockers and pre-incubated with XOD. Then the LR extract was incubated without XOD, and with XOD, allopurinol-blocked XOD and febuxostat-blocked XOD before ultrafiltration LC-MS was performed. By comparing the chromatographic profiles of the incubation samples, not only the ligands, but also the binding position of these ligands with XOD could be determined. Finally, three compounds, pinosylvin, pinocembrin and methoxy-5-hydroxy-trans-stilbene, were identified as potential XOD inhibitors and the binding modes of these three compounds were shown to be similar to those of febuxostat. To verify the XOD inhibitory activity of the screened compounds, the microplate method and molecular docking in silico were used to evaluate the enzyme inhibitory activities and the binding positions with XOD. The results showed that the developed method could screen for XOD ligands in LR extracts and also determine the binding positions of the ligands. To our knowledge, this is the first report of the XOD inhibitory activity of these three compounds.


Assuntos
Inibidores Enzimáticos , Lindera/química , Extratos Vegetais/química , Xantina Oxidase/antagonistas & inibidores , Cromatografia Líquida/métodos , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/metabolismo , Espectrometria de Massas/métodos , Simulação de Acoplamento Molecular , Raízes de Plantas/química , Ultrafiltração/métodos , Xantina Oxidase/química , Xantina Oxidase/metabolismo
13.
J Pharm Biomed Anal ; 173: 75-85, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31121457

RESUMO

The phytochemical composition of different extracts obtained from stinking chamomile (Anthemis cotula L.) was investigated. Ethanol was used as solvent and accelerated solvent extraction (ASE), microwave assisted extraction (MAE), maceration, soxhlet extraction (SE), and ultrasound assisted extraction (UAE) were applied on plant material. Comparison of the phytochemical contents, antioxidant, and enzyme inhibitory properties were performed. The most abundant sesquiterpene in the extracts was anthecotuloide, while the most present phenolics were caffeoyl quinic acid, quercetin, and kaempferol derivatives. UAE extract was the most efficient in the extraction of sesquiterpenoids and polyphenols. Considering the assays on antioxidant activity and enzyme inhibition, ASE extract showed highest phenolic content (62.92 mg gallic acid equivalent/g extract). Likewise, this extract showed highest radical scavenging (103.44 mg trolox equivalent [TE]/g extract and 155.70 mg TE/g extract, for DPPH and ABTS assays respectively) and reducing power potential (435.32 and 317.89 mg TE/g extract, for CUPRAC and FRAP assays, respectively). The different extracts showed similar results in the enzyme inhibition assays suggesting that the extraction methods used have no effect on observed enzyme activities. Novelty of our findings are the inhibitory action of the ethanol extract of A. cotula aerial parts on key enzymes associated with Alzheimer's disease (acetyl cholinesterase, butyryl cholinesterase), type 2 diabetes (α-amylase, α-glucosidase), and skin hyperpigmentation disorders (tyrosinase). Data collected from the present work further appraises the multiple potential biological properties of stinking chamomile suggesting the need for further investigation on its constituents.


Assuntos
Anthemis/química , Fracionamento Químico/métodos , Extratos Vegetais/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Depuradores de Radicais Livres/análise , Depuradores de Radicais Livres/isolamento & purificação , Depuradores de Radicais Livres/farmacologia , Lactonas/análise , Lactonas/isolamento & purificação , Micro-Ondas , Componentes Aéreos da Planta/química , Extratos Vegetais/análise , Extratos Vegetais/farmacologia , Polifenóis/análise , Polifenóis/isolamento & purificação , Sesquiterpenos/análise , Sesquiterpenos/isolamento & purificação , Solventes/química , Ondas Ultrassônicas
14.
Eur J Med Chem ; 173: 154-166, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30995568

