Soy Isoflavones Daidzein Research Articles

Daidzein nanosuspension combined with 5-fluorouracil induces apoptosis and alleviate inflammation in Caco-2 colorectal adenocarcinoma cells

Colorectal cancer is the third most common malignancy worldwide and the second most common cause of cancer deaths. Alternative treatment strategies, including nano-based formulations, show remarkable promise to improve the efficacy of chemotherapy. The soy isoflavone daidzein has attracted attention for its anti-cancer activity. The aim of this study was to investigate the effects of daidzein nanosuspension with 5-fluorouracil on apoptosis and inflammation induced in human colorectal adenocarcinoma cells (Caco-2). Cytotoxicity, total antioxidant/oxidant status (TAS, TOS), and the expression of proteins associated with apoptosis and inflammation were analyzed by the MTT, ELISA and qRT-PCR, respectively. Daidzein nanosuspension showed a strong cytotoxicity (IC50 = 240.3 μM for daidzein, IC50 = 35.34 μM for daidzein nanosuspension) compared to daidzein. The combination of 5-fluorouracil and daidzein nanosuspension treatment showed a synergistic antiproliferative effect without increasing oxidative stress. The combination showed a significant increase in p53 levels and a significant decrease in Bcl-2, IL-6, TNF-α and MMP-9 levels. qRT-PCR results showed that the combinations of daidzein and DZ-NS with 5-FU regulated these parameters in Caco-2 cells. In conclusion, daidzein nanosuspension with 5-fluorouracil treatment reduced the proliferation and inflammation and increased apoptosis of Caco-2 cells without oxidative stress. The current study shows that this combination also elucidates several signaling pathways, including effects on oxidative stress, apoptosis, and inflammation. The integration of this nanotechnology approach into traditional chemotherapy will form the basis of an innovative strategy.

A Gnotobiotic Mouse Model with Divergent Equol-Producing Phenotypes: Potential for Determining Microbial-Driven Health Impacts of Soy Isoflavone Daidzein.

The implications of soy consumption on human health have been a subject of debate, largely due to the mixed evidence regarding its benefits and potential risks. The variability in responses to soy has been partly attributed to differences in the metabolism of soy isoflavones, compounds with structural similarities to estrogen. Approximately one-third of humans possess gut bacteria capable of converting soy isoflavone daidzein into equol, a metabolite produced exclusively by gut microbiota with significant estrogenic potency. In contrast, lab-raised rodents are efficient equol producers, except for those raised germ-free. This discrepancy raises concerns about the applicability of traditional rodent models to humans. Herein, we designed a gnotobiotic mouse model to differentiate between equol producers and non-producers by introducing synthetic bacterial communities with and without the equol-producing capacity into female and male germ-free mice. These gnotobiotic mice display equol-producing phenotypes consistent with the capacity of the gut microbiota received. Our findings confirm the model's efficacy in mimicking human equol production capacity, offering a promising tool for future studies to explore the relationship between endogenous equol production and health outcomes like cardiometabolic health and fertility. This approach aims to refine dietary guidelines by considering individual microbiome differences.

Abstract WP263: Associations of Equol-Producing Status With Cardioembolic Stroke in Japanese Patients With Stroke

Introduction: Equol, a metabolite of a soy isoflavone daidzein transformed by the gut microbiota, is associated with the amelioration of cardiovascular diseases and cognitive impairment. Approximately 50% of Asians and 30% of Westerners may be equol producers depending on the presence of equol-producing gut microbiota. It is still unknown whether equol-producing status is associated with development of stroke and its prognosis. Methods: In this single-center, retrospective study, we investigated the serum concentration of equol in 200 patients with stroke hospitalized in the National Cerebral and Cardiovascular Center (NCVC) from September 2019 to October 2021, and 103 healthy subjects registered in the Suita Study, a population-based cohort study at NCVC from November 2016 to September 2018. The concentration of equol was measured using liquid chromatography-mass spectrometry. Results: Eighty-five patients with stroke (43.5%) and 50 healthy subjects (48.5%) were equol producers ( p =0.32). However, patients with cardioembolic stroke (CES) had a significantly lower frequency of the equol producers (28.9%) compared to healthy subjects ( p =0.04), as well as patients with other stroke subtypes (48.0%) ( p =0.04). The prevalence of atrial fibrillation was significantly lower in equol producers than that in non-producers (26.3% vs. 47.3%, p =0.04) among patients with ischemic stroke. Additionally, equol-producing status was an independent predictor for a favorable outcome (modified Rankin scale 0-2) at discharge for ischemic stroke in a multivariable logistic regression analysis (odds ratio 2.75, 95% confidential interval 1.05-7.27, p =0.04). Discussion: CES, atrial fibrillation, and clinical outcome may be influenced by equol-producing status. These findings could provide new insights into the racial difference in the prevalence of CES between Asians and Westerners.

