Identification of Helicobacter pylori-carcinogenic TNF-alpha-inducing protein inhibitors via daidzein derivatives through computational approaches.

Gastric cancer is considered a class 1 carcinogen that is closely linked to infection with Helicobacter pylori (H. pylori), which affects over 1 million people each year. However, the major challenge to fight against H. pylori and its associated gastric cancer due to drug resistance. This research gap had led our research team to investigate a potential drug candidate targeting the Helicobacter pylori-carcinogenic TNF-alpha-inducing protein. In this study, a total of 45 daidzein derivatives were investigated and the best 10 molecules were comprehensively investigated using in silico approaches for drug development, namely pass prediction, quantum calculations, molecular docking, molecular dynamics simulations, Lipinski rule evaluation, and prediction of pharmacokinetics. The molecular docking study was performed to evaluate the binding affinity between the target protein and the ligands. In addition, the stability of ligand-protein complexes was investigated by molecular dynamics simulations. Various parameters were analysed, including root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration (Rg), hydrogen bond analysis, principal component analysis (PCA) and dynamic cross-correlation matrix (DCCM). The results has confirmed that the ligand-protein complex CID: 129661094 (07) and 129664277 (08) formed stable interactions with the target protein. It was also found that CID: 129661094 (07) has greater hydrogen bond occupancy and stability, while the ligand-protein complex CID 129664277 (08) has greater conformational flexibility. Principal component analysis revealed that the ligand-protein complex CID: 129661094 (07) is more compact and stable. Hydrogen bond analysis revealed favourable interactions with the reported amino acid residues. Overall, this study suggests that daidzein derivatives in particular show promise as potential inhibitors of H. pylori.

Overexpression of GmPAL Genes Enhances Soybean Resistance Against Heterodera glycines.

Soybean cyst nematode (Heterodera glycines, soybean cyst nematode [SCN]) disease adversely affects the yield of soybean and leads to billions of dollars in losses every year. To control the disease, it is necessary to study the resistance genes of the plant and their mechanisms. Isoflavonoids are secondary metabolites of the phenylalanine pathway, and they are synthesized in soybean. They are essential in plant response to biotic and abiotic stresses. In this study, we reported that phenylalanine ammonia-lyase (PAL) genes GmPALs involved in isoflavonoid biosynthesis, can positively regulate soybean resistance to SCN. Our previous study demonstrated that the expression of GmPAL genes in the resistant cultivar Huipizhi (HPZ) heidou are strongly induced by SCN. PAL is the rate-limiting enzyme that catalyzes the first step of phenylpropanoid metabolism, and it responds to biotic or abiotic stresses. Here, we demonstrate that the resistance of soybeans against SCN is suppressed by PAL inhibitor l-α-(aminooxy)-ß-phenylpropionic acid (L-AOPP) treatment. Overexpression of eight GmPAL genes caused diapause of nematodes in transgenic roots. In a petiole-feeding bioassay, we identified that two isoflavones, daidzein and genistein, could enhance resistance against SCN and suppress nematode development. This study thus reveals GmPAL-mediated resistance against SCN, information that has good application potential. The role of isoflavones in soybean resistance provides new information for the control of SCN. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Exploring functional genes' correlation with (S)-equol concentration and new daidzein racemase identification.