RESUMO

Aminoacyl-tRNA synthetases (aaRSs) catalyse the ATP-dependent coupling of an amino acid to its cognate tRNA. Being vital for protein translation aaRSs are considered a promising target for the development of novel antimicrobial agents. 5'-O-(N-aminoacyl)-sulfamoyl adenosine (aaSA) is a non-hydrolysable analogue of the aaRS reaction intermediate that has been shown to be a potent inhibitor of this enzyme family but is prone to chemical instability and enzymatic modification. In an attempt to improve the molecular properties of this scaffold we synthesized a series of base substituted aaSA analogues comprising cytosine, uracil and N3-methyluracil targeting leucyl-, tyrosyl- and isoleucyl-tRNA synthetases. In in vitro assays seven out of the nine inhibitors demonstrated Kiapp values in the low nanomolar range. To complement the biochemical studies, X-ray crystallographic structures of Neisseria gonorrhoeae leucyl-tRNA synthetase and Escherichia coli tyrosyl-tRNA synthetase in complex with the newly synthesized compounds were determined. These highlighted a subtle interplay between the base moiety and the target enzyme in defining relative inhibitory activity. Encouraged by this data we investigated if the pyrimidine congeners could escape a natural resistance mechanism, involving acetylation of the amine of the aminoacyl group by the bacterial N-acetyltransferases RimL and YhhY. With RimL the pyrimidine congeners were less susceptible to inactivation compared to the equivalent aaSA, whereas with YhhY the converse was true. Combined the various insights resulting from this study will pave the way for the further rational design of aaRS inhibitors.


Assuntos
Aminoacil-tRNA Sintetases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Nucleosídeos/farmacologia , Pirimidinas/farmacologia , Aminoacil-tRNA Sintetases/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/síntese química , Escherichia coli/citologia , Escherichia coli/enzimologia , Estrutura Molecular , Nucleosídeos/análise , Nucleosídeos/síntese química , Pirimidinas/análise , Pirimidinas/síntese química , Relação Estrutura-Atividade
15.
Int J Biol Macromol ; 132: 766-771, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30953721

RESUMO

The inhibitory effect of glucose on the activity of α­amylase used for starch hydrolysis was explored in this study. Four gelatinized corn starch dispersions (5 g/100 mL) containing different glucose concentrations (0.5, 1.0, 2.0 and 4.0 g/100 mL) and a control without added glucose were subjected to enzymatic hydrolysis with α­amylase (0.33 IU/mL) for 2 h. The hydrolysis kinetics showed that the limiting hydrolysis advance was reduced as glucose concentration increased. A Michaelis-Menten scheme was used for developing a mathematical model of the hydrolysis kinetics. The mathematical model predicted that the maximum hydrolysis value was consequence of the inhibition of the enzyme activity by the initial glucose load added to the gelatinized starch dispersions and by the glucose produced by amylolytic action. FTIR analysis of the Amide I band showed that glucose disrupted the secondary structure of the α­amylase, an effect that could be related to the inhibition of the enzymatic activity.


Assuntos
Inibidores Enzimáticos/farmacologia , Glucose/farmacologia , Modelos Biológicos , Amido/química , alfa-Amilases/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/análise , Géis , Glucose/análise , Hidrólise , Cinética , Estrutura Secundária de Proteína , alfa-Amilases/química
16.
Artigo em Inglês | MEDLINE | ID: mdl-30901732

