Suppression of arachidonic acid metabolism and nitric oxide formation by kudzu isoflavones in murine macrophages

Inhibitory effect of kudzu isoflavones on arachidonic acid metabolism and nitric oxide (NO) production in lipopolysaccharide activated RAW 264.7 macrophages were investigated. Isoflavone aglycones, such as daidzein, genistein, biochanin A, and formononetin significantly suppressed arachidonic acid release (50 microM). Biochanin A, which displayed the most active inhibition on arachidonic acid release in HT-29 human colon cancer cells, exhibited its most potent suppression in RAW 264.7 cell (by 86%) without showing cytotoxicity. However, isoflavone glucosides, puerarin and daidzin, showed lower inhibitory activities on the release of arachidonic acid and its metabolites. In NO formation, biochanin A showed marked inhibition, by 62% (50 microM), followed by genistein, daidzein, formononetin, and daidzin, 56, 39, 33, and 8%, respectively. 5,7-Dihydroxyl group in the A-ring of isoflavones could be a key functional group responsible for the strong inhibitory activity of biochanin A and genistein on NO production. These activities may contribute to the antiinflammatory and anticarcinogenic properties of kudzu isoflavones.     

The red clover isoflavone irilone is largely resistant to degradation by the human gut microbiota

Intestinal bacteria may influence bioavailability and physiological activity of dietary isoflavones. We therefore investigated the ability of human intestinal microbiota to convert irilone and genistein in vitro. In contrast to genistein, irilone was largely resistant to transformation by fecal slurries of ten human subjects. The fecal microbiota converted genistein to dihydrogenistein, 6′‐hydroxy‐O‐desmethylangolensin, and 2‐(4‐hydroxyphenyl)‐propionic acid. However, considerable interindividual differences in the rate of genistein degradation and the pattern of metabolites formed from genistein were observed. Only one metabolite, namely dihydroirilone, was formed from irilone in minor amounts. In further experiments, Eubacterium ramulus, a prevalent flavonoid‐degrading species of the human gut, was tested for transformation of irilone. In contrast to genistein, irilone was not converted by E. ramulus. Irilone only differs from genistein by a methylenedioxy group attached to the A‐ring of the isoflavone skeleton. This substitution obviously restricts the degradability of irilone by human intestinal bacteria.     

Content composition and antioxidant activity of isoflavones in commercial and homemade soymilk and tofu

BACKGROUND: Isoflavones, found in soymilk and tofu, are one of the phytochemicals in soy‐based products that may promote good health. Homemade tofu and various homemade soymilk samples were made using different soaking, grinding, and cooking methods. The homemade samples were compared to commercial tofu and soymilk for total isoflavone content and composition as well as their antioxidant capacity. All samples were freeze‐dried and extracted with a 58% acetonitrile solution which was subsequently used to determine the isoflavone content by reverse‐phase high‐performance liquid chromatography. The antioxidant activity of extracts was determined using a modified 2,2‐azino‐bis‐(3‐ethylbenzthiazoline‐6‐sulfonic acid) method and total antioxidant capacity was reported as ascorbic acid equivalents. RESULTS: The total isoflavone, aglycone, and antioxidant levels were significantly higher in homemade soymilk and tofu (1571 µg) than in commercial samples. Homemade soymilk made by the extended boiling method yielded the highest total isoflavone (2567 µg) and glucoside (1525 µg) content. A strong positive correlation was observed between the total isoflavone, aglycone conjugates, and genistein series concentration and antioxidant capacity of soymilk. CONCLUSION: Increased moist heating time yielded the highest concentration of total isoflavones as well as aglycone conjugates and the genistein series. Increasing the duration of boiling can increase the isoflavone content of both homemade and commercial soymilk and tofu. Copyright © 2007 Society of Chemical Industry     

Red clover (Trifolium pratense L.) isoflavones: determination of concentrations by plant stage,flower colour,plant part and cultivar

