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1.
J Chromatogr A ; 1647: 462131, 2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-33971520

RESUMO

Three modes of facilitating mini-scale-liquid-liquid-extraction (msLLE) prior to automated integration with full evaporation dynamic headspace (FEDHS) extraction were evaluated in this work. For msLLE, 1.2 mL of dichloromethane (DCM) was added to a conical-bottomed vial containing 7 mL of aqueous sample. The solution was then subjected to three different mixing modes, namely vortex-assistance (where a "whirlpool" was created in the solution), agitation-assistance (where the vial was rotated in circular motion) and quickMix-assistance (where the vial was shaken at a high speed). Vortex-assistance was performed manually while the other two modes were automated using a commercial autosampler. Following this, the DCM extract was transferred automatically to another vial and was then vaporized and sent through a Tenax TA sorbent tube in the FEDHS step. Due to the stronger π interaction between the sorbent and the analytes of interest, the analytes were selectively concentrated while the DCM vapor passed through unhampered. After FEDHS, the analytes were thoroughly desorbed into a gas chromatography-mass spectrometric system for analysis. The applicability of this procedure was validated in the extraction of six chlorinated benzenes (CBs) (1,2-dichlorobenzene, 1,3-dichlorobenzene, 1,4-dichlorobenzene, 1,2,4-trichlorobenzene, 1,2,4,5-tetrachlorobenzne and hexachlorobenzene) from aqueous samples. The quickMix-assisted msLLE-FEDHS approach achieved good absolute extraction recoveries (between 74.2% and 88.7%), low limits of detection (between 0.0006 and 0.0116 µg/L), good linearity (r2≥0.9920), good repeatability (between 1.9% and 8.4%, and good reproducibility (between 9.0% and 13.6%). It was found to be superior to the methods published by the United States Environmental Protection Agency. Five consecutive fully automated quickMix-assisted-msLLE-FEDHS-GC-MS runs spanned only ca. 4 hr.


Assuntos
Clorobenzenos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Extração Líquido-Líquido/métodos , Clorobenzenos/análise , Clorobenzenos/química , Clorobenzenos/isolamento & purificação , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Estados Unidos , United States Environmental Protection Agency
2.
Mar Drugs ; 19(4)2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33915855

RESUMO

The pressurized liquid extraction (PLE) technique was used, for the first time, to obtain protein extracts with antioxidant activity from side streams (muscle, heads, viscera, skin, and tailfins) of gilthead sea bream (Sparus aurata) in order to give added value to these underutilized matrices. Extraction conditions previously optimized for sea bass (Dicentrarchus labrax) side streams were applied. Protein recovery percentages were 22% (muscle), 33% (heads), 78% (viscera), 24% (skin), and 26% (tailfins), which represented an increase of 1.2-4.5-fold compared to control samples (extraction by stirring). The SDS-PAGE profiles revealed that PLE-assisted extraction influenced protein molecular weight distribution of the obtained extracts. PLE conditions also allowed increasing the antioxidant capacity measured by both Trolox equivalent antioxidant capacity (TEAC; 1.3-2.4 fold) and oxygen radical absorbance capacity (ORAC; 1.9-6.4) assays for all fish extracts. Inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-qTOF-MS) were used to investigate the presence of toxic metals and mycotoxins in sea bream side streams. The levels of As, Hg, Cd, and Pb were below those established by authorities for fish muscle for human consumption (except for Cd in viscera samples). Through a nontargeted screening approach, no mycotoxins or related metabolites were detected for all sea bream side streams. This study contributes to the research on the valorization of fish processing side streams using environmentally friendly technology.


Assuntos
Antioxidantes/farmacologia , Proteínas de Peixes/farmacologia , Extração Líquido-Líquido , Dourada/metabolismo , Animais , Antioxidantes/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Proteínas de Peixes/isolamento & purificação , Manipulação de Alimentos , Metais/isolamento & purificação , Peso Molecular , Micotoxinas/isolamento & purificação , Capacidade de Absorbância de Radicais de Oxigênio , Pressão , Espectrometria de Massas por Ionização por Electrospray , Resíduos
3.
Molecules ; 26(7)2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33916390

