In Silico and In Vitro multiple analysis approach for screening naturally derived ligands for red seabream aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AHR) is a key ligand-dependent transcription factor that mediates the toxic effects of compounds such as dioxin. Recently, natural ligands of AHR, including flavonoids, have been attracting physiological and toxicological attention as they have been reported to regulate major biological functions such as inflammation and anti-cancer by reducing the toxic effects of dioxin. Additionally, it is known that natural AHR ligands can accumulate in wildlife tissues, such as fish. However, studies in fish have investigated only a few ligands in experimental fish species, and the AHR response of marine fish to natural AHR ligands of various other structures has not been thoroughly investigated. To explore various natural AHR ligands in marine fish, which make up the most fish, it is necessary to develop new screening methods that consider the specificity of marine fish. In this study, we investigated the response of natural ligands by constructing in vitro and in silico experimental systems using red seabream as a model species. We attempted to develop a new predictive model to screen potential ligands that can induce transcriptional activation of red seabream AHR1 and AHR2 (rsAHR1 and rsAHR2). This was achieved through multiple analyses using in silico/ in vitro data and Tox21 big data. First, we constructed an in vitro reporter gene assay of rsAHR1 and rsAHR2 and measured the response of 10 representatives natural AHR ligands in COS-7 cells. The results showed that FICZ, Genistein, Daidzein, I3C, DIM, Quercetin and Baicalin induced the transcriptional activity of rsAHR1 and rsAHR2, while Resveratrol and Retinol did not induce the transcriptional activity of rsAHR isoforms. Comparing the EC50 values of the respective compounds in rsAHR1 and rsAHR2, FICZ, Genistein, and Daidzein exhibited similar isoform responses, but I3C, Baicalin, DIM and Quercetin show the isoform-specific responses. These results suggest that natural AHR ligands have specific profiling and transcriptional activity for each rsAHR isoform. In silico analysis, we constructed homology models of the ligand binding domains (LBDs) of rsAHR1 and rsAHR2 and calculated the docking energies (U_dock values) of natural ligands with measured in vitro transcriptional activity and dioxins reported in previous studies. The results showed a significant correlation (R2=0.74(rsAHR1), R2=0.83(rsAHR2)) between docking energy and transcriptional activity (EC50) value, suggesting that the homology model of rsAHR1 and rsAHR2 can be utilized to predict the potential transactivation of ligands. To broaden the applicability of the homology model to diverse compound structures and validate the correlation with transcriptional activity, we conducted additional analyses utilizing Tox21 big data. We calculated the docking energy values for 1860 chemicals in both rsAHR1 and rsAHR2, which were tested for transcriptional activation in Tox21 data against human AHR. By comparing the U_dock energy values between 775 active compounds and 1085 inactive compounds, a significant difference (p<0.001) was observed between the U_dock energy values in the two groups, suggesting that the U_dock value can be applied to distinguish the activation of compounds. Furthermore, we observed a significant correlation (R2=0.45) between the AC50 of Tox21 database and U_dock values of human AHR model. In conclusion, we calculated equations to translate the results of an in silico prediction model for ligand screening of rsAHR1 and rsAHR2 transactivation. This ligand screening model can be a powerful tool to quantitatively estimate AHR transactivation of major marine agents to which red seabream may be exposed. The study introduces a new screening approach for potential natural AHR ligands in marine fish, based on homology model-docking energy values of rsAHR1 and rsAHR2, with implications for future agonist development and applications bridging in silico and in vitro data.

Profiling of Metabolites in a Fermented Soy Dietary Supplement Reinforces its Role in the Management of Intestinal Inflammation.

SCOPE: Gastro-AD (GAD) is a soy flour derived product that undergoes an industrial fermentation with Lactobacillus delbrueckii R0187 and has demonstrated clinical effects in gastroesophageal reflux and peptic ulcer symptom resolution. The aim of this study is to describe and link GAD's metabolomic profile to plausible mechanisms that manifest and explain the documented clinical outcomes. METHODS AND RESULTS: 1H NMR spectroscopy with multivariate statistical analysis is used to characterize the prefermented soy flour and GAD products. The acquired spectra are screened using various resources and the molecular assignments are confirmed using total correlation spectroscopy (TOCSY). Peaks corresponding to different metabolites are integrated and compared between the two products for relative changes. HPLC and GC are used to quantify some specific molecules. NMR analyses demonstrate significant changes in the composition of various assigned bioactive moieties. HPLC and GC analysis demonstrate deglycation of isoflavones after fermentation, resulting in estrogenically active secondary metabolites that have been previously shown to help to reduce inflammation. CONCLUSION: The identification of bioactive molecules, such as genistein and SCFAs, capable of modulating anti-inflammatory signaling cascades in the stomach's gastric and neuroendocrine tissues can explain the reported biological effects in GAD and is supported by in vivo data.

