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.

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.

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.

Molecular mechanism of naringenin regulation on flavonoid biosynthesis to improve the salt tolerance in pigeon pea (Cajanus cajan (Linn.) Millsp.).

Flavonoids are important secondary metabolites in the plant growth and development process. As a medicinal plant, pigeon pea is rich in secondary metabolites. As a flavonoid, there are few studies on the regulation mechanism of naringenin in plant stress resistance. In our study, we found that naringenin can increase the pigeon pea's ability to tolerate salt and influence the changes that occur in flavonoids including naringenin, genistein and biochanin A. We analyzed the transcriptome data after 1 mM naringenin treatment, and identified a total of 13083 differentially expressed genes. By analyzing the metabolic pathways of these differentially expressed genes, we found that these differentially expressed genes were enriched in the metabolic pathways of phenylpropanoid biosynthesis, starch and sucrose metabolism and so on. We focused on the analysis of flavonoid biosynthesis related pathways. Among them, the expression levels of enzyme genes CcIFS, CcCHI and CcCHS in the flavonoid biosynthesis pathway had considerably higher expression levels. By counting the number of transcription factors and the binding sites on the promoter of the enzyme gene, we screened the transcription factors CcMYB62 and CcbHLH35 related to flavonoid metabolism. Among them, CcMYB62 has a higher expression level than the others. The hairy root transgene showed that CcMYB62 could induce the upregulation of CcCHI, and promote the accumulation of naringenin, genistein and biochanin A. Our study revealed the molecular mechanism of naringenin regulating flavonoid biosynthesis under salt stress in pigeon pea, and provided an idea for the role of flavonoids in plant resistance to abiotic stresses.

Bifidobacterium breve MCC1274 Supplementation Increased the Plasma Levels of Metabolites with Potential Anti-Oxidative Activity in APP Knock-In Mice.

BACKGROUND: We previously reported the effects of a probiotic strain, Bifidobacterium breve MCC1274, in improving cognitive function in preclinical and clinical studies. Recently, we demonstrated that supplementation of this strain led to decreased amyloid-ß production, attenuated microglial activation, and suppressed inflammation reaction in the brain of APP knock-in (AppNL - G - F) mice. OBJECTIVE: In this study, we investigated the plasma metabolites to reveal the mechanism of action of this probiotic strain in this Alzheimer's disease (AD)-like model. METHODS: Three-month-old mice were orally supplemented with B. breve MCC1274 or saline for four months and their plasma metabolites were comprehensively analyzed using CE-FTMS and LC-TOFMS. RESULTS: Principal component analysis showed a significant difference in the plasma metabolites between the probiotic and control groups (PERMANOVA, p = 0.03). The levels of soy isoflavones (e.g., genistein) and indole derivatives of tryptophan (e.g., 5-methoxyindoleacetic acid), metabolites with potent anti-oxidative activities were significantly increased in the probiotic group. Moreover, there were increased levels of glutathione-related metabolites (e.g., glutathione (GSSG)_divalent, ophthalmic acid) and TCA cycle-related metabolites (e.g., 2-Oxoglutaric acid, succinic acid levels) in the probiotic group. Similar alternations were observed in the wild-type mice by the probiotic supplementation. CONCLUSION: These results suggest that the supplementation of B. breve MCC1274 enhanced the bioavailability of potential anti-oxidative metabolites from the gut and addressed critical gaps in our understanding of the gut-brain axis underlying the mechanisms of the probiotic action of this strain in the improvement of cognitive function.

Selenium increases photosynthetic capacity, daidzein biosynthesis, nodulation and yield of peanuts plants (Arachis hypogaea L.).

This study aimed to investigate the roles of selenium (Se) application on the profile of photosynthetic pigments, oxidant metabolism, flavonoids biosynthesis, nodulation, and its relation to agronomic traits of peanut plants. Two independent experiments were carried out: one conducted in soil and the other in a nutrient solution. When the plants reached the V2 growth stage, five Se doses (0, 7.5, 15, 30, and 45 µg kg-1) and four Se concentrations (0, 5, 10, and 15 µmol L-1) were supplied as sodium selenate. The concentration of photosynthetic pigments, activity of antioxidant enzymes and the concentration of total sugars in peanut leaves increased in response to Se fertilization. In addition, Se improves nitrogen assimilation efficiency by increasing nitrate reductase activity which results in a higher concentration of ureides, amino acids and proteins. Se increases the synthesis of daidzein and genistein in the root, resulting in a greater number of nodules and concentration and transport of ureides to the leaves. Se-treated plants showed greater growth, biomass accumulation in shoots and roots, yield and Se concentration in leaves and grains. Our results contribute to food security and also to increase knowledge about the effects of Se on physiology, biochemistry and biological nitrogen fixation in legume plants.

Gastroprotective effects of Caragana ambigua stocks on ethanol-induced gastric ulcer in rats supported by LC-MS/MS characterization of formononetin and biochanin A.

BACKGROUND: Caragana ambigua has been the part of the dietary routines of the regional people in south-west Pakistan and has traditionally been used for the treatment of diabetes there. There is an increased production of reactive oxygen species in diabetics, leading to gastrointestinal disorders. Natural antioxidants exhibit gastroprotective effects owing to their free-radical scavenging action. C. ambigua possesses appreciable phenolic and flavonoid content; thus, it has the potential to protect against gastrointestinal disorders (e.g. gastric ulcer). RESULTS: This study reports the anti-ulcer potential of C. ambigua. Four different fractions (chloroform, ethyl acetate, butanol, and aqueous) of plant were compared against omeprazole. Ulcer index, ulcer inhibition percentage, gastric pH and volume, total acidity, gastric protein, gastric wall mucus, and histopathology of gastric walls of rats were assessed. All fractions exhibited a reduction in ulcer index and promotion of percentage of ulcer inhibition compared with the ulcer control group. Furthermore, the fractions revealed a significant (P < 0.001) diminution in gastric volume and total acidity with an increase in pH. Among the fractions investigated, the chloroform fraction unveiled the most promising anti-ulcer activity, which is comparable to omeprazole. Liquid chromatography-tandem mass spectrometry screening of fractions revealed the presence of formononetin and biochanin A (isoflavones reported to have anti-ulcer properties) in the chloroform fraction. CONCLUSION: This study establishes that C. ambigua possesses significant potential in reducing gastric ulcer progression. Formononetin and biochanin A are chiefly responsible for the stated bioactivity due to the fact that these compounds were solely present in the chloroform fraction. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effect of Genistein Supplementation on Exercise-Induced Inflammation and Oxidative Stress in Mice Liver and Skeletal Muscle.

Background and objectives: The purpose of this study was to investigate the influences of oral high-dose genistein (GE) administration on exercise-induced oxidative stress, inflammatory response and tissue damage. Materials and Methods: Thirty-two mice were randomly divided into control group (Con; sedentary/0.5% CMC-Na), GE administrated group (GE; sedentary/GE dosed), exercise group (Ex; exercise/0.5% CMC-Na), or GE administrated plus exercise group (GE + Ex; exercise/GE dosed), mice in the GE and GE + Ex group were given GE orally at the dose of 200 mg/kg weight. Results: Plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT) levels, liver interleukin (IL)-6, IL-1ß, superoxide dismutase 1 (SOD1), catalase (CAT), hemeoxygenase-1 (HO-1) gene expression levels and skeletal muscle IL-6, nuclear factor erythroid 2-related factor (Nrf2), and HO-1 gene expression levels increased immediately after exhaustive exercise. GE supplementation increased liver protein carbonyl concentrations. On the other hand, GE supplementation significantly decreased SOD1, CAT gene expression levels in the liver and Nrf2, and HO-1 gene expression levels in the skeletal muscles. Conclusions: Acute exercise induced organ damage, inflammation, and oxidative stress in skeletal muscles and the liver. However, a single dose of GE supplementation before exercise did not lead to favorable antioxidant and anti-inflammatory effects in this study.

Streptomyces genisteinicus sp. nov., a novel genistein-producing actinomycete isolated from a Chinese medicinal plant and proposal of Streptomyces michiganensis Corbaz et al. 1957 as a later heterotypic synonym of Streptomyces xanthochromogenes Arishima et al. 1956.