BACKGROUND: Red clover (Trifolium pratense L.), a widely used fodder, has recently received considerable interest as a valuable source of isoflavones for many health‐related applications. The aim of this study was to analyse the concentrations of four isoflavones, daidzein, genistein, formononetin and biochanin A, in extracts of leaves, stems, roots and different coloured flowers from red clover by high‐performance liquid chromatography and to determine the antifungal activities of the extracts. RESULTS: Among greenhouse‐grown samples the highest amounts of daidzein and genistein were found in petioles (0.11–0.28 and 0.30–0.54 mg g^?1 respectively), while leaves were rich in formononetin and biochanin A (5.57–9.05 and 10.94–14.59 mg g^?1 respectively). High formononetin concentrations were found in roots, but very little biochanin A. Among field‐grown samples the highest concentrations of daidzein and genistein were detected in stems collected in August (0.24 and 0.55 mg g^?1 respectively), while the highest amounts of formononetin and biochanin A were found in young leaves collected in June (7.47 and 9.69 mg g^?1 respectively). Most of the extracts inhibited the growth of Drechslera teres, while the root extract showed the strongest effect against Microdochium nivale. CONCLUSION: High levels of bioactive compounds found not only in inflorescences but also in roots, leaves and stems suggest that red clover may be an inexpensive and valuable resource for many applications. Copyright © 2007 Society of Chemical Industry     

Production of an Isoflavone Concentrate from Red Clover Flowers

Red clover flowers are a rich source of isoflavones in the forms of biochanin A, formononetin, genistein, and genistin. A simple process was developed to recover isoflavones from red clover flowers as a nutraceutical product. Isoflavones were 1st extracted in alkaline water and insoluble polyvinylpolypyrrolidone (PVP) was then added to the extract for adsorbing isoflavones. The adsorbed isoflavones were subsequently eluted with ethanol and dried. The main steps of this process were optimized for the product yield and quality. The final product was an isoflavone concentrate, containing more than 20 wt% isoflavones, which represented a recovery of greater than 50% of the total isoflavone amount in red clover flowers.     

Soy isoflavone genistein induces cell death in breast cancer cells through mobilization of endogenous copper ions and generation of reactive oxygen species

Scope: Worldwide geographical variation in cancer incidence indicates a correlation between dietary habits and cancer risk. Epidemiological studies have suggested that populations with high isoflavone intake through soy consumption have lower rates of breast, prostate, and colon cancer. Isoflavone genistein in soybean is considered a potent chemopreventive agent against cancer. Although several mechanisms have been proposed, a clear anticancer action mechanism of genistein is still not known. Methods and results: Here, we show that the cytotoxic action of genistein against breast cancer cells involves mobilization of endogenous copper. Further, whereas the copper specific chelator neocuproine is able to inhibit the apoptotic potential of genistein, the molecules which specifically bind iron (desferroxamine mesylate) and zinc (histidine) are relatively ineffective in causing such inhibition. Also, genistein‐induced apoptosis in these cells is inhibited by scavengers of reactive oxygen species (ROS) implicating ROS as effector elements leading to cell death. Conclusions: As copper levels are known to be considerably elevated in almost all types of cancers, in this proof‐of‐concept study we show that genistein is able to target endogenous copper leading to prooxidant signaling and consequent cell death. We believe that such a mechanism explains the anticancer effect of genistein as also its preferential cytotoxicity towards cancer cells.     

Lesser in vitro anaerobic cecal isoflavone disappearance was associated with greater apparent absorption of daidzein and genistein in Golden Syrian hamsters

Our hypothesis in this study was that in vitro disappearance of isoflavones from fecal or cecal contents of Golden Syrian hamsters paralleled the apparent absorption of these compounds, comparable with previous findings from in vitro human fecal incubations. Two studies were conducted to test this idea: one on in vitro fecal (study 1, n = 20/sex) and the other on in vitro cecal contents (study 2, n = 10/sex) ability to degrade isoflavones. According to HPLC analysis, urinary isoflavone excretion was significantly less by 2-4 fold in males compared with females in both studies. Fecal isoflavone excretion was not significantly different between sexes or isoflavones (study 1) and was <0.5% of ingested dose. In vitro anaerobic fecal isoflavone degradation rate constants from study 1 were minimal with no significant correlation between urinary and fecal isoflavone excretion. However, in vitro anaerobic cecal isoflavone degradation rate constants (study 2) were greater and significantly correlated with urinary excretion of daidzein (R = 0.90; p = 0.01) and genistein (R = 0.93; p = 0.004), but not glycitein (R = 0.50; p = 0.3). Both male and female hamsters showed a pattern of urinary isoflavone excretion similar to that found in humans (daidzein > genistein). Hamster in vitro cecal isoflavone degradation rate constants seemed to be analogous to human in vitro fecal isoflavone degradation rate constants for genistein and daidzein. The sex difference in isoflavone excretion in hamsters and the instability in glycitein excretion across studies coupled with the paucity of human data on this isoflavone deserve further investigation.     