RESUMO

In the present study, a simple and environmentally friendly extraction method based on natural deep eutectic solvents (NADESs) was established to extract four bioactive steroidal saponins from Dioscoreae Nipponicae Rhizoma (DNR). A total of twenty-one types of choline chloride, betaine, and L-proline based NADESs were tailored, and the NADES composed of 1:1 molar ratio of choline chloride and malonic acid showed the best extraction efficiency for the four steroidal saponins compared with other NADESs. Then, the extraction parameters for extraction of steroidal saponins by selected tailor-made NADES were optimized using response surface methodology and the optimal extraction conditions are extraction time, 23.5 min; liquid-solid ratio, 57.5 mL/g; and water content, 54%. The microstructure of the DNR powder before and after ultrasonic extraction by conventional solvents (water and methanol) and the selected NADES were observed using field emission scanning electron microscope. In addition, the four steroidal saponins were recovered from NADESs by D101 macroporous resin with a satisfactory recovery yield between 67.27% and 79.90%. The present research demonstrates that NADESs are a suitable green media for the extraction of the bioactive steroidal saponins from DNR, and have a great potential as possible alternatives to organic solvents for efficiently extracting bioactive compounds from natural products.


Assuntos
Dioscorea/química , Extração Líquido-Líquido/métodos , Compostos Fitoquímicos/isolamento & purificação , Saponinas/isolamento & purificação , Colina/química , Análise Fatorial , Química Verde , Malonatos/química , Estrutura Molecular , Compostos Fitoquímicos/química , Extratos Vegetais/química , Rizoma/química
4.
Molecules ; 26(7)2021 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-33916551

RESUMO

Vaccinium dunalianum Wight, usually processed as a traditional folk tea beverage, is widely distributed in the southwest of China. The present study aimed to investigate the antioxidant, α-glucosidase and pancreatic lipase inhibitory activities of V.dunalianum extract and isolate the bioactive components. In this study, the crude extract (CE) from the buds of V. dunalianum was prepared by the ultrasound-assisted extraction method in 70% methanol and then purified with macroporous resin D101 to obtain the purified extract (PM). Five fractions (Fr. A-E) were further obtained by MPLC column (RP-C18). Bioactivity assays revealed that Fr. B with 40% methanol and Fr. D with 80% methanol had better antioxidant with 0.48 ± 0.03 and 0.62 ± 0.01 nM Trolox equivalent (TE)/mg extract for DPPH, 0.87 ± 0.02 and 1.58 ± 0.02 nM TE/mg extract for FRAP, 14.42 ± 0.41 and 19.25 ± 0.23 nM TE/mg extract for ABTS, and enzyme inhibitory effects with IC50 values of 95.21 ± 2.21 and 74.55 ± 3.85 for α-glucosidase, and 142.53 ± 11.45 and 128.76 ± 13.85 µg/mL for pancreatic lipase. Multivariate analysis indicated that the TPC and TFC were positively related to the antioxidant activities. Further phytochemical purification led to the isolation of ten compounds (1-10). 6-O-Caffeoylarbutin (7) showed significant inhibitory effects on α-glucosidase and pancreatic lipase enzymes with values of 38.38 ± 1.84 and 97.56 ± 7.53 µg/mL, and had the highest antioxidant capacity compared to the other compounds.


Assuntos
Antioxidantes/isolamento & purificação , Arbutina/análogos & derivados , Ácidos Cafeicos/isolamento & purificação , Flavonoides/isolamento & purificação , Inibidores de Glicosídeo Hidrolases/isolamento & purificação , Lipase/química , Vaccinium/química , alfa-Glucosidases/química , Antioxidantes/química , Arbutina/química , Arbutina/isolamento & purificação , Benzotiazóis/antagonistas & inibidores , Benzotiazóis/química , Compostos de Bifenilo/química , Ácidos Cafeicos/química , Flavonoides/química , Inibidores de Glicosídeo Hidrolases/química , Lipase/antagonistas & inibidores , Lipase/metabolismo , Extração Líquido-Líquido/métodos , Metanol/química , Picratos/química , Extratos Vegetais/química , Folhas de Planta/química , Solventes/química , Sonicação , Ácidos Sulfônicos/antagonistas & inibidores , Ácidos Sulfônicos/química , alfa-Glucosidases/metabolismo
5.
Molecules ; 26(7)2021 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-33916639