Safety Assessment and Hepatic-Renal Protection of Cajanus cajan (L.) Millsp. Root and Its Soy Isoflavone Contents.

Cajanus cajan (L.) Millsp., also known as pigeon pea, has roots that have exhibited much pharmacological potential. The present study was conducted to assess the safe dose of the ethanolic extract of C. cajan roots (EECR95) and to analyze the main soy isoflavones contents. In vitro, we investigated the mutagenicity and cytotoxic effect of EECR95 on Salmonella typhimurium-TA98 and TA100 (by Ames tests) and RAW 264.7, L-929, and HGF-1 cell lines (by MTT tests) for 24 h of incubation. We found no mutagenic or cytotoxic effects of EECR95. After administration of 0.2 or 1.0 g/kg bw of EECR95 to both male and female Wistar rats for 90 days, there were no significant adverse effects on the behaviors (body weight, water intake, and food intake), organ/tissue weights, or immunohistochemical staining, and the urine and hematological examinations of the rats were within normal ranges. EECR95 potentially decreases renal function markers in serum (serum uric acid, BUN, CRE, and GLU) or liver function markers (cholesterol, triglyceride, and glutamic-pyruvate-transaminase (GPT)). We also found that EECR95 contained five soy isoflavones (genistein, biochanin A, daidzein, genistin, and cajanol), which may be related to its hepatorenal protection. Based on the high dose (1.0 g/kg bw) of EECR95, a safe daily intake of EECR95 for human adults is estimated to be 972 mg/60 kg person/day.

Enhanced Cholesterol-Lowering and Antioxidant Activities of Soymilk by Fermentation with Lactiplantibacillus plantarum KML06.

This study aimed to evaluate the cholesterol-lowering and antioxidant activities of soymilk fermented with probiotic Lactobacillaceae strains and to investigate the production of related bioactive compounds. Lactiplantibacillus plantarum KML06 (KML06) was selected for the fermentation of soymilk because it has the highest antioxidant, cholesterol-lowering, and β-glucosidase activities among the 10 Lactobacillaceae strains isolated from kimchi. The genomic information of strain KML06 was analyzed. Moreover, soymilk fermented with KML06 was evaluated for growth kinetics, metabolism, and functional characteristics during the fermentation period. The number of viable cells, which was similar to the results of radical scavenging activities and cholesterol assimilation, as well as the amount of soy isoflavone aglycones, daidzein, and genistein, was the highest at 12 h of fermentation. These results indicate that soymilk fermented with KML06 can prevent oxidative stress and cholesterol-related problems through the production of soy isoflavone aglycones.

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.

Microbial Metabolism of the Soy Isoflavones Daidzein and Genistein in Postmenopausal Women: Human Intervention Study Reveals New Metabotypes.

Soy isoflavones belong to the group of phytoestrogens and are associated with beneficial health effects but are also discussed to have adverse effects. Isoflavones are intensively metabolized by the gut microbiota leading to metabolites with altered estrogenic potency. The population is classified into different isoflavone metabotypes based on individual metabolite profiles. So far, this classification was based on the capacity to metabolize daidzein and did not reflect genistein metabolism. We investigated the microbial metabolite profile of isoflavones considering daidzein and genistein. Isoflavones and metabolites were quantified in the urine of postmenopausal women receiving a soy isoflavone extract for 12 weeks. Based on these data, women were clustered in different isoflavone metabotypes. Further, the estrogenic potency of these metabotypes was estimated. Based on the excreted urinary amounts of isoflavones and metabolites, the metabolite profiles could be calculated, resulting in 5 metabotypes applying a hierarchical cluster analysis. The metabotypes differed in part strongly regarding their metabolite profile and their estimated estrogenic potency.