红车轴草提取物中异黄酮成分的分析

目的:以高效液相色谱法对红车轴草中四种主要异黄酮物质(大豆黄素、染料木素、鹰嘴豆芽素B和鹰嘴豆芽素A)的含量进行测定。方法:高效液相色谱法的条件:反相C18柱(250mm×4.6mm),MeOH-CH3CN-H2O(3.7:1.3:5,V:V:V)为流动相,流速1.0ml/min,进样量20μl,柱温为室温,检测波长为260nm。样品前处理方法:称取一定量的红车轴草提取物样品加盐酸甲醇溶液(42:500,V:V)40ml,于85℃水浴中回流水解2h,冷却后用甲醇定容到50ml,匀后用0.45μm滤膜过滤,取滤液。结果:经高效液相色谱法测定,以保留时间定性,以峰面积定量,得出红车轴草(1#)中四种主要异黄酮物质的含量分别为:大豆黄素(0.43%)、染料木素(0.35%)、鹰嘴豆芽素B(4.79%)和鹰嘴豆芽素A(1.32%);而红车轴草(2#)中四种主要异黄酮物质的含量分别为:大豆黄素(0.60%)、染料木素(0.64%)、鹰嘴豆芽素B(9.29%)和鹰嘴豆芽素A(2.63%)。     

红车轴草不同部位中异黄酮含量的测定

采用反相高效液相色谱法对红车轴草不同部位(花、茎和叶)中四种主要异黄酮(鸡豆黄素A、芒柄花黄素、染料木素和大豆黄素)的含量进行了测定,结果表明:以占干物质计,4种异黄酮总量,叶中含量最高(0.856%),茎次之(0.403%),花中含量较低(0.258%)。鸡豆黄素A和芒柄花黄素是红车轴草中两种最主要的异黄酮,主要分布在叶中,其次是茎,花中含量较低。染料木素和大豆黄素,无论是花、茎还是叶,含量均较低。     

Effect of free or protein-associated soy isoflavones on the antioxidant status in rats

BACKGROUND: The objective of this study was to investigate the effect of chronic ingestion of free and protein‐associated soy isoflavones on the antioxidant status in male Wistar rats. Free isoflavone (iso), protein‐associated soy isoflavone (iso + prot) and soy protein (prot) extracts were administered for 30 days by gavage to the rats at a dosage of 1 mg aglycone isoflavones per 200 g body weight, adjusted daily, and the prot group was given the same concentration of soy protein received by the iso + prot group. Antioxidant capacity of plasma, thiobarbituric acid‐reactive substance (TBARS) and glutathione (GSH) levels and catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities in plasma, erythrocytes and tissues and gene expression levels in liver and kidney were evaluated. RESULTS: Chronic ingestion of free but not of protein‐associated soy isoflavones nor of solely soy protein increased plasma antioxidant capacity and GPx activity in erythrocytes. Soy protein increased CAT activity and gene expression in liver. SOD activity in erythrocytes was increased by all treatments. CONCLUSION: The overall results confirm that dietary soy isoflavones have a positive effect on antioxidant status, enhancing antioxidant capacity of plasma and antioxidant enzymes in various tissues, but the effects are dependent on the form of administration and on a complex mechanism of antioxidant status balance on the organism. Copyright © 2010 Society of Chemical Industry     

Functional attributes of soybean seeds and products,with reference to isoflavone content and antioxidant activity

The concentrations of isoflavones, especially daidzein and genistein, were determined by high performance liquid chromatography in 4 soybean cultivars and 26 soybean products. The total isoflavone content of the soybean cultivars was in the range of 525–986 mg per kg, and for soy products it was 32.9–795 mg per kg. Amongst the soybean products, the isoflavone content decreased in the order: soy sprouts, soy seeds, soy flour, soy milk, soy meals and soy sauce. Significant differences in the concentration of genistein and daidzein were observed between the commercial soy products and also within the soybean cultivars. The antioxidant activity of soybean and soy products correlated well with total phenolic content (TPC) and total isoflavones (TI), whereas TPC showed higher correlation with TI.     

