大豆异黄酮微生物转化研究进展

大豆异黄酮是大豆在其生长过程中形成的一类次生代谢产物,具有抗氧化、抗癌、减少骨质流失、降低心脑血管发病率等多种生理功能。目前已知,被摄人机体的大豆异黄酮将被肠道微生物菌群转化为具有更高、更广生物学活性的不同产物。因此,大豆异黄酮对人体的有益调节作用强弱并不简单取决于摄人机体的净含量的多少,更在于被摄人机体的大豆异黄酮将如何被肠道菌群转化。本文从大豆异黄酮的组成与功能、大豆异黄酮体内吸收、代谢及微生物转化、转化产物的活性以及高效合成等方面进行了系统综述,对大豆异黄酮微生物生物转化研究现状和存在问题进行分析总结,并对今后发展趋势进行展望,旨在推动高活性大豆异黄酮微生物转化产物的研究与开发。     

X-ray single-crystal analysis of (-)-(S)-equol isolated from rat's feces

The biological effects of soy isoflavones have attracted considerable interest in recent years, leading to numerous studies on dietary intake and epidemiology. (-)-(S)-equol (3) is a metabolite produced in vivo from the isoflavone daidzein, a kind of soy phytoestrogen, by the action of gut microflora. It has higher biological activity than its precursor. Here, we wish to report the isolation and first X-ray crystal structure of 3 from the feces of rats fed a soy-isoflavone-containing diet.     

Increased aggressive behavior and decreased affiliative behavior in adult male monkeys after long-term consumption of diets rich in soy protein and isoflavones

Estrogen produced by aromatization of gonadal androgen has an important facilitative role in male-typical aggressive behavior that is mediated through its interaction with estrogen receptors (ER) in the brain. Isoflavones found in soybeans and soy-based dietary supplements bind ER and have dose- and tissue-dependent effects on estrogen-mediated responses. Yet, effects of isoflavone-rich diets on social and aggressive behavior have not been studied. We studied the effects of long-term (15 months) consumption of diets rich in soy isoflavones on spontaneous social behavior among adult male cynomolgus macaques (Macaca fascicularis) (n = 44) living in nine stable social groups. There were three experimental conditions which differed only by the source of dietary protein: casein and lactalbumin (no isoflavones), soy protein isolate containing 0.94 mg isoflavones/g protein, and soy protein isolate containing 1.88 mg isoflavones/g protein. In the monkeys fed the higher amount of isoflavones, frequencies of intense aggressive (67% higher) and submissive (203% higher) behavior were elevated relative to monkeys fed the control diet (P's < 0.05). In addition, the proportion of time spent by these monkeys in physical contact with other monkeys was reduced by 68%, time spent in proximity to other monkeys was reduced 50%, and time spent alone was increased 30% (P's < 0.02). There were no effects of treatment on serum testosterone or estradiol concentrations or the response of plasma testosterone to exogenous gonadotropin-releasing hormone (GnRH). The results indicate that long-term consumption of a diet rich in soy isoflavones can have marked influences on patterns of aggressive and social behavior.     

Fecal Bacterial Community Changes Associated with Isoflavone Metabolites in Postmenopausal Women after Soy Bar Consumption

Soy isoflavones and their metabolism by intestinal microbiota have gained attention because of potential health benefits, such as the alleviation of estrogen/hormone-related conditions in postmenopausal women, associated with some of these compounds. However, overall changes in gut bacterial community structure and composition in response to addition of soy isoflavones to diets and their association with excreted isoflavone metabolites in postmenopausal women has not been studied. The aim of this study was to determine fecal bacterial community changes in 17 postmenopausal women after a week of diet supplementation with soy bars containing isoflavones, and to determine correlations between microbial community changes and excreted isoflavone metabolites. Using DGGE profiles of PCR amplified 16S rRNA genes (V3 region) to compare microbial communities in fecal samples collected one week before and one week during soy supplementation revealed significant differences (ANOSIM p<0.03) before and after soy supplementation in all subjects. However, between subjects comparisons showed high inter-individual variation that resulted in clustering of profiles by subjects. Urinary excretion of isoflavone (daidzein) metabolites indicated four subjects were equol producers and all subjects produced O-desmethylangolensin (ODMA). Comparison of relative proportions of 16S rRNA genes from 454 pyrosequencing of the last fecal samples of each treatment session revealed significant increases in average proportions of Bifidobacterium after soy consumption, and Bifidobacterium and Eubacterium were significantly greater in equol vs non-S-(-)equol producers. This is the first in vivo study using pyrosequencing to characterize significant differences in fecal community structure and composition in postmenopausal women after a week of soy diet-supplementation, and relate these changes to differences in soy isoflavones and isoflavone metabolites.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00244907","term_id":"NCT00244907"}}NCT00244907     

