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1.
Nat Commun ; 12(1): 6085, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34667183

RESUMO

Isoflavonoids comprise a class of plant natural products with great nutraceutical, pharmaceutical and agricultural significance. Their low abundance in nature and structural complexity however hampers access to these phytochemicals through traditional crop-based manufacturing or chemical synthesis. Microbial bioproduction therefore represents an attractive alternative. Here, we engineer the metabolism of Saccharomyces cerevisiae to become a platform for efficient production of daidzein, a core chemical scaffold for isoflavonoid biosynthesis, and demonstrate its application towards producing bioactive glucosides from glucose, following the screening-reconstruction-application engineering framework. First, we rebuild daidzein biosynthesis in yeast and its production is then improved by 94-fold through screening biosynthetic enzymes, identifying rate-limiting steps, implementing dynamic control, engineering substrate trafficking and fine-tuning competing metabolic processes. The optimized strain produces up to 85.4 mg L-1 of daidzein and introducing plant glycosyltransferases in this strain results in production of bioactive puerarin (72.8 mg L-1) and daidzin (73.2 mg L-1). Our work provides a promising step towards developing synthetic yeast cell factories for de novo biosynthesis of value-added isoflavonoids and the multi-phased framework may be extended to engineer pathways of complex natural products in other microbial hosts.


Assuntos
Flavonoides/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vias Biossintéticas , Isoflavonas/metabolismo , Engenharia Metabólica
2.
Molecules ; 26(20)2021 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-34684855

RESUMO

Vitexin is a C-glucoside flavone that exhibits a wide range of pharmaceutical activities. However, the poor solubility of vitexin limits its applications. To resolve this limitation, two glycoside hydrolases (GHs) and four glycosyltransferases (GTs) were assayed for glycosylation activity toward vitexin. The results showed that BtGT_16345 from the Bacillus thuringiensis GA A07 strain possessed the highest glycosylation activity, catalyzing the conversion of vitexin into new compounds, vitexin-4'-O-ß-glucoside (1) and vitexin-5-O-ß-glucoside (2), which showed greater aqueous solubility than vitexin. To our knowledge, this is the first report of vitexin glycosylation. Based on the multiple bioactivities of vitexin, the two highly soluble vitexin derivatives might have high potential for pharmacological usage in the future.


Assuntos
Apigenina/metabolismo , Glucosídeos/metabolismo , Bacillus thuringiensis/metabolismo , Catálise , Flavonas/metabolismo , Glicosilação , Glicosiltransferases/metabolismo , Isoflavonas/metabolismo , Solubilidade
3.
Nutrients ; 13(8)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34444848

RESUMO

Flavonoids are a major group of dietary plant polyphenols and have a positive health impact, but their modification and degradation in the human gut is still widely unknown. Due to the rise of metagenome data of the human gut microbiome and the assembly of hundreds of thousands of bacterial metagenome-assembled genomes (MAGs), large-scale screening for potential flavonoid-modifying enzymes of human gut bacteria is now feasible. With sequences of characterized flavonoid-transforming enzymes as queries, the Unified Human Gastrointestinal Protein catalog was analyzed and genes encoding putative flavonoid-modifying enzymes were quantified. The results revealed that flavonoid-modifying enzymes are often encoded in gut bacteria hitherto not considered to modify flavonoids. The enzymes for the physiologically important daidzein-to-equol conversion, well studied in Slackiaisoflavoniconvertens, were encoded only to a minor extent in Slackia MAGs, but were more abundant in Adlercreutzia equolifaciens and an uncharacterized Eggerthellaceae species. In addition, enzymes with a sequence identity of about 35% were encoded in highly abundant MAGs of uncultivated Collinsella species, which suggests a hitherto uncharacterized daidzein-to-equol potential in these bacteria. Of all potential flavonoid modification steps, O-deglycosylation (including derhamnosylation) was by far the most abundant in this analysis. In contrast, enzymes putatively involved in C-deglycosylation were detected less often in human gut bacteria and mainly found in Agathobacter faecis (formerly Roseburia faecis). Homologs to phloretin hydrolase, flavanonol/flavanone-cleaving reductase and flavone reductase were of intermediate abundance (several hundred MAGs) and mainly prevalent in Flavonifractor plautii. This first comprehensive insight into the black box of flavonoid modification in the human gut highlights many hitherto overlooked and uncultured bacterial genera and species as potential key organisms in flavonoid modification. This could lead to a significant contribution to future biochemical-microbiological investigations on gut bacterial flavonoid transformation. In addition, our results are important for individual nutritional recommendations and for biotechnological applications that rely on novel enzymes catalyzing potentially useful flavonoid modification reactions.