Antioxidant potential and radioprotective effect of soy isoflavone against gamma irradiation induced oxidative stress

The in vitro antioxidant potential and in vivo radioprotective ability of soy isoflavones was studied. Male Wistar rats were orally administered with soybean isoflavones (60 mg/kg) for 21 days followed by gamma irradiation exposure. Survival studies in rats exposed at 10 Gy and endogenous spleen colony forming unit assay (CFU) at 6.0 Gy were performed in order to find radioprotective and immunomodulatory nature of the compound. The rat liver post mitochondrial supernatant and erythrocytes were used to measure lipid peroxidation (LPO) and glutathione (GSH) content along with various antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) after gamma irradiation exposure at 2.0 Gy. Pretreatment with soy isoflavone, prior to gamma irradiation resulted in the increased survival rate of the animals as compared to irradiated group. CFU counts in the isoflavone treated group followed by gamma irradiation at 6 Gy were significantly high as compared to control and the irradiated group, showing immunomodulatory nature of the isoflavones. Pretreatment with isoflavones also significantly reduced the LPO, enhanced the activity of antioxidant enzymes and improved haematological and histological parameters. The present study suggests that supplementation with isoflavone has potent antioxidant activity and act as probable radioprotector against gamma radiation induced oxidative damage.     

Bioactive action of β‐glucosidase enzyme of Bifidobacterium longum upon isoflavone glucosides present in soymilk

Bioconversion of isoflavone glucosides and antioxidant activity by probiotic strain (Bifidobacterium longum) during soymilk fermentation was investigated, as well as partial characterisation of the produced enzyme β‐glucosidase. The enzyme has higher affinity for genistin than for other substrates assayed. Maximum activity occurred at 42 °C and at pH 6.0; keeping 70–80% of activity for 60 days stored at low temperatures. Bifidobacterium longum grew well in soymilk (8.26 log CFU mL^?1 and pH of 3.9 at 24 h) and were produced in good quantities of organic acids. High hydrolysis degree of isoflavone glucosides (81.2%) was observed at 24 h. Enhancements in bioactivity were assessed in fermented soymilk by monitoring the radical‐scavenging activity, antioxidant activity and DNA protective action. The use of probiotic Bifidobacterium strain as β‐glucosidase producer increased bioactive isoflavone content and demonstrated that this enzyme plays a key role in the bioavailability of soymilk isoflavones, reducing the bioconversion time compared to other studies.     

Bioavailability of Isoflavone Metabolites After Korean Fermented Soybean Paste (Doenjang) Ingestion in Estrogen‐Deficient Rats

Doenjang (DJ), a fermented soybean product used in soups, stews, and sauces, contains high quality proteins, fats, vitamins, minerals, and other functional ingredients, including isoflavones and saponins. This study investigated whether DJ improves the bioavailability of isoflavones compared to boiled soybean (BS) in sham‐operated or ovariectomized (OVX) rats. We also examined the effects of ovariectomy on the differences in bioavailability of isoflavones. BS and DJ were administered in sham‐operated and OVX rats, and blood samples were collected. Twenty‐six isoflavone‐derived metabolites were identified. Pharmacokinetic analysis revealed that T_1/2 values of the individual isoflavone metabolites were most different in sham and OVX rats, even after the same sample treatment; however, T_max values were significant different in a few metabolites such as daidzein 4′‐glucuronide, daidzein 4′‐sulfate, 2‐(4‐hydroxyphenyl)propionic acid, and benzoic acid. For most of the individual metabolites, C_max was higher in both sham and OVX rats administered BS than those administered DJ. The AUC was generally lower in OVX rats than in sham rats. The AUC of daidzein and genistein in BS‐fed sham rats was approximately 1.7‐fold higher than those administered DJ, whereas glycitein was detected only in the DJ group. No significant differences in AUC of daidzein and genistein were observed between BS and DJ administration in OVX rats, although the total isoflavone content of DJ was lower; thus, DJ‐mediated isoflavone bioavailability was more effective in OVX rats. Similar tendencies were observed for phase II and gut‐mediated metabolites. These results suggested that DJ enhanced isoflavone bioavailability under estrogen deficiency, even when the total isoflavone content was decreased by fermentation.     