Regulation of soy isoflavones on weight gain and fat percentage: evaluation in a Chinese Guangxi minipig model

This study was conducted to determine the effects of soy isoflavones on changes in body and tissue weight and on insulin-like factor I (IGF-I) and peroxisome proliferator-activated receptor-γ (PPARγ) gene and protein expression in muscle and adipose tissues in Chinese Guangxi minipig, as a model for studying human nutrition. A total of 72 male Chinese Guangxi minipigs were fed basal diet (control, Con), low dose of soy isoflavones and high dose of soy isoflavones (HSI). The results showed that HSI increased the body weight (BW) gain and fat percentage of minipigs (P < 0.05). In addition, the serum concentrations of IGF-I and interleukin-6 were increased by high levels of soy isoflavones (P < 0.05). Furthermore, a diet containing soy isoflavones enhanced IGF-I mRNA expression levels in longissimus muscle, but decreased these levels in perirenal fat. However, the mRNA and protein expression levels of PPARγ in longissimus muscle and subcutaneous adipose tissue were both increased when compared with the Con. The data indicated that soy isoflavones regulated the BW gain and fat percentage of Chinese Guangxi minipigs, which also showed changes in IGF-I system and PPARγ. However, further research is required to clarify the causative relationship.     

饲喂雌鼠大豆异黄酮对后代雌性生殖性状的影响

目的研究雌性小鼠饲喂异黄酮后对其后代雌鼠生殖性状的影响。方法分别在雌鼠从断奶到妊娠前和妊娠期间两个不同阶段的日粮中添加不同剂量的大豆异黄酮（0、50、400 mg/kg）。记录F1代的出生窝产仔数、出生窝重、雌鼠的阴道最早开张时间和最早见栓时间,以及后代雌鼠性成熟期和体成熟期生殖器官重量、血清雌激素的含量。结果雌鼠妊娠期饲喂含50、400 mg/kg大豆异黄酮的饲料后F1代的窝产仔数显著高于对照组（P〈0.05）;雌鼠妊娠期采食400 mg/kg大豆异黄酮饲料,其后代雌鼠初次配种时间明显延迟（P〈0.05）;45、65日龄体重显著低于对照组,65日龄子宫重显著低于对照组,卵巢重显著高于对照组;血清中雌激素含量显著低于对照组（P〈0.05）,妊娠期间采食50 mg/kg大豆异黄酮饲料,其F1代雌鼠仅窝产仔数和45日龄时血清中雌激素含量受到影响;而雌鼠从断奶到怀孕期间饲喂含大豆异黄酮饲料的F1代在各项指标与对照组均差异无显著性。结论雌鼠妊娠期间饲喂含400 mg/kg大豆异黄酮的饲料会显著影响其雌性后代的初情期、生殖器官发育、血清雌激素含量等生殖生理性状,而在非妊娠期饲喂含大豆异黄酮饲料对F1代无显著影响。     

Soy isoflavones and cognitive function

There is growing interest in the physiological functions of soy isoflavones, especially in whether they affect cognitive function and have beneficial effects on neurodegenerative diseases. Here we review the recent evidence from clinical and experimental studies supporting a role for soy isoflavones in cognitive function. Soy isoflavones may mimic the actions and functions of estrogens on brain, and they have been shown to have positive effects on the cognitive function in females; however, studies on their effects on spatial memory have not provided consistent results in males. Although data from humans, cultures, and animal models are currently insufficient for elucidating the metabolism of soy isoflavone actions on cognitive function and the nervous system, we suggest two putative pathways; (1) an estrogen receptor-mediated pathway and (2) via the inhibition of tyrosine kinase, in particular by genistein, which is one of the soy isoflavones. Although soy isoflavones appear to have a positive effect on brain function, further research is needed to determine not only the efficacy but also the safety of soy isoflavones on the nervous system and cognitive function.     