Assuntos
Proteínas de Bactérias/metabolismo , Flavonoides/metabolismo , Microbioma Gastrointestinal/fisiologia , Fenômenos Fisiológicos da Nutrição/fisiologia , Simulação por Computador , Equol/metabolismo , Genoma Bacteriano , Humanos , Isoflavonas/metabolismo , Metagenoma , Peptídeo Hidrolases/metabolismo , Proteólise
4.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203212

RESUMO

Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated isoflavonoids for inhibition of firefly luciferase. These natural compounds are often studied using luciferase reporter-gene assays. We used a quantitative structure-activity relationship (QSAR) model to compare the results of in silico predictions with a newly developed in vitro assay that enables concomitant detection of inhibition of firefly and Renilla luciferases. The QSAR model predicted a moderate to high likelihood of firefly luciferase inhibition for all of the 11 isoflavonoids investigated, and the in vitro assays confirmed this for seven of them: daidzein, genistein, glycitein, prunetin, biochanin A, calycosin, and formononetin. In contrast, none of the 11 isoflavonoids inhibited Renilla luciferase. Molecular docking calculations indicated that isoflavonoids interact favorably with the D-luciferin binding pocket of firefly luciferase. These data demonstrate the importance of reporter-enzyme inhibition when studying the effects of such compounds and suggest that this in vitro assay can be used to exclude false-positives due to firefly or Renilla luciferase inhibition, and to thus define the most appropriate reporter gene.


Assuntos
Genes Reporter/fisiologia , Isoflavonas/metabolismo , Luciferases de Renilla/metabolismo , Animais , Vaga-Lumes , Genes Reporter/genética , Isoflavonas/química , Luciferases de Renilla/química , Estrutura Secundária de Proteína
5.
PLoS One ; 16(7): e0254190, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34214105

RESUMO

Several isoflavonoids are well known for their ability to act as soybean phytoalexins. However, the overall effects of the soybean-Aspergillus oryzae interaction on metabolism remain largely unknown. The aim of this study is to reveal an overview of nutritive and metabolic changes in germinated and A. oryzae-elicited soybeans. The levels of individual nutrients were measured using the ustulation, ashing, Kjeldahl, and Folch methods. The levels of individual amino acids were measured using high-performance liquid chromatography. Low-molecular-weight compounds were measured through metabolome analysis using liquid chromatography-mass spectrometry. Although the levels of individual nutrients and amino acids were strongly influenced by the germination process, the elicitation process had little effect on the change in the contents of individual nutrients and amino acids. However, after analyzing approximately 700 metabolites using metabolome analysis, we found that the levels of many of the metabolites were strongly influenced by soybean-A. oryzae interactions. In particular, the data indicate that steroid, terpenoid, phenylpropanoid, flavonoid, and fatty acid metabolism were influenced by the elicitation process. Furthermore, we demonstrated that not the germination process but the elicitation process induced daidzein prenylation, suggesting that the soybean-A. oryzae interactions produce various phytoalexins that are valuable for health promotion and/or disease prevention.


Assuntos
Aspergillus oryzae/metabolismo , Isoflavonas/metabolismo , Metaboloma/fisiologia , Prenilação/fisiologia , Soja/metabolismo , Aminoácidos/metabolismo , Fermentação/fisiologia , Flavonoides/metabolismo , Germinação/fisiologia , Nutrientes/metabolismo , Extratos Vegetais/metabolismo
6.
Biochem Biophys Res Commun ; 569: 61-65, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34229124

RESUMO

For the beneficial pharmacological properties of isoflavonoids and their related glycoconjugates, there is increasingly interest in their enzymatic conversion. In this study, a novel ß-glucosidase gene isolated from metagenomic library of mangrove sediment was cloned and overexpressed in Escherichia coli BL21(DE3). The purified recombination ß-glucosidase, designated as r-Bgl66, showed high catalytic activity for soy isoflavone glycosides. It converted soy isoflavone flour extract with the productivities of 0.87 mM/h for daidzein, 0.59 mM/h for genistein and 0.42 mM/h for glycitein. The kcat/Km values for daidzin, genistin and glycitin were 208.73, 222.37 and 288.07 mM-1 s-1, respectively. In addition, r-Bgl66 also exhibited the characteristic of glucose-tolerance, and the inhibition constant Ki was 471.4 mM. These properties make it a good candidate in the enzymatic hydrolysis of soy isoflavone glycosides. This study also highlights the utility of metagenomic approach in discovering novel ß-glucosidase for soy isoflavone glycosides hydrolysis.


Assuntos
Avicennia/crescimento & desenvolvimento , Glicosídeos/metabolismo , Isoflavonas/metabolismo , Metagenoma/genética , Microbiologia do Solo , beta-Glucosidase/metabolismo , Biocatálise/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Biblioteca Gênica , Sedimentos Geológicos/microbiologia , Glucose/metabolismo , Glucose/farmacologia , Hidrólise , Cinética , Proteínas Recombinantes/metabolismo , Soja/metabolismo , beta-Glucosidase/genética , beta-Glucosidase/isolamento & purificação
7.
FEMS Microbiol Lett ; 368(13)2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34173644

RESUMO

Equol is the isoflavone-derived metabolite with the greatest estrogenic and antioxidant activity. It is produced from daidzein by fastidious and oxygen-susceptible intestinal bacteria, which hinders their use at an industrial scale. Therefore, expressing the equol production machinery into easily-cultivable hosts would expedite the heterologous production of this compound. In this work, four genes (racemase, tdr, ddr and dzr) coding for key enzymes involved in equol production in Adlercreutzia equolifaciens DSM19450T were synthesized and cloned in a pUC-derived vector (pUC57-equol) that was introduced in Escherichia coli. Recombinant clones of E. coli produced equol in cultures supplemented with daidzein (equol precursor) and dihydrodaidzein (intermediate compound). To check whether equol genes were expressed in Gram-positive bacteria, the pUC57-equol construct was cloned into the low-copy-number vector pIL252, and the new construct (pIL252-pUC57-equol) introduced into model strains of Lacticaseibacillus casei and Lactococcus lactis. L. casei clones carrying pIL252-pUC57-equol produced a small amount of equol from dihydrodaidzein but not from daidzein, while L. lactis recombinant clones produced no equol from either of the substrates. This is the first time that A. equolifaciens equol genes have been cloned and expressed in heterologous hosts. E. coli clones harboring pUC57-equol could be used for biotechnological production of equol.


Assuntos
Actinobacteria/genética , Proteínas de Bactérias/genética , Equol/biossíntese , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Escherichia coli/genética , Isoflavonas/metabolismo
8.
J Sci Food Agric ; 101(13): 5314-5324, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34032287

RESUMO

Soy contains many bioactive phytochemicals, such as isoflavones, which have the effect of preventing many cancers. Some studies have shown the beneficial effect of soy-based food and isoflavone intake on gastric cancer (GC), while others claimed no effect. Therefore, whether the beneficial effect of soy-based food is related to its fermentation or whether its protective effect comes from isoflavones still remains inconclusive. Our aim was to investigate the relationship between total soybean, fermented soybean, non-fermented soybean and isoflavone intake, and the risk of GC. Ten cohort studies and 21 case-control studies involving 916 354 participants were included. The association between soy-based food and isoflavone intake and the risk of GC was calculated with the pooled relative risks (RRs) for the highest versus lowest intake categories. The results showed that isoflavone intake might be a protective factor to GC, but the result was not statistically significant (RR = 0.92; 95% CI: 0.79-1.07). However, total soybean intake could significantly decrease the risk of GC by 36% (RR = 0.64; 95% CI: 0.51-0.80), which might be credited to non-fermented soybean products (RR = 0.79; 95% CI: 0.71-0.87). In contrast, high intake of fermented soybean products could increase the risk of GC (RR = 1.19; 95% CI: 1.02-1.38). High intake of total soybean and non-fermented soybean products could reduce the risk of GC, and high intake of fermented soybean products could increase the risk, which indicated that the beneficial effect of soy-based food might be related to its non-fermentation. However, high intake of isoflavones may not be associated with the incidence of GC. © 2021 Society of Chemical Industry.