Daidzein phosphorylates and activates 5-lipoxygenase via the MEK/ERK pathway: a mechanism for inducing the production of 5-lipoxygenase metabolite that inhibit influenza virus intracellular replication

We previously reported that the soy isoflavone daidzein (Dz) suppresses the intracellular replication of influenza virus and that arachidonic acid-derived oxidation product via lipid oxidase 5-lipoxygenase (5-LOX) is involved in its antiviral effect. The activation of 5-LOX by Dz triggers anti-influenza activity; however, the mechanism of activation of 5-LOX remains unclear. Therefore, in this study, we aimed to clarify the activation mechanism using human monocyte-derived THP-1 cells differentiated using phorbol 12-myristate 13-acetate. THP-1 cells expressed 5-LOX endogenously and Dz did not induce 5-LOX expression. However, 8 h after treatment with Dz, the amount of 5-hydroxyeicosatetraenoic acid (5-HETE), an arachidonic acid oxidation product via 5-LOX, increased significantly suggesting that the enzyme is activated regardless of changes in 5-LOX protein levels. Intracellular Ca2+ content, ATP concentration, 5-LOX protein phosphorylation, and 5-LOX intracellular localization are known 5-LOX activation factors. The intracellular Ca2+ and ATP concentrations were not affected by Dz treatment. The enzymatic activity of 5-LOX is regulated by the phosphorylation of three serine residues and four tyrosine residues. Pretreatment with inhibitors of each kinase revealed that Dz-induced 5-HETE production was suppressed by the MEK/ERK inhibitor. 5-LOX in which the Ser663 residue was phosphorylated was found to be increased in the nuclear fraction of Dz-treated THP-1 cells. Furthermore, immunocytochemistry showed that 5-LOX translocates to the nuclear envelope following Dz treatment. These results indicate that Dz activates 5-LOX by phosphorylating Ser663 via the MEK/ERK pathway. Thus, these results demonstrate that Dz exerts anti-influenza virus activity via the MEK/ERK signal transduction pathway.

Potential Protective Mechanisms of S-equol, a Metabolite of Soy Isoflavone by the Gut Microbiome, on Cognitive Decline and Dementia.

S-equol, a metabolite of soy isoflavone daidzein transformed by the gut microbiome, is the most biologically potent among all soy isoflavones and their metabolites. Soy isoflavones are phytoestrogens and exert their actions through estrogen receptor-β. Epidemiological studies in East Asia, where soy isoflavones are regularly consumed, show that dietary isoflavone intake is inversely associated with cognitive decline and dementia; however, randomized controlled trials of soy isoflavones in Western countries did not generally show their cognitive benefit. The discrepant results may be attributed to S-equol production capability; after consuming soy isoflavones, 40–70% of East Asians produce S-equol, whereas 20–30% of Westerners do. Recent observational and clinical studies in Japan show that S-equol but not soy isoflavones is inversely associated with multiple vascular pathologies, contributing to cognitive impairment and dementia, including arterial stiffness and white matter lesion volume. S-equol has better permeability to the blood–brain barrier than soy isoflavones, although their affinity to estrogen receptor-β is similar. S-equol is also the most potent antioxidant among all known soy isoflavones. Although S-equol is available as a dietary supplement, no long-term trials in humans have examined the effect of S-equol supplementation on arterial stiffness, cerebrovascular disease, cognitive decline, or dementia.