Systematic Engineering of Genistein Biosynthetic Pathway through Genetic Regulators and Combinatorial Enzyme Screening.

Microbial production of genistein, an isoflavonoid primarily found in soybeans, is gaining prominence in the food industry due to its significant nutritional and health benefits. However, challenges arise in redesigning strains due to intricate regulatory nodes between cell growth and genistein production and in systematically exploring core enzymes involving genistein biosynthesis. To address this, this study devised a strategy that simultaneously and precisely rewires flux at both acetyl-CoA and malonyl-CoA nodes toward genistein synthesis. In particular, naringenin, the primary precursor of genistein, was accumulated 2.6 times more than the unoptimized strain through transcriptional repressor-based genetic regulators. Building upon this, a combination of isoflavone synthase and cytochrome P450 reductase with the remarkable conversion of naringenin to genistein was screened from enzyme homologue libraries. The integrated metabolic engineering strategy yields the highest reported production (98 mg/L of genistein) to date, providing a framework for the biosynthesis of diverse flavonoids, including genistein.

Polyphenol exposure of mothers and infants assessed by LC-MS/MS based biomonitoring in breast milk.

Exposure to polyphenols is relevant throughout critical windows of infant development, including the breastfeeding phase. However, the quantitative assessment of polyphenols in human breast milk has received limited attention so far, though polyphenols may positively influence infant health. Therefore, a targeted LC-MS/MS assay was developed to investigate 86 analytes representing different polyphenol classes in human breast milk. The sample preparation consisted of liquid extraction, salting out, freeze-out, and a dilution step. Overall, nearly 70% of the chemically diverse polyphenols fulfilled all strict validation criteria for full quantitative assessment. The remaining analytes did not fulfill all criteria at every concentration level, but can still provide useful semi-quantitative insights into nutritional and biomedical research questions. The limits of detection for all analyzed polyphenols were in the range of 0.0041-87 ng*mL-1, with a median of 0.17 ng*mL-1. Moreover, the mean recovery was determined to be 82% and the mean signal suppression and enhancement effect was 117%. The developed assay was applied in a proof-of-principle study to investigate polyphenols in breast milk samples provided by twelve Nigerian mothers at three distinct time points post-delivery. In total, 50 polyphenol analytes were detected with almost half being phenolic acids. Phase II metabolites, including genistein-7-ß-D-glucuronide, genistein-7-sulfate, and daidzein-7-ß-D-glucuronide, were also detected in several samples. In conclusion, the developed method was demonstrated to be fit-for-purpose to simultaneously (semi-) quantify a wide variety of polyphenols in breast milk. It also demonstrated that various polyphenols including their biotransformation products were present in breast milk and therefore likely transferred to infants where they might impact microbiome development and infant health.

Effect of exogenous genistein on osteogenic differentiation of adipose-derived mesenchymal stem cells in laying hens.

Previous literature revealed that genistein might play a preventive role in osteoporosis. Therefore, we aimed to evaluate the effect of genistein on the osteogenic potency of laying hens' adipose-derived stem cells (LHASCs). The viability of LHASCs after isolation was investigated on tissue culture plastic (TCP) under exposure to genistein up to 50 µg/mL by MTT assay. Our preliminary result revealed that LHASCs cultured under genistein exposure up to 20 µg/mL are feasible. Then, we evaluated the osteogenic induction of LHASCs under exposure to 0, 10, and 20 µg/mL genistein. The Alizarin Red staining confirmed the calcium deposition. Our findings showed that osteogenic differentiation under exposure to 20 µg/mL genistein led to higher ALP activity and more calcium content. We then tried to see the probable additive effect of the genistein-plus Poly-L-lactic acid (PLLA) scaffold on the cell viability and osteogenic capacity of LHASCs. For this, cells were cultured on a PLLA scaffold and exposed to 20 µg/mL genistein. Cell growth rate, as indicated by the MTT assay, revealed no differences between the groups. LHASCs cultured on a genistein-plus PLLA scaffold showed higher ALP activity and more calcium content. The expressions of Osteocalcin, COL1A2, ALP, and Runx2 genes were increased in the genistein-plus PLLA group as compared with PLLA and TCP groups. Adequate proliferation rates and higher expression of osteogenic markers provide genistein as a suitable substrate to support the proliferation and differentiation of LHASCs. Genistein supports osteogenic induction as a further positive effect if such a PLLA scaffold is available.