With its estrogenic activity, (S)-equol plays an important role in maintaining host health and preventing estrogen-related diseases. Exclusive production occurs through the transformation of soy isoflavones by intestinal bacteria, but the reasons for variations in (S)-equol production among different individuals and species remain unclear. Here, fecal samples from humans, pigs, chickens, mice, and rats were used as research objects. The concentrations of (S)-equol, along with the genetic homology and evolutionary relationships of (S)-equol production-related genes [daidzein reductase (DZNR), daidzein racemase (DDRC), dihydrodaidzein reductase (DHDR), tetrahydrodaidzein reductase (THDR)], were analyzed. Additionally, in vitro functional verification of the newly identified DDRC gene was conducted. It was found that approximately 40% of human samples contained (S)-equol, whereas 100% of samples from other species contained (S)-equol. However, there were significant variations in (S)-equol content among the different species: rats > pigs > chickens > mice > humans. The distributions of the four genes displayed species-specific patterns. High detection rates across various species were exhibited by DHDR, THDR, and DDRC. In contrast, substantial variations in detection rates among different species and individuals were observed with respect to DZNR. It appears that various types of DZNR may be associated with different concentrations of (S)-equol, which potentially correspond to the regulatory role during (S)-equol synthesis. This enhances our understanding of individual variations in (S)-equol production and their connection with functional genes in vitro. Moreover, the newly identified DDRC exhibits higher potential for (S)-equol synthesis compared to the known DDRC, providing valuable resources for advancing in vitro (S)-equol production. IMPORTANCE: (S)-equol ((S)-EQ) plays a crucial role in maintaining human health, along with its known capacity to prevent and treat various diseases, including cardiovascular diseases, metabolic syndromes, osteoporosis, diabetes, brain-related diseases, high blood pressure, hyperlipidemia, obesity, and inflammation. However, factors affecting individual variations in (S)-EQ production and the underlying regulatory mechanisms remain elusive. This study examines the association between functional genes and (S)-EQ production, highlighting a potential correlation between the DZNR gene and (S)-EQ content. Various types of DZNR may be linked to the regulation of (S)-EQ synthesis. Furthermore, the identification of a new DDRC gene offers promising prospects for enhancing in vitro (S)-EQ production.

Network pharmacology study on fermented soybeans for the prevention of Alzheimer's disease in older individuals.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the disruption of synaptic communication among millions of neurons. Recent research has highlighted the potential therapeutic effectiveness of natural polyphenolic compounds in addressing AD. Soybeans are abundant in polyphenols, and their polyphenolic composition undergoes significant alteration through fermentation by Eurotium cristatum. Through comprehensive database searches, we identified active components within fermented soybean polyphenols and genes associated with AD. Subsequently, we utilized Venn diagrams to analyze the overlap between AD-related genes and these components. Furthermore, we visualized the network between intersecting targets and proteins using Cytoscape software. The anti-AD effects of soybeans were further explored through comprehensive analysis, including protein-protein interaction analysis, pathway enrichment analysis, and molecular docking studies. Our investigation unveiled 6-hydroxydaidzein as a major component of fermented soybean polyphenols, shedding light on its potential therapeutic significance in combating AD. The intersection between target proteins of fermented soybeans and disease-related targets in AD comprised 34 genes. Protein-protein interaction analysis highlighted key potential targets, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycogen synthase kinase 3 beta (GSK3B), amyloid precursor protein (APP), cyclin-dependent kinase 5 (CDK5), and beta-site APP cleaving enzyme 1 (BACE1). Molecular docking results demonstrated a robust binding effect between major components from fermented soybeans and the aforesaid key targets implicated in AD treatment. These findings suggest that fermented soybeans demonstrate a degree of efficacy and present promising prospects in the prevention of AD.

Toxic Features and Metabolomic Intervention of Glabrene, an Impurity Found in the Pharmaceutical Product of Glabridin.

Glabridin is a widely used product in the cosmetics and pharmaceutical industry, which is generally isolated and purified from Licorice (Glycyrrhiza glabra) extract in industrial production. It has wide clinical applications, but significant toxicity has also been reported. The purity of glabridin raw material is generally between 90% and 98%. We have identified a toxic impurity, glabrene, in the industrial product glabridin. Our investigation using an AB wild-type zebrafish toxicity test showed that glabrene has a significant lethal effect with an LC10 of 2.8 µM. Glabrene induced obvious malformation and disrupted cartilage development in zebrafish larvae. Furthermore, the compound significantly reduced larval mobility and caused damage to brain neural tissues. Metabolic pathway analysis and neurotransmitter quantification via ELISA indicated abnormal activation of the phenylalanine metabolic pathway, resulting in elevated dopamine and acetylcholine levels in vivo. These findings provide insights into the potential risks of glabrene contamination and offer a new reference point for enhancing safety measures and quality controls in licorice-derived products.