Isoflavones daidzin and daidzein inhibit lipopolysaccharide-induced inflammation in RAW264.7 macrophages

BackgroundInflammation contributes to various diseases and soybeans and legumes are shown to reduce inflammation. However, the bioactive ingredients involved and mechanisms are not completely known. We hypothesized that soy isoflavones daidzin and daidzein exhibit anti-inflammatory effect in lipopolysaccharides (LPS)-stimulated RAW264.7 macrophage cell model and that activation mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways may mediate the effect.MethodsCell viability and nitric oxide (NO) level were determined by 3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Griess reagent respectively. ELISA kits and Western blotting respectively assessed the generations of pro-inflammatory cytokines and protein expressions of signaling molecules. p65 nuclear translocation was determined by immunofluorescence assay.ResultsThe in vitro results showed that both isoflavones did not affect cell viability at the concentrations being tested and significantly reduced levels of NO, pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor-α (TNF-α), and inflammatory indicators such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in RAW264.7 cells. Daidzin and daidzein partially suppressed MAPK signaling pathways, reducing the phosphorylation of p38 and ERK; whilst phosphorylation of JNK was mildly but not significantly decreased. For the involvement of NF-κB signaling pathways, daidzin only reduced the phosphorylation of p65 whereas daidzein effectively inhibited the phosphorylation of IKKα/β, IκBα and p65. Daidzin and daidzein inhibited p65 nuclear translocation, comparable with dexamethasone (positive control).ConclusionThis study supports the anti-inflammatory effects of isoflavones daidzin and daidzein, which were at least partially mediated through inactivation of MAPK and/or NF-κB signaling pathways in macrophages.

1175-P: Association between Equol-Producer and Type 2 Diabetes Mellitus in a Japanese Elderly Population

Objective: Equol (Eq) , a metabolite produced from soy isoflavone daidzein by intestinal bacteria, exhibits estrogen activity and may exert effects on glucose metabolism. Eq levels vary extensively depending on the capability for Eq production and amounts of daidzein consumed. We examined the relationship between Eq levels and type 2 diabetes mellitus. Research Design and Methods: Among 1,395 outpatients with diabetes at our hospital, 147 subjects (age: 70-89 years) were recruited (DM group) . Also, 147 age-/sex-matched non-DM subjects were recruited from 1,113 individuals who underwent routine health examinations (non-DM group) . As the estrogenic effect of Eq becomes apparent after menopause, the subjects were age restricted. Eq producers (EqPr) were determined based on the logarithm of the ratio of urinary Eq to the concentration of daidzein with a cut-off value of −1.75. Results: Urinary daidzein (p=0.03) and Eq concentrations (p=0.01) were significantly lower in the DM group versus the non-DM group. The proportion of EqPr differed significantly between the groups (38.8% and 53.1%, respectively; p=0.01) . However, analyses with sex-based stratification showed such difference only in women; the proportion of EqPr was 31.4% and 52.8%, respectively (p&amp;lt;0.01) . Reversely, there were significantly fewer DM subjects among EqPr versus non-EqPr (p=0.01) . Among women, EqPr had significantly less DM than non-EqPr (p&amp;lt;0.01) . However, such differences were not observed in men. Correlation analyses showed a significant positive correlation between urinary Eq and daidzein concentrations only in EqPr (r=0.55, p&amp;lt;0.01) . Conclusion: In postmenopausal women, EqPr were less diabetic; this status appears to be protective against DM. Eq levels increased in parallel with the amount of daidzein consumed in EqPr, but not in non-EqPr. Therefore, Eq supplements and/or enteric bacteria transplantation, rather than dietary intervention, may be effective in preventing DM in non-EqPr. Disclosure K.Hamaura: None. H.Murakami: None. A.Tamura: None. Y.Nishiya: None. S.Mizushiri: None. M.Daimon: None.

Maximizing the Estrogenic Potential of Soy Isoflavones through the Gut Microbiome: Implication for Cardiometabolic Health in Postmenopausal Women.