Genistein alleviates chronic heat stress-induced lipid metabolism disorder and mitochondrial energetic dysfunction by activating the GPR30-AMPK-PGC-1α signaling pathways in the livers of broiler chickens.

The objective of this study was to investigate the preventive effects and mechanisms of genistein (GEN) on production performance and metabolic disorders in broilers under chronic heat stress (HS). A total of 120 male 3-wk-old Ross broilers were randomly assigned to 5 groups: a thermoneutral zone (TN) group maintained at normal temperature (21°C ± 1°C daily), an HS group subjected to cyclic high temperature (32°C ± 1°C for 8 h daily), and 3 groups exposed to HS with varying doses of GEN (50, 100, or 150 mg/kg diet). The experimental period lasted for 3 wk. Here, HS led to a decline in growth performance parameters and hormone secretion disorders (P < 0.05), which were improved by 100 and 150 mg/kg GEN treatment (P < 0.05). Moreover, the HS-induced increases in the liver index (P < 0.01) and abdominal fat rate (P < 0.05) were attenuated by 150 mg/kg GEN (P < 0.05). The HS-induced excessive lipid accumulation in the liver and serum (P < 0.01) was ameliorated after 100 and 150 mg/kg GEN treatment (P < 0.05). Furthermore, the HS-induced decreases in lipolysis-related mRNA levels and increases in lipid synthesis-related mRNA levels in the liver (P < 0.01) were effectively blunted after 100 and 150 mg/kg GEN treatment (P < 0.05). Importantly, the HS-stimulated hepatic mitochondrial energetic dysfunction and decreases in the mRNA or protein levels of peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α), nuclear respiratory factor 1, and mitochondrial transcription factor A in the liver were ameliorated by 150 mg/kg GEN (P < 0.05). Moreover, 50 to 150 mg/kg GEN treatment resulted in a significant increase in the mRNA or protein levels of G protein-coupled estrogen receptor (GPR30), AMP-activated protein kinase (AMPK) α1, phosphorylated AMPKα, and phosphorylated acetyl-CoA carboxylase α. Collectively, GEN alleviated metabolic disorders and hepatic mitochondrial energetic dysfunction under HS, possibly through the activation of GPR30-AMPM-PGC-1α pathways. These data provide a sufficient basis for GEN as an additive to alleviate HS in broilers.

Intercropping of Narrow-Leafed Lupin (Lupinus angustifolius L.) and Barley (Hordeum vulgare L.) Affects the Flavonoid Composition of Both Crops.

Barley (Hordeum vulgare L.) is a common cereal crop in agricultural production and is often included in legume-cereal intercropping. Flavonoids, a major class of secondary metabolites found in barley, are involved in plant defense and protection. However, the effect of intercropping on barley flavonoids remains unknown. Herein, an intercropping system involving barley and lupin (Lupinus angustifolius L.) was studied. Intercropping increased the level of luteolin in lupin roots. Lupin-barley intercropping considerably increased genistein, rutin, and apigenin in barley shoots. Genistein and apigenin were also detected in intercropped barley roots and rhizosphere soil. The three flavonoids have been reported as defense compounds, suggesting that lupin triggers a defense response in barley to strengthen its survival ability.

Genistein ameliorates starvation-induced porcine follicular granulosa cell apoptosis.

In brief: Genistein contributes to granulosa cell (GC) survival by two routes: one is that genistein induced p-AMPK and inhibited p-mTOR, which induces LC3 activation and autophagy; the other is that genistein inhibited caspase-3 and its cleavage, which induces PARP1 activation and PARylation. Abstract: Genistein is an isoflavone which is beneficial for health, but little is known regarding its function on granulosa cell fate during follicular atresia. In the present study, we established an in vitro model of porcine follicular granulosa cell apoptosis by serum deprivation and showed that treatments with 1 µM and 10 µM genistein significantly reduced the apoptotic rate of granulosa cells compared to the blank control (P < 0.05). These results suggest that genistein at micromolar levels alleviates serum deprivation-induced granulosa cell apoptosis, and the ameliorative effect of genistein on granulosa cell apoptosis is likely to be able to inhibit nutrient depletion-induced follicular atresia. Further experimental results revealed that the expression of the autophagic marker protein LC3II in 100 nM-10 µM genistein treatment increased in a dose-dependent manner and was higher than the control (P < 0.05). Genistein also dose dependently promoted the phosphorylation of AMPK (adenosine 5'-monophosphate-activated protein kinase) in granulosa cells. Poly(ADP-ribose) (pADPr) formation in genistein-treated groups was also notably higher than in the controls (P < 0.05). Collectively, genistein alleviates serum deprivation-induced granulosa cells in vitro through enhancing autophagy, which involving AMPK activation and PARylation signaling. However, further study should be carried out to investigate the role of the aforementioned signaling on this process.

Oxidation of Naringenin, Apigenin, and Genistein by Human Family 1 Cytochrome P450 Enzymes and Comparison of Interaction of Apigenin with Human P450 1B1.1 and Scutellaria P450 82D.1.

Naringenin, an initial synthesized flavanone in various plant species, is further utilized for production of many biologically active flavonoids, e.g., apigenin, eriodictyol, and genistein, by various plant enzymes including cytochrome P450s (P450s or CYPs). We examined how these flavonoids are oxidized by human P450 family 1 and 2A enzymes. Naringenin was principally oxidized at the 3'-position to form eriodictyol by CYP1 enzymes more efficiently than by CYP2A enzymes, and the resulting eriodictyol was further oxidized to two penta-hydroxylated products. In contrast to plant P450 enzymes, these human P450s did not mediate the desaturation of naringenin and eriodictyol to give apigenin and luteolin, respectively. Apigenin was oxidized at the C3' and C6 positions to form luteolin and scutellarein by these P450s. CYP1B1.1 and 1B1.3 had high activities in apigenin 6-hydroxylation with a homotropic cooperative manner, as has been observed previously in chrysin 6-hydroxylation (Nagayoshi et al., Chem. Res. Toxicol. 2019, 32, 1268-1280). Molecular docking analysis suggested that CYP1B1 had two apigenin binding sites and showed similarities in substrate recognition sites to plant CYP82D.1, one of the enzymes in catalyzing apigenin and chrysin 6-hydroxylations in Scutellaria baicalensis. The present results suggest that human CYP1 enzymes and CYP2A13 in some reactions have important roles in the oxidation of naringenin, eriodictyol, apigenin, and genistein and that human CYP1B1 and Scutellaria CYP82D.1 have similarities in their SRS regions, catalyzing 6-hydroxylation of both apigenin and chrysin.

Genistein enhances NAD+ biosynthesis by upregulating nicotinamide phosphoribosyltransferase in adipocytes.

A decrease in the NAD+ level in adipocytes causes adipose-tissue dysfunction, leading to systemic glucose, and lipid metabolism failure. Therefore, it is necessary to develop small molecules and nutraceuticals that can increase NAD+ levels in adipocytes. Genistein, a nutraceutical derived from soybeans, has various physiological activities and improves glucose and lipid metabolism. In this study, we aimed to unravel the effects of genistein on the NAD+ level in adipocytes and the underlying molecular mechanisms. Genistein enhanced NAD+ biosynthesis by increasing the expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in NAD+ biosynthesis. A pull-down assay using genistein-immobilized beads revealed prohibitin 1 (PHB1) as a target protein of genistein. The knockdown of Phb1 suppressed the genistein-induced increase in NAMPT expression and NAD+ level in adipocytes. Genistein-bound PHB1 contributed to the stabilization of the transcription factor CCAAT/enhancer-binding protein ß through the activation of extracellular signal-regulated kinase, resulting in increased NAMPT expression at the transcriptional level. Genistein induced the dephosphorylation of peroxisome proliferator-activated receptor at serine 273 and increased the level of the insulin-sensitizing adipokine adiponectin in adipocytes, whereas the knockdown of Nampt and Phb1 abolished these genistein-mediated effects. Our results proved the potential efficacy of genistein in increasing the NAD+ level and restoring metabolic function in adipocytes. Furthermore, we identified PHB1, localized to the plasma membrane, as a novel candidate target protein for increased expression of NAMPT in adipocytes. Overall, these findings will assist in developing NAD+-boosting nutraceuticals to alleviate metabolic dysfunctions in adipose tissues.