Bioinformatics Identification and Expression Analysis of Acetyl-CoA Carboxylase Reveal Its Role in Isoflavone Accumulation during Soybean Seed Development.

Isoflavones belong to the class of flavonoid compounds, which are important secondary metabolites that play a crucial role in plant development and defense. Acetyl-CoA carboxylase (ACCase) is a biotin-dependent enzyme that catalyzes the conversion of Acetyl-CoA into Malonyl-CoA in plants. It is a key enzyme in fatty acid synthesis and also catalyzes the production of various secondary metabolites. However, information on the ACC gene family in the soybean (Glycine max L. Merr.) genome and the specific members involved in isoflavone biosynthesis is still lacking. In this study, we identified 20 ACC family genes (GmACCs) from the soybean genome and further characterized their evolutionary relationships and expression patterns. Phylogenetic analysis showed that the GmACCs could be divided into five groups, and the gene structures within the same groups were highly conserved, indicating that they had similar functions. The GmACCs were randomly distributed across 12 chromosomes, and collinearity analysis suggested that many GmACCs originated from tandem and segmental duplications, with these genes being under purifying selection. In addition, gene expression pattern analysis indicated that there was functional divergence among GmACCs in different tissues. The GmACCs reached their peak expression levels during the early or middle stages of seed development. Based on the transcriptome and isoflavone content data, a weighted gene co-expression network was constructed, and three candidate genes (Glyma.06G105900, Glyma.13G363500, and Glyma.13G057400) that may positively regulate isoflavone content were identified. These results provide valuable information for the further functional characterization and application of GmACCs in isoflavone biosynthesis in soybean.

Enzymatic Fructosylation of Phenolic Compounds: A New Alternative for the Development of Antidiabetic Drugs.

Enzymatic fructosylation has emerged as a strategy to enhance the hydrophilicity of polyphenols by introducing sugar moieties, leading to the development of phenolic glycosides, which exhibit improved solubility, stability, and biological activities compared to their non-glycosylated forms. This study provides a detailed analysis of the interactions between five phenolic fructosides (4MFPh, MFF, DFPh, MFPh, and MFPu) and twelve proteins (11ß-HS1, CRP, DPPIV, IRS, PPAR-γ, GK, AMPK, IR, GFAT, IL-1ß, IL-6, and TNF-α) associated with the pathogenesis of T2DM. The strongest interactions were observed for phlorizin fructosides (DFPh) with IR (-16.8 kcal/mol) and GFAT (-16.9 kcal/mol). MFPh with 11ß-HS1 (-13.99 kcal/mol) and GFAT (-12.55 kcal/mol). 4MFPh with GFAT (-11.79 kcal/mol) and IR (-12.11 kcal/mol). MFF with AMPK (-9.10 kcal/mol) and PPAR- γ (-9.71 kcal/mol), followed by puerarin and ferulic acid monofructosides. The fructoside group showed lower free energy binding values than the controls, metformin and sitagliptin. Hydrogen bonding (HB) was identified as the primary interaction mechanism, with specific polar amino acids such as serin, glutamine, glutamic acid, threonine, aspartic acid, and lysine identified as key contributors. ADMET results indicated favorable absorption and distribution characteristics of the fructosides. These findings provide valuable information for further exploration of phenolic fructosides as potential therapeutic agents for T2DM.

Daidzein Hydroxylation by CYP81E63 Is Involved in the Biosynthesis of Miroestrol in Pueraria mirifica.