RESUMO

Valorization of vegetable oil waste residues is gaining importance due to their high protein and polyphenol contents. Protease inhibitors (PIs), proteins from these abundantly available waste residues, have recently gained importance in treating chronic diseases. This research aimed to use canola meal of genetically diverse Brassica napus genotypes, BLN-3347 and Rivette, to identify PIs with diverse functionalities in therapeutic and pharmacological applications. The canola meal PI purification steps involved: native PAGE and trypsin inhibition activity, followed by ammonium sulfate fractionation, anion exchange, gel filtration, and reverse-phase chromatography. The purified PI preparations were characterized using SDS-PAGE, isoelectric focusing (IEF), and N terminal sequencing. SDS-PAGE analysis of PI preparations under native reducing and nonreducing conditions revealed three polymorphic PIs in each genotype. The corresponding IEF of the genotype BLN-3347, exhibited three acidic isoforms with isoelectric points (pI) of 4.6, 4.0, and 3.9, while Rivette possessed three isoforms, exhibiting two basic forms of pI 8.65 and 9.9, and one acidic of pI 6.55. Purified PI preparations from both the genotypes displayed dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE) inhibition activities; the BLN-3347 PI preparation exhibited a strong inhibitory effect with lower IC50 values (DPP-IV 37.42 µg/mL; ACE 129 µg/mL) than that from Rivette (DPP-IV 67.97 µg/mL; ACE 376.2 µg/mL). In addition to potential human therapy, these highly polymorphic PIs, which can inhibit damaging serine proteases secreted by canola plant pathogens, have the potential to be used by canola plant breeders to seek qualitative trait locus (QTLs) linked to genes conferring resistance to canola diseases.


Assuntos
Anti-Hipertensivos/farmacologia , Brassica napus/química , Dipeptidil Peptidase 4/química , Inibidores Enzimáticos/farmacologia , Hipoglicemiantes/farmacologia , Peptidil Dipeptidase A/química , Sequência de Aminoácidos , Anti-Hipertensivos/química , Anti-Hipertensivos/isolamento & purificação , Brassica napus/genética , Brassica napus/metabolismo , Dipeptidil Peptidase 4/metabolismo , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Genótipo , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/isolamento & purificação , Focalização Isoelétrica , Cinética , Extração Líquido-Líquido/métodos , Peptidil Dipeptidase A/metabolismo , Extratos Vegetais/química
6.
Int J Mol Sci ; 22(9)2021 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-33923223

RESUMO

Herpes Simplex Virus Type-1 (HSV-1) forms progeny in the nucleus within distinct membrane-less inclusions, the viral replication compartments (VRCs), where viral gene expression, DNA replication, and packaging occur. The way in which the VRCs maintain spatial integrity remains unresolved. Here, we demonstrate that the essential viral transcription factor ICP4 is an intrinsically disordered protein (IDP) capable of driving protein condensation and liquid-liquid phase separation (LLPS) in transfected cells. Particularly, ICP4 forms nuclear liquid-like condensates in a dose- and time-dependent manner. Fluorescence recovery after photobleaching (FRAP) assays revealed rapid exchange rates of EYFP-ICP4 between phase-separated condensates and the surroundings, akin to other viral IDPs that drive LLPS. Likewise, HSV-1 VRCs revealed by EYFP-tagged ICP4 retained their liquid-like nature, suggesting that they are phase-separated condensates. Individual VRCs homotypically fused when reaching close proximity and grew over the course of infection. Together, the results of this study demonstrate that the HSV-1 transcription factor ICP4 has characteristics of a viral IDP, forms condensates in the cell nucleus by LLPS, and can be used as a proxy for HSV-1 VRCs with characteristics of liquid-liquid phase-separated condensates.


Assuntos
Regulação Viral da Expressão Gênica , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Animais , Núcleo Celular/metabolismo , Chlorocebus aethiops , Herpes Simples/genética , Herpes Simples/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas Intrinsicamente Desordenadas/genética , Extração Líquido-Líquido , Transição de Fase , Células Vero
7.
Molecules ; 26(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33917024

RESUMO

Beta glucan (ß-glucan) has promising bioactive properties. Consequently, the use of ß-glucan as a food additive is favored with the dual-purpose potential of increasing the fiber content of food products and enhancing their health properties. Our aim was to evaluate the biological activity of ß-glucan (antimicrobial, antitoxic, immunostimulatory, and anticancer) extracted from Saccharomyces cerevisiae using a modified acid-base extraction method. The results demonstrated that a modified acid-base extraction method gives a higher biological efficacy of ß-glucan than in the water extraction method. Using 0.5 mg dry weight of acid-base extracted ß-glucan (AB extracted) not only succeeded in removing 100% of aflatoxins, but also had a promising antimicrobial activity against multidrug-resistant bacteria, fungi, and yeast, with minimum inhibitory concentrations (MIC) of 0.39 and 0.19 mg/mL in the case of resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, respectively. In addition, AB extract exhibited a positive immunomodulatory effect, mediated through the high induction of TNFα, IL-6, IFN-γ, and IL-2. Moreover, AB extract showed a greater anticancer effect against A549, MDA-MB-232, and HepG-2 cells compared to WI-38 cells, at high concentrations. By studying the cell death mechanism using flow-cytometry, AB extract was shown to induce apoptotic cell death at higher concentrations, as in the case of MDA-MB-231 and HePG-2 cells. In conclusion, the use of a modified AB for ß-glucan from Saccharomyces cerevisiae exerted a promising antimicrobial, immunomodulatory efficacy, and anti-cancer potential. Future research should focus on evaluating ß-glucan in various biological systems and elucidating the underlying mechanism of action.