Soy isoflavones have been suggested as an alternative treatment for managing postmenopausal symptoms and promoting long-term health due to their structural similarity to mammalian estrogen and ability to bind to estrogen receptors. Among all soy isoflavones and their metabolites, (S)-equol is known for having the strongest estrogenic activity. Equol is a metabolite of the soy isoflavone daidzein produced through intestinal bacterial metabolism. However, more than half of the human population is not able to produce equol due to the lack of equol-producing bacteria in their gastrointestinal tract. The interpersonal variations in the gut microbiome complicate the interpretation of data collected from humans. Furthermore, because rodents are efficient equol-producers, translatability between rodent models and humans is challenging. Herein, we first summarized the current knowledge of the microbial conversion of daidzein to equol, its relation to health, and proposed the need for developing model systems by which equol production can be manipulated while controlling other known confounding factors. Determining the necessity of equol-producing capacity within a gut microbial community when consuming soy as a functional ingredient, and identifying strategies to maximize equol production by modulating the gut microbiome, may provide future therapeutic approaches to improve the health of postmenopausal women.

Soy isoflavone metabolite equol inhibits cancer cell proliferation in a PAP associated domain containing 5-dependent and an estrogen receptor-independent manner

Isoflavone is a species of polyphenol found mainly in soy and soy products. Many studies have demonstrated its estrogen receptor (ER)-dependent action. Equol is an intestinal metabolite of a major soy isoflavone daidzein. We aimed to elucidate the mechanism for ER-independent actions of equol. Equol has been shown to inhibit proliferation of HeLa human cervical cancer cells and mouse melanoma B16 cells in an ER-independent manner. Using functional genetic screening, PAP associated domain containing 5 (PAPD5), which is a non-canonical poly(A) polymerase, was identified as an essential molecule in the ER-independent action. While peroral administration of equol inhibited tumor growth of control B16 cells subcutaneously inoculated in mice, it had little effect on the growth of PAPD5-ablated B16 cells. Intriguingly, equol progressed tumor growth of the PAPD5-ablated human breast cancer MCF-7 cells, which have high ERα expression. Equol has been found to induce polyadenylation of snoRNAs in a PAPD5-depdendent manner. Furthermore, peroral equol administration increased microRNA miR-320a expression in tumors. Together, these results suggest that equol may have a dual effect on ER-positive cancer cells, acting with, antiproliferative activity through PAPD5 and exhibiting proliferative activity via ERα and the former could be associated with miR-320a.

Soy and Frequent Dairy Consumption with Subsequent Equol Production Reveals Decreased Gut Health in a Cohort of Healthy Puerto Rican Women.

The U.S. Hispanic female population has one of the highest breast cancer (BC) incidence and mortality rates, while BC is the leading cause of cancer death in Puerto Rican women. Certain foods may predispose to carcinogenesis. Our previous studies indicate that consuming combined soy isoflavones (genistein, daidzein, and glycitein) promotes tumor metastasis possibly through increased protein synthesis activated by equol, a secondary dietary metabolite. Equol is a bacterial metabolite produced in about 20–60% of the population that harbor and exhibit specific gut microbiota capable of producing it from daidzein. The aim of the current study was to investigate the prevalence of equol production in Puerto Rican women and identify the equol producing microbiota in this understudied population. Herein, we conducted a cross-sectional characterization of equol production in a clinically based sample of eighty healthy 25–50 year old Puerto Rican women. Urine samples were collected and evaluated by GCMS for the presence of soy isoflavones and metabolites to determine the ratio of equol producers to equol non-producers. Furthermore, fecal samples were collected for gut microbiota characterization on a subset of women using next generation sequencing (NGS). We report that 25% of the participants were classified as equol producers. Importantly, the gut microbiota from equol non-producers demonstrated a higher diversity. Our results suggest that healthy women with soy and high dairy consumption with subsequent equol production may result in gut dysbiosis by having reduced quantities (diversity) of healthy bacterial biomarkers, which might be associated to increased diseased outcomes (e.g., cancer, and other diseases).