Effect of soy isoflavones on breast cancer recurrence and death for patients receiving adjuvant endocrine therapy

Background

The intake of soy isoflavones among women with breast cancer has become a public health concern, because these compounds have weak estrogenic effects. There is little clinical evidence about their safety for patients with breast cancer who are receiving adjuvant endocrine therapy.

Methods

For patients who underwent surgery for breast cancer between August 2002 and July 2003 and who were receiving adjuvant endocrine therapy, we examined associations between dietary intake of soy isoflavones and recurrence of breast cancer and death. We measured dietary intake of soy isoflavones at baseline using a validated food frequency questionnaire. We estimated hazard ratios (HRs) and 95% confidence intervals (CIs) by means of multivariable Cox proportional hazards regression models. We further stratified the analyses by hormonal receptor status and endocrine therapy.

Results

The median follow-up period for the 524 patients in this study was 5.1 years. Among premenopausal patients, the overall death rate (30.6%) was not related to intake of soy isoflavones (HR = 1.05, 95% CI 0.78–1.71 for the highest quartile [> 42.3 mg/day] v. the lowest quartile [< 15.2 mg/day], p for trend = 0.87). Relative to post-menopausal patients in the lowest quartile of soy isoflavone intake, the risk of recurrence for post-menopausal patients in the highest quartile was significantly lower (HR = 0.67, 95% CI 0.54–0.85, p for trend = 0.02). Inverse associations were observed in patients with estrogen and progesterone receptor positive disease and those receiving anastrozole therapy.

Interpretation

High dietary intake of soy isoflavones was associated with lower risk of recurrence among post-menopausal patients with breast cancer positive for estrogen and progesterone receptor and those who were receiving anastrozole as endocrine therapy.A variety of health benefits in terms of cancer and cardiovascular disease have been attributed to the consumption of soy foods, primarily because of soy isoflavones.¹ Three primary isoflavones account for virtually all of the isoflavones in soy beans: genistein (about 50%), daidzein (about 40%) and glycitein (about 10%). The chemical structure of soy isoflavones is similar to that of estrogens. The isoflavones are therefore considered to be possible selective estrogen receptor modulators, which may bind to estrogen receptors and selectively stimulate or inhibit estrogen-like action in various tissues.² Given that soy-based foods are now more frequently consumed than was previously the case, both as an alternative approach to treating the symptoms of menopause and for promoting cardiovascular health,^3,4 concerns have arisen about the intake of these compounds by patients with hormone-sensitive breast cancer, in whom tumour growth depends largely on estrogen. Tamoxifen and anastrozole are commonly used as adjuvant endocrine therapy for hormone-sensitive breast cancer, and these drugs are effective in preventing recurrence and prolonging survival.^5,6 In some experimental studies the inhibitory effects of tamoxifen on growth of implanted mammary tumours were negated by dietary administration of soy isoflavones,^7,8 but in other rodent cancer models, soy food appeared to enhance the beneficial effects of tamoxifen.⁹ Little is known about the potential effects of consuming soy isoflavones for patients with breast cancer who are receiving adjuvant endocrine therapy. We used data for a cohort of postoperative patients with breast cancer who were receiving adjuvant endocrine therapy to examine the relation between intake of soy isoflavones and recurrence of breast cancer and death.     

Effects of chronic exposure to soy meal containing diet or soy derived isoflavones supplement on semen production and reproductive system of male rabbits

Soy and derivative diets deliver large doses of isoflavones to human and animals throughout their lifespan, including gestation. Epidemiologic and experimental data suggest that the consumption of soybean containing foods may protect against cardiovascular disease and decrease breast, prostate and endometrial cancer risk. Based on animal and in vitro studies, however, concerns have been raised that consumption of isoflavones may cause potential adverse effects on the reproductive tract and behavior. The aim of this study was to investigate the effects of chronic consumption of a soy meal containing diet or soy isoflavones supplement on the morphology of reproductive organs, semen quality, age that males reached puberty, and sexual behavior of male rabbits. With this purpose, 16 female rabbits were randomly assigned to receive: (1) a soy- and alfalfa-free diet; (2) a soy- and alfalfa-free diet supplemented with 5mg/kg body wt./day of soy isoflavones; (3) a soy- and alfalfa-free diet supplemented with 20mg/kg body wt./day of soy isoflavones; (4) a diet containing 18% of soy meal, throughout the gestation and lactation. After weaning, male offspring received the same diet, which was given to the respective mother. The age that males reached puberty, semen characteristics and sexual behavior were evaluated in these animals. At 33 weeks of age, the reproductive organs were submitted to histological evaluation. Rabbits, which received large amounts of isoflavones (20mg/kg body wt./day) had a lesser food intake, body weight and semen volume. Spermatogenesis, morphology of male genital organs and sexual behavior did not differ significantly from the control group. We conclude that chronic dietary treatment with soy based diet or soy isoflavones have no adverse effects on the observed reproductive patterns of male rabbits.     