Assuntos
Extração Líquido-Líquido , Saccharomyces cerevisiae/química , beta-Glucanas/isolamento & purificação , beta-Glucanas/farmacologia , Ácidos , Anti-Infecciosos/química , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/isolamento & purificação , Fatores Imunológicos/farmacologia , Imunomodulação/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Extração Líquido-Líquido/métodos , Testes de Sensibilidade Microbiana , Espectroscopia de Infravermelho com Transformada de Fourier , beta-Glucanas/química
8.
Molecules ; 26(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33917117

RESUMO

Intrinsically disordered proteins (IDPs) are critical players in the dynamic control of diverse cellular processes, and provide potential new drug targets because their dysregulation is closely related to many diseases. This review focuses on several medicinal studies that have identified low-molecular-weight inhibitors of IDPs. In addition, clinically relevant liquid-liquid phase separations-which critically involve both intermolecular interactions between IDPs and their posttranslational modification-are analyzed to understand the potential of IDPs as new drug targets.


Assuntos
Proteínas de Transporte/metabolismo , Descoberta de Drogas , Proteínas Intrinsicamente Desordenadas/metabolismo , Transdução de Sinais , Animais , Biomarcadores , Descoberta de Drogas/métodos , Humanos , Proteínas Intrinsicamente Desordenadas/antagonistas & inibidores , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/isolamento & purificação , Extração Líquido-Líquido/métodos , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
9.
Int J Mol Sci ; 22(7)2021 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-33805474

RESUMO

Understanding the interaction of ions with organic receptors in confined space is of fundamental importance and could advance nanoelectronics and sensor design. In this work, metal ion complexation of conformationally varied thiacalix[4]monocrowns bearing lower-rim hydroxy (type I), dodecyloxy (type II), or methoxy (type III) fragments was evaluated. At the liquid-liquid interface, alkylated thiacalixcrowns-5(6) selectively extract alkali metal ions according to the induced-fit concept, whereas crown-4 receptors were ineffective due to distortion of the crown-ether cavity, as predicted by quantum-chemical calculations. In type-I ligands, alkali-metal ion extraction by the solvent-accessible crown-ether cavity was prevented, which resulted in competitive Ag+ extraction by sulfide bridges. Surprisingly, amphiphilic type-I/II conjugates moderately extracted other metal ions, which was attributed to calixarene aggregation in salt aqueous phase and supported by dynamic light scattering measurements. Cation-monolayer interactions at the air-water interface were monitored by surface pressure/potential measurements and UV/visible reflection-absorption spectroscopy. Topology-varied selectivity was evidenced, towards Sr2+ (crown-4), K+ (crown-5), and Ag+ (crown-6) in type-I receptors and Na+ (crown-4), Ca2+ (crown-5), and Cs+ (crown-6) in type-II receptors. Nuclear magnetic resonance and electronic absorption spectroscopy revealed exocyclic coordination in type-I ligands and cation-π interactions in type-II ligands.


Assuntos
Complexos de Coordenação/química , Éteres de Coroa/química , Íons/metabolismo , Fenóis/química , Sulfetos/química , Ar , Alquilação , Cálcio/metabolismo , Complexos de Coordenação/metabolismo , Éteres de Coroa/síntese química , Éteres de Coroa/metabolismo , Difusão Dinâmica da Luz , Íons/química , Extração Líquido-Líquido , Espectroscopia de Ressonância Magnética , Metais/química , Conformação Molecular , Fenóis/metabolismo , Solventes/química , Espectrofotometria Ultravioleta , Sulfetos/metabolismo , Água/química
10.
Anal Methods ; 13(16): 1955-1964, 2021 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-33913942

RESUMO

Honey is widely consumed worldwide, however, this food can be contaminated by chemical contaminants, such as the insecticide dichlorodiphenyltrichloroethane (DDT). Despite legal restrictions on DDT use, this organochlorine pesticide has been detected in honey collected in several developed and developing countries, representing risks to human health, animals, and the environment due to its high environmental persistence, potential carcinogenicity, and ecotoxicological effects. Thus, the development of an analytical method for DDT monitoring in this matrix is important to ensure food security. Therefore, this study aimed to optimize and validate a simple, low-cost, and efficient method using the liquid-liquid extraction with low-temperature purification (LLE-LTP) to determine DDT in honey samples by high-performance liquid chromatography with diode array detector (HPLC-DAD). The proposed method was validated according to SANTE guidelines, being considered selective, precise, accurate, and linear in the range of 8.0-160 µg kg-1. The limits of detection (LOD) and quantification (LOQ) achieved were 4.0 and 8.0 µg kg-1, respectively. This LOQ value is lower than the maximum residue limit established by the Brazilian and European Union legislation. Therefore, the LLE-LTP combined to HPLC-DAD allows the routine analysis of DDT in honey samples and can be widely applied in studies to monitor this pesticide, especially in developing countries, where DDT use is still allowed.


Assuntos
Mel , Animais , Brasil , Cromatografia Líquida de Alta Pressão , DDT/análise , Mel/análise , Humanos , Extração Líquido-Líquido , Temperatura
11.
Molecules ; 26(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806573

RESUMO

In this work, for the first time, Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) method was developed for qualitative and quantitative analysis of veterinary antibiotics (cephalosporins, diaminopyrimidines, fluoro(quinolones), lincosamides, macrolides, penicillins, pleuromutilins, sulfonamides, tetracyclines, and sulfones) in hen eggshells. The sample preparation method is based on a liquid-liquid extraction with a mixture of metaphosphoric acid, ascorbic acid, EDTA disodium salt dihydrate, and acetonitrile. The chromatographic separation was performed on Luna® Omega Polar C18 10 column in gradient elution mode and quantitated in an 8 min run. Validation such as linearity, selectivity, precision, recovery, matrix effect, limit of quantification (LOQ), and limit of detection (LOD) was found to be within the acceptance criteria of the validation guidelines of the Commission Decision 2002/657/EC and EUR 28099 EN. Average recoveries ranged from 81-120%. The calculated LOQ values ranged from 1 to 10 µg/kg, the LOD values ranged from 0.3 to 4.0 µg/kg, depending on analyte. The developed method has been successfully applied to the determination of antibacterial compounds in hen eggshell samples obtained from different sources. The results revealed that enrofloxacin, lincomycin, doxycycline, and oxytetracycline were detected in hen eggshell samples.


Assuntos
Antibacterianos/análise , Cromatografia Líquida de Alta Pressão/métodos , Casca de Ovo/química , Extração Líquido-Líquido/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Antibacterianos/isolamento & purificação , Galinhas
12.
Food Chem ; 355: 129577, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-33799236

RESUMO

An excellent high-efficiency natural deep eutectic solvent (NADES, ChCl-MA) was screened out and integrated with pulse-ultrasonication technique for extracting phenolic compounds from Carya cathayensis Sarg. peels (CCSPs). Single factor experiment combined with response surface methodology (RSM) using Box-Behnken design (BBD) were employed to investigate significant factors and optimize their influence on extraction of phenolic compounds. Significant synergistic effect triggered by ChCl-MA based pulse-ultrasonication over other methods used alone were proved by comparative study concerning a variety of bioactive components and antioxidant activities. The second-order kinetic model was developed and validated (R2 > 0.99) to describe the extraction process and its mechanism; and second-order kinetic extraction rate constant (k), saturation concentration (Cs), and initial extraction rate (h) were calculated. FT-IR, DSC and SEM results further demonstrated synergistic effect and influence during extraction. Overall, this study provided a green and high-efficiency alternative for the recovery of various phenolics compounds from plant source by-products.