White Kwao Krua (Pueraria candollei var. mirifica), a Thai medicinal plant, is a rich source of phytoestrogens, especially isoflavonoids and chromenes. These phytoestrogens are well known; however, their biosynthetic genes remain largely uncharacterized. Cytochrome P450 (P450) is a large protein family that plays a crucial role in the biosynthesis of various compounds in plants, including phytoestrogens. Thus, we focused on P450s involved in the isoflavone hydroxylation that potentially participates in the biosynthesis of miroestrol. Three candidate P450s were isolated from the transcriptome libraries by considering the phylogenetic and expression data of each tissue of P. mirifica. The candidate P450s were functionally characterized both in vitro and in planta. Accordingly, the yeast microsome harboring PmCYP81E63 regiospecifically exhibited either 2' or 3' daidzein hydroxylation and genistein hydroxylation. Based on in silico calculation, PmCYP81E63 had higher binding energy with daidzein than with genistein, which supported the in vitro result of the isoflavone specificity. To confirm in planta function, the candidate P450s were then transiently co-expressed with isoflavone-related genes in Nicotiana benthamiana. Despite no daidzein in the infiltrated N. benthamiana leaves, genistein and hydroxygenistein biosynthesis were detectable by liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Additionally, we demonstrated that PmCYP81E63 interacted with several enzymes related to isoflavone biosynthesis using bimolecular fluorescence complementation studies and a yeast two-hybrid analysis, suggesting a scheme of metabolon formation in the pathway. Our findings provide compelling evidence regarding the involvement of PmCYP81E63 in the early step of the proposed miroestrol biosynthesis in P. mirifica.

Dual-function C2H2-type zinc-finger transcription factor GmZFP7 contributes to isoflavone accumulation in soybean.

Isoflavones are a class of secondary metabolites produced by legumes and play important roles in human health and plant stress tolerance. The C2H2 zinc-finger transcription factor (TF) functions in plant stress tolerance, but little is known about its function in isoflavone regulation in soybean (Glycine max). Here, we report a C2H2 zinc-finger TF gene, GmZFP7, which regulates isoflavone accumulation in soybean. Overexpressing GmZFP7 increased the isoflavone concentration in both transgenic hairy roots and plants. By contrast, silencing GmZFP7 expression significantly reduced isoflavone levels. Metabolomic and qRT-PCR analysis revealed that GmZFP7 can increase the flux of the phenylpropanoid pathway. Furthermore, dual-luciferase and electrophoretic mobility shift assays showed that GmZFP7 regulates isoflavone accumulation by influencing the expression of Isoflavone synthase 2 (GmIFS2) and Flavanone 3 ß-hydroxylase 1 (GmF3H1). In this study, we demonstrate that GmZFP7 contributes to isoflavone accumulation by regulating the expression of the gateway enzymes (GmIFS2 and GmF3H1) of competing phenylpropanoid pathway branches to direct the metabolic flux into isoflavone. A haplotype analysis indicated that important natural variations were present in GmZFP7 promoters, with P-Hap1 and P-Hap3 being the elite haplotypes. Our findings provide insight into how GmZFP7 regulates the phenylpropanoid pathway and enhances soybean isoflavone content.

Anti-neuroinflammatory Effects of Active Compound SPA1413 via Suppression of the MAPK and JAK/STAT Signaling Pathways.

Isoflavones and their derivatives possess neuroprotective activities against neurological disorders. Recently, the active compound SPA1413 (dehydroequol) derived from S-equol, an isoflavone-derived metabolite produced by human intestinal bacteria, was identified as a potent anti-amyloidogenic and neuroinflammatory candidate against Alzheimer's disease. However, its detailed modes of action, associated signaling pathways, and comparison with potential isoflavone derivatives have not yet been studied. Hence, the current study aimed to identify signaling pathways associated with SPA1413 using lipopolysaccharides (LPS)-stimulated BV2 cells as the experimental model via biological assays, Western blotting, and quantitative (q)RT-PCR. The results indicate that the SPA1413 anti-neuroinflammatory effect arises due to suppression of the nitric oxide (NO), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and mitogen-activated protein kinase (MAPK) signaling networks, including those of p38 and c-Jun N-terminal kinase (JNK). Interestingly, SPA1413 inhibited IL-11 through the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway. In addition, SPA1413 inhibited neuronal cell death by reducing LPS-activated microglia in neuronal N2a cells. Our findings suggest that SPA1413 may act as a strong anti-neuroinflammatory candidate by suppressing the MAPK and JAK/STAT signaling pathways.