Phenolic Antioxidants of Soy Protein Hydrolyzates

Soy protein hydrolyzate (SPH) was investigated to identify the phenolic compounds responsible for antioxidant activity. Three isoflavones–genistein, daidzein, and glycitein–were found in SPH. These are the same isoflavones that have been identified in raw soybeans. No isoflavone glycosides were found in SPH. The phenolic acids found in SPH were caffeic, ferulic, p-coumaric, syringic, vanillic, gentisic, and p-hydroxybenzoic acids. The compounds were identified by TLC, GLC and UV spectra. The isoflavones were confirmed by their mass spectra.     

Preparation and characterisation of glyceollin‐enriched soya bean protein using solid‐state fermentation

Glyceollins, stress‐induced phytoalexins from parent soya bean isoflavones, were elicited with Aspergillus oryzae. This solid‐state fermentation facilitated the conversion of isoflavone glycosides into aglycones and glyceollins, which could mainly enrich in soya bean protein isolate (SPI) during protein preparation due to protein–polyphenol interactions. Glyceollin‐enriched SPI exhibited less flavour volatiles, higher solubility, lower whiteness and higher antioxidant activity than unfermented SPI. Fermented SPI was more easily to be digested during in vitro pepsin–trypsin digestion. This may be attributed to partial degradation of protein, especially α′ and α subunits of β‐conglycinin and acidic subunit of glycinin. The antioxidant activity of digestive products derived from fermented SPI was obviously enhanced with increasing digestion time due to simultaneous release of antioxidant peptides and glyceollins involved in the interior of protein molecule. These results suggest an effective technique for producing a nutrient‐enhanced SPI as novel functional ingredients applied in food industry.     

Characterisation and comparison of phenols,flavonoids and isoflavones of soymilk and their correlations with antioxidant activity

To investigate the relationship between bioactive compounds and antioxidant property, total phenolic content (TPC), total flavonoid content (TFC), individual and total isoflavone content of soymilk and their correlations with oxygen‐radical‐absorbing capacity (ORAC), ferric reducing antioxidant power (FRAP) and 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging capacity values were evaluated and compared. Results showed that TPC, TFC, isoflavones (including concentration and profiles) and antioxidation activity significantly varied among ten tested soybean cultivars. Significant correlations were established between DPPH and TFC (r = 0.553, P < 0.01), DPPH and TPC (r = 0.753, P < 0.01), FRAP and TFC (r = 0.599, P < 0.01) and FRAP and TPC (r = 0.616, P < 0.01). Positive linear correlations were found between subtotal, total isoflavones and ORAC. Unlike other isoflavone monomers, aglycone isoflavones correlated positively with DPPH and FRAP significantly. DPPH well correlated with FRAP, whereas neither DPPH nor FRAP correlated with ORAC. TPC, TFC, individual and total isoflavone values are potentially useful for soymilk antioxidant activity assessment.     