Assuntos
Isoflavonas/metabolismo , Substâncias Protetoras/metabolismo , Soja/metabolismo , Neoplasias Gástricas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Isoflavonas/análise , Masculino , Pessoa de Meia-Idade , Substâncias Protetoras/análise , Fatores de Risco , Sementes/química , Sementes/metabolismo , Alimentos de Soja/análise , Soja/química , Neoplasias Gástricas/epidemiologia , Neoplasias Gástricas/prevenção & controle , Adulto Jovem
9.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33809928

RESUMO

Based on their nutrient composition, soybeans and related foods have been considered to be nutritious and healthy for humans. Particularly, the biological activity and subsequent benefits of soy products may be associated with the presence of isoflavone in soybeans. As an alternative treatment for menopause-related symptoms, isoflavone has gained much popularity for postmenopausal women who have concerns related to undergoing hormone replacement therapy. However, current research has still not reached a consensus on the effects of isoflavone on humans. This overview is a summary of the current literature about the processing of soybeans and isoflavone types (daidzein, genistein, and S-equol) and supplements and their extraction and analysis as well as information about the utilization of isoflavones in soybeans. The processes of preparation (cleaning, drying, crushing and dehulling) and extraction of soybeans are implemented to produce refined soy oil, soy lecithin, free fatty acids, glycerol and soybean meal. The remaining components consist of inorganic constituents (minerals) and the minor components of biologically interesting small molecules. Regarding the preventive effects on diseases or cancers, a higher intake of isoflavones is associated with a moderately lower risk of developing coronary heart disease. It may also reduce the risks of breast and colorectal cancer as well as the incidence of breast cancer recurrence. Consumption of isoflavones or soy foods is associated with reduced risks of endometrial and bladder cancer. Regarding the therapeutic effects on menopausal syndrome or other diseases, isoflavones have been found to alleviate vasomotor syndromes even after considering placebo effects, reduce bone loss in the spine and ameliorate hypertension and in vitro glycemic control. They may also alleviate depressive symptoms during pregnancy. On the other hand, isoflavones have not shown definitive effects regarding improving cognition and urogenital symptoms. Because of lacking standardization in the study designs, such as the ingredients and doses of isoflavones and the durations and outcomes of trials, it currently remains difficult to draw overall conclusions for all aspects of isoflavones. These limitations warrant further investigations of isoflavone use for women's health.


Assuntos
Isoflavonas/administração & dosagem , Menopausa/efeitos dos fármacos , Fitoestrógenos/administração & dosagem , Extratos Vegetais/administração & dosagem , Soja/química , Animais , Fracionamento Químico , Suplementos Nutricionais , Avaliação Pré-Clínica de Medicamentos , Fogachos/tratamento farmacológico , Humanos , Isoflavonas/química , Isoflavonas/isolamento & purificação , Isoflavonas/metabolismo , Redes e Vias Metabólicas , Fitoestrógenos/química , Fitoestrógenos/isolamento & purificação , Fitoestrógenos/metabolismo , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Soja/metabolismo , Análise Espectral , Relação Estrutura-Atividade , Síndrome
10.
Molecules ; 26(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923487

RESUMO

The aryl hydrocarbon receptor (AhR) is a transcription factor deeply implicated in health and diseases. Historically identified as a sensor of xenobiotics and mainly toxic substances, AhR has recently become an emerging pharmacological target in cancer, immunology, inflammatory conditions, and aging. Multiple AhR ligands are recognized, with plant occurring flavonoids being the largest group of natural ligands of AhR in the human diet. The biological implications of the modulatory effects of flavonoids on AhR could be highlighted from a toxicological and environmental concern and for the possible pharmacological applicability. Overall, the possible AhR-mediated harmful and/or beneficial effects of flavonoids need to be further investigated, since in many cases they are contradictory. Similar to other AhR modulators, flavonoids commonly exhibit tissue, organ, and species-specific activities on AhR. Such cellular-context dependency could be probably beneficial in their pharmacotherapeutic use. Flavones, flavonols, flavanones, and isoflavones are the main subclasses of flavonoids reported as AhR modulators. Some of the structural features of these groups of flavonoids that could be influencing their AhR effects are herein summarized. However, limited generalizations, as well as few outright structure-activity relationships can be suggested on the AhR agonism and/or antagonism caused by flavonoids.


Assuntos
Flavonoides/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Humanos , Isoflavonas/metabolismo , Compostos Fitoquímicos/metabolismo , Polifenóis/metabolismo , Fatores de Transcrição/metabolismo
11.
FEMS Microbiol Lett ; 368(8)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33930123

RESUMO

Equol, which produced from daidzein (one of the principal isoflavones), is recognized to be the most resultful in stimulating an estrogenic and antioxidant response. The daidzein transformation was studied during fermentation of five growth media inoculated with feces from a healthy human, and a daidzein conversion strain was isolated. To enrich the bacterial population involved in daidzein metabolism in a complex mixture, fecal samples were treated with antibiotics. The improved propidium monoazide combined with the quantitative polymerase chain reaction (PMAxx-qPCR) assay showed that the ampicillin treatment of samples did result in a reduction of the total visible bacteria counts by 52.2% compared to the treatment without antibiotics. On this basis, the newly isolated rod-shaped, Gram-positive anaerobic bacterium, named strain Y11 (MN560033), was able to metabolize daidzein to equol under anaerobic conditions, with a conversion ratio (equol ratio: the amount of equol produced/amount of supplemented daizein) of 0.56 over 120 h. The 16S rRNA partial sequence of the strain Y11 exhibited 99.8% identity to that of Slackia equolifaciens strain DZE (NR116295). This study will provide new insights into the biotransformation of equol from daidzein by intestinal microbiota from the strain-level and explore the possibility of probiotic interventions.


Assuntos
Bactérias Anaeróbias/classificação , Equol/metabolismo , Bacilos Gram-Positivos/classificação , Isoflavonas/metabolismo , Bactérias Anaeróbias/isolamento & purificação , Bactérias Anaeróbias/metabolismo , Técnicas de Tipagem Bacteriana , Biotransformação , DNA Bacteriano/genética , Fezes/microbiologia , Bacilos Gram-Positivos/isolamento & purificação , Bacilos Gram-Positivos/metabolismo , Humanos , Intestinos/microbiologia , Testes de Sensibilidade Microbiana , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
12.
Cancer Sci ; 112(5): 1772-1784, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33682294

RESUMO

Traditional Chinese medicine treatment of diseases has been recognized, but the material basis and mechanisms are not clear. In this study, target prediction of the antigastric cancer (GC) effect of Guiqi Baizhu (GQBZP) and the analysis of potential key compounds, key targets, and key pathways for the therapeutic effects against GC were carried out based on the method of network analysis and Kyoto Encyclopedia of Genes and Genomes enrichment. There were 33 proteins shared between GQBZP and GC, and 131 compounds of GQBZP had a high correlation with these proteins, indicating that the PI3K-AKT signaling pathway might play a key role in GC. From these studies, we selected human epidermal growth factor receptor 2 (HER2) and programmed cell death 1-ligand 1 (PD-L1) for docking; the results showed that 385 and 189 compounds had high docking scores with HER2 and PD-L1, respectively. Six compounds were selected for microscale thermophoresis (MST). Daidzein/quercetin and isorhamnetin/formononetin had the highest binding affinity for HER2 and PD-L1, with Kd values of 3.7 µmol/L and 490, 667, and 355 nmol/L, respectively. Molecular dynamics simulation studies based on the docking complex structures as the initial conformation yielded the binding free energy between daidzein/quercetin with HER2 and isorhamnetin/formononetin with PD-L1, calculated by molecular mechanics Poisson-Boltzmann surface area, of -26.55, -14.18, -19.41, and -11.86 kcal/mol, respectively, and were consistent with the MST results. In vitro experiments showed that quercetin, daidzein, and isorhamnetin had potential antiproliferative effects in MKN-45 cells. Enzyme activity assays showed that quercetin could inhibit the activity of HER2 with an IC50 of 570.07 nmol/L. Our study provides a systematic investigation to explain the material basis and molecular mechanism of traditional Chinese medicine in treating diseases.


Assuntos
Antígeno B7-H1/metabolismo , Medicamentos de Ervas Chinesas/metabolismo , Proteínas de Neoplasias/metabolismo , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/metabolismo , Antígeno B7-H1/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Medicamentos de Ervas Chinesas/uso terapêutico , Humanos , Isoflavonas/metabolismo , Isoflavonas/farmacologia , Simulação de Acoplamento Molecular/métodos , Proteínas de Neoplasias/química , Fosfatidilinositol 3-Quinases/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quercetina/análogos & derivados , Quercetina/metabolismo , Quercetina/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/química , Transdução de Sinais , Neoplasias Gástricas/tratamento farmacológico
13.
J Chromatogr Sci ; 59(5): 412-418, 2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33723580

RESUMO

Calycosin and formononetin were efficiently extracted from Astragali Radix and purified by high-speed countercurrent chromatography. Calycosin and formononetin could be hydrolyzed from calycosin-7-glucoside and ononin, respectively. The best extraction conditions were realized by single factor and orthogonal experiments, which were 100% ethanol, 2.5 mol/L hydrochloric acid, 1:40 ratio of solid to liquid, extracted 2 h and one time. The two-phase solvent system of n-hexane-ethyl acetate-ethanol-water (3:5:3:5, v/v) was selected for the purification of calycosin, and 1.3 mg calycosin (the purity was 95.8% and the recovery was 85.9%) was obtained from 264.9-mg crude extraction. The two-phase solvent system of n-hexane-ethyl acetate-ethanol-water (4:5:4:5, v/v) was selected for the purification of formononetin, and 2.0 mg formononetin (the purity was 98.9% and the recovery was 84.4%) was obtained from 248.9-mg crude extraction. Their structures were identified by HPLC, melting points, UV, FTIR, ESI-MS, 1H NMR and 13C NMR spectrum. According to the antioxidant activity assay, the scavenging abilities of calycosin to 1,1-diphenyl-2-picrylhydrazyl and hydroxyl free radicals (·OH) were stronger. The scavenging effect of formononetin was not demonstrated.


Assuntos
Distribuição Contracorrente/métodos , Medicamentos de Ervas Chinesas/química , Isoflavonas/isolamento & purificação , Extração Líquido-Líquido/métodos , Astragalus propinquus , Cromatografia Líquida de Alta Pressão , Sequestradores de Radicais Livres/análise , Sequestradores de Radicais Livres/isolamento & purificação , Sequestradores de Radicais Livres/metabolismo , Isoflavonas/análise , Isoflavonas/metabolismo
14.
Int J Biol Macromol ; 180: 252-261, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33741369

RESUMO

Strong inhibition of the human UDP-glucuronosyltransferase enzymes (UGTs) may lead to undesirable effects, including hyperbilirubinaemia and drug/herb-drug interactions. Currently, there is no good way to examine the inhibitory effects and specificities of compounds toward all the important human UGTs, side-by-side and under identical conditions. Herein, we report a new, broad-spectrum substrate for human UGTs and its uses in screening and characterizing of UGT inhibitors. Following screening a variety of phenolic compound(s), we have found that methylophiopogonanone A (MOA) can be readily O-glucuronidated by all tested human UGTs, including the typical N-glucuronidating enzymes UGT1A4 and UGT2B10. MOA-O-glucuronidation yielded a single mono-O-glucuronide that was biosynthesized and purified for structural characterization and for constructing an LC-UV based MOA-O-glucuronidation activity assay, which was then used for investigating MOA-O-glucuronidation kinetics in recombinant human UGTs. The derived Km values were crucial for selecting the most suitable assay conditions for assessing inhibitory potentials and specificity of test compound(s). Furthermore, the inhibitory effects and specificities of four known UGT inhibitors were reinvestigated by using MOA as the substrate for all tested UGTs. Collectively, MOA is a broad-spectrum substrate for the human UGTs, which offers a new and practical tool for assessing inhibitory effects and specificities of UGT inhibitors.


Assuntos
Benzodioxóis/metabolismo , Inibidores Enzimáticos/farmacologia , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/metabolismo , Isoflavonas/metabolismo , Animais , Benzodioxóis/química , Cães , Avaliação Pré-Clínica de Medicamentos/métodos , Interações Medicamentosas , Inibidores Enzimáticos/metabolismo , Feminino , Glucuronídeos/química , Glucuronídeos/metabolismo , Glucuronosiltransferase/química , Humanos , Isoflavonas/química , Cinética , Macaca fascicularis , Masculino , Camundongos , Microssomos Hepáticos/metabolismo , Coelhos , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
15.
J Sci Food Agric ; 101(12): 5124-5131, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33608899

RESUMO

BACKGROUND: Okara is a major agri-industrial by-product of the tofu and soymilk industries. Employing food-wastes as substrates for the green production of natural functional compounds is a recent trend that addresses the dual concepts of sustainable production and a zero-waste ecosystem. RESULTS: Extracts of unfermented okara and okara fermented with Rhizopus oligosporus were obtained using ethanol as extraction solvent, coupled with ultrasound sonication for enhanced extraction. Fermented extracts yielded significantly better results for total phenolic content (TPC) and total flavonoid content (TFC) than unfermented extracts. A qualitative liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) analysis revealed a shift from glucoside forms to respective aglycone forms of the detected isoflavones, post-fermentation. Since the aglycone forms have been associated with numerous health benefits, a quantitative high-performance liquid chromatography (HPLC) analysis was performed. Fermented okara extracts had daidzein and genistein concentrations of 11.782 ± 0.325 µg mL-1 and 10.125 ± 1.028 µg mL-1 , as opposed to that of 6.7 ± 2.42 µg mL-1 and 4.55 ± 0.316 µg mL-1 in raw okara extracts, respectively. Lastly, the detected isoflavones were mapped to their metabolic pathways, to understand the biochemical reactions triggered during the fermentation process. CONCLUSION: Fermented okara may be implemented as a sustainable solution for production of natural bioactive isoflavonoids genistein and daidzein. © 2021 Society of Chemical Industry.