Hepatotoxic effect of dietary phytoestrogens on juvenile cultured Russian sturgeon (Acipenser gueldenstaedtii).

In the last two decades, much controversy has grown over the use of soybean products in aquafeeds, especially for carnivorous fish like sturgeons. One point of discussion is the effect of soybean phytoestrogens on fish health. There are many aspects of phytoestrogen utilization in aquafeeds, therefore, the aim of this study is to verify if common legume phytoestrogens can affect juvenile cultured sturgeon erythrocyte and hepatocyte genotoxicity and cause liver pathology. Russian sturgeons were fed from 100 till 365 dph1 with daidzein, genistein, and coumestrol supplemented diets in concentrations: 10, 0.05 and 0.001 g kg-1 of feed, respectively. The SCGE2 method combined with qPCR of three genes involved in DNA repair and genome maintenance, namely cyp1a1, gaad45a and p53 were analyzed. The results were compared with histopathological evaluation of liver tissue. In fish fed with coumestrol supplemented diet, DNA strand damage was the highest in both erythrocytes and hepatocytes, however, simultaneously the lowest level of oxidative DNA damage was found. Additionally, slightly elevated expression of the p53 gene was observed along with a decreased number of apoptotic hepatocytes, which suggests that low concentration of coumestrol may support DNA repair mechanisms in the liver. Although, daidzein showed a preventive effect only against fibrosis. Isoflavones did not show a significant effect on DNA damage in studied cells. Genistein was found to increase macro- and microvesicular steatosis, portal hepatitis and fibrosis, indicating its negative role in the development of liver injuries. Daidzein alleviated some sturgeon liver damage, especially macrovesicular steatosis and interface hepatitis. However, it increased hepatocyte apoptosis, which may suggest daidzein potentially inducing liver injury, though not manifested by other histopathological lesions. Therefore, it can be concluded that at given concentrations, the tested phytoestrogens did not show clearly hepatoprotective effect in sturgeons.

Unveiling the Remarkable Antioxidant Activity of Plant-Based Fish and Seafood Analogs through Electrochemical Sensor Analysis.

The global consumption of vegan foods is experiencing an expressive upward trend, underscoring the critical need for quality control measures based on nutritional and functional considerations. This study aimed to evaluate the functional quality of caviar and salmon analog food inks based on pulses combined with nano ingredients and produced in our laboratory (LNANO). The primary objective of this work was to determine the total antioxidant compounds contained in these samples using a voltammetric technique with a glassy carbon electrode. The samples underwent ethanolic extraction (70%) with 1 h of stirring. The voltammograms were acquired in a phosphate buffer electrolyte, pH 3.0 with Ag/AgCl (KCl 3 mol L-1) as the reference electrode and platinum wire as the auxiliary electrode. The voltammograms revealed prominent anodic current peaks at 0.76-0.78 V, which are attributed to isoflavones. Isoflavones, known secondary metabolites with substantial antioxidant potential commonly found in pulses, were identified. The total isoflavone concentrations obtained ranged from 31.5 to 64.3 mg Eq genistein 100 g-1. The results not only validated the efficacy of the electrochemical sensor for quantifying total antioxidant compounds in the samples but also demonstrated that the concentration of total isoflavones in caviar and salmon analogs fell within the expected limits.

Protective effects of genistein on the production performance and lipid metabolism disorders in laying hens with fatty liver hemorrhagic syndrome by activation of the GPER-AMPK signaling pathways.