RESUMO

As a novel non-purine xanthine oxidase inhibitor, WSJ-557 is a potential drug for gout. To determine the WSJ-557 concentration in plasma and various tissues of rats, a fast and sensitive method was first established by the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in this paper. The liquid-liquid extraction of ethyl acetate was adopted for the sample preparation, and carbamazepine was taken as the internal standard. In the process of chromatographic separation, MRM transitions for WSJ-557 and carbamazepine (internal standard, IS) were m/z 316.1 → 260.0 and m/z 237.0 → 194.0, correspondingly. The great linearity of WSJ-557 in all bio-samples was found in the corresponding concentration range (r > 0.99). The intra- and inter-day precision (RSD%) were below 9.5% in various tissues and plasma, whose accuracy (RE%) was within ±9.2%. This method was resoundingly employed to the WSJ-557 study on rat pharmacokinetics and tissue distribution after the intravenous administration and oral administration. The average absolute bioavailability (F) of WSJ-557 was 6.48%. The highest distribution level of gastric and intestinal tissues indicated that WSJ-557 was first absorbed in the stomach and intestine. Moreover, this analytical method provides a significant approach for the further development and investigation of WSJ-557.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Inibidores Enzimáticos/farmacocinética , Espectrometria de Massas em Tandem/métodos , Xantina Oxidase/antagonistas & inibidores , Animais , Disponibilidade Biológica , Estabilidade de Medicamentos , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/química , Feminino , Limite de Detecção , Modelos Lineares , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Distribuição Tecidual
17.
ACS Chem Biol ; 14(4): 715-724, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30831024

RESUMO

In cancers, increased fucosylation (attachment of fucose sugar residues) on cell-surface glycans, resulting from the abnormal upregulation of the expression of specific fucosyltransferase enzymes (FUTs), is one of the most important types of glycan modifications associated with malignancy. Fucosylated glycans on cell surfaces are involved in a multitude of cellular interactions and signal regulation in normal biological processes, as well as in disease. For example, sialyl LewisX is a fucosylated cell-surface glycan that is abnormally abundant in some cancers where it has been implicated in facilitating metastasis, allowing circulating tumor cells to bind to the epithelial tissue within blood vessels and invade into secondary sites by taking advantage of glycan-mediated interactions. To identify inhibitors of FUT enzymes as potential cancer therapeutics, we have developed a novel high-throughput assay that makes use of a fluorogenically labeled oligosaccharide as a probe of fucosylation. This probe, which consists of a 4-methylumbelliferyl glycoside, is recognized and hydrolyzed by specific glycoside hydrolase enzymes to release fluorescent 4-methylumbelliferone, yet when the probe is fucosylated prior to treatment with the glycoside hydrolases, hydrolysis does not occur and no fluorescent signal is produced. We have demonstrated that this assay can be used to measure the inhibition of FUT enzymes by small molecules, because blocking fucosylation will allow glycosidase-catalyzed hydrolysis of the labeled oligosaccharide to produce a fluorescent signal. Employing this assay, we have screened a focused library of small molecules for inhibitors of a human FUT enzyme involved in the synthesis of sialyl LewisX and demonstrated that our approach can be used to identify potent FUT inhibitors from compound libraries in microtiter plate format.


Assuntos
Inibidores Enzimáticos/análise , Fucose/química , Fucosiltransferases/antagonistas & inibidores , Fucosiltransferases/metabolismo , Ensaios de Triagem em Larga Escala , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Glicosilação , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Triazóis/química , Triazóis/metabolismo
18.
Mikrochim Acta ; 186(3): 190, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30771090

RESUMO

A copper(II)-based two-dimensional metal-organic framework with nanosheet structure (CuBDC NS) that possesses peroxidase (POx) mimicking activity was prepared. In the presence of hydrogen peroxide, the system catalyses the oxidation of terephthalic acid to a blue-fluorescent product (excitation = 315 nm; emission = 425 nm). Pyrophosphate has a very strong affinity for Cu2+ ion and blocks the POx-mimicking activity of the CuBDC NS. If, however, inorganic pyrophosphatase is present, the POx mimicking activity is gradually restored because pyrophosphate is hydrolyzed. The findings were used to design a method for the determination of the activity of inorganic pyrophosphatase by fluorometry. Fluorescence increases linearly in the 1-50 mU·mL-1 inorganic pyrophosphatase activity range. The limit of detection is 0.6 mU·mL-1 (S/N = 3). Graphical abstract A copper(II)-based two-dimensional metal-organic framework (CuBDC NS) is described that possesses POx-mimicking activity. Inorganic pyrophosphate (PPi) was hydrolyzed to phosphate in the presence of inorganic pyrophosphatase (PPase). Hence, it cannot coordinate with Cu2+ in CuBDC NS, its structure was well-conserved to catalyses the oxidation of terephthalic acid (H2BDC) to produce a blue fluorescent product (oxBDC) in the presence of hydrogen peroxide (H2O2).


Assuntos
Materiais Biomiméticos/química , Inibidores Enzimáticos/análise , Pirofosfatase Inorgânica/sangue , Estruturas Metalorgânicas/química , Difosfatos/química , Ensaios Enzimáticos/métodos , Fluorescência , Fluorometria/métodos , Humanos , Peróxido de Hidrogênio/química , Pirofosfatase Inorgânica/química , Limite de Detecção , Peroxidase/química , Ácidos Ftálicos/química
19.
Int J Mol Sci ; 20(4)2019 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-30781686

RESUMO

Recent studies revealed the role of lipase in the pathogenicity of Malassezia restricta in dandruff and seborrheic dermatitis (D/SD). The lipase from M. restricta (Mrlip1) is considered a potential target for dandruff therapy. In this work, we performed structure-based virtual screening in Zinc database to find the natural bioactive inhibitors of Mrlip1. We identified three compounds bearing superior affinity and specificity from the Traditional Chinese Medicine database (~60,000 compounds), and their binding patterns with Mrlip1 were analyzed in detail. Additionally, we performed three sets of 100 ns MD simulations of each complex in order to understand the interaction mechanism of Mrlip1 with known inhibitor RHC80267 and the newly identified compounds such as ZINC85530919, ZINC95914464 and ZINC85530320, respectively. These compounds bind to the active site cavity and cause conformational changes in Mrlip1. The Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) studies suggested that the average binding energy was stronger in the case of Mrlip1-ZINC85530919 and Mrlip1-ZINC95914464. The selected natural inhibitors might act as promising lead drugs against Mrlip1. Further, the present study will contribute to various steps involved in developing and creating potent drugs for several skin diseases including dandruff.


Assuntos
Inibidores Enzimáticos/análise , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Lipase/antagonistas & inibidores , Malassezia/enzimologia , Simulação de Dinâmica Molecular , Domínio Catalítico , Ligações de Hidrogênio , Ligantes , Lipase/química , Lipase/metabolismo , Simulação de Acoplamento Molecular , Análise de Componente Principal , Estrutura Secundária de Proteína , Solventes , Termodinâmica
20.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1110-1111: 116-123, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30802754

RESUMO

BLU-554 is a potent, highly selective oral FGFR4 inhibitor. A bioanalytical assay for quantification of BLU-554 in mouse plasma and six tissue homogenates (brain, kidney, liver, lung, small intestine, and spleen) was developed and validated using liquid chromatography with tandem mass spectrometric detection and with erlotinib as internal standard. After protein precipitation with acetonitrile in a 96-well format and separation on an XBridge® Peptide BEH C18 column by gradient elution using 0.2% (v/v) ammonium hydroxide (in water) and methanol, analytes were ionized by positive electrospray and monitored in the selected reaction monitoring mode by triple quadrupole mass spectrometry. The assay was validated in a 1-1000 ng/ml concentration range using calibration in mouse plasma. Precisions (intra-day and inter-day) were in the range 2.8-10.1% and accuracies were in between 88.5 and 96.6% for all levels in all matrices. The assay was successfully applied for a pilot pharmacokinetic and tissue distribution study in wild-type mice.


Assuntos
Cromatografia Líquida/métodos , Inibidores Enzimáticos/sangue , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Espectrometria de Massas em Tandem/métodos , Animais , Estabilidade de Medicamentos , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Análise dos Mínimos Quadrados , Limite de Detecção , Masculino , Camundongos , Reprodutibilidade dos Testes
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA