genistin and daidzin

Post-menopausal women under treatment with levothyroxine for their medical conditions may take concomitantly dietary supplements containing soy isoflavones in combination to treat their post-menopausal symptoms. The aim of this study was to investigate the effect of a fixed combination of soy isoflavones on the oral bioavailability of levothyroxine in post-menopausal female volunteers.. 12 healthy post-menopausal female, who were on stable oral levothyroxine as replacement/supplementation therapy for hypothyroidism, received a single recommended oral dose of a food supplement containing 60 mg of soy isoflavones (>19% genistin and daidzin) concomitantly with (test) and 6 h later (reference) the administration of levothyroxine in a randomized, open label, crossover fashion. Plasma concentrations of levothyroxine and soy isoflavones (daidzin, daidzein, genistin, genistein, S-equol) were determined by LC-MS/MS. Pharmacokinetic (PK) parameters were determined by non-compartmental analysis. No effect of soy isoflavones was assumed if the 90% confidence intervals (CIs) for the estimated ratio test/reference was included in the acceptance limits 0.80-1.25 for PK parameters Cmax and AUCt.. The test/reference ratios Cmax and AUCt of levothyroxine were very close to unity (1.02 and 0.99, respectively) and the corresponding 90% CIs (0.99-1.04 and 0.88-1.12, respectively) fell entirely within the acceptance bioequivalence limits.. The combination of soy isoflavones used in the present investigation does not affect the rate and extent of levothyroxine absorption when administered concomitantly in post-menopausal women.

Topics: Administration, Oral; Biological Availability; Cross-Over Studies; Dietary Supplements; Equol; Female; Glycine max; Humans; Isoflavones; Middle Aged; Postmenopause; Therapeutic Equivalency; Thyroxine

Isoflavone supplements, consumed by women experiencing menopausal symptoms, are suggested to have positive effects on menopause-related adiposity and cardiovascular disease risk profile, but discussions about their safety are still ongoing.. The objective was to study the effects of an 8-wk consumption of 2 different isoflavone supplements compared with placebo on whole-genome gene expression in the adipose tissue of postmenopausal women.. This double-blind, randomized, placebo-controlled crossover intervention consisted of 2 substudies, one with a low-genistein (LG) supplement (56% daidzein + daidzin, 16% genistein + genistin, and 28% glycitein + glycitin) and the other with a high-genistein (HG) supplement (49% daidzein + daidzin, 41% genistein + genistin, and 10% glycitein + glycitin). Both supplements provided ∼ 100 mg isoflavones/d (aglycone equivalents). After the 8-wk isoflavone and placebo period, whole-genome arrays were performed in subcutaneous adipose tissue of postmenopausal women (n = 26 after LG, n = 31 after HG). Participants were randomized by equol-producing phenotype, and data analysis was performed per substudy for equol producers and nonproducers separately.. Gene set enrichment analysis showed downregulation of expression of energy metabolism-related genes after LG supplementation (n = 24) in both equol-producing phenotypes and oppositely regulated expression for equol producers (down) and nonproducers (up) after HG supplementation (n = 31). Expression of inflammation-related genes was upregulated in equol producers but downregulated in nonproducers, independent of supplement type. Only 4.4-7.0% of the genes with significantly changed expression were estrogen responsive. Body weight, adipocyte size, and plasma lipid profile were not affected by isoflavone supplementation.. Effects of isoflavones on adipose tissue gene expression were influenced by supplement composition and equol-producing phenotype, whereas estrogen-responsive effects were lacking. LG isoflavone supplementation resulted in a caloric restriction-like gene expression profile for both producer phenotypes and pointed toward a potential beneficial effect, whereas both supplements induced anti-inflammatory gene expression in equol producers. The study was registered at clinicaltrials.gov as NCT01556737.

Topics: Adipose Tissue; Adiposity; Aged; Cross-Over Studies; Dietary Supplements; Double-Blind Method; Equol; Female; Gene Expression; Genistein; Humans; Isoflavones; Middle Aged; Netherlands; Nutritional Status; Postmenopause; Surveys and Questionnaires

A multicentric, open, prospective, observational and no-randomized clinical trial was carried out in Spain with 190 postmenopausal women receiving a soy preparation rich in isoflavones (PHYTO SOYA, capsules containing 17.5 mg isoflavones). The main object of the present study was to investigate its efficacy in alleviating the symptomatology derived from the lack of estrogen, mainly hot flushes, but also other symptoms such as sleep disorder, anxiety, depression, vaginal dryness, loss of libido and bone pain. Each patient received 35 mg isoflavones per day in two doses. During the four months' treatment, a statistically significant decrease in the number of hot flushes with PHYTO SOYA was experienced by 80.82% women; only 5,48% patients did not improve with the treatment. The average reduction was 47.8%, which is equivalent to 4 hot flushes. All the other studied parameters also showed a statistically significant decrease. No severe side-effects were reported and tolerance was excellent. Treatment with PHYTO SOYA resulted in a significant improvement of the symptomatology that accompanies the lack of estrogen during menopause.

Topics: Anxiety; Blood Pressure; Chromatography, High Pressure Liquid; Climacteric; Depression; Estrogens, Non-Steroidal; Female; Glycine max; Hot Flashes; Humans; Isoflavones; Menopause; Metrorrhagia; Middle Aged; Molecular Structure; Pain; Phytoestrogens; Phytotherapy; Plant Extracts; Plant Preparations; Prospective Studies; Sleep Wake Disorders; Spectrum Analysis; Statistics as Topic; Surveys and Questionnaires; Treatment Outcome

Analyses of metabolite secretions by field-grown plants remain scarce. We analyzed daidzein secretion by field-grown soybean. Daidzein secretion was higher during early vegetative stages than reproductive stages, a trend that was also seen for hydroponically grown soybean. Daidzein secretion was up to 10 000-fold higher under field conditions than hydroponic conditions, leading to a more accurate simulation of rhizosphere daidzein content.

Topics: Genistein; Glucosides; Glycine max; Hydroponics; Isoflavones; Organ Specificity; Plant Leaves; Plant Roots; Rhizosphere

To detect major soy isoflavone glycosides, namely daidzin (DZ) and genistin (GEN), novel open sandwich fluorescence-linked immunosorbent assay (os-FLISA) was developed by taking advantage of enhanced interactions between variable regions of heavy (VH) and light chain (VL) domains in the presence of an antigen. The VH and VL genes were expressed in Escherichia coli as a chimera protein with green fluorescence protein (AcGFP1) and maltose-binding protein (MBP), respectively. Comprehensive characterization of os-FLISA displayed nearly the same specificity as parental DZ- and GEN-specific monoclonal antibody, demonstrating the potential of the developed assay for detection of both DZ and GEN. Their detectable range in this system exhibited at 0.1-12.5 μg mL

Topics: Antibodies, Monoclonal; Escherichia coli; Fluorescence; Food Analysis; Glycine max; Green Fluorescent Proteins; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Immunosorbent Techniques; Isoflavones; Limit of Detection; Maltose-Binding Proteins; Recombinant Proteins; Reproducibility of Results

This study investigated the effects of persistent ultraviolet B (UV-B) irradiation on isoflavone accumulation in soybean sprouts. Three malonyl isoflavones were increased by UV-B. Malonylgenistin specifically accumulated upon UV-B exposure, whereas the other isoflavones were significantly increased under both dark conditions and UV-B exposure. The results of isoflavone accumulation to UV-B irradiation time were observed as following: acetyl glycitin rapidly increased and then gradually decreased; malonyl daidzin and malonyl genistin were highly accumulated within an intermediate period; genistein and daidzin were gradually maximized; daidzin, glycitin, genistein, and malonyl glycitin did not increase; and glycitin, acetyl daidzin, and acetyl genistin exhibited trace amounts. Transcriptional analysis of isoflavonoid biosynthetic genes demonstrated that most metabolic genes were highly activated in response to UV-B 24 and UV-B 36 treatments. In particular, it was found that GmCHS6, GmCHS7, and GmCHS8 genes among the eight known genes encoding chalcone synthase were specifically related to UV-B response.

Topics: Acyltransferases; Gene Expression Regulation, Plant; Genistein; Glucosides; Glycine max; Isoflavones; Kinetics; Seedlings; Time; Ultraviolet Rays

Lignans and flavonoids are found in plants in their glycosylated forms and need to be hydrolyzed to aglycones to become bioavailable. Putative β-glucosidase genes from Lactobacillus mucosae INIA P508 were inserted into the plasmid pNZ:TuR. The strain Lactococcus lactis MG1363 harboring the plasmid pNZ:TuR.glu913 showed high β-glucosidase activity and was able to transform secoisolariciresinol diglucoside (SDG) into secoisolariciresinol (SECO). Lactic acid bacteria and Bifidobacterium strains harboring pNZ:TuR.glu913 were incubated with a soy beverage supplemented with flax seed extracts. SDG was almost completely consumed by the transformed strains, while concentration of SECO greatly increased. Moreover, these strains showed high deglycosylation of the isoflavone glycosides daidzin and genistin. In addition, other lignan and flavonoid aglycones were produced, i.e. matairesinol, pinoresinol, quercetin, and eriodyctiol. These deglycosylase activities were maintained when this glucosidase gene was cloned in a food grade vector, pLEB590, and transformed into L. lactis MG1363. This is the first report of the use of a food grade plasmid that confers the ability to efficiently catalyze the deglycosylation of lignans, isoflavonoids, flavones, and flavanones. The recombinant bacteria of this study would be of value for the development of fermented vegetal foods enriched in bioavailable forms of lignans and flavonoids.

Topics: beta-Glucosidase; Bifidobacterium; Flavonoids; Food Microbiology; Gene Expression; Glycosylation; Isoflavones; Lactobacillus; Lactococcus lactis; Lignans; Plasmids

Soybean (Glycine max L.) is a good source of natural antioxidants and commonly consumed as fermented products such as cheonggukjang, miso, tempeh, and sufu in Asian countries. The aim of the current study was to examine the influence of novel endophytic bacterial strain,

Topics: Amino Acids; Antioxidants; Bacillus amyloliquefaciens; Fermentation; Food Hypersensitivity; Genistein; Glycine max; Isoflavones; Nutritive Value; Phenols

Soy-based foods are consumed for their health beneficial effects, implying that the population is exposed to soy isoflavones in the diet. Herein, male rats at 21, 35, and 75 days of age were maintained either on a casein control diet, soybean meal (SBM), or control diet supplemented with daidzin and genistin (G + D) for 14 days. Feeding of SBM and G + D diets decreased testicular testosterone (T) secretion regardless of age. Altered androgen secretion was due to decreased (P < 0.05) Star and Hsd17β protein in the testes and was associated with increased (P < 0.05) Lhβ and Fshβ subunit protein expression in pituitary glands. Second, male rats were fed either a casein control diet, control diet + daidzin, control diet + genistin, or control diet + genistin + daidzin (G + D). Compared to control, feeding of all isoflavone-containing diets decreased (P < 0.05) testicular T concentrations, and more so in the G + D diet group. Interestingly, Esr1 and androgen receptor protein and pituitary Fshβ with Lhβ subunit protein were increased (P < 0.05) by feeding of genistin and G + D diets, but not the daidzin diet. However, daidzein and genistein both caused a concentration dependent inhibition (P < 0.05) of T secretion by Leydig cells in vitro with IC50 of 184 ηM and 36 ηM, respectively. Results demonstrated that altered testicular steroidogenic capacity and pituitary FSHβ and LHβ subunit expression due to soy-based diets result from specific actions by genistein and daidzein. Experiments to assess effects of isoflavone regulation of intratesticular androgen concentrations on male fertility are warranted.

Topics: Aging; Androgens; Animal Feed; Animals; Diet; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation; Isoflavones; Leydig Cells; Male; Neurosecretory Systems; Random Allocation; Rats; Soybean Proteins

Topics: Anticarcinogenic Agents; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Female; Glycine max; Humans; Isoflavones; MCF-7 Cells

Semen Sojae Preparatum is one of the most widely used traditional Chinese medicines. A reliable and accurate high-performance liquid chromatography with diode array detection method has been developed and validated for the quantitative determination of the ten bioactive compounds contained in Semen Sojae Preparatum. The samples were first extracted by pressurized liquid extraction using 80% ethanol at 100°C for 15 min and three static extraction cycles. Chromatographic separation was conducted on a C18 column using a mobile phase consisting of water and acetonitrile under gradient elution, and the detection wavelength was set at 210 nm. The samples were further analyzed on a high-performance liquid chromatography with time-of-flight mass spectrometry system to confirm the determination results. All the ten analytes were well separated, and the calibration curves showed good linearity. The intra- and interday precisions were evaluated in terms of relative standard deviation values within the ranges of 0.20-1.43% and 0.40-4.78%, respectively. The recoveries for the ten analytes were all in the ranges of 96.2-104.3%, with relative standard deviation values < 3.85%. The established high-performance liquid chromatography method could serve as a reliable and accurate method for the quality evaluation of Semen Sojae Preparatum from different origins.

Topics: Calibration; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Genistein; Isoflavones; Mass Spectrometry; Molecular Structure; Semen; Time Factors

The aim of this study was to characterize β-glucosidase from Citrobacter koser GXW-1 isolated from soil and to improve the enzyme by molecular modification.. A bacterial strain with β-glucosidase activity was screened from the soil around Wuming sugar mill in Guangxi by esculin-ferric ammonium citrate selecting plate. The 16S rDNA of the strain was obtained and analyzed. By searching GenBank database, the genes encoding β-glucosidase from the same genus Citrobacter were found. These sequences were aligned. Then, a gene encoding β-glucosidase was amplified by PCR. The recombinant plasmid pQE-cbgl was constructed. The recombinant protein was purified with Ni-NTA. The enzyme properties of the recombinant protein CBGL were studied in detail. At last, the wild enzyme CBGL was reformed by error-prone PCR and site-directed random mutagenesis.. C. koser GXW-1 with β-glucosidase activity was isolated from the soil. A gene encoding β-glucosidase was cloned from the wild strain GXW-1. The properties of CBGL were identified. Its optimal pH and temperature were 6.0 and 45℃. Its Km and Vmax value were (11.280±1.073) mmol/L and (0.1704±0.0073) μmol/(mg·min), respectively. Its Ki values was (66.84±3.40) mmol/L. CBGL can hydrolyze α-pNPG, stevioside, daidzin and genistin. CBGL was modified by error-prone PCR and site directed random mutagenesis. A positive mutant W147F was obtained successfully. Its Vmax was 2.54 times that of the wild enzyme CBGL.. CBGL not only can hydrolyze β-glycosidic bond, but also can hydrolyze the α-glycosidic bond in α-pNPG. Furthermore, CBGL can hydrolyze stevioside, daidzin and genistin. These characteristics indicate that the β-glucosidase CBGL has important applications in theoretical research and in industry.

Topics: Bacterial Proteins; beta-Glucosidase; Citrobacter; Diterpenes, Kaurane; Enzyme Stability; Glucosides; Hydrogen-Ion Concentration; Isoflavones; Kinetics; Phylogeny; Soil Microbiology; Substrate Specificity; Temperature

Two phenols, bakuchiol (1) and 3-hydroxybakuchiol (2), and two isoflavone glycosides, daidzin (3) and genistin (4) were isolated from Otholobium mexicanum J. W. Grimes (Fabaceae). Moreover, the ability of the raw extract and isolated metabolites to inhibit the enzymes α-amylase and α-glucosidase was evaluated in vitro. In the α-amylase assay, the methanolic extract exhibited a moderate inhibitory activity with an IC₅₀ of 470 μg/mL, while inhibition percentages of bakuchiol (1), 3-hydroxybakuchiol (2), and daidzin (3) were less than 25% at the maximum dose tested (1 μM). Genistin (4) exhibited a poor activity with an IC₅₀ of 805 μM. In the α-glucosidase assay, the methanolic extract exhibited a strong inhibitory activity with an IC₅₀ value of 32 μg/mL, while 3-hydroxybakuchiol (2) exhibited a moderate inhibitory activity with an IC₅₀ of 345 μM. Daidzin (3) and genistin (4) exhibited lower inhibitory activity with IC₅₀ values of 564 μM and 913 μM, respectively. Bakuchiol (1) exhibited a poor inhibitory activity with an inhibition percentage less than 10% at the maximum dose tested (1 mM).

Topics: alpha-Amylases; alpha-Glucosidases; Animals; Enzyme Inhibitors; Fabaceae; Hypoglycemic Agents; Isoflavones; Plant Extracts; Swine

Daidzin (DZ) and genistin (GEN) are two major soy isoflavone glycosides isolated from soybeans. Soy products containing isoflavones have recently been widely accepted for commercial use. However, the Japanese Government has suggested that soy isoflavone intake should be limited because of their estrogenic effects due to their interactions with estrogen receptors. In this study, we established a one-step indirect competitive immunochromatographic assay (ICA) for rapid and sensitive detection of total isoflavone glycosides (DZ and GEN) using gold nanoparticles conjugated with a monoclonal antibody against DZ. This assay was able to be completed in 15min following the immersion of a test strip in an analyte solution. Furthermore, the limit of detection for the total amount of isoflavone glycosides was ∼125ngmL(-1). Considering that the major soy isoflavone glycosides found in soy products are DZ and GEN, this study demonstrates the potential use of ICA for the assessments of over consumption of isoflavones in soy supplements and foods, which would increase the safe dietary intake of soy products.

Topics: Antibodies, Monoclonal; Chromatography, Affinity; Glycine max; Glycosides; Gold Colloid; Isoflavones

A plastic composite support (PCS) bioreactor was implemented to evaluate the effects on isoflavone deglycosylation in black soymilk fermented by Rhizopus oligosporus NTU 5.. Evaluation for the optimal PCS for mycelia immobilisation was conducted, which led to the significant results that the most mycelium weight (0.237 g per PCS, P < 0.05) is held by an S-type PCS; therefore, it was selected for black soymilk fermentation. It was found that the PCS fermentation system without pH control exhibits better efficiency of isoflavone bioconversion (daidzin to daidzein, and genistin to genistein) than the one with pH control at pH 6.5. As for the long-run fermentation, those without pH control indeed accelerate the isoflavone bioconversion by continuously releasing β-glucosidase into soymilk. Deglycosylation can be completed in 8 to 24 h and sustained for at least 34 days as 26 batches. The non-pH-control fermentation system also exhibits the highest total phenolic content (ranged from 0.147 to 0.340 mg GAE mL(-1) sample) when compared to the pH-controlled and suspended ones. Meanwhile, the black soymilk from the 22nd batch with 8 h fermentation demonstrated the highest DPPH radical scavenging effect (54.7%).. A repeated-batch PCS fermentation system was established to accelerate the deglycosylation rate of isoflavone in black soymilk. © 2015 Society of Chemical Industry.

Topics: Antioxidants; beta-Glucosidase; Biphenyl Compounds; Fermentation; Food Handling; Genistein; Glycosides; Humans; Hydrogen-Ion Concentration; Isoflavones; Picrates; Rhizopus; Soy Milk

Daidzin, genistin, and glycitein are major isoflavone compounds in soybean that are indispensable nutrients in traditional Chinese foods. Generally, strategies for detecting and separating soy isoflavones have been based on HPLC and chromatographic techniques, which are tedious and time-consuming procedures. In the present study, we developed an ELISA-based approach for daidzin detection using a broad-specificity monoclonal antibody (clone number: AA9) with an effective detection range of 10-10 000 ng/mL. Subsequently, we prepared an immunoaffinity column by coupling the monoclonal antibody AA9 to CNBr-activated Sepharose 4B. Our results demonstrate that the immunoaffinity column can efficiently and specifically extract daidzin, glycitein, and genistin from numerous structurally similar soy isoflavones in leguminous plants, thereby providing a new method for the extraction of target components from similar compounds in natural products.

Topics: Chromatography, Affinity; Enzyme-Linked Immunosorbent Assay; Glycine max; Isoflavones; Time Factors

Isoflavone profile is greatly affected by heating process. However, kinetic analyses of isoflavone conversion and degradation using a continuous industry processing method have never been characterized. In this study, Proto soybean was soaked and blanched at 80 °C for 2 min and then processed into soymilk, which underwent UHT (ultra-high temperature) at 135 to 150 °C for 10 to 50 s with a pilot plant-scale Microthermics processor. The isoflavone profile was determined at different time/temperature combinations. The results showed that all isoflavone forms exhibited distinct changing patterns over time. In the soymilk under UHT conditions, the degradation (disappearance) of malonyldaizin and malonylgenistin exhibited first-order kinetics with activation energies of 59 and 84 kj/mole, respectively. At all UHT temperatures, malonylgenistin showed higher rate constants than malonyldaidzin. However, malonylglycitin changed irregularly under these UHT temperatures. The increase of genistin, daidzin, glycitein and acetlydaidzin during heating demonstrated zero-order kinetics and the rate constants increased with temperature except for the conditions of 145 to 150 °C for 50 s. Overall, genistein series exhibited higher stability than daidzein series. Under all UHT conditions, total isoflavone decreased from 12% to 24%.

Topics: Food Handling; Glucosides; Glycine max; Hot Temperature; Humans; Isoflavones; Kinetics; Soy Milk

Dietary isoflavones, daidzein and genistein are of huge interest in the nutraceutical field due to their practical application to postmenopause complications. This study is the first report an efficient method to prepare isoflavone rich soybean leaves (soyleaves) which is an edible food stuff in Asian countries. The preharvest treatment of ethylene highly stimulated the level of isoflavone in soyleaves. Annotation and quantification of metabolites were determined by UPLC-Q-TOF-MS and HPLC. Phenolic metabolites of soyleaves are mostly kaempferol glycosides, but not dietary isoflavones. The accumulated isoflavones by ethylene treatment were determined to be daidzin 1, genistin 2, malonyldaidzin 3 and malonylgenistin 4, which were easily hydrolyzed to daidzein and genistein by β-glucosidase. Total content of dietary isoflavones was increased up to 13854 μg/g. The most suitable condition was estimated to be 250 μg/g ethylene or 200 μg/g ethephon (ethylene donor) treatment at the R3 growth stage. The ratio of daidzein and genistein glycosides was approximately 5 to 3. The accumulated isoflavonoid biosynthesis pathway genes were identified within the transcriptome of soyleaves tissues at 1 day after treatment of ethephon. The quantitative RT-PCR analysis of these genes indicated significantly higher expression of CHS, CHI, IFS, HID, IF7GT, and IF7MaT compared to control leaves. These findings suggest that ethylene activates a set of structural genes involved in isoflavonoid biosynthesis, thereby leading to enhanced production of isoflavones in soybean plants.

Topics: beta-Glucosidase; Chromatography, High Pressure Liquid; DNA, Complementary; Ethylenes; Genistein; Glucosides; Glycine max; Glycosides; Isoflavones; Plant Leaves; RNA, Plant; Tandem Mass Spectrometry

To test the hypothesis that the plant stress related elicitor cis-jasmone (cJ) provides protection in soybean pods against the seed-sucking stink bug pest, Euschistus heros, the growth of E. heros on cJ-treated pods was investigated using three soybean cultivars differing in insect susceptibility, i.e. BRS 134 (susceptible), IAC 100 (resistant) and Dowling (resistant). E. heros showed reduced weight gain when fed cJ-treated Dowling, whereas no effect on weight gain was observed when fed other treated cultivars. Using analysis of variance, a three factor (cultivar x treatment x time) interaction was observed with concentrations of the flavonoid glycosides daidzin and genistin, and their corresponding aglycones, daidzein and genistein. There were increases in genistein and genistin concentrations in cJ-treated Dowling at 144 and 120 h post treatment, respectively. Higher concentrations of malonyldaidzin and malonylgenistin in Dowling, compared to BRS 134 and IAC 100, were observed independently of time, the highest concentrations being observed in cJ-treated seeds. Levels of glycitin and malonylglycitin were higher in BRS 134 and IAC 100 compared to Dowling. Canonical variate analysis indicated daidzein (in the first two canonical variates) and genistein (in the first only) as important discriminatory variables. These results suggest that cJ treatment leads to an increase in the levels of potentially defensive isoflavonoids in immature soybean seeds, but the negative effect upon E. heros performance is cultivar-dependent.

Topics: Animals; Cyclopentanes; Feeding Behavior; Flavonoids; Genistein; Glucosides; Glycine max; Heteroptera; Isoflavones; Oxylipins; Seeds

The present study describes the biotransformation of a commercially available crude extract of soy isoflavones, which contained significant amounts of the soy isoflavone glycosides daidzin and genistin, by recombinant

Topics: Antioxidants; Bacillus megaterium; Biotransformation; Biphenyl Compounds; Escherichia coli; Glycine max; Glycosides; Isoflavones; Monophenol Monooxygenase; Picrates

Lactobacillus rhamnosus C6 strain showed higher β-glucosidase activity as well as biotransformation of isoflavones from glycones (daidzin and genistin) to aglycones (daidzein and genistein) in soymilk. However, L. rhamnosus C2 and Lactobacillus casei NCDC297 also exhibited similar activity during soymilk fermentation. These three strains can be selected for the development of functional fermented soy foods enriched with aglycone forms of isoflavones, such as soy yoghurt, soy cheese, soy beverages and soy dahi.. The study determined β-glucosidase activity of probiotic Lactobacillus cultures for bioconversion of isoflavones to aglycones in fermenting soymilk medium. Soymilk was fermented with six strains (L. rhamnosus C6 and C2, L. rhamnosus NCDC19 and NCDC24 and L. casei NCDC17 and NCDC297) at 37 °C for 12 h. The highest β-glucosidase activity and isoflavone bioconversion after 12 h occurred by L. rhamnosus C6 culture during fermentation in soymilk. Increased isoflavone aglycone content in fermented soymilk is likely to improve the biological functionality of soymilk (e.g. antioxidant activity, alleviation of hormonal disorders in postmenopausal women, etc.).. Lactobacillus rhamnosus C6 culture can be used for the development of functional fermented soy-based products.

Topics: beta-Glucosidase; Fermentation; Functional Food; Humans; Isoflavones; Lacticaseibacillus casei; Lacticaseibacillus rhamnosus; Lactobacillus; Probiotics; Soy Milk

Soy isoflavones are known as major bioactive compounds in soybean (Glycine max), which is an indispensable food. Despite their utility, the consumption of isoflavones has recently been limited because they exhibit oestrogenic and topoisomerase II inhibitory effects. To assess their intake limitation, accurate, sensitive, and effective quantitative analyses are necessary. In this study, we produced the monoclonal antibody (MAb) against daidzin (DZ) and applied it to an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the simultaneous determination of DZ and genistin (GEN), which are known as two major soy isoflavone glycosides in soy products. Using the DZ-MAb, we developed a sensitive icELISA method, where the limit of detection for DZ and GEN was 1.95ng/ml. Several validation analyses revealed that the icELISA is sufficiently accurate and sensitive to be used to assess the overconsumption of soy isoflavones, which would lead to the safe dietary intake of soy products.

Topics: Antibodies, Monoclonal; Enzyme-Linked Immunosorbent Assay; Glycine max; Glycosides; Hydrolysis; Isoflavones; Plant Extracts; Sensitivity and Specificity

Trypsin is a serine protease that has been proposed as a potential therapeutic target for metabolic disorders and malignancy diseases, thus the identification of biomolecular interactions of compounds to trypsin could be of great therapeutic importance. In this study, trypsin was immobilized on a monolithic silica capillary column via sol-gel. The binding properties of four small molecules (daidzin, genistin, matrine and oxymatrine) to trypsin were examined using the trypsin affinity columns by frontal analysis. The results indicate that the matrine (dissociation constant, Kd = 7.904 μM) has stronger interaction with trypsin than the oxymatrine (Kd = 8.204 μM), whereas daidzin and genistin were nearly have no affinity with trypsin. The results demonstrated that the frontal affinity chromatography can be used for the direct determination of protein-protease inhibitor binding interactions and have several significant advantages, including easy fabricating, reproducible, minimal technological requirements and potential to become a reliable alternative for quantitative studies of biomolecular interactions.

Topics: Alkaloids; Chromatography, Affinity; Enzymes, Immobilized; Isