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1.
Food Chem ; 303: 125310, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31473456

RESUMO

Tyrosinase is a metalloenzyme involved in o-hydroxylation of monophenols and oxidation of o-diphenols to o-quinones, with formation of brown or black pigments (melanines). Tyrosinase inhibitors are of great interest in medicine and cosmetics (skin whitening compounds), but also in food and beverage industry (antibrowning agents). Here we report on the activity as mushroom tyrosinase inhibitors of a series of hydroxyphenyl thiosemicarbazones (1-5): one of them revealed an inhibitory activity stronger than kojic acid, used as reference. Enzymatic inhibition activity was confirmed by colorimetric measurements on small wheels of Fuji apples treated with the hydroxyphenyl thiosemicarbazones. The mechanism of action of compounds 1-5 was investigated by molecular modelling and by studying in solution their speciation with Cu(II) ions, the ions in the active site of the enzyme. Finally, compounds 1-5 were tested on human fibroblasts: they are not cytotoxic and they do not activate cells in a pro-inflammatory way.


Assuntos
Agaricales/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Reação de Maillard/efeitos dos fármacos , Monofenol Mono-Oxigenase/antagonistas & inibidores , Tiossemicarbazonas/química , Tiossemicarbazonas/farmacologia , Domínio Catalítico , Humanos , Cinética , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/metabolismo , Oxirredução/efeitos dos fármacos
2.
J Sci Food Agric ; 100(5): 1998-2006, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31849067

RESUMO

BACKGROUND: In this investigation, the chemical and biological profiles of three Turkish Crataegus species (Crataegus orientalis, Crataegus szovitsii and Crataegus tanacetifolia) were studied in order to provide the first comprehensive characterization and their health-promoting potential. In this respect, polyphenolic profiles were evaluated using ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. The in vitro antioxidant activities of the Crataegus samples were evaluated by using free-radical scavenging, phosphomolybdenum, ferrous-ion chelating, and reducing power assays. The inhibitory activities against α-glucosidase, amylase, cholinesterases (acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)), and tyrosinase were also established. RESULTS: The untargeted metabolomic approach highlighted the effect of both extraction types and species on the phenolic profiles of different Crateagus tissues (i.e. leaves and twigs). The methanolic extracts showed the maximum radical scavenging and reducing activity in all test systems, whereas for ferrous-ion chelating assays the decocted and infused extracts showed the highest activity. Only the methanolic extracts were effective against AChE and BChE. The extract tested showed remarkable inhibitory effects against tyrosinase and α-glucosidase, whereas all the extracts exhibited modest inhibition against α-amylase. Overall, the twig extracts of the three species studied showed superior antioxidant and enzyme inhibitory activities. CONCLUSION: On the basis of these results, the three Crataegus species can be classified as potent bioresources for high-value phytochemicals, which warrant further investigations for developing novel nutraceuticals. © 2019 Society of Chemical Industry.


Assuntos
Crataegus/química , Inibidores Enzimáticos/química , Extratos Vegetais/química , Antioxidantes/metabolismo , Butirilcolinesterase/química , Crataegus/metabolismo , Inibidores Enzimáticos/metabolismo , Metabolômica , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/química , Fenóis/química , Fenóis/metabolismo , Extratos Vegetais/metabolismo , Turquia , alfa-Amilases/antagonistas & inibidores , alfa-Amilases/química , alfa-Glucosidases/química
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.
Molecules ; 24(18)2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31510069

RESUMO

In this study, an in vitro tyrosinase inhibition assay in combination with ultra performance liquid chromatography-orbitrap mass spectrometry (UPLC-orbitrap-MS) was developed for the rapid screening and identification of tyrosinase modulators from roots of Angelica keiskei. Of the 15 candidates considered, nine chalcones, xanthoangelols (1), B (2), D (3), E (4), G (5), H (6), 4-hydroxyderricin (7), xanthokeismin B (8) and (2E)-1-[4-hydroxy-2-(2-hydroxy-2-propanyl)-2,3-dihydro-1-benzofuran-7-yl]-3-(4-hydroxyphenyl)-2-propen-1-one (9), five coumarins, umbelliferone (10), selinidin (11), isopimpinellin (12), phellopterin (13) and xanthyletin (14), and one other compound, ashitabaol A (15), were distinguished between the test samples and the controls with statistical significance, and the structure of each compound was determined by comparing with in-house standards and the literature. Among these, six compounds, xanthoangelol (1), xanthoangelol D (3), xanthoangelol H (6), 4-hydroxyderricin (7), laserpitin (16) and isolaserpitin (17), were isolated from roots of A. keiskei. Of the compounds isolated, compounds 1, 7 and 16 were subjected to tyrosinase inhibitory assay, and the IC50 values were 15.87 ± 1.21, 60.14 ± 2.29 and >100 µM, respectively. The present study indicated that the combination of in vitro tyrosinase inhibition assay coupled with UPLC-MS/MS could be widely applied to the rapid screening of active substances from various natural resources.


Assuntos
Angelica/química , Inibidores Enzimáticos/química , Monofenol Mono-Oxigenase/química , Raízes de Plantas/química , Chalcona/análogos & derivados , Chalcona/química , Chalconas/química , Cromatografia Líquida , Cumarínicos/química , Inibidores Enzimáticos/farmacologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Espectrometria de Massas em Tandem , Umbeliferonas/química
5.
Molecules ; 24(15)2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31370334

RESUMO

Hyperpigmentation is considered by many to be a beauty problem and is responsible for photoaging. To treat this skin condition, medicinal cosmetics containing tyrosinase inhibitors are used, resulting in skin whitening. In this study, taraxerol methyl ether (1), spinasterol (2), 6-hydroxyflavanone (3), (+)-dihydrokaempferol (4), 3,4-dihydroxybenzoic acid (5), taraxerol (6), taraxerone (7), and lupeol acetate (8) were isolated from Manilkara zapota bark. Their chemical structures were elucidated by analysis of their nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) data, and by comparing them with data found in the literature. The in vitro antityrosinase, antioxidant, and cytotoxic activities of the isolated compounds (1-8) were evaluated. (+)-Dihydrokaempferol (4) exhibited higher monophenolase inhibitory activity than both kojic acid and α-arbutin. However, it showed diphenolase inhibitory activity similar to kojic acid. (+)-Dihydrokaempferol (4) was a competitive inhibitor of both monophenolase and diphenolase activities. It exhibited the strongest 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and ferric reducing antioxidant power (FRAP) activities of the isolated compounds. Furthermore, (+)-dihydrokaempferol (4) also demonstrated potent cytotoxicity in breast carcinoma cell line (BT474), lung bronchus carcinoma cell line (Chago-K1), liver carcinoma cell line (HepG2), gastric carcinoma cell line (KATO-III), and colon carcinoma cell line (SW620). These results suggest that M. zapota bark might be a good potential source of antioxidants and tyrosinase inhibitors for applications in cosmeceutical products.


Assuntos
Manilkara/química , Monofenol Mono-Oxigenase/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Compostos Fitoquímicos/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Arbutina/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Flavonoides/química , Flavonoides/isolamento & purificação , Humanos , Hidroxibenzoatos/química , Hidroxibenzoatos/isolamento & purificação , Monofenol Mono-Oxigenase/química , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/química , Ácido Oleanólico/isolamento & purificação , Compostos Fitoquímicos/química , Pironas/química , Estigmasterol/análogos & derivados , Estigmasterol/química , Estigmasterol/isolamento & purificação
6.
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
7.
Colloids Surf B Biointerfaces ; 182: 110350, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31326622

RESUMO

Centaurea pumilio was the subject of phytochemical and biological studies, and its extract was used in the green synthesis of silver nanoparticles (AgNPs). Liquid chromatography/electrospray ionization mass spectrometry allowed the tentative identification of twenty-nine phytoconstituents of C. pumilio methanolic extract (CME), while column chromatography led to the identification of eight phenolic compounds. The neutral red uptake method showed the safety of CME and AgNPs on skin cells (HaCaT cell lines), while their high antioxidant potentials were demonstrated based on their oxygen radical absorbance capacity, and these results were confirmed in vivo. Additionally, CME and AgNPs had promising abilities to retard the ageing process and combat dark spots by potently inhibiting collagenase, elastase and tyrosinase, in addition to antimicrobial activity against skin infection-causing strains, especially Staphylococcus aureus, which was further confirmed by the significant phagocytic activity of neutrophils via engulfment. This study presents C. pumilio as a candidate for healthy skin.


Assuntos
Anti-Infecciosos/química , Antioxidantes/química , Centaurea/química , Inibidores Enzimáticos/química , Flavonoides/química , Glicosídeos/química , Nanopartículas Metálicas/química , Animais , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Candida albicans/crescimento & desenvolvimento , Linhagem Celular , Colagenases/química , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Células Epiteliais , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Glicosídeos/isolamento & purificação , Glicosídeos/farmacologia , Humanos , Nanopartículas Metálicas/administração & dosagem , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/química , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Elastase Pancreática/antagonistas & inibidores , Elastase Pancreática/química , Fagocitose/efeitos dos fármacos , Componentes Aéreos da Planta/química , Extratos Vegetais/química , Cultura Primária de Células , Ratos , Ratos Wistar , Prata/química , Pele/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento
8.
J Agric Food Chem ; 67(32): 9060-9069, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31339696

RESUMO

Glutathione S-transferases (GSTs) play an active role in the development of drug resistance by numerous cancer cells, including melanoma cells, which is a major cause of chemotherapy failure. As part of our continuous effort to explore why dietary polyphenols bearing the catechol moiety (dietary catechols) show usually anticancer activity, catechol-type diphenylbutadiene (3,4-DHB) was selected as a model of dietary catechols to probe whether they work as pro-oxidative chemosensitizers via GST inhibition in melanoma cells. It was found that, in human melanoma A375 cells, 3,4-DHB is easily converted to its ortho-quinone via copper-containing tyrosinase-mediated two-electron oxidation along with generation of reactive oxygen species (ROS) derived from the oxidation; the resulting ortho-quinone and ROS are responsible for its ability to sensitize the cisplatin-resistant cells by inhibiting GST, followed by induction of apoptosis in an ASK1-JNK/p38 signaling cascade and mitochondria-dependent pathway. This work provides further evidence to support that dietary catechols exhibit antimelanoma activity by virtue of their tyrosinase-dependent pro-oxidative role and gives useful information for designing polyphenol-inspired GST inhibitors and sensitizers in chemotherapy against melanoma.


Assuntos
Antineoplásicos/farmacologia , Butadienos/farmacologia , Catecóis/farmacologia , Inibidores Enzimáticos/farmacologia , Glutationa Transferase/antagonistas & inibidores , Melanoma/enzimologia , Monofenol Mono-Oxigenase/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptose , Butadienos/química , Butadienos/metabolismo , Catecóis/química , Catecóis/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Glutationa Transferase/metabolismo , Humanos , Melanoma/metabolismo , Melanoma/fisiopatologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Químicos , Monofenol Mono-Oxigenase/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
9.
Food Chem ; 300: 125209, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31344629

RESUMO

Turbot can induce allergy in susceptible individuals due to the presence of parvalbumin (PV), a major fish allergen. This study aimed at evaluating the digestibility and the ability of PV to elicit the release of cellular degranulation, following treatment with tyrosinase (PV-Tyr), caffeic acid (PV-CA) and in combination (PV-Tyr/CA), using in vitro digestion and RBL-2H3 (passive rat basophil leukemia) cell line. The digestion assay products revealed that the stability of PV in simulated gastric fluid (SGF) was stronger, while in simulated intestinal fluid (SIF) was rather weak. Western blot analysis revealed that the IgG-binding abilities of the cross-linked PV were markedly reduced. Moreover, crosslinking hampered the release of cellular degranulation process in RBL-2H3 cell lines. PV-Tyr/CA showed highly significant reduction in the release rate of ß-hexosaminidase (66.02%), histamine (35.01%), tryptase (29.25%), cysteinyl leukotrienes (29.72%), prostaglandin D2 (34.96%), IL-4 (43.99%) and IL-13 (38.93%) and shown potential in developing hypoallergenic fish products.


Assuntos
Ácidos Cafeicos/química , Citocinas/metabolismo , Hipersensibilidade Alimentar/imunologia , Monofenol Mono-Oxigenase/química , Parvalbuminas/química , Alérgenos/química , Alérgenos/farmacocinética , Animais , Degranulação Celular/efeitos dos fármacos , Linhagem Celular , Digestão , Proteínas de Peixes da Dieta/química , Linguados , Suco Gástrico , Histamina/metabolismo , Humanos , Parvalbuminas/imunologia , Parvalbuminas/farmacologia , Ratos , beta-N-Acetil-Hexosaminidases/metabolismo
10.
Talanta ; 204: 525-532, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31357329

RESUMO

In this work, we present a smartphone-based multiplexed enzymatic biosensor utilizing the unique colorimetric properties of the poly(aniline-co-anthranilic acid) (ANI-co-AA) composite film coupled with horseradish peroxidase (HRP), glucose oxidase (GOx), horseradish peroxidase-glucose oxidase (GOx-HRP) and tyrosinase (Tyr) enzymes. The enzymes are immobilized on the composite polymer film by adsorption and they catalyze a reversible redox color change of the host polymer from green to blue in the presence of their substrate. A smartphone was applied as color detector, for image acquisition and data handling. A ColorLab® android application, free of charge software application, was used to enable easy and clear display of the sensors' response indicating remarkable changes in the optical features. The results were confirmed by the spectrophotometric measurements. The developed colorimetric enzymatic biosensors were studied and optimized in relation to different experimental parameters. Moreover, the colorimetric enzymatic biosensors were applied to food and pharmaceutical analysis. It has been shown by these studies that the colorimetric biosensors are promising as quick and simple tests for handheld analysis in various fields.


Assuntos
Catecóis/análise , Glucose/análise , Peróxido de Hidrogênio/análise , Agaricales/enzimologia , Armoracia/enzimologia , Técnicas Biossensoriais/métodos , Colorimetria/instrumentação , Colorimetria/métodos , Enzimas Imobilizadas/química , Sucos de Frutas e Vegetais/análise , Glucose Oxidase/química , Peroxidase do Rábano Silvestre/química , Limite de Detecção , Monofenol Mono-Oxigenase/química , Polímeros/química , Pyrus/química , Reprodutibilidade dos Testes , Smartphone , Vinho/análise
11.
Int J Biol Macromol ; 136: 1034-1041, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31233796

RESUMO

Many skin disorders and diseases are related to tyrosinase activity, in particular, due to the vital role played by this enzyme in the melanogenic process. Although numerous natural and synthetic tyrosinase inhibitors have been published, substantial efforts have been made to understand the influence of tyrosinase inhibition on the viability of melanoma cells. Here, we assess the impact of two keto-derivatives: 2-acetyl-furan (F1), furfural-acetone (F2), and two carboxyl-derivatives: 2-furan-acrylic acid (F3), 5-methyl-2-furan-acrylic acid (F4), on the mushroom tyrosinase (mTYR) activity, by applying spectroscopic, kinetic and theoretical techniques. From an exploratory and theoretical point of view, results indicated that albeit all furans bind tightly to and inhibit mTYR very efficient, carboxyl-furan derivatives presented best inhibitory activities than keto- derivatives and performed the inhibition competitively and reversible. Moreover, we examined the influence of carboxyl derivative on the viability of melanoma cells. Results expose differential toxicity of these furan derivatives, which indicates a piece of evidence that furan inhibition activity may be related to its toxicity against B16F10 cells.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Furanos/farmacologia , Melanoma/patologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Agaricales/enzimologia , Animais , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/metabolismo , Furanos/metabolismo , Humanos , Camundongos , Simulação de Acoplamento Molecular , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/metabolismo , Conformação Proteica
12.
J Sci Food Agric ; 99(13): 6001-6010, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31225640

RESUMO

BACKGROUND: In this study, we aimed to evaluate the influence of different extraction procedures [decoction, homogenizer-assisted extraction (HAE), infusion, maceration, Soxhlet and ultrasound-assisted extraction (UAE)] on the chemical profiling and biological properties of methanol and water extracts of Pulicaria dysenterica (L.) Bernh. The chemical profiles of the extracts were evaluated by high-performance liquid chromatography coupled to electrospray ionization and time-of-flight mass spectrometry (HPLC-ESI-TOF-MS). The antioxidant properties and enzymes (lipase, α-amylase, α-glucosidase, tyrosinase and cholinesterases) inhibitory potential of the extracts were evaluated. RESULTS: The chemical profiles were dependent on the type of extraction methods as well as on the type of solvent. The methanolic extracts showed higher levels of total phenolic, flavonoid, and phenolic acid content, while the highest total flavonol content was observed in the HAE-water extract. Forty different compounds were identified from P. dysenterica. In relation to the potential in vitro anti-diabetic effects, the highest activity against the studied key enzymes was observed for the macerated extract (α-amylase: 0.58 ± 0.03 and α- glucosidase: 1.65 ± 0.03 mmol ACAE g-1 ). The HAE-methanol extract was the most potent inhibitor of cholisterases, whereas the highest activities against tyrosinase were observed for UAE-methanol extract, followed by macerated and Soxhlet. The inhibitory activity of the studied extracts against lipase were in the order: soxhlet > macerated> HAE-methanol > UAE-methanol. CONCLUSION: This study has established scientific baseline data on the therapeutic properties of P. dysentrica, thereby advocating the need for further investigations in an endeavour to develop novel pharmaceuticals from this plant. © 2019 Society of Chemical Industry.


Assuntos
Extratos Vegetais/química , Pulicaria/química , Antioxidantes/química , Antioxidantes/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Flavonoides/química , Flavonoides/isolamento & purificação , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/isolamento & purificação , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/química , Análise Multivariada , Fenóis/química , Fenóis/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray , alfa-Amilases/antagonistas & inibidores , alfa-Amilases/química , alfa-Glucosidases/química
13.
Anal Chim Acta ; 1073: 45-53, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31146835

RESUMO

A novel and ultrasensitive electrochemiluminescent (ECL) bioassay has been developed for the detection of atrazine (ATZ), whereby bifunctional S, N-codoped carbon dots (S, N-CDs) and activated mesoporous bicarbons (BCs) have been innovatively integrated to synergistically amplify the ECL signal. When endogenous dissolved O2 is used as a coreactant, its sluggish reduction hinders the enhancement of ECL intensity of the luminophore, thus restricting its further application in bioanalysis. Here, bifunctional S, N-CDs severe as not only the ECL luminophore but the coreaction accelerator of dissolved O2 to generate more intermediates to boost the coreaction without using any other coreactant and coreaction accelerator. The as-formed nanoarchitectures of BCs possess enlarged surface area as the nanocarriers. They could provide adequate active sites for immobilization of tyrosinase (Tyr), which greatly prompts the ECL bioassay applications. Such a smart integration of bifunctional S, N-CDs, activated mesoporous BCs and the enzyme inhibition reaction achieves a unique and attractive high-performance signal-on ECL bioassay, realizing ultrasensitive detection of ATZ. Under the optimal condition, this bioassay exhibits two linear ranges, from 0.0001 to 0.01 µg L-1 and 0.01-20 µg L-1 with a detection limit of 0.08 ng L-1 at signal to noise ratio of 3. The as-fabricated assay is sensitive and economical, opening a new way for the enhancement of ECL signal output and a versatile strategy for ultrasensitive ECL bioanalysis.


Assuntos
Atrazina/análise , Técnicas Biossensoriais , Técnicas Eletroquímicas , Medições Luminescentes , Monofenol Mono-Oxigenase/química , Nanopartículas/química , Atrazina/metabolismo , Carbono/química , Monofenol Mono-Oxigenase/metabolismo , Nanopartículas/metabolismo , Tamanho da Partícula , Porosidade , Pontos Quânticos/química , Propriedades de Superfície
14.
Anal Bioanal Chem ; 411(20): 5277-5285, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31161325

RESUMO

Rapid, highly sensitive detection of tau protein and other neurodegenerative biomarkers remains a significant hurdle for diagnostic tests for Alzheimer's disease. In this work, we developed a novel tyrosinase (TYR)-induced tau aptamer-tau-tau antibody (anti-tau) sandwich fluorescence immunoassay to detect tau protein that used dopamine (DA)-functionalized CuInS2/ZnS quantum dots as the fluorophore. CuInS2/ZnS core/shell quantum dots with high luminescence, low toxicity, and excellent biocompatibility were successfully fabricated and decorated with DA through amide conjugation. Meanwhile, TYR was conjugated with anti-tau by a click reaction. When DA-functionalized CuInS2/ZnS quantum dots were added to the sandwich system, TYR catalyzed the transformation of DA to dopamine quinone, which acted as an effective electron acceptor and triggered fluorescence quenching. The fluorescence intensity of the immunoassay based on DA-functionalized CuInS2/ZnS quantum dots shows good performance in terms of linearity with the logarithm of tau protein concentration, with a linear concentration range from 10 pM to 200 nM. This work is the first to use a TYR-induced fluorescence immunoassay for the rapid detection of tau protein, paving a new way for the detection of disease biomarkers. Graphical abstract.


Assuntos
Cobre/química , Imunofluorescência/métodos , Índio/química , Monofenol Mono-Oxigenase/química , Pontos Quânticos/química , Selênio/química , Sulfetos/química , Compostos de Zinco/química , Proteínas tau/análise , Cristalografia por Raios X , Dopamina/análogos & derivados , Dopamina/química , Microscopia Eletrônica de Transmissão , Análise Espectral/métodos
15.
Dalton Trans ; 48(20): 6899-6909, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31038147

RESUMO

A Cu(i) fully fluorinated O-donor monodentate alkoxide complex, K[Cu(OC4F9)2], was previously shown to form a trinuclear copper-dioxygen species with a {Cu3(µ3-O)2} core, TOC4F9, upon reactivity with O2 at low temperature. Herein is reported a significantly expanded kinetic and mechanistic study of TOC4F9 formation using stopped-flow spectroscopy. The TOC4F9 complex performs catalytic oxidase conversion of hydroquinone (H2Q) to benzoquinone (Q). TOC4F9 also demonstrated hydroxylation of 2,4-di-tert-butylphenolate (DBP) to catecholate, making TOC4F9 the first trinuclear species to perform tyrosinase (both monooxygenase and oxidase) chemistry. Resonance Raman spectra were also obtained for TOC4F9, to our knowledge, the first such spectra for any T species. The mechanism and substrate reactivity of TOC4F9 are compared to those of its bidentate counterpart, TpinF, formed from K[Cu(pinF)(PR3)]. The monodentate derivative has both faster initial formation and more diverse substrate reactivity.


Assuntos
Cobre/química , Hidrocarbonetos Fluorados/química , Monofenol Mono-Oxigenase/química , Catálise , Temperatura Baixa , Cinética , Ligantes , Modelos Moleculares , Estrutura Molecular , Oxirredução , Relação Estrutura-Atividade
16.
Biosens Bioelectron ; 136: 128-131, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31078022

RESUMO

Liposomal photoelectrochemical (PEC) bioanalysis holds enormous potential for future sensitive PEC bioanalysis. With tyrosinase (Tyr) and TiO2 as representative enzyme and electrode, respectively, this communication reports the elegant use of Tyr-loaded liposomes (TLL) toward in situ sensitization of the electrode and thereby the realization of ultrasensitive PEC immunoassay. Specifically, Tyr-encapsulated and detection antibody-functionalized liposomes were first prepared and used as the signal probe. The subsequent sandwich immunobinding could confine the functional liposomes, which were then lysed with surfactant to release the encapsulated Tyr. The free Tyr could then initiate the transformation of tyrosine to dopa, the latter could bind with the undercoordinated Ti sites, forming the stable dopa-Ti charge transfer complex and thus generating enhanced anodic photocurrent under visible light for signaling. Since different semiconductors and enzymes may be adapted into this format, this work is expected to stimulate more interest in the enzyme-induced activation of semiconductors for advanced liposomal PEC bioanalysis.


Assuntos
Imunoensaio/métodos , Lipossomos , Monofenol Mono-Oxigenase/química , Semicondutores , Di-Hidroxifenilalanina/metabolismo , Luz , Titânio , Tirosina/metabolismo
17.
J Agric Food Chem ; 67(19): 5486-5495, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31012315

RESUMO

Our previous research showed that Pleurotus eryngii and Pleurotus ostreatus were effective fungi for pretreatment of industrial hemp stalks to improve enzymatic saccharification. The secretomes of these two fungi were analyzed to search for the effective enzyme cocktails degrading hemp lignin during the pretreatment process. In total, 169 and 155 proteins were identified in Pleurotus eryngii and Pleurotus ostreatus, respectively, and 50% of the proteins involved in lignocellulose degradation were CAZymes. Because most of the extracellular proteins secreted by fungi are glycosylated proteins, the N-linked glycosylation of enzymes could be mapped. In total, 27 and 24 N-glycosylated peptides were detected in Pleurotus eryngii and Pleurotus ostreatus secretomes, respectively. N-Glycosylated peptides of laccase, GH92, exoglucanase, phenol oxidase, α-galactosidase, carboxylic ester hydrolase, and pectin lyase were identified. Deglycosylation could decrease enzymatic saccharification of hemp stalks. The activities of laccase, α-galactosidase, and phenol oxidase and the thermal stability of laccase were reduced after deglycosylation.


Assuntos
Cannabis/microbiologia , Proteínas Fúngicas/metabolismo , Pleurotus/enzimologia , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Galactosidases/química , Galactosidases/genética , Galactosidases/metabolismo , Glicosilação , Lacase/química , Lacase/genética , Lacase/metabolismo , Lignina/metabolismo , Monofenol Mono-Oxigenase/química , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Peroxidases/química , Peroxidases/genética , Peroxidases/metabolismo , Caules de Planta/microbiologia , Pleurotus/classificação , Pleurotus/genética , Pleurotus/crescimento & desenvolvimento , Polissacarídeo-Liase/química , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Transporte Proteico
18.
Org Biomol Chem ; 17(18): 4543-4553, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-30994696

RESUMO

We investigate the self-assembly of a palmitoylated (C16-chain at the N terminus) peptide fragment in comparison to the unlipidated peptide EELNRYY, a fragment of the gut hormone peptide PYY3-36. The lipopeptide C16-EELNRYY shows remarkable pH-dependent self-assembly above measured critical aggregation concentrations, forming fibrils at pH 7, but micelles at pH 10. The parent peptide does not show self-assembly behaviour. The lipopeptide forms hydrogels at sufficiently high concentration at pH 7, the dynamic mechanical properties of which were measured. We also show that the tyrosine functionality at the C terminus of EELNRYY can be used to enzymatically produce the pigment melanin. The enzyme tyrosinase oxidises tyrosine into 3,4-dihydroxyphenylalanine (DOPA), DOPA-quinone and further products, eventually forming eumelanin. This is a mechanism of photo-protection in the skin, for this reason controlling tyrosinase activity is a major target for skin care applications and EELNRYY has potential to be developed for such uses.


Assuntos
Lipopeptídeos/química , Melaninas/síntese química , Monofenol Mono-Oxigenase/química , Oligopeptídeos/química , Fragmentos de Peptídeos/química , Peptídeo YY/química , Sequência de Aminoácidos , Corantes Fluorescentes/química , Hidrogéis/química , Concentração de Íons de Hidrogênio , Lipopeptídeos/metabolismo , Micelas , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeo YY/metabolismo , Conformação Proteica em Folha beta , Multimerização Proteica , Pirenos/química , Tirosina/química
19.
Biosens Bioelectron ; 132: 279-285, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30884314

RESUMO

Highly sensitive phenol biosensor was developed by using well-dispersed carbon nanotubes (CNTs) in enzyme solution and adding CNTs in enzyme electrodes. First, the intact CNTs were dispersed in aqueous tyrosinase (TYR) solution, and TYR molecules were precipitated and crosslinked to prepare the sample of enzyme adsorption, precipitation and crosslinking (EAPC). EAPC exhibited 10.5- and 5.4-fold higher TYR activity per mg of CNTs as compared to enzyme adsorption (EA) and enzyme adsorption/crosslinking (EAC), respectively. EAPC retained 29% of its initial activity after incubation at 40 °C for 128 h, while EA and EAC showed no residual activities, respectively. In biosensing a model phenolic compound of catechol, the sensitivities of EA, EAC and EAPC electrodes on glassy carbon electrode (GCE) were 34, 281 and 675 µA/mM/cm2, respectively. When 90 w/w% CNTs were added to the enzyme electrodes, the sensitivities of EA, EAC, and EAPC electrodes were 146, 427, and 1160 µA/mM/cm2, respectively, and the EAPC electrode showed a 2.3-fold increase in sensitivity upon CNT addition. Catechol and phenol could also be detected by EAPC on the screen-printed electrode (SPE), with sensitivities of 1340 and 1170 µA/mM/cm2, respectively. The sensitivity of EAPC-SPE for phenol detection in the effluent from real municipal wastewater treatment plant was 1100 µA/mM/cm2. The sensitivity of EAPC-SPE retained 74% of its initial sensitivity after incubation at 40 °C for 12 h. The combination of EAPC immobilization and CNT addition has great potential for application in the development of sensitive enzyme biosensors for various analytes and phenols in water environments.


Assuntos
Agaricales/enzimologia , Técnicas Biossensoriais/métodos , Enzimas Imobilizadas/química , Monofenol Mono-Oxigenase/química , Nanotubos de Carbono/química , Fenóis/análise , Poluentes Químicos da Água/análise , Catecóis/análise , Reagentes para Ligações Cruzadas/química , Eletrodos , Limite de Detecção , Modelos Moleculares , Nanotubos de Carbono/ultraestrutura , Fenol/análise , Águas Residuárias/análise
20.
Int J Biol Macromol ; 131: 309-314, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30872058

RESUMO

The present paper aimed to obtain the polysaccharides with potent tyrosinase inhibitory activity from chestnut kernel. The ultrasound extracted polysaccharide fractions 1-1 (UEP1-1) and UEP2-1 were obtained by successive ultrasound-assisted-extraction, DEAE-52 and Sephadex G-100 chromatography. The Fourier transform infrared spectrum revealed that both UEP1-1 and UEP2-1 had the characteristic absorption peaks of polysaccharides. The degree of esterification of UEP1-1 (52.4%) and UEP2-1 (49.0%) was higher than that of the crude UEP (33.0%), indicating that the gel properties of the polysaccharides changed after purification. The x-ray diffraction patterns of UEP1-1 and UEP2-1 suggested that they were semi-crystalline polymers with a degree of crystallinity of 23.9 and 35.8%, respectively. The weight-average molecular weight (Mw) of UEP1-1 and UEP2-1 was 75.4 and 59.9 kDA, respectively. The monosaccharide composition of UEP1-1 and UEP2-1 was glucose, galactose, arabinose, mannose, xylose, rhamnose and fructose with different proportions. UEP1-1 displayed a dose-dependent inhibitory effect on tyrosinase activity, and the inhibition mode was found to be competitive.


Assuntos
Fagaceae/química , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/química , Polissacarídeos/química , Polissacarídeos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Peso Molecular , Monossacarídeos/química , Polissacarídeos/isolamento & purificação , Análise Espectral
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