大豆异黄酮抗肿瘤效应研究进展(综述)

大豆异黄酮是大豆生长过程中形成的一类次生代谢产物,对人体具有多种生理功能。研究表明,大豆异黄酮具有抗肿瘤、预防肿瘤作用,是一种很有潜力的癌症化学预防剂,能显著降低多种恶性肿瘤的发病率。现将近几年大豆异黄酮抗肿瘤效应及其机制的研究进展综述如下,以期为大豆异黄酮的深入研究及开发利用提供参考。     

Molecular mechanisms of action of the soy isoflavones includes activation of promiscuous nuclear receptors. A review

Consumption of soy has been demonstrated to reduce circulating cholesterol levels, most notably reducing low-density lipoprotein (LDL) cholesterol levels in hypercholesterolemic individuals. The component or components that might be responsible for this effect is still a matter of debate or controversy among many researchers. Candidate agents include an activity of soy protein itself, bioactive peptides produced during the digestive process, or the soy isoflavones. Although soy intake may provide other health benefits including preventative or remediative effects on cancer, osteoporosis and symptoms of menopause, this review will focus on isoflavones as agents affecting lipid metabolism. Isoflavones were first discovered as a bioactive agent disrupting estrogen action in female sheep, thereby earning the often-used term 'phytoestrogens'. Subsequent work confirmed the ability of isoflavones to bind to estrogen receptors. Along with the cholesterol-lowering effect of soy intake, research that is more recent has pointed to a beneficial antidiabetic effect of soy intake, perhaps mediated by soy isoflavones. The two common categories of antidiabetic drugs acting on nuclear receptors known as peroxisome proliferator activated receptors (PPARs) are the fibrates and glitazones. We and others have recently asked the research question 'do the soy isoflavones have activities as either "phytofibrates" or "phytoglitazones"?' Such an activity should be able to be confirmed both in vivo and in vitro. In both the in vivo and in vitro cases, this action has indeed been confirmed. Further work suggests a possible action of isoflavones similar to the nonestrogenic ligands that bind the estrogen-related receptors (ERRs). Recently, these receptors have been demonstrated to contribute to lipolytic processes. Finally, evaluation of receptor activation studies suggests that thyroid receptor activation may provide additional clues explaining the metabolic action of isoflavones. The recent advances in the discovery and evaluation of the promiscuous nuclear receptors that bind many different chemical ligands should prove to help explain some of the biological effects of soy isoflavones and other phytochemicals.     

Metabolism of isoflavones in human subjects

Isoflavones form a group of plant compounds that occur mainly in legumes, soy being the most important source in human diet. The high levels of isoflavones in the diet have been associated with a lowered risk for hormone-dependent diseases, including breast and prostate cancers, osteoporosis and cardiovascular disease. The metabolism of isoflavones in humans has been studied to a certain extent, but detailed studies are lacking. This paper reviews the current knowledge on metabolism of isoflavones and presents some preliminary results of a comprehensive soy feeding study, in which the phase I metabolites of soy isoflavones, daidzein, genistein and glycitein, were identified by GC-MS. This revised version was published online in June 2006 with corrections to the Cover Date.     

Soy,Red Clover,and Isoflavones and Breast Cancer: A Systematic Review

Background

Soy and red clover isoflavones are controversial due to purported estrogenic activity and possible effects on breast cancer. We conducted a systematic review of soy and red clover for efficacy in improving menopausal symptoms in women with breast cancer, and for potential impact on risk of breast cancer incidence or recurrence.