The aim of this study was to evaluate the beneficial effects and potential mechanisms of genistein (GEN) on production performance impairments and lipid metabolism disorders in laying hens fed a high-energy and low-protein (HELP) diet. A total of 120 Hy-line Brown laying hens were fed with the standard diet and HELP diet supplemented with 0, 50, 100, and 200 mg/kg GEN for 80 d. The results showed that the declines in laying rate (P < 0.01), average egg weight (P < 0.01), and egg yield (P < 0.01), and the increase of the ratio of feed to egg (P < 0.01) induced by HELP diet were markedly improved by 100 and 200 mg/kg of GEN treatment in laying hens (P < 0.05). Moreover, the hepatic steatosis and increases of lipid contents (P < 0.01) in serum and liver caused by HELP diet were significantly alleviated by treatment with 100 and 200 mg/kg of GEN in laying hens (P < 0.05). The liver index and abdominal fat index of laying hens in the HELP group were higher than subjects in the control group (P < 0.01), which were evidently attenuated by dietary 50 to 200 mg/kg of GEN supplementation (P < 0.05). Dietary 100 and 200 mg/kg of GEN supplementation significantly reduced the upregulations of genes related to fatty acid transport and synthesis (P < 0.01) but enhanced the downregulations of genes associated with fatty acid oxidation (P < 0.01) caused by HELP in the liver of laying hens (P < 0.05). Importantly, 100 and 200 mg/kg of GEN supplementation markedly increased G protein-coupled estrogen receptor (GPER) mRNA and protein expression levels and activated the AMP-activated protein kinase (AMPK) signaling pathway in the liver of laying hens fed a HELP diet (P < 0.05). These data indicated that the protective effects of GEN against the decline of production performance and lipid metabolism disorders caused by HELP diet in laying hens may be related to the activation of the GPER-AMPK signaling pathways. These data not only provide compelling evidence for the protective effect of GEN against fatty liver hemorrhagic syndrome in laying hens but also provide the theoretical basis for GEN as an additive to alleviate metabolic disorders in poultry.

Fatty liver hemorrhagic syndrome (FLHS) is a nutritional and metabolic disease that seriously threatens the health and performance of laying hens, which is characterized by hepatic steatosis and lipid metabolism disorders. As an isoflavone phytoestrogen, genistein (GEN) exerts many beneficial functions, including alleviating lipid metabolism disorders and anti-inflammatory properties. However, further research is needed on the protective effect and potential mechanism of GEN on the FLHS in laying hens. Here, we found that GEN treatment improved liver injury and decline of production performance in laying hens with FLHS. Moreover, GEN treatment alleviated hepatic steatosis and lipid metabolism disorders through reducing the expression levels of mRNA related to fatty acid transport and synthesis and enhancing the mRNA expression levels of factors associated with fatty acid oxidation in FLHS layers, which may be achieved by activation of the G protein-coupled estrogen receptor­adenosine 5'-monophosphate (AMP)-activated protein kinase signaling pathways. These data not only provide compelling evidence for the protective effects and mechanisms of GEN against FLHS in laying hens but also provide the theoretical basis for GEN to alleviate other metabolic disorders in poultry.

Genistein protects benzotriazole ultraviolet stabilizer UV-234-induced hepatotoxicity by modulating ROS/Nrf2 and NF-κB signaling in yellow catfish (Pelteobagrus fulvidraco).

Benzotriazole ultraviolet stabilizers (BUVSs) are a group of anthropogenic chemicals widely used in commodities and industrial products, posing a potential threat to aquatic organisms. However, limited data are available on the toxicity effects of BUVSs in the liver, and no data are available on effective therapeutic strategies. In this study, we exployed aimed to explore the hepatotoxicity of 2-(benzotriazol-2-yl)-4,6-bis(2-phenylpropan-2-yl)phenol (UV-234) and reveal the preventive function of Genistein. At first, yellow catfish (Pelteobagrus fulvidraco) exposed to UV-234 (10 µg/L) showed up-regulated serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) and hepatic reactive oxygen species (ROS) overproduction, along with significantly reduced activities of antioxidants enzymes and nuclear factor erythroid-derived 2-related factor 2 (Nrf2) basal levels. In contrast, 100 mg/kg diet of Genistein improve the hepatic antioxidative capability of fish via activating Nrf2 pathway. Furthermore, we confirmed that UV-234 exposure could induce nuclear factor-κB (NF-κB)-driven inflammatory response, as evidenced by the hepatic inflammatory cells infiltration, lower levels of plasma complement C3 (C3) and complement C4 (C4) as well as higher mRNA levels of NF-κB and inflammatory cytokines. Conversely, feeding UV-234-exposed fish on Genistein-supplemented diets attenuated above adverse effects. Meanwhile, we confirmed that Genistein supplement protected liver apoptosis induced by UV-234 via suppressing up-regulated expression levels of pro-apoptotic genes (Bax, caspase3). In summary, our findings revealed that Genistein positively regulates the Nrf2-mediated antioxidant defenses and reduce NF-κB-driven inflammatory response, thus indirectly inhibiting hepatic damage induced by UV-234 in yellow catfish (Pelteobagrus fulvidraco).