Assuntos
Carya/química , Extração Líquido-Líquido/métodos , Fenóis/química , Extratos Vegetais/química , Solventes/química , Antioxidantes/química , Carya/metabolismo , Frutas/química , Frutas/metabolismo , Fenóis/isolamento & purificação , Sonicação , Espectroscopia de Infravermelho com Transformada de Fourier
13.
Nat Commun ; 12(1): 2114, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33837182

RESUMO

Lack of detailed knowledge of SARS-CoV-2 infection has been hampering the development of treatments for coronavirus disease 2019 (COVID-19). Here, we report that RNA triggers the liquid-liquid phase separation (LLPS) of the SARS-CoV-2 nucleocapsid protein, N. By analyzing all 29 proteins of SARS-CoV-2, we find that only N is predicted as an LLPS protein. We further confirm the LLPS of N during SARS-CoV-2 infection. Among the 100,849 genome variants of SARS-CoV-2 in the GISAID database, we identify that ~37% (36,941) of the genomes contain a specific trio-nucleotide polymorphism (GGG-to-AAC) in the coding sequence of N, which leads to the amino acid substitutions, R203K/G204R. Interestingly, NR203K/G204R exhibits a higher propensity to undergo LLPS and a greater effect on IFN inhibition. By screening the chemicals known to interfere with N-RNA binding in other viruses, we find that (-)-gallocatechin gallate (GCG), a polyphenol from green tea, disrupts the LLPS of N and inhibits SARS-CoV-2 replication. Thus, our study reveals that targeting N-RNA condensation with GCG could be a potential treatment for COVID-19.


Assuntos
Substituição de Aminoácidos/efeitos dos fármacos , Catequina/análogos & derivados , Proteínas do Nucleocapsídeo/genética , Replicação Viral/efeitos dos fármacos , /virologia , Catequina/farmacologia , Genoma Viral/genética , Humanos , Extração Líquido-Líquido , Proteínas do Nucleocapsídeo/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Replicação Viral/genética
14.
Molecules ; 26(8)2021 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-33919479

RESUMO

In the present work, a novel sample preparation method, micro salting-out assisted matrix solid-phase dispersion (µ-SOA-MSPD), was developed for the determination of bisphenol A (BPA) and bisphenol B (BPB) contaminants in bee pollen. The proposed method was designed to combine two classical sample preparation methodologies, matrix solid-phase dispersion (MSPD) and homogenous liquid-liquid extraction (HLLE), to simplify and speed-up the preparation process. Parameters of µ-SOA-MSPD were systematically investigated, and results indicated the significant effect of salt and ACN-H2O extractant on the signal response of analytes. In addition, excellent clean-up ability in removing matrix components was observed when primary secondary amine (PSA) sorbent was introduced into the blending operation. The developed method was fully validated, and the limits of detection for BPA and BPB were 20 µg/kg and 30 µg/kg, respectively. Average recoveries and precisions were ranged from 83.03% to 94.64% and 1.76% to 5.45%, respectively. This is the first report on the analysis of bisphenol contaminants in bee pollen sample, and also on the combination of MSPD and HLLE. The present method might provide a new strategy for simple and fast sample preparation of solid and semi-solid samples.


Assuntos
Compostos Benzidrílicos/isolamento & purificação , Fenóis/isolamento & purificação , Pólen/química , Animais , Abelhas/química , Compostos Benzidrílicos/química , Compostos Benzidrílicos/toxicidade , Cromatografia Líquida de Alta Pressão , Humanos , Extração Líquido-Líquido , Fenóis/química , Fenóis/toxicidade , Pólen/toxicidade , Extração em Fase Sólida
15.
Molecules ; 26(8)2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33920003

RESUMO

Recently, we proposed a new sample preparation method involving reduced solvent and sample usage, based on dehydration homogeneous liquid-liquid extraction (DHLLE) for the screening of volatiles and semi-volatiles from honey. In the present research, the method was applied to a wide range of honeys (21 different representative unifloral samples) to determine its suitability for detecting characteristic honey compounds from different chemical classes. GC-FID/MS disclosed 130 compounds from different structural and chemical groups. The DHLLE method allowed the extraction and identification of a wide range of previously reported specific and nonspecific marker compounds belonging to different chemical groups (including monoterpenes, norisoprenoids, benzene derivatives, or nitrogen compounds). For example, DHLLE allowed the detection of cornflower honey chemical markers: 3-oxo-retro-α-ionols, 3,4-dihydro-3-oxoedulan, phenyllactic acid; coffee honey markers: theobromine and caffeine; linden honey markers: 4-isopropenylcyclohexa-1,3-diene-1-carboxylic acid and 4-(2-hydroxy-2-propanyl)cyclohexa-1,3-diene-1-carboxylic acid, as well as furan derivatives from buckwheat honey. The obtained results were comparable with the previously reported data on markers of various honey varieties. Considering the application of much lower volumes of very common reagents, DHLLE may provide economical and ecological advantages as an alternative sample preparation method for routine purposes.


Assuntos
Métodos Analíticos de Preparação de Amostras , Mel/análise , Extração Líquido-Líquido/métodos , Compostos Orgânicos Voláteis/análise , Desidratação , Cromatografia Gasosa-Espectrometria de Massas , Extração em Fase Sólida , Solventes/química , Ultrassom
16.
Molecules ; 26(5)2021 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33803489

RESUMO

Poor adherence to antihypertensive drug therapy is a well-recognized problem and can be assessed by mass spectrometry-based analyses of body fluids. However, contrary statements exist whether drug quantification in blood or qualitative screening in urine is more suitable. The present pilot study aimed to further elucidate the power of blood plasma drug concentrations for adherence monitoring by developing and validating a quantification procedure for nine antihypertensive drugs (amlodipine, bisoprolol, candesartan, canrenone, carvedilol, metoprolol, olmesartan, torasemide, and valsartan) in blood plasma using liquid-liquid extraction and an ultra-high-performance liquid chromatography-ion trap mass spectrometry analysis. The procedure should then be used for an adherence assessment and compared with the results of an established qualitative urine screening. Selectivity, carryover, matrix effect, accuracy, precision, dilution integrity, and stability were successfully validated, except for amlodipine. The applicability was demonstrated by analyzing 19 plasma samples containing 28 antihypertensive drugs and comparing the measured concentrations with calculated dose-dependent reference plasma concentration ranges. The interpretation of plasma concentrations was found to be more sophisticated and time-consuming than that of urine screening results, and adherence could not be assessed in two cases (10%) due to measured plasma concentrations below the lower limit of quantification. However, 14 out of 19 subjects were classified as adherent (75%) and three as nonadherent (15%), in contrast to 19 (100%) that were claimed to be adherent based on the results of the qualitative urine screening. Nevertheless, further data is needed to estimate whether plasma quantification is superior in terms of assessing adherence to antihypertensive medication.


Assuntos
Anti-Hipertensivos/análise , Anti-Hipertensivos/uso terapêutico , Monitoramento de Medicamentos/métodos , Anlodipino/uso terapêutico , Anti-Hipertensivos/sangue , Benzimidazóis/uso terapêutico , Compostos de Bifenilo/uso terapêutico , Líquidos Corporais/química , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Hipertensão/tratamento farmacológico , Imidazóis/uso terapêutico , Extração Líquido-Líquido/métodos , Metoprolol/uso terapêutico , Cooperação do Paciente/estatística & dados numéricos , Projetos Piloto , Plasma/química , Valores de Referência , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos , Tetrazóis/uso terapêutico
17.
J Chromatogr A ; 1643: 462080, 2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33799073

RESUMO

Acorus tatarinowii Schott is a traditional Chinese medicine used to treat memory and cognitive dysfunction. Because of their efficacy and lower toxic effects, research on α- and ß-asarone, the phytoconstituents, has attracted attention owing to their remarkable pharmacological activities. Silver ion coordination complexation high-speed counter-current chromatography was used to separate these isomers from A. tatarinowii extract, coupled with accelerated solvent extraction. Accelerated solvent extraction parameters were investigated with single-factor and orthogonal testing. A two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (2:1:2:1, v/v) with 0.50 mol/L silver ions was selected for separation. From 2.0 g crude extract, 1.4 g of ß-asarone and 0.09 g of α-asarone were obtained with purities over 98% by sequential sample loading in 20 h. The isolated compounds were identified by electrospray ionization mass spectrometry, 1H and 13C NMR. Silver ions significantly increased the separation factor and retention of the stationary phase. The chromatographic behavior indicated that cis-configuration was more strongly complexed with the silver ion. This was further demonstrated with the help of computational analysis. In conclusion, the established method could be employed to separate other cis-trans or E/Z isomers that form coordination complexes.


Assuntos
Acorus/química , Anisóis/análise , Distribuição Contracorrente/métodos , Acorus/metabolismo , Anisóis/isolamento & purificação , Teoria da Densidade Funcional , Isomerismo , Extração Líquido-Líquido , Espectroscopia de Ressonância Magnética , Extratos Vegetais/química , Prata/química , Espectrometria de Massas por Ionização por Electrospray
18.
J Chromatogr A ; 1645: 462108, 2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-33857674

RESUMO

The solvation parameter model affords a useful tool to model distribution properties of neutral compounds in biphasic separation systems. Common applications include column characterization and method development in gas chromatography; reversed-phase, micellar and hydrophilic interaction liquid chromatography; supercritical fluid chromatography; and micellar electrokinetic chromatography. The characterization of the distribution properties of liquid-liquid partition systems is another major application of this model. This tutorial is aimed at establishing good practices for the application of the model to separation systems. Suitable experimental protocols to determine system constants by multiple linear regression analysis and descriptors by the Solver method are presented; statistical tools to evaluate model quality are discussed; and model-specific data analysis tools based on system maps and correlation diagrams are described.