Assuntos
Genisteína/metabolismo , Isoflavonas/metabolismo , Rhizopus/metabolismo , Alimentos de Soja/análise , Resíduos/análise , Fermentação , Manipulação de Alimentos , Genisteína/análise , Isoflavonas/análise , Metabolômica , Extratos Vegetais/análise , Extratos Vegetais/metabolismo , Sementes/química , Sementes/metabolismo , Sementes/microbiologia , Alimentos de Soja/microbiologia , Soja/química , Soja/metabolismo , Soja/microbiologia
16.
Food Chem ; 347: 128981, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-33444886

RESUMO

The interaction between ovalbumin (OVA) and isoflavonoid glabridin (GB) was investigated using spectroscopic and molecular docking techniques. Fluorescence spectroscopy revealed that GB was bound to OVA mainly due to hydrogen bonding and hydrophobic forces. FT-IR spectroscopy showed that the combination of GB and OVA resulted in a decrease in the ß-sheet content of OVA and an increase in the α-helix and extended-chain content. All these experimental results were supported and clarified by molecular docking simulations. GB binding was able to inhibit chemical denaturant-induced structural changes in OVA as observed by intrinsic tryptophan and ANS fluorescence. Moreover, GB-OVA complex increased the aqueous solubility of GB by about 4.45 times at pH 7.0. These results provided insights into the interaction between GB and OVA that contributes to the utilization of GB in the food and pharmaceutical industries.


Assuntos
Isoflavonas/química , Simulação de Acoplamento Molecular , Ovalbumina/química , Fenóis/química , Sítios de Ligação , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Isoflavonas/metabolismo , Nanoestruturas/química , Ovalbumina/metabolismo , Tamanho da Partícula , Fenóis/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Desnaturação Proteica , Ureia/química
17.
Plant Cell Rep ; 40(3): 517-528, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33389047

RESUMO

KEY MESSAGE: Isoflavones are not involved in rhizobial signaling in red clover, but likely play a role in defense in the rhizosphere. Red clover (Trifolium pratense) is a high-quality forage legume, well suited for grazing and hay production in the temperate regions of the world. Like many legumes, red clover produces a number of phenylpropanoid compounds including anthocyanidins, flavan-3-ols, flavanols, flavanones, flavones, and isoflavones. The study of isoflavone biosynthesis and accumulation in legumes has come into the forefront of biomedical and agricultural research due to potential for medicinal, antimicrobial, and environmental implications. CRISPR/Cas9 was used to knock out the function of a key enzyme in the biosynthesis of isoflavones, isoflavone synthase (IFS1). A hemizygous plant carrying a 9-bp deletion in the IFS1 gene was recovered and was intercrossed to obtain homozygous mutant plants. Levels of the isoflavones formononetin, biochanin A and genistein were significantly reduced in the mutant plants. Wild-type and mutant plants were inoculated with rhizobia to test the effect of the mutation on nodulation, but no significant differences were observed, suggesting that these isoflavones do not play important roles in nodulation. Gene expression profiling revealed an increase in expression of the upstream genes producing the precursors for IFS1, namely, phenylalanine ammonium lyase and chalcone synthase, but there were no significant differences in IFS1 gene expression or in the downstream genes in the production of specific isoflavones. Higher expression in genes involved in ethylene response was observed in the mutant plants. This response is normally associated with biotic stress, suggesting that the plants may have been responding to cues in the surrounding rhizosphere due to lower levels of isoflavones.


Assuntos
Isoflavonas/metabolismo , Oxigenases/genética , Proteínas de Plantas/genética , Trifolium/genética , Trifolium/metabolismo , Sistemas CRISPR-Cas , Deleção de Genes , Regulação da Expressão Gênica de Plantas , Genisteína/metabolismo , Isoflavonas/genética , Oxigenases/metabolismo , Proteínas de Plantas/metabolismo , Nodulação/genética , Plantas Geneticamente Modificadas , Rhizobium/fisiologia , Rizosfera
18.
Phytother Res ; 35(1): 180-197, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32780464

RESUMO

Functional foods have nutritional properties and organic functions, which are beneficial to health. Certain types of functional food components are so-called phytoestrogens, non-steroidal compounds derived from the metabolism of precursors contained in plants, which originate secondary metabotypes known to induce biological responses and by mimicry or modulating the action of endogenous estrogen. These molecules are involved in several physiological and pathological processes related to reproduction, bone remodeling, skin, cardiovascular, nervous, immune systems, and metabolism. This review aimed to present an overview of phytoestrogens regarding their chemical structure, actions, and effects in the organism given several pathologies. Several studies have demonstrated beneficial phytoestrogen actions, such as lipid profile improvement, cognitive function, menopause, oxidative stress, among others. Phytoestrogens effects are not completely elucidated, being necessary future research to understand the exact action mechanisms, whether they are via estrogen receptor or whether other hidden mechanisms produce these effects. Thus, this review makes a general approach to the phytoestrogen actions, beneficial effects, risk and limitations. However, the complexities of biological effects after ingestion of phytoestrogens and the differences in their metabolism and bioavailability indicate that interpretation of either risk or benefits needs to be made with caution.


Assuntos
Fitoestrógenos/farmacologia , Antioxidantes/farmacologia , Doenças Cardiovasculares , Cognição , Dieta , Feminino , Humanos , Isoflavonas/metabolismo , Lipídeos/sangue , Menopausa , Fármacos Neuroprotetores/farmacologia , Osteoporose , Estresse Oxidativo , Fitoestrógenos/química , Plantas , Receptores de Estrogênio/metabolismo
19.
Food Chem ; 343: 128553, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33176956

RESUMO

This study investigated the bioconversion and bioaccessibility of soy isoflavones produced in sogurt fermented with S. thermophilus and L. bulgaricus during in vitro digestion. The highest survivability of S. thermophilus (6.49 log cfu/mL) and L. bulgaricus (6.48 log cfu/mL) was in oral phase. In gastric phase, the total aglycones of sogurt (26.73 g/L) increased up to 20 times than control (1.21 g/L), with a significant increase in daidzein (17.05 g/L) and genistein (9.68 g/L). Addition of 8U of ß-glucosidase into soymilk significantly increased the conversion of isoflavone in ENTII (daidzein: 0.46 g/L; genistein: 0.18 g/L) than in ENTI (daidzein: 0.33 g/L; genistein: 0.20 g/L). The particle size analysis and confocal micrographs of digesta also suggest the size of fat and protein in gastric phase to be smaller than in intestinal phase. The results indicate the prospective to develop soy-based fermented products capable of releasing high isoflavone in the digestive system.


Assuntos
Isoflavonas/metabolismo , Iogurte/análise , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Digestão , Genisteína/metabolismo , Isoflavonas/análise , Lactobacillus delbrueckii/crescimento & desenvolvimento , Lactobacillus delbrueckii/metabolismo , Leite de Soja/metabolismo , Streptococcus thermophilus/crescimento & desenvolvimento , Streptococcus thermophilus/metabolismo , beta-Glucosidase/metabolismo
20.
Chembiochem ; 22(7): 1223-1231, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33237595

RESUMO

ß-Glucosidases are used in the food industry to hydrolyse glycosidic bonds in complex sugars, with enzymes sourced from extremophiles better able to tolerate the process conditions. In this work, a novel ß-glycosidase from the acidophilic organism Alicyclobacillus herbarius was cloned and heterologously expressed in Escherichia coli BL21(DE3). AheGH1 was stable over a broad range of pH values (5-11) and temperatures (4-55 °C). The enzyme exhibited excellent tolerance to fructose and good tolerance to glucose, retaining 65 % activity in the presence of 10 % (w/v) glucose. It also tolerated organic solvents, some of which appeared to have a stimulating effect, in particular ethanol with a 1.7-fold increase in activity at 10 % (v/v). The enzyme was then applied for the cleavage of isoflavone from isoflavone glucosides in an ethanolic extract of soy flour, to produce soy isoflavones, which constitute a valuable food supplement, full conversion was achieved within 15 min at 30 °C.


Assuntos
Alicyclobacillus/enzimologia , Isoflavonas/metabolismo , Soja/química , beta-Glucosidase/metabolismo , Domínio Catalítico , Estabilidade Enzimática , Escherichia coli/metabolismo , Glicosídeos/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Isoflavonas/química , Cinética , Estrutura Terciária de Proteína , Soja/metabolismo , Temperatura , beta-Glucosidase/química , beta-Glucosidase/genética
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