Improvement of the functional value of green soybean (edamame) using germination and tempe fermentation: A comparative metabolomics study.

Green soybean, also known as edamame, is a legume with high nutritional and functional value. Despite its growing popularity and potential health benefits, the functionality of green soybean has not been thoroughly studied. Previous research on the functionality of green soybean has largely focused on a limited number of specific, well-studied, bioactive metabolites, without comprehensively investigating the metabolome of this legume. Additionally, very few studies have explored the improvement of the functional value of green soybean. This study aimed to investigate the metabolome profile of green soybean, identify bioactive metabolites, and to further explore the potential improvement of the identified bioactive metabolites using germination and tempe fermentation. A total of 80 metabolites were annotated from green soybean using GC-MS and HPLC-PDA-MS. Among them, 16 important bioactive metabolites were identified: soy isoflavones daidzin, glycitin, genistin, malonyl daidzin, malonyl genistin, malonyl glycitin, acetyl daidzin, acetyl genistin, acetyl glycitin, daidzein, glycitein, and genistein, as well as other metabolites including 3,4-dihydroxybenzoic acid, 3-hydroxyanthranillic acid, 3-hydroxy-3-methylglutaric acid (meglutol), and 4-aminobutyric acid (GABA). Germination and tempe fermentation techniques were employed to potentially improve the concentrations of these bioactive metabolites. While showing improvements in amino acid contents, germination process did not improve bioactive metabolites significantly. In contrast, tempe fermentation was found to significantly increase the concentrations of daidzein, genistein, glycitein, acetyl genistin, acetyl daidzin, 3-hydroxyanthranillic acid, and meglutol (>2-fold increase with p < 0.05) while also improving amino acid levels. This study highlights the potentials of germination and fermentation to improve the functionality of legumes, particularly green soybean.

Nimbin analogs stimulate glucose uptake and glycogen storage in the insulin signalling cascade by enhancing the IRTK, PI3K and Glut-4 mechanism in myotubes.

BACKGROUND: Diabetes Mellitus is a metabolic disorder characterized by insulin dysfunction or failure of the pancreatic ß-cells to produce insulin resulting in hyperglycemia. Adverse effects of hyperglycemic conditions continue to be common, reducing treatment adherence. Intensified therapies are required for the constant loss of endogenous islet reserve. AIM: This study aimed to evaluate the effect of Nimbin semi-natural analogs (N2, N5, N7, and N8) from A. indica on high glucose-induced ROS and apoptosis with insulin resistance in L6 myotubes evaluated along with Wortmannin and Genistein inhibitors and the expression of key genes in the insulin signalling pathway. MATERIALS AND METHODS: The analogs were screened for anti-oxidant and anti-diabetic activity using cell-free assays; The ability of analogs to suppress ROS and prevent apoptosis induced by High glucose and uptake glucose and glycogen storage in L6 myotubes was evaluated using DCFH-DA, AO-PI and 2NBDG staining. Further, the glucose uptake was performed in the presence of Insulin Receptor Tyrosine Kinase (IRTK) inhibitors, and the expression of key genes PI3K, Glut-4, GS and IRTK in the insulin signalling pathway were evaluated. KEY FINDINGS: The Nimbin analogs were not toxic to the L6 cells, and the analogs could scavenge ROS and suppress cellular damage induced due to high glucose. Enhanced glucose uptake was observed in N2, N5 and N7 compared to N8. The maximum activity of optimum concentration was found to be 100 µM. The N2, N5 and N7 showed an increase in IRTK, which is equivalent to insulin at a concentration of 100 µM. The IRTK inhibitor with Genistein (50 µM) confirmed the presence of IRTK-dependent glucose transport activation; it also supports the expression of key genes PI3K, Glut-4, GS and IRTK. As a result of PI3K activation, N2, N5, and N7 exhibited the insulin-mimetic effect by enhancing glucose uptake and glycogen conversion regulating glucose metabolism. SIGNIFICANCE: N2, N5 and N7 could therapeutically benefit against insulin resistance by glucose metabolism modulation, insulin secretion, ß-cell stimulation, inhibition of gluconeogenic enzymes and ROS protection.

EGF-receptor phosphorylation and downstream signaling are activated by genistein during subacute liver damage.