A novel genistein-producing actinobacterial strain, designated strain CRPJ-33T, was isolated from the healthy leaves of a medicinal plant Xanthium sibiricum collected from Hunan Province, PR China. 16S rRNA gene sequence analysis indicated strain CRPJ-33T belonged to the genus Streptomyces and had 99.7, 99.0, 98.9, 98.9, 98.8 and 98.7% sequence similarities to Streptomyces zhihengii YIM T102T, Streptomyces eurocidicus NRRL B-1676T, Streptomyces xanthochromogenes NRRL B-5410T, Streptomyces michiganensis NBRC 12797T, Streptomyces mauvecolor LMG 20100T and Streptomyces lavendofoliae NBRC 12882T, respectively. Phylogenetic analysis of 16S rRNA gene sequences showed that strain CRPJ-33T was most closely related to S. zhihengii YIM T102T. However, digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between them were much less than the recommended threshold values. Furthermore, differential comparisons of the phenotypic characteristics were enough to distinguish strain CRPJ-33T from S. zhihengii YIM T102T. Meanwhile, the ANI and dDDH values or MLSA distances between strain CRPJ-33T and other type strains, which exhibited ≥98.7 % 16S rRNA gene sequence similarities to strain CRPJ-33T, were far away from the recommended threshold values. Based on these results, it is thought that strain CRPJ-33T should represent a novel species of the genus Streptomyces, for which the name Streptomyces genisteinicus sp. nov. is proposed. The type strain is CRPJ-33T (=MCCC 1K04965T=JCM 34526T). In addition, the phenotypic, chemotaxonomic and genotypic characteristics, as well as phylogenetic information revealed that the type strains of S. xanthochromogenes and S. michiganensis should belong to same genomic species. Consequently, it is proposed that S. michiganensis is a heterotypic synonym of S. xanthochromogenes for which an emended description is given.

A metabolomics approach to evaluate post-fermentation enhancement of daidzein and genistein in a green okara extract.

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.

Dietary isoflavone-induced, estrogen receptor-ß-mediated proliferation of Caco-2 cells is modulated by gallic acid.

Dietary isoflavones and their biotransformation products (from food fermentation) are estrogen mimics which activate estrogen receptors (ER)α and ERß. In silico molecular modelling is used to determine theoretical binding energies of genistein, daidzein and hydroxylated biotransformation products, and to investigate structure-binding energy relationships with ERß. Results suggest that ligand hydroxyl arrangement determines binding energy and influences binding affinity. Caco-2 cells (ERß expressing) are used to study the proliferative effect of genistein, daidzein and their hydroxylated biotransformation products. Isoflavones/biotransformation products showed weaker enhancement of Caco-2 proliferation than 17ß-estradiol. The EC50s of isoflavones/biotransformation products agreed with in silico-predicted binding affinity order. Hydroxylated biotransformation products studied showed greater Caco-2 proliferative effects than the parent isoflavones except 8-hydroxygenistein, probably due to unfavourable ERß interactions caused by 8-hydroxygenistein's extra hydroxyl. Caco-2 pre-treatment with UDP-glucose dehydrogenase inhibitor gallic acid promoted genistein/8-hydroxygenistein-mediated proliferation. This is probably due to a reduced isoflavone glucuronidation to form low estrogenicity glucuronides. Findings are discussed in the context of dietary isoflavones/gallic acid and effects on proliferation of ERß-expressing gut cancer cells.

Dietary genistein supplementation protects against lipopolysaccharide-induced intestinal injury through altering transcriptomic profile.

Genistein is abundant in the corn-soybean meal feed. Little information is available about the effect of dietary genistein on the intestinal transcriptome of chicks, especially when suffering from intestinal injury. In this study, 180 one-day-old male ROSS 308 broiler chickens were randomly allocated to 3 groups, with 4 replicates (cages) of 15 birds each. The treatments were as follows: chicks received a basal diet (CON), a basal diet and underwent lipopolysaccharide-challenge (LPS), or a basal diet supplemented with 40 mg/kg genistein and underwent LPS-challenge (GEN). LPS injection induced intestinal injury and inflammatory reactions in the chicks. Transcriptomic analysis identified 7,131 differently expressed genes (3,281 upregulated and 3,851 downregulated) in the GEN group compared with the LPS group (P adjusted value < 0.05, |fold change| > 1.5), which revealed that dietary genistein exposure altered the gene expression profile and signaling pathways in the ileum of LPS-treated chicks. Furthermore, dietary genistein improved intestinal morphology, mucosal immune function, tight junction, antioxidant activity, apoptotic process, and growth performance, which were adversely damaged by LPS injection. Therefore, adding genistein into the diet of chicks can alter RNA expression profile and ameliorate intestinal injury in LPS-challenged chicks, thereby improving the growth performance of chicks with intestinal injury.

Dietary Genistein Reduces Methylglyoxal and Advanced Glycation End Product Accumulation in Obese Mice Treated with High-Fat Diet.

Our previous study has found that dietary genistein could ameliorate high-fat diet (HFD)-induced obesity and especially lower methylglyoxal (MGO) and advanced glycation end product (AGE) accumulation in healthy mice exposed to genistein and HFD. However, it is still unclear whether dietary genistein intervention has a similar beneficial effect in obese mice. In this study, the mice were induced with obesity after being fed a HFD for nine weeks before being administered with two doses of genistein, 0.1% (G 0.1) and 0.2% (G 0.2), in the HFD for additional 19 weeks. After 19 week treatment, genistein supplementation reduced body and liver weights, plasma and liver MGO levels, and kidney AGE levels in mice. Mechanistically, genistein upregulated the expressions of glyoxalase I and II and aldose reductase to detoxify MGO, and genistein and its microbial metabolites, dihydrogenistein and 6'-hydroxy-O-demethylangolensin, were able to trap endogenous MGO via formation of MGO conjugates. Taken together, our results provide novel insights into the antiobesity and antiglycation roles of dietary genistein in obese subjects.

Biotransformation of phytoestrogens from soy in enzymatically characterized liver microsomes and primary hepatocytes of Atlantic salmon.

Efficient aquaculture is depending on sustainable protein sources. The shortage in marine raw materials has initiated a shift to "green aquafeeds" based on staple ingredients such as soy and wheat. Plant-based diets entail new challenges regarding fish health, product quality and consumer risks due to the possible presence of chemical contaminants, natural toxins and bioactive compounds like phytoestrogens. Daidzein (DAI), genistein (GEN) and glycitein (GLY) are major soy isoflavones with considerable estrogenic activities, potentially interfering with the piscine endocrine system and affecting consumers after carry-over. In this context, information on isoflavone biotransformation in fish is crucial for risk evaluation. We have therefore isolated hepatic fractions of Atlantic salmon (Salmo salar), the most important species in Norwegian aquaculture, and used them to study isoflavone elimination and metabolite formation. The salmon liver microsomes and primary hepatocytes were characterized with respect to phase I cytochrome P450 (CYP) and phase II uridine-diphosphate-glucuronosyltransferase (UGT) enzyme activities using specific probe substrates, which allowed comparison to results in other species. DAI, GEN and GLY were effectively cleared by UGT. Based on the measurement of exact masses, fragmentation patterns, and retention times in liquid chromatography high-resolution mass spectrometry, we preliminarily identified the 7-O-glucuronides as the main metabolites in salmon, possibly produced by UGT1A1 and UGT1A9-like activities. In contrast, the production of oxidative metabolites by CYP was insignificant. Under optimized assay conditions, only small amounts of mono-hydroxylated DAI were detectable. These findings suggested that bioaccumulation of phytoestrogens in farmed salmon and consumer risks from soy-containing aquafeeds are unlikely.

Translating In Vitro Acrolein-Trapping Capacities of Tea Polyphenol and Soy Genistein to In Vivo Situation is Mediated by the Bioavailability and Biotransformation of Individual Polyphenols.

