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J Chromatogr A ; 1637: 461733, 2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33385745


A hydrophilic interaction (HILIC) ultra-high performance liquid chromatography (UHPLC) with triple quadrupole tandem mass spectrometry (MS/MS) method was developed and validated for the quantification of 21 free amino acids (AAs). Compared to published reports, our method renders collectively improved sensitivity with lower limit of quantification (LLOQ) at 0.5~42.19 ng/mL with 0.3 µL injection volume (or equivalently 0.15~12.6 pg injected on column), robust linear range from LLOQ up to 3521~5720 ng/mL (or 1056 ~ 1716 pg on column) and a high throughput with total time of 6 min per sample, as well as easier experimental setup, less maintenance and higher adaptation flexibility. Ammonium formate in the mobile phase, though commonly used in HILIC, was found unnecessary in our experimental setup, and its removal from mobile phase was key for significant improvement in sensitivity (4~74 times higher than with 5 mM ammonium formate). Addition of 10 (or up to100 mM) hydrochloric acid (HCl) in the sample diluent was crucial to keep response linearity for basic amino acids of histidine, lysine and arginine. Different HCl concentration (10~100 mM) in sample diluent also excreted an effect on detection sensitivity, and it is of importance to keep the final prepared sample and calibrators in the same HCl level. Leucine and isoleucine were distinguished using different transitions. Validated at seven concentration levels, accuracy was bound within 75~125%, matrix effect generally within 90~110%, and precision error mostly below 2.5%. Using this newly developed method, the free amino acids were then quantified in a total of 544 African indigenous vegetables (AIVs) samples from African nightshades (AN), Ethiopian mustards (EM), amaranths (AM) and spider plants (SP), comprising a total of 8 identified species and 43 accessions, cultivated and harvested in USA, Kenya and Tanzania over several years, 2013~2018. The AN, EM, AM and SP were distinguished based on free AAs profile using machine learning methods (ML) including principle component analysis, discriminant analysis, naïve Bayes, elastic net-regularized logistic regression, random forest and support vector machine, with prediction accuracy achieved at ca. 83~97% on the test set (train/test ratio at 7/3). An interactive ML platform was constructed using R Shiny at for modeling train-test simulation and category prediction of unknown AIV sample(s). This new method presents a robust and rapid approach to quantifying free amino acids in plants for use in evaluating plants, biofortification, botanical authentication, safety, adulteration and with applications to nutrition, health and food product development.

Aminoácidos/análise , Cromatografia Líquida de Alta Pressão/métodos , Aprendizado de Máquina , Espectrometria de Massas em Tandem/métodos , Verduras/química , Teorema de Bayes , Humanos , Interações Hidrofóbicas e Hidrofílicas , Análise de Componente Principal , Reprodutibilidade dos Testes
Plant Cell Physiol ; 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32931553


Choline, as a precursor of glycine betaine (GB) and phospholipids, is known to play roles in plant tolerance to salt stress, but the downstream metabolic pathways regulated by choline conferring salt tolerance are still unclear for non-GB accumulating species. The objectives were to examine how choline affect salt tolerance in a non-GB accumulating grass species and to determine major metabolic pathways of choline regulating salt tolerance involving GB or lipid metabolism. Kentucky bluegrass (Poa pratensis) plants were subjected to salt stress (100 mM NaCl) with or without foliar application of choline chloride (1 mM) in a growth chamber. Choline or GB alone and the combined application increased leaf photochemical efficiency, relative water content, and osmotic adjustment, and reduced leaf electrolyte leakage. Choline application had no effects on endogenous GB content and GB-synthesis genes did not show responses to choline under non-stress and salt stress conditions. GB was not detected in Kentucky bluegrass leaves. Lipidomic analysis revealed an increase in content of monogalactosyl diacylglycerol, phosphatidylcholine and phosphatidylethanolamine, and a decrease of phosphatidic acid content by choline application in plants exposed to salt stress. Choline-mediated lipid reprogramming could function as a dominant salt tolerance mechanism in non-GB accumulating grass species.

Rapid Commun Mass Spectrom ; 34(19): e8882, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32644256


RATIONALE: The safe consumption of Solanum scabrum and S. nigrum berries (SNBs) depends on a reliable and rapid chemical screen for the testing of the fruit and/or final food and industrial products for the presence and level of toxic glycoalkaloids. Such a rapid and sensitive screen could also be used by those involved in food safety and forensics, industry, research labs and those in agriculture production, breeding and food processing. Significant variation in the content and composition of glycoalkaloids across SNBs has been reported. To facilitate high-throughput targeted analysis, this work overcame the slow scan speed of a traditional triple quadruple mass spectrometry (QqQ) method by development of a pseudo-MS3 method. METHODS: In-source fragmentation functioned as a pseudo-MS or pseudo-hydrolysis to trim down the structurally diverse and complex glycosides into five types of aglycone ions, which were then analyzed using multiple reaction monitoring (MRM). Characteristic product ions were selected based on the aglycone skeleton and substitution pattern and associated fragmentation pathway. RESULTS: A compact method with only 15 MRM transitions were developed for high-throughput screening of very diverse glycoalkaloids. Glycosides of the same aglycone type were readily identified in the same transition window without the need for mass spectra interpretation. Validated using solamargine, the sole available standard, the accuracy was 99.7-101.3%, the intra- and inter-day precision were, respectively, 2.5-5.0% and 8.0-9.2%, and the lower limit of detection and quantification were, respectively, 3.1 and 10.2 ng/mL (with 1 µL injection volume). CONCLUSIONS: The peudo-MS3 method allowed for high-throughput targeted analysis with compact MRM transitions to address a large number of glycoalkaloids with diverse structures. This method could serve to meet the most heavy-duty demand for rapid inspection of glycoalkaloids in SNBs. This method can be adopted and used by those involved in food safety and forensics, in developing food and industrial products and in genetics and breeding.

Steroids ; 124: 29-34, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28549802


5α-Reductase is a key enzyme responsible for dihydrotestosterone biosynthesis and has been recognized as an important target for discovering new drugs against benign prostatic hyperplasia (BPH). In this study, a series of novel steroidal androst-3,5-diene-3-carboxylic acids have been designed and synthesized. Biological evaluations were performed on their 5α-reductase inhibitory activities by both in vitro enzyme inhibition assay and in vivo by prostate weighing method. Results showed that most of them displayed excellent 5α-reductase inhibitory potency. Detailed evaluation indicated that most of the compounds displayed slightly higher inhibition potency towards type 2 isozyme. Among all the compounds, 16a was found to be the most potential inhibitor with the IC50 of 0.25µM and 0.13µM against type 1 and 2 isozymes respectively. In vivo 5a-reductase inhibitory evaluation of 16a also showed a more significant reduction effect (p<0.001) in rat prostate weight than epristeride. Furthermore, the results of in silico ADME study indicated that compound 16a exhibited good pharmacokinetic properties. Thus, 16a could serve as promising lead candidates for further study.

Inibidores de 5-alfa Redutase/síntese química , Inibidores de 5-alfa Redutase/farmacologia , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/farmacologia , Colestenona 5 alfa-Redutase/metabolismo , Desenho de Fármacos , Inibidores de 5-alfa Redutase/química , Inibidores de 5-alfa Redutase/farmacocinética , Animais , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacocinética , Técnicas de Química Sintética , Simulação por Computador , Masculino , Ratos
Bioorg Med Chem Lett ; 27(12): 2668-2673, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28460819


The estrogen receptor (ER) has played an important role in breast cancer development and progression and is a central target for anticancer drug discovery. In order to develop novel selective ERα modulators (SERMs), we designed and synthesized 18 novel 3-aryl-4-anilino-2H-chromen-2-one derivatives based on previously reported lead compounds. The biological results indicated that most of the compounds presented potent ERα binding affinity and possessed better anti-proliferative activities against MCF-7 and Ishikawa cell lines than the positive control tamoxifen. The piperidyl substituted compounds such as 16d and 18d demonstrated strong ERα binding affinities and excellent anti-proliferative activities respectively. Compound 18d displayed the most potent ERα binding affinity with RBA value of 2.83%, while 16d exhibited the best anti-proliferative activity against MCF-7 cells with IC50 value of 4.52±2.47µM. Further molecular docking studies were also carried out to investigate binding pattern of the newly synthesized compounds with ERα. All these results together with the structure-activity relationships (SARs) indicated that these 3-aryl-4-anilino-2H-chromen-2-one derivatives with basic side chain could serve as promising leads for further optimization as novel SERMs.

Antineoplásicos/farmacologia , Cromonas/farmacologia , Desenho de Fármacos , Receptor alfa de Estrogênio/antagonistas & inibidores , Simulação de Acoplamento Molecular , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromonas/síntese química , Cromonas/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Receptor alfa de Estrogênio/metabolismo , Humanos , Células MCF-7 , Estrutura Molecular , Relação Estrutura-Atividade
Bioorg Med Chem Lett ; 27(4): 867-874, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28110871


Various 3-substituted 4-anilino-coumarin derivatives have been designed, synthesized and their anti-proliferative properties have been studied. The in vitro cytotoxicity screening was performed against MCF-7, HepG2, HCT116 and Panc-1 cancer cell lines by MTT assay. Most of the synthesized compounds exhibited comparable anti-proliferative activity to the positive control 5-Fluorouracil against these four tested cancer cell lines. Among the different substituents at C-3 position of coumarin scaffold, 3-trifluoroacetyl group showed the most promising results. Especially, compounds 33d (IC50=16.57, 5.45, 4.42 and 5.16µM) and 33e (IC50=20.14, 6.71, 4.62 and 5.62µM) showed excellent anti-proliferative activities on MCF-7, HepG2, HCT116 and Panc-1 cell lines respectively. In addition, cell cycle analysis and apoptosis activation revealed that 33d induced G2/M phase arrest and apoptosis in MCF-7 cells in a dose-dependent manner. Low toxicity of compounds 33d and 33e was observed against human umbilical vein endothelial cells (HUVECs), suggesting their acceptable safety profiles in normal cells. Furthermore, the results of in silico ADME studies indicated that both 33d and 33e exhibited good pharmacokinetic properties.

Antineoplásicos/síntese química , Cumarínicos/química , Desenho de Fármacos , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cumarínicos/farmacologia , Dano ao DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Células HCT116 , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Células MCF-7 , Relação Estrutura-Atividade