The epidermal growth factor receptor (EGFR) plays an important role on hepatic protection in acute and chronic liver injury. The aim of this study was to investigate the role of genistein on EGFR expression, phosphorylation and signaling pathways in experimental subacute liver damage induced by carbon tetrachloride (CCl4). We used male Wistar rats that were randomly divided into four groups: (1) Control; (2) Genistein 5 mg/kg per oral; (3) Subacute liver damage induced by CCl4 4 mg/kg subcutaneously; and (4) Animals received CCl4 and genistein at the dosage indicated. The effect of genistein on EGFR expression, phosphorylation and signaling pathways were investigated by western blot and densitometric analyses. Histological changes were evaluated on slices stained with Hematoxylin-Eosin and Masson´s trichromic, as well as an immunohistochemical analysis for proliferating cell nuclear antigen (PCNA). Additionally, pro-inflammatory cytokines and liver enzymes were quantified. Our study showed that genistein increased EGFR expression, EGFR-specific tyrosine residues phosphorylation (pY1068-EGFR and pY84-EGFR), signal transducer and activator of transcription phosphorylation (pSTAT5), protein kinase B phosphorylation (pAKT) and PCNA in animals with CCl4-induced subacute liver damage. It was found a significant reduction of pro-inflammatory cytokines in serum from animals with subacute liver damage treated with genistein. Those effects were reflected in an improvement in the architecture and liver function. In conclusion, genistein can induce a transactivation of EGFR leading to downstream cell signaling pathways as early events associated with regeneration and hepatoprotection following subacute liver damage.

Adding genistein or luteolin decreased the yield of citrinin and without reducing pigments in yam solid-fermentation by Monascus.

BACKGROUND: Chinese yam fermented by Monascus, namely red mold dioscorea (RMD), has the potential of treating diseases. However, the production of citrinin limits the application of RMD. In the present study, the fermentation process of Monascus was optimized by adding genistein or luteolin to reduce citrinin yield. RESULTS: The results showed that citrinin in 25 g of Huai Shan yam was reduced by 48% and 72% without affecting the pigment yield by adding 0.2 g of luteolin or genistein, respectively, to a 250-mL conical flask after fermentation for 18 days at 28 °C, whereas the addition of luteolin increased the content of yellow pigment by 1.3-fold. Under optimal conditions, citrinin in 20 g of iron bar yam decreased by 55% and 74% after adding 0.2 g of luteolin or genistein. Luteolin also increased yellow pigment content by 1.2-fold. Ultra HPLC coupled to quadrupole time-of-flight mass spectrometry was used for the preliminary analysis of Monascus fermentation products. It was found that the amino acid types in RMD are similar to those in yams, but there are fewer polysaccharides and fatty acids. CONCLUSION: The results obtained in the present study showed that the addition of genistein or luteolin could reduce citrinin on the premise of increasing pigment yield, which laid a foundation for the better use of yams in Monascus fermentation. © 2023 Society of Chemical Industry.

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.

Genistein attenuates renal ischemia-reperfusion injury via ADORA2A pathway.

BACKGROUND: Studies have shown oxidative stress and apoptosis are the main pathogenic mechanisms of renal ischemia/reperfusion (IR) injury (IRI). Genistein, a polyphenolic non-steroidal compound, has been extensively explored in oxidative stress, inflammation and apoptosis. Our research aims to reveal the potential role of genistein on renal IRI and its potential molecular mechanism both in vivo and in vitro. METHODS: In vivo experiments, mice were pretreated with or without genistein. Renal pathological changes and function, cell proliferation, oxidative stress and apoptosis were measured. In vitro experiments, overexpression of ADORA2A and knockout of ADORA2A cells were constructed. Cells proliferation, oxidative stress and apoptosis were analyzed. RESULTS: Our results in vivo showed that the renal damage induced by IR was ameliorated by genistein pretreatment. Moreover, ADORA2A was activated by genistein, along with inhibition of oxidative stress and apoptosis. The results in vitro showed that genistein pretreatment and ADORA2A overexpression reversed the increase of apoptosis and oxidative stress in NRK-52E cells induced by H/R, while the knockdown of ADORA2A partially weakened this reversal from genistein treatment. CONCLUSIONS: Our results demonstrated that genistein have a protective effect against renal IRI by inhibiting oxidative stress and apoptosis via activating ADORA2A, presenting its potential use for the treatment of renal IRI.