8-Prenyl daidzein and 8-prenyl genistein from germinated soybean modulate inflammatory response in activated macrophages.

Soy isoflavones have been shown to have anti-inflammatory properties; however, the anti-inflammatory effects of isoflavone metabolites produced during soybean germination remain unclear. We found that the daidzein and genistein derivatives, 8-prenyl daidzein (8-PD) and 8-prenyl genistein (8-PG), demonstrated a more potent effect than daidzein and genistein on repressing inflammatory responses in macrophages. Although IkB protein levels were unaltered, 8-PD and 8-PG repressed nuclear factor kappa B (NF-κB) activation, which was associated with reduced ERK1/2, JNK, and p38 MAPK activation and suppressed mitogen- and stress-activated kinase 1 phosphorylation. Inflammatory responses induced by the medium containing hypertrophic adipocyte secretions were successfully suppressed by 8-PD and 8-PG treatment. In the ex vivo study, 8-PD and 8-PG significantly inhibited proinflammatory C-C motif chemokine ligand 2 (CCL2) secretion from the adipose tissues of mice fed a long-term high-fat diet. The data suggest that 8-PD and 8-PG could regulate macrophage activation under obesity conditions.

A high-resolution mass spectrometry-based methodology for characterization and identification of methylophiopogonanone B metabolites from cryopreserved hepatocytes and liver microsomes.

Methylophiopogonanone B (MOB), one of the homoisoflavonoids isolated from Ophiopogon japonicus, has been demonstrated to possess antioxidative and antitumor activities. The aim of this work was to investigate the metabolism of MOB using liver microsomes and hepatocytes. MOB was individually incubated with rat, monkey, and human hepatocytes to generate the metabolites. To investigate the bioactivation pathways, MOB was incubated with liver microsomes in the presence of glutathione (GSH). All the metabolites were detected and identified using LC with a quadrupole Orbitrap mass spectrometer. Under the current conditions, nine metabolites were identified in hepatocyte incubations. Of these metabolites, M7 derived from hydroxylation was identified as the most abundant metabolite in hepatocyte incubation. MOB was metabolized via demethylation, hydroxylation, and glucuronidation. In liver microsomes, five GSH conjugates were detected and identified. MOB was subjected to bioactivation through demethylation yielding M9, which further formed quinone-methide and ortho-quinone intermediates, followed by GSH conjugation. This work is the first to study the metabolism of MOB, which will help us understand its disposition and efficacy.

Pharmacokinetics, pharmacodynamics, toxicity, and formulations of daidzein: An important isoflavone.

Daidzein, 7-hydroxy-3-(4-hydroxyphenyl)-4H-chromen-4-one is a naturally occurring compound present in leguminous plants, especially in soybeans. Chemically it belongs to the isoflavone class and possesses high nutritive value. Daidzein acts on estrogen receptor and is non-steroidal in nature hence it can also be called as non-steroidal phytoestrogenic compound. Daidzein has been studied by many researchers for its pharmacological activities. Daidzein metabolites were also studied in detail for their health benefits. Researchers have developed novel formulations of daidzein in the past few years to improve its aqueous solubility and bioavailability. Self-emulsified daidzein, poly(lactic-co-glycolic) acid daidzein nanoparticles, nanoemulsion, nanoemulsion gel, and co-crystals are a few of them. The present review provides detailed information on the chemistry, drug development aspects, pharmacokinetics, and pharmacodynamics of daidzein. A literature search was performed using various datasets like PubMed, EBSCO, ProQuest Scopus, and selected websites including the National Institutes of Health and the World Health Organization. Daidzein has a wide range of pharmacodynamic properties in the treatment of cancer, neurodegenerative disorders, cardiac disorders, diabetes and its complication, osteoporosis, and skin disorders. The pharmacokinetic, pharmacodynamics, and drug development aspects of daidzein will help researchers to design further research work on daidzein in the future.