Assuntos
Cromatografia Gasosa , Cromatografia Líquida , Extração Líquido-Líquido , Modelos Químicos , Modelos Lineares
19.
Arch Insect Biochem Physiol ; 106(3): e21771, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33644898

RESUMO

Antimicrobial proteins (AMPs) are small, cationic proteins that exhibit activity against bacteria, viruses, parasites, fungi as well as boost host-specific innate immune responses. Insects produce these AMPs in the fat body and hemocytes, and release them into the hemolymph upon microbial infection. Hemolymph was collected from the bacterially immunized fifth instar larvae of tasar silkworm, Antheraea mylitta, and an AMP was purified by organic solvent extraction followed by size exclusion and reverse-phase high-pressure liquid chromatography. The purity of AMP was confirmed by thin-layer chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The molecular mass was determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry as 14 kDa, and hence designated as AmAMP14. Peptide mass fingerprinting of trypsin-digested AmAMP14 followed by de novo sequencing of one peptide fragment by tandem mass spectrometry analysis revealed the amino acid sequences as CTSPKQCLPPCK. No homology was found in the database search and indicates it as a novel AMP. The minimum inhibitory concentration of the purified AmAMP14 was determined against Escherichia coli, Staphylococcus aureus, and Candida albicans as 30, 60, and 30 µg/ml, respectively. Electron microscopic examination of the AmAMP14-treated cells revealed membrane damage and release of cytoplasmic contents. All these results suggest the production of a novel 14 kDa AMP in the hemolymph of A. mylitta to provide defense against microbial infection.


Assuntos
Peptídeos Catiônicos Antimicrobianos , Hemolinfa/metabolismo , Proteínas de Insetos/isolamento & purificação , Mariposas/metabolismo , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Candida albicans/efeitos dos fármacos , Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Escherichia coli/efeitos dos fármacos , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacologia , Larva/metabolismo , Extração Líquido-Líquido/métodos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos
20.
J Chromatogr A ; 1642: 462032, 2021 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-33714769

RESUMO

Liquid-liquid extraction is one of the most widely used and simplest sample preparation techniques. However, consumption of large volumes of organic solvent and manual handling are two major drawbacks of this technique. A multifunction autosampler syringe is introduced which permits automated liquid-liquid extraction in an enclosed operating environment, with low consumption of organic solvents. The device described herein features a micromixer function in addition to common autosampler syringe features like accurate and precise aspirating and dispensing. To test the functionality of the micromixer syringe, manual extraction of caffeine from a tea infusion and semi-automated extraction of dichloroethane from water were carried out. Excellent recoveries of caffeine from a tea infusion (89% recovery with 1.3% RSD) and dichloroethane from water (107% recovery with 10% RSD) were obtained. Two automated workflows were tested using the micromixer syringe mounted in a laboratory autosampler. Standalone automated micro liquid-liquid extraction was performed for sample preparation of selected polychlorinated biphenyl (PCB) congeners prior to comprehensive two-dimensional gas chromatography - electron capture detection analysis. Extraction of PCBs using the described approach used substantially less solvent than a validated solid-phase extraction approach whilst delivering equivalent results for samples with high-level PCBs. Finally, fully automated extraction and GC-MS analysis of polynuclear aromatic hydrocarbons (PAHs) from water samples was performed. Mean recoveries of extraction for PCB and PAH analysis were > 70% using 4 min automated liquid-liquid extractions.


Assuntos
Extração Líquido-Líquido/instrumentação , Extração Líquido-Líquido/métodos , Compostos Orgânicos/isolamento & purificação , Água/química , Cromatografia Gasosa-Espectrometria de Massas , Compostos Orgânicos/análise , Bifenilos Policlorados/análise , Bifenilos Policlorados/isolamento & purificação , Hidrocarbonetos Policíclicos Aromáticos/análise , Hidrocarbonetos Policíclicos Aromáticos/isolamento & purificação , Solventes , Seringas , Poluentes Químicos da Água/análise
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