Soy isoflavone-caused shunting of the corticosteroidogenesis pathways in andropausal subjects: Top-down impulse for the optimal supplementation design

In a series of our previous works, we revealed the beneficial effects of applied soy isoflavones (genistein or daidzein) on the wide context of corticosteroidogenesis in vivo, in a rat model of the andropause. Soy isoflavones decreased the circulating levels of pituitary adrenocorticotropic hormone, inhibited aldosterone secretion, as well as corticosterone production and secretion, but stimulated dehydroepiandrosterone secretion, all in andropausal rats. In vitro studies indicate that the mechanism underlying these hormonal changes relies on inhibition of the pituitary tyrosine kinase and adrenocortical 3β-hydroxysteroid dehydrogenase enzymes by soy isoflavones. Although the clinical studies are in their infancy, the opinion is that genistein and daidzein have therapeutic potential for the safe treatment of ageing-caused androgen deprivation and glucocorticoid excess with related metabolic/hemodynamic issues in males. Our accumulated experience and knowledge in the field of biomedical effects of plant polyphenols have provided a platform for potential recommending the agenda to organize and accelerate experimental research aimed at producing the optimal supplementation. We hypothesize that an in vivo approach should first be exploited in the sequence of investigative steps, followed by in vitro studies and synchronously conducted molecular docking analyses. In vivo research, besides establishing the margin of exposure safety or adjustment of the correct polyphenol dose, enables identification and quantification of the metabolites of applied polyphenols in the blood. Subsequent in vitro exploitation of the metabolites and related docking analyses provide clarification of the molecular mechanisms of action of applied polyphenols. Chemical modification of the polyphenol structure or coupling it with nanoparticles might be the next step in optimizing the design of supplementation. Selected, intact or chemically-modified polyphenol molecules should be included in preclinical studies on a more closely-related species, while clinical studies would finally assess the safety and effectiveness of a polyphenol-based remedial strategy. The final supplement represents a product of an appropriate technological process, conducted in accordance with the recommendations derived from the preceding research.

Administration of Cholic Acid Inhibits Equol Production from Daidzein in Mice.

Equol (Eq) is a metabolite of soy isoflavone daidzein (De) produced by the intestinal microbiota. The clinical effectiveness of soy isoflavone is considered to depend on the individual ability of Eq production. Previous studies have demonstrated that habitual dietary patterns may influence the production of Eq. For example, high Eq producers consumed less fat as a percentage of energy than low Eq producers. However, the inhibitory factors of Eq production are unknown. Recently, it was reported that bile acids induced by high-fat diet consumption may be a host-related factor controlling the composition of the intestinal microbiota. In this study, we investigated the effect of cholic acid (CA) administration, a mimic of the microbiota altered by a high-fat diet, on Eq production in mice. CA administration significantly decreased the levels of the De metabolites Eq, dihydrodaidzein, and O-desmethylangolensin in the serum of mice. However, CA administration did not affect the total molar concentration of De and its metabolites. Moreover, CA administration increased the levels of secondary bile acids, particularly deoxycholic acid (DCA), which has strong antibacterial activity in the cecum contents of mice. Thus, CA administration may increase the levels of DCA, a secondary bile acid, resulting in inhibition of Eq production. These findings may help to reveal the factors inhibiting Eq production and enhance the clinical effectiveness of isoflavone intake.

Detection of conjugated soy metabolites in urinary and tissue samples after methanol extraction

Glucuronide conjugates of soy isoflavone metabolites genistein, daidzein, glycitein and equol in urinary samples from women, female pigs and female rats were pretreated with methanol instead of solid phase extraction or liquid-liquid extraction after enzymatic digestion. The samples were analyzed using an LC-ESI-MS/MS ion trap system. Analysis of rat liver sample using this procedure was also attempted. By comparison the elution sequences with results from previous studies, glucuronide conjugates including 7-gluc-Dai, 7-gluc-Gly, 4′-gluc-Dai, 7-gluc-Gen, then 4′-gluc-Gen were identified sequentially in women urine. However, 5-gluc-Gen was also observed in female porcine urine. In rat urine, sul-Dai was observed after 7-gluc-Dai, while 7-gluc-Equ and 4′-gluc-Equ were observed after 7-gluc-Gen. The metabolite profiles in rat liver tissue samples were found with 7-gluc-Dai and 7-gluc-Gen. However, glucuronide conjugates of equol and sulfate conjugate of daidzein were only found in rat urine. Interestingly, quantification studies performed by LC-APCI/MS and LC-ECD showed that the aglycone and conjugated glycitein were both found in porcine and rat urine, but only conjugated glycitein was found in woman urine.

Dietary daidzein decreases food intake accompanied with delayed gastric emptying in ovariectomized rats.

We previously found that equol, a metabolite of intestinal bacterial conversion from soy isoflavone daidzein, has female-specific anorectic effects. In the present study, we used seven-week-old female ovariectomized (OVX) Sprague Dawley rats to test the hypothesis that the anorectic effect of dietary daidzein may be attributed to delayed gastric emptying. Results suggest that dietary daidzein delays gastric emptying and that it has an anorectic effect with residual gastric contents, but not without gastric contents. Dietary equol significantly decreased daily food intake in the OVX rats without sleeve gastrectomy, but not in those with sleeve gastrectomy, suggesting that the accumulation of food in the stomach is required for the anorectic effect of equol to occur. These results support the hypothesis that the anorectic effect of dietary daidzein is attributed to delayed gastric emptying.

DO EQUOL’S C-RING HYDROGENS CONTRIBUTE TO FREE RADICAL SCAVENGING?

Free radical scavenging potency of physiologically active equol molecule, derived by gut microbiota from soy isoflavone daidzein, was investigated by using M06-2X/6-311++G(d,p) level of theory, accompanied with the TST and Eckart tunneling corrections for the estimation of rate constants. Phenolic hydrogens of equol are recognized as much more abstractable than C-ring hydrogens. This finding is opposite to the one very recently suggested, but in accordance with well-known facts related to the flavonoid chemistry and phenolic O−H vs C−H reactivity.

Soy Metabolism by Gut Microbiota from Patients with Precancerous Intestinal Lesions.

Background: Colorectal cancer (CRC) requires the presence of a variety of factors predisposing a tumorigenic milieu. Excluding familial clustering and hereditary CRC syndromes, the development of sporadic CRC from precancerous lesions is influenced by tissue inflammation, modulation of intestinal immunity, hormones, dietary habits and gut microbiota composition. As concerning the last two aspects, the intestinal presence of equol, the most biologically active metabolite of the soy isoflavone daidzein and the presence of a genetic determinant of gut microbiota able to metabolize daidzein, seem to lower the CRC risk. It has been hypothesized that the anaerobic microorganisms of the Bacteroides genus play a role in equol production. Aim: To evaluate the presence of (i) anaerobic gut microbiota and (ii) the urinary levels of soy isoflavones (daidzein, genistein and equol) in patients with and without precancerous lesions, challenged with a daidzein-rich soy extract. Methods: Consecutive subjects undergoing colonoscopy participated to the study. Feces were collected from all patients one week before colonoscopy for gut microbiota studies. After the endoscopy examination and the histological evaluation, 40 subjects, 20 with sporadic colorectal adenomas (SCA/P group) and 20 without proliferative lesions (control group) were enrolled for the study. Urine levels of soy isoflavones daidzein, genistein and their metabolite equol, were determined by high performance liquid chromatographic (HPLC) analysis and gut microbiota analysis was performed by Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) procedure. Results: Seventeen different bacterial species were identified in the fecal samples of the forty subjects participating to the study. Ten bacterial species resulted anaerobic Gram-negative bacteria, all belonging to the Bacteroides genus. A significant difference of bacteria species was evidenced in the fecal samples of the two groups of subjects. Particularly important was the evidence of Parabacteroides distasonis, Clostridium clostridioforme and Pediococcus pentasaceus only in control fecal samples, such as the presence of Bacteroides fragilis and Prevotella melaningenica only in SCA/P fecal samples. Concerning the soy isoflavones levels, no statistically significant differences were revealed in the genistein and daidzein urinary levels between the two groups of subjects. On the contrary, urinary equol levels were undetectable in ten SCA/P subjects and in two controls; moreover, when present, the levels of urinary equol were significantly lower in SCA/P subjects compared to controls (0.24 ± 0.27 mg/24 hrs vs. 21.25 ± 4.3 mg/24 hrs, respectively, p = 1.12 × 10−6). Conclusions: Our results suggest that the presence of anaerobic Bacteroides in the colon, and the production of equol from soy, could determine a milieu able to contrast the development of colonic mucosa proliferative lesions.