Isoflavone profiles,phenol content,and antioxidant activity of soybean seeds as influenced by cultivar and growing location in Ohio

Isoflavone levels and isoflavone chemical composition in soybeans vary between planting locations although the exact factors which control isoflavone biosynthesis are unclear. We compared levels of 12 isoflavones in soybean seeds of six cultivars grown in four different locations in Ohio in 2002 as determined by high‐performance liquid chromatography. Antioxidant activity contained in plant‐based foods can improve food oxidative stability and phenolics and isoflavones have proven active in food systems. Radical scavenging activity was assessed using the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical. Total phenolics (TPCs) were determined by using Folin–Ciocalteu reagent. Total isoflavones (TIs) varied five‐fold (1573–7710 nmol g^?1) between seeds from the various location–cultivar combinations. One location (Wooster, Ohio) produced seeds containing half the isoflavones as the other locations tested apparently due to poor growing conditions. The cultivars could be divided into two groups based on TI, one having approximately 50% more isoflavones. Surprisingly, across the entire data set, with increasing TI, the proportion of isoflavones accounted for by the daidzein family increased due primarily to malonyl daidzin. DPPH scavenging did not differ significantly by location or cultivar (P > 0.05) and did not correlate with TPC or TI. Profiling soybean isoflavones could help elucidate how isoflavone biosynthesis is regulated and lead to better disease resistance of soybean crops and soy foods with greater health benefits. Copyright © 2007 Society of Chemical Industry     

Nutrikinetics of Isoflavone Metabolites After Fermented Soybean Product (Cheonggukjang) Ingestion in Ovariectomized Mice

1 Scope

Cheonggukjang (CGJ) is a soybean‐based quick‐fermented food popular in Korea that contains a variety of biologically active compounds including isoflavones and saponins. Isoflavone bioavailability may be important for the bone health of postmenopausal women; therefore, the aim of this study is to evaluate the influence of fermentation on the isoflavone metabolite nutrikinetic profile after single dose CGJ or unfermented soybean administration in ovariectomized (OVX) and sham mice.

2 Methods and results

We identify 34 isoflavone metabolites using UPLC–QTOF‐MS and analyze their nutrikinetics at different time points (0.25, 0.5, 1, 2, 4, 8, 16, and 24 h) to understand their fermentation‐ and OVX‐mediated time‐dependent concentration changes. Nutrikinetics analysis shows that genistein, daidzein, genistein 4′‐sulfate, dihydrodaidzein sulfate, equol 4′‐sulfate, and equol‐7‐glucuronide are present at high concentrations in all groups based on area‐under‐the‐curve analysis. OVX mice appear to show lower isoflavone bioavailability than mice in the sham group. CGJ enhances various isoflavone metabolite bioavailability including genistein, 3‐hydroxygenistein, and equol 7‐glucuronide, compared to the unfermented soybean‐treated group. Among these metabolites, intact isoflavones, 3‐hydroxygenistein, genistein 4′‐sulfate, and equol 7‐glucuronide promote osteoblastogenesis and inhibit osteoclast formation.

3 Conclusions

CGJ has good isoflavone bioavailability and may be beneficial for the bone health of postmenopausal women.     

酱油渣异黄酮抗氧化功效成分纯化研究

以酱油渣为原料,采用乙醇提取法提取酱油渣中大豆异黄酮,对提取条件进行了优化。比较了醇沉法、等电点沉淀法、浓缩离心法、正丁醇法、二氯甲烷法、乙酸乙酯萃取法等6种方法对异黄酮粗提物的纯化效果。通过高效液相鉴定了纯化产物的异黄酮单体组成,并采用邻苯三酚氧化法和DPPH法评价了异黄酮纯化物的体外抗氧化活性。结果表明：优化条件下提取的异黄酮粗提物中异黄酮质量分数为1.21%、提取率为（85.36±0.09）%。6种纯化方法中,浓缩离心法所得异黄酮产物中异黄酮纯度为7.75%,去除蛋白质效果最佳;二氯甲烷萃取法所得异黄酮产物中异黄酮纯度为27.74%,去除总糖和灰分效果最佳;两者联用后所得纯化产物的异黄酮纯度为44.88%,其主要由染料木素、大豆黄素和大豆素组成,质量分数分别为67.37%、9.33%、23.30%。在质量浓度为1 mg/mL时,异黄酮纯化物对超氧阴离子、DPPH自由基的清除率分别为67.65%、89.18%,显著高于异黄酮粗提物（P＜0.05）,其IC50分别为0.34、0.26 mg/mL,说明异黄酮纯化物具有一定的抗氧化活性,有望作为一种潜在的天然抗氧化剂应用于功能性食品中。