SCOPE: Acrolein (ACR) is a highly toxic unsaturated aldehyde. Humans are both endogenously and exogenously exposed to ACR. Long-term exposure to ACR leads to various chronic diseases. Dietary polyphenols have been reported to be able to attenuate ACR-induced toxicity in vitro via formation of ACR-polyphenol conjugates. However, whether in vitro ACR-trapping abilities of polyphenols can be maintained under in vivo environments is still unknown. METHODS AND RESULTS: Two most commonly consumed dietary polyphenols, (-)-epigallocatechin-3-gallate (EGCG) from tea and genistein from soy, are evaluated for their anti-Acrolein behaviors both in vitro and in mice. Tea EGCG exerts a much higher capacity to capture ACR than soy genistein in vitro. But translation of in vitro anti-ACR activity into in vivo is mainly mediated by bioavailability and biotransformation of individual polyphenols. It is found that 1) both absorbed EGCG and genistein can trap endogenous ACR by forming mono-ACR adducts and eventually be excreted into mouse urine; 2) both absorbed EGCG and genistein can produce active metabolites, methyl-EGCG (MeEGCG) and orobol, to scavenge endogenous ACR; 3) both MeEGCG and non-absorbed EGCG show ability to trap ACR in the gut; 4) considerable amounts of microbial metabolites of genistein display enhanced anti-ACR capacity both in the body and in the gut, compared to genistein; and 5) biotransformation of genistein is able to boost its in vivo anti-ACR capacity, compared to EGCG. CONCLUSION: The findings demonstrate that in vivo anti-ACR ability of dietary polyphenols cannot be reflected solely based on their in vitro ability. The bioavailability and biotransformation of individual polyphenols, and especially the gut microbiome, contribute to in vivo anti-ACR ability of dietary polyphenols.

Genistein potentiates Centchroman induced antineoplasticity in breast cancer via PI3K/Akt deactivation and ROS dependent induction of apoptosis.

AIMS: Recently, strategies of cancer treatment using combination of agents with distinct molecular mechanism(s) of action are considered more promising due to its high efficacy and reduced systemic toxicity. The study is aimed to improve the efficacy of selective estrogen receptor modulator, Centchroman (CC) by combination with the phytoestrogen Genistein (GN). METHODS: Cytotoxicity was evaluated by Sulforhodamine B assay. Cell cycle analysis was done through flow cytometry. Further, Apoptosis was analyzed using Annexin V/PI staining, tunel assay and electron microscopic examination and verified using western blot analysis. In order to validate the in vitro results, in vivo analysis was performed using 4T1-syngeneic mouse model. KEY FINDINGS: In this study, we report that the dietary isoflavone genistein (GN) synergistically improved antineoplasticity of CC in breast cancer by arresting cells at G2/M phase culminating in ROS dependent apoptosis. The combination of CC plus GN caused dysregulation of Bax and Bcl-2 ratio inducing mitochondrial dysfunction, activation of Caspase-3/7, -9 and PARP cleavage. Further, combination significantly suppresses phosphorylation of PI3K/Akt/NF-κB, enhancing apoptosis. Additionally, combination markedly reduced tumor growth compared to CC and GN alone in mouse 4T1 breast tumor model. SIGNIFICANCE: Together, these studies suggest that GN represents a potential adjunct molecule whose role in CC induced apoptosis deserves attention.

Biochanin A protects against angiotensin II-induced damage of dopaminergic neurons in rats associated with the increased endophilin A2 expression.

The brain renin-angiotensin system plays a vital role in the modulation of the neuroinflammatory responses and the progression of dopaminergic (DA) degeneration. Angiotensin II (Ang II) induces microglia activation via angiotensin II type 1 receptor (AT1R), which in turn affects the function of DA neurons. Endophilin A2 (EPA2) is involved in fast endophilin-mediated endocytosis and quickly endocytoses several G-protein-coupled receptor (GPCR), while AT1R belongs to GPCR family. Therefore, we speculated that EPA2 may modulate microglia activation via endocytosing AT1R. Biochanin A is an O-methylated isoflavone, classified as a kind of phytoestrogen due to its chemical structure that is similar to mammalian estrogens. In this study, we investigated the protective effects of biochanin A on Ang II-induced DA neurons damage in vivo, and molecular mechanisms. The results showed that biochanin A treatment for 7 days attenuated the behavioral dysfunction, inhibited the microglial activation, and prevented DA neuron damage in Ang II-induced rats. Furthermore, biochanin A increased EPA2 expression and decreased the expression of AT1R, gp91phox, p22 phox, NLRP3, ASC, Caspase-1, IL-1ß, IL-6, IL-18, and TNF-α. In summary, these results suggest that biochanin A exerts protective effects in Ang II-induced model rats, and the mechanisms may involve inhibition of inflammatory responses, an increase in EPA2 expression and a decrease in AT1R expression.

Postnatal genistein administration selectively abolishes sexual dimorphism in specific hypothalamic dopaminergic system in mice.

As demonstrated in previous studies, early postnatal genistein (GEN) administration to mice pups of both sexes, at doses similar to that of infant soy-based formulas, may affect the development of some steroid-sensitive neuronal circuits (i.e. nitrergic and vasopressinergic systems), causing irreversible alterations in adults. Here, we investigated the hypothalamic and mesencephalic dopaminergic system (identified with tyrosine hydroxylase immunohistochemistry). GEN administration (50 mg/kg) to mice of both sexes during the first week of postnatal life specifically affected tyrosine hydroxylase immunohistochemistry in the hypothalamic subpopulation of neurons, abolishing their sexual dimorphism. On the contrary, we did not observe any effects in the mesencephalic groups. Due to the large involvement of dopamine in circuits controlling rodent sexual behavior and food intake, these results clearly indicate that the early postnatal administration of GEN may irreversibly alter the control of reproduction, of energetic metabolism, and other behaviors. These results suggest the need for a careful evaluation of the use of soy products in both human and animal newborns.

The Role of Dietary Phytoestrogens and the Nuclear Receptor PPARγ in Adipogenesis: An in Vitro Study.

BACKGROUND: Phytoestrogens, naturally occurring plant chemicals, have long been thought to confer beneficial effects on human cardiovascular and metabolic health. However, recent epidemiological studies, have yielded conflicting outcomes, in which phytoestrogen consumption was both positively and negatively correlated with adiposity. Interestingly, several dietary phytoestrogens are known to stimulate or inhibit the activity of the peroxisome proliferator-activated receptor gamma (PPARγ), a key physiological regulator of adipogenesis. OBJECTIVE: The objective of this study was to test the hypothesis that the pro- or anti-adipogenic activity of phytoestrogen chemicals is related to the ability to activate PPARγ in adipocytes. METHODS: The effects of resveratrol and the soy isoflavones genistein and daidzein on adipogenesis were examined in cell-based assays using the 3T3-L1 cell model. In parallel, ligand-mediated alterations in PPARγ target gene expression were measured by quantitative polymerase chain reaction. The agonist/antagonist activities of phytoestrogens on PPARγ were further assessed by quantifying their ability to affect recruitment of transcriptional cofactors to the receptor. RESULTS: Resveratrol displayed significant anti-adipogenic activities as exhibited by the ability to antagonize PPARγ-dependent adipocyte differentiation, down-regulate genes involved in lipid metabolism, block cofactor recruitment to PPARγ, and antagonize the effects of the PPARγ agonist rosiglitazone. In contrast, genistein and daidzein functioned as PPARγ agonists while also displaying pro-adipogenic activities. CONCLUSIONS: These data provide biological evidence that the pro- or anti-obesity effects of phytoestrogens are related to their relative agonist/antagonist activity on PPARγ. Thus, PPARγ-activation assays may enable the screening of dietary components and identification of agents with adipogenic activities. https://doi.org/10.1289/EHP3444.

Isoflavones.

Phytoestrogens are naturally occurring nonsteroidal phenolic plant compounds that, due to their molecular structure and size, resemble vertebrate steroids estrogens. This review is focused on plant flavonoids isoflavones, which are ranked among the most estrogenic compounds. The main dietary sources of isoflavones for humans are soybean and soybean products, which contain mainly daidzein and genistein. When they are consumed, they exert estrogenic and/or antiestrogenic effects. Isoflavones are considered chemoprotective and can be used as an alternative therapy for a wide range of hormonal disorders, including several cancer types, namely breast cancer and prostate cancer, cardiovascular diseases, osteoporosis, or menopausal symptoms. On the other hand, isoflavones may also be considered endocrine disruptors with possible negative influences on the state of health in a certain part of the population or on the environment. This review deals with isoflavone classification, structure, and occurrence, with their metabolism, biological, and health effects in humans and animals, and with their utilization and potential risks.