Methods

We searched MEDLINE, Embase, the Cochrane Library, and AMED from inception to March 2013 for human interventional or observational data pertaining to the safety and efficacy of soy and red clover isoflavones in patients with or at risk of breast cancer.

Results

Of 4179 records, we included a total of 131 articles: 40 RCTs, 11 uncontrolled trials, and 80 observational studies. Five RCTs reported on the efficacy of soy for hot flashes, showing no significant reductions in hot flashes compared to placebo. There is lack of evidence showing harm from use of soy with respect to risk of breast cancer or recurrence, based on long term observational data. Soy intake consistent with that of a traditional Japanese diet (2-3 servings daily, containing 25-50mg isoflavones) may be protective against breast cancer and recurrence. Human trials show that soy does not increase circulating estradiol or affect estrogen-responsive target tissues. Prospective data of soy use in women taking tamoxifen does not indicate increased risk of recurrence. Evidence on red clover is limited, however existing studies suggest that it may not possess breast cancer-promoting effects.

Conclusion

Soy consumption may be associated with reduced risk of breast cancer incidence, recurrence, and mortality. Soy does not have estrogenic effects in humans. Soy intake consistent with a traditional Japanese diet appears safe for breast cancer survivors. While there is no clear evidence of harm, better evidence confirming safety is required before use of high dose (≥100mg) isoflavones can be recommended for breast cancer patients.     

Role of soy isoflavones in the hypotriglyceridemic effect of soy protein in the rat

The present study was undertaken to determine whether isoflavones present in soy protein isolate contribute to the triglyceride-lowering effect of the protein relative to casein. Plasma triglyceride concentrations, their secretion rate into blood circulation, and post-heparin plasma lipoprotein lipase activity (a major determinant of intravascular catabolism of triglycerides) were measured in the fasted state in male Sprague-Dawley rats fed for 21 days one of three experimental diets varying in protein source (20% weight/weight): soy protein isolate, casein or casein to which 1.82 mg/g isoflavones (genistein and daidzein) were added to match the isoflavone content of soy protein isolate. Body weight gain was slightly lower in soy protein fed rats than in casein fed rats, but this effect was not statistically significant (P = 0.22). Casein plus isoflavones diet induced intermediary weight gain. A decrease in plasma total triglycerides was observed in rats fed soy protein and casein plus isoflavones compared with casein (P < 0.05), and there was a tendency to a positive correlation between weight gain and plasma triglyceride concentrations (r = 0.35, P = 0.06). However, no significant effect was observed on hepatic triglyceride concentrations, triglyceride secretion rate by the liver and post-heparin plasma lipoprotein lipase activity. These results show that soy protein isolate, in comparison with casein, has a hypotriglyceridemic effect in the rat and suggest that isoflavones may be responsible, at least in part, for this effect. The lowering effect of soy protein isolate and isoflavones on plasma triglyceride concentrations may be mediated by an alteration in energy balance, and possibly by the hepatic production of lipoproteins more susceptible to intravascular hydrolysis. Subtle but sustained changes in triglyceride secretion and post-heparin plasma lipoprotein lipase activity may also be implicated.     

Circulating isoflavonoid levels in CD-1 mice: effect of oral versus subcutaneous delivery and frequency of administration

The CD-1 mouse is a commonly used animal model to understand the biological effects of early-life exposure to soy isoflavones in infants. Most studies using CD-1 mice have administered isoflavones by daily subcutaneous injection, while infants receive oral feeds every few hours. The study objectives were to compare the total serum levels of genistein (GEN), daidzein (DAI) and the DAI metabolites equol and O-desmethyl-angolensin (O-DMA), after subcutaneous injection and oral dosing and to determine if frequency of oral administration results in different circulating levels of isoflavones using the CD-1 mouse model. From postnatal days 1 to 5, pups randomly received corn oil or soy isoflavones (total daily dose, 0.010 mg DAI+0.025 mg GEN) by subcutaneous injection once a day, orally once a day or orally every 4 hours. On postnatal day 5, 1 h posttreatment, mice were killed and serum was collected. Mice treated with soy isoflavones had higher (P<.05) serum GEN (female: 1895–3391 ng/ml and male: 483–578 ng/ml) and DAI (female: 850–1580 ng/ml and male: 248–322 ng/ml) concentrations versus control (5–20 ng/ml) mice, regardless of route or frequency of administration, and were similar among dosing strategies. Total serum concentrations of GEN and DAI were higher (P<.05) among females (GEN: 2714 ± 393 ng/ml and DAI: 1205 ± 164 ng/ml) than males (GEN: 521 ± 439 ng/ml and DAI: 288 ± 184 ng/ml) across treatment groups. Serum equol and O-DMA concentrations were negligible (<3 ng/ml) across groups. In conclusion, different routes of delivery and frequency of administration resulted in similar total serum levels of GEN, DAI¸ equol or O-DMA.     

The effects of soy isoflavones on obesity

Over the last decades, the prevalence of obesity and related diseases has increased rapidly in the Western world. Obesity is a disorder of energy balance and is associated with hyper-insulinemia, insulin resistance, and abnormalities in lipid metabolism, and it is one of the most important risk factors in the development of Type II diabetes, cardiovascular disease, atherosclerosis, and certain cancers. Because of the lower frequency of these diseases in Asian countries, attention has been turned toward the Asian diet, which consists highly of soy and soy-based products. The health benefits associated with soy consumption have been linked to the content of isoflavones, the main class of the phytoestrogens. As a result of their structural similarities to endogenous estrogens, isoflavones elicit weak estrogenic effects by competing with 17beta-estradiol (E2) for binding to the intranuclear estrogen receptors (ERs) and exert estrogenic or antiestrogenic effects in various tissues. The estrogenic activities of soy isoflavones are thought to play an important role in their health-enhancing properties. Additionally, the isoflavones have been proved to exert non-ER-mediated effects through numerous other pathways. Genistein, daidzein, and glycitein are the principal isoflavones in soy. Genistein is the most thoroughly examined of these, because it is the most prevalent isoflavone in soy and the most active of these compounds, because of its higher binding affinity for the ER. Genistein and daidzein can be obtained in high levels in humans under certain nutritional conditions, and epidemiologic and laboratory data suggest that these compounds could have health benefits in human obesity. This review will focus on the latest results of research on isoflavones and their effect on obesity in cell cultures, rodents, and humans.     

Dietary soy isoflavones inhibit activation of rat platelets

Isoflavones (isoflavonoids) have been proposed to be the active compounds that contribute to decreased mortality from chronic diseases in populations that consume large amounts of soy products. Diets containing soy protein with and without isoflavones were fed to rats to determine if these compounds could exert in vivo effects on physiologic markers of platelet activation. Three methods were employed to monitor platelet activation: measurement of electronic mean platelet volume, which is an indicator of shape change; monitoring of collagen-induced production of reactive oxygen signals (hydrogen peroxide); and determination of increases in phosphorylation of protein tyrosine residues after collagen stimulation. Apparent volumes were significantly smaller for platelets from rats fed isoflavones, suggesting that these platelets were in a more disc-like, quiescent state compared with platelets from rats fed the isoflavone-reduced diet (means +/- SEM, 5.37 +/- 0.08 vs. 5.70 +/- 0.06 fL, n = 6/group, P < 0.008). Results from the other functional tests were consistent with this finding. Platelet production of hydrogen peroxide was found be significantly lower 1, 3, and 5 minutes after addition of collagen for rats fed isoflavones versus rats fed the isoflavone-reduced diet (n = 6/group, P < 0.004). Phosphorylated tyrosine residues in platelet proteins after stimulation also were shown to be significantly lower in the platelets exposed to dietary isoflavones (n = 5/group, P < 0.047). These combined results indicate that soy isoflavones can alter early-event signaling networks that result in less activated platelets and may partially explain the beneficial effects of dietary soy against human heart disease.     

Reduction of leptin secretion by soy isoflavonoids in murine adipocytes in vitro

Daidzein and genistein are the main aglycones of soy isoflavonoid, and have many useful activities in vitro and in vivo. However, equol, a metabolite of daidzein in vivo, has attracted attention due to its stronger activity than that of the naturally occurring isoflavonoids. We subjected the soy isoflavonoids, including the naturally occurring (S)-equol, to mouse adipocytes, and compared the inhibitory activity on the leptin secretion. Equol, daidzein and genistein inhibited the leptin secretion, whereas O-desmethylangolensin had a lower activity. The inhibitory activity of the isoflavones was not affected by the addition of an iNOS inhibitor and an estrogen.