Puerarin Prevents Cadmium-Induced Neuronal Injury by Alleviating Autophagic Dysfunction in Rat Cerebral Cortical Neurons.

Autophagic dysfunction is one of the main mechanisms of cadmium (Cd)-induced neurotoxicity. Puerarin (Pue) is a natural antioxidant extracted from the medicinal and edible homologous plant Pueraria lobata. Studies have shown that Pue has neuroprotective effects in a variety of brain injuries, including Cd-induced neuronal injury. However, the role of Pue in the regulation of autophagy to alleviate Cd-induced injury in rat cerebral cortical neurons remains unclear. This study aimed to elucidate the protective mechanism of Pue in alleviating Cd-induced injury in rat cerebral cortical neurons by targeting autophagy. Our results showed that Pue alleviated Cd-induced injury in rat cerebral cortical neurons in vitro and in vivo. Pue activates autophagy and alleviates Cd-induced autophagic blockade in rat cerebral cortical neurons. Further studies have shown that Pue alleviates the Cd-induced inhibition of autophagosome-lysosome fusion, as well as the inhibition of lysosomal degradation. The specific mechanism is related to Pue alleviating the inhibition of Cd on the expression levels of the key proteins Rab7, VPS41, and SNAP29, which regulate autophagosome-lysosome fusion, as well as the lysosome-related proteins LAMP2, CTSB, and CTSD. In summary, these results indicate that Pue alleviates Cd-induced autophagic dysfunction in rat cerebral cortical neurons by alleviating autophagosome-lysosome fusion dysfunction and lysosomal degradation dysfunction, thereby alleviating Cd-induced neuronal injury.

Development of Therapeutic Agent for Osteoarthritis via Inhibition of KIAA1199 Activity: Effect of Ipriflavone In Vivo.

This study aimed to clarify the effects of ipriflavone, which effectively reduces KIAA1199 activity, on osteoarthritis (OA) development and progression in an in vivo OA mouse model. The OA model mice were divided into the ipriflavone (200 mg/kg/day) group and the control group. OA onset and progression were evaluated with the Mankin score, and KIAA1199 expression and hyaluronan (HA) accumulation were analyzed by immunostaining. The molecular weight of HA in the cartilage tissue and serum HA concentration were analyzed by chromatography and competitive HA enzyme-linked immunoassay. The effects of ipriflavone on the bovine cartilage explant culture under the influence of IL-1ß were also investigated. In the ipriflavone group, Safranin-O stainability was well-preserved, resulting in significant reduction of the Mankin score (p = 0.027). KIAA1199 staining positivity decreased and HA stainability was preserved in the ipriflavone group. The serum HA concentration decreased, and the molecular weight of HA in the cartilage tissue increased in the ipriflavone group. The results of the cartilage explant culture indicated that ipriflavone could reduce GAG losses and increase the molecular weight of HA. Thus, ipriflavone may have an inhibitory effect on OA development/progression. Ipriflavone could be a therapeutic drug for OA by targeting KIAA1199 activity.

Extraction of Isoflavones, Alpha-Hydroxy Acids, and Allantoin from Soybean Leaves-Optimization by a Mixture Design of the Experimental Method.

Soybeans are commonly known as a valuable source of biologically active compounds including isoflavones as well as allantoin and alpha-hydroxy acids. Since these compounds exhibit skin therapeutic effects, they are widely used in the cosmetic and pharmaceutical industries. The presented paper shows the optimization of three solvent systems (ethanol, water, and 1,3-propanediol) to increase the extraction efficiency of isoflavones (daidzin, genistin, 6″-O-malonyldaidzin, 6″-O-malonylglycitin, 6″-O-malonylgenistin), allantoin, and alpha-hydroxy acids (citric acid, malic acid) from soybean leaves. A simplex centroid mixture design for three solvents with interior points was applied for the experimental plan creation. Based on the obtained results of metabolite extraction yield in relation to solvent composition, polynomial regression models were developed. All models were significant, with predicted R-squared values between 0.77 and 0.99, while in all cases the model's lack of fit was not significant. The optimal mixture composition enabling the maximization of extraction efficiency was as follows: 32.9% ethanol, 53.9% water, and 13.3% propanediol (v/v/v). Such a mixture composition provided the extraction of 99%, 91%, 100%, 92%, 99%, 70%, 92%, and 69% of daidzin, genistin, 6″-O-malonyldaidzin, 6″-O-malonylglycitin, 6″-O-malonylgenistin, allantoin, citric acid, and malic acid, respectively. The solvent mixture composition developed provides a good extraction efficiency of the metabolites from soybean leaves and high antioxidant properties.

Comparisons of Physicochemical Properties, Bacterial Diversities, Isoflavone Profiles and Antioxidant Activities on Household and Commercial doenjang.

In this study, the physicochemical properties (pH, acidity, salinity, and soluble protein), bacterial diversities, isoflavone contents, and antioxidant activities of doenjang (fermented soy paste), household doenjang (HDJ), and commercial doenjang (CDJ), were assessed and compared. The values of pH 5.14-5.94 and acidity 1.36-3.03%, indicated a similar level in all doenjang. The salinity was high in CDJ at 12.8-14.6%, and the protein contents (25.69-37.54 mg/g) were generally high in HDJ. Forty-three species were identified from the HDJ and CDJ. The main species were verified to be Bacillus amyloliquefaciens (B. amyloliquefaciens), B. amyloliquefaciens subsp. plantarum, Bacillus licheniformis, Bacillus sp. and Bacillus subtilis. Comparing the ratios of isoflavone types, the HDJ has an aglycone ratio of >80%, and 3HDJ indicates a ratio of isoflavone to aglycone of 100%. In the CDJ, except 4CDJ, glycosides account for a high proportion of more than 50%. The results of antioxidant activities and DNA protection effects were variedly confirmed regardless of HDJs and CDJs. Through these results, it is judged that HDJs have a variety of bacterial species compared to CDJs, and these are biologically active and converted from glycoside to aglycone. Bacterial distribution and isoflavone contents could be used as basic data.

Formononetin Inhibits Mast Cell Degranulation to Ameliorate Compound 48/80-Induced Pseudoallergic Reactions.

Formononetin (FNT) is a plant-derived isoflavone natural product with anti-inflammatory, antioxidant, and anti-allergic properties. We showed previously that FNT inhibits immunoglobulin E (IgE)-dependent mast cell (MC) activation, but the effect of FNT on IgE-independent MC activation is yet unknown. Our aim was to investigate the effects and possible mechanisms of action of FNT on IgE-independent MC activation and pseudoallergic inflammation. We studied the effects of FNT on MC degranulation in vitro with a cell culture model using compound C48/80 to stimulate either mouse bone marrow-derived mast cells (BMMCs) or RBL-2H3 cells. We subsequently measured ß-hexosaminase and histamine release, the expression of inflammatory factors, cell morphological changes, and changes in NF-κB signaling. We also studied the effects of FNT in several in vivo murine models of allergic reaction: C48/80-mediated passive cutaneous anaphylaxis (PCA), active systemic anaphylaxis (ASA), and 2,4-dinitrobenzene (DNCB)-induced atopic dermatitis (AD). The results showed that FNT inhibited IgE-independent degranulation of MCs, evaluated by a decrease in the release of ß-hexosaminase and histamine and a decreased expression of inflammatory factors. Additionally, FNT reduced cytomorphological elongation and F-actin reorganization and attenuated NF-κB p65 phosphorylation and NF-κB-dependent promoter activity. Moreover, the administration of FNT alleviated pseudoallergic responses in vivo in mouse models of C48/80-stimulated PCA and ASA, and DNCB-induced AD. In conclusion, we suggest that FNT may be a novel anti-allergic drug with great potential to alleviate pseudoallergic responses via the inhibition of IgE-independent MC degranulation and NF-κB signaling.

Nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry metabolomics studies on non-organic soybeans versus organic soybeans (Glycine max), and their fermentation by Rhizopus oligosporus.

BACKGROUND: Soybeans (Glycine max) are high in proteins and isoflavones, which offer many health benefits. It has been suggested that the fermentation process enhances the nutrients in the soybeans. Organic foods are perceived as better than non-organic foods in terms of health benefits, yet little is known about the difference in the phytochemical content that distinguishes the quality of organic soybeans from non-organic soybeans. This study investigated the chemical profiles of non-organic (G, T, U, UB) and organic (C, COF, A, R, B, Z) soybeans (G. max [L.] Merr.) and their metabolite changes after fermentation with Rhizopus oligosporus. RESULTS: A clear separation was only observed between non-organic G and organic Z, which were then selected for further investigation in the fermentation of soybeans (GF and ZF). All four groups (G, Z, GF, ZF) were analyzed using nuclear magnetic resonance (NMR) spectroscopy along with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this way a total of 41 and 47 metabolites were identified respectively, with 12 in common. A clear variation (|log1.5 FC| > 2 and P < 0.05) was observed between Z and ZF: most of the sugars and isoflavone glycosides were found only in Z, while more amino acids and organic acids were found in ZF. An additional four metabolites clustered as C-glycosylflavonoids were discovered from MS/MS-based molecular networking. CONCLUSION: Chemical profiles of non-organic and organic soybeans exhibited no significant difference. However, the metabolite profile of the unfermented soybeans, which were higher in sugars, shifted to higher amino acid and organic acid content after fermentation, thereby potentially enhancing their nutritional value. © 2022 Society of Chemical Industry.

Association between urinary zinc excretion and isoflavone-metabolizing enterotypes among Japanese females: a cross-sectional study.

BACKGROUND: Zinc absorption and competition among gut bacteria have been reported in animal studies. Thus, gut bacteria may modify zinc availability in humans. Metabolism of intestinal bacteria is known to be necessary for the activation of several phytoconstituents in the body. For example, equol, a typical substance of soybean isoflavone, is produced by intestinal bacteria metabolizing daidzein and the enterotype is one of distinct ones among Japanese population. The difference in the intestinal microflora can modify the bioavailability of zinc. In this study, we examined urinary zinc concentrations in adult female equol producers (EQPs). METHODS: Urine samples from women participating in health examinations in Miyagi, Okinawa, Kyoto, Kochi, and Hokkaido prefectures were used; from total 17,484 samples, approximately 25 samples were randomly selected for each age group from 30 to 60 years per region (subsample: n = 520), and 520 samples with available urinary zinc concentration (determined by flame atomic absorption analysis) and enterobacterial type were analyzed. EQP was defined as log(equol/daidzein) ≥ -1.42, and urinary concentrations were corrected for creatinine concentration. Urinary zinc concentrations were compared by Student's t-test and multiple regression analyses. RESULTS: The geometric mean urinary zinc concentration (µg/g-Cr) was lower in EQP than in non-EQP (p = 0.0136 by t-test after logarithm transformation). On the other hand, there was no correlation between urinary zinc concentration with daidzein (r = -0.0495, P = 0.436) and equol concentrations (r = -0.0721, P = 0.256). There was a significant negative association between urinary zinc concentration and EQP (ß = -0.392, P = 0.0311) after adjusting with other potential confounding variables, such as daidzein intake. CONCLUSIONS: The results suggest that gut bacteria that produce equol are involved in the metabolism of zinc. Based on previous studies, the bacteria that affect the metabolism of both substances are thought to be Enterococcus. Future studies are expected to identify specific intestinal bacteria for zinc availability and understand individual differences in the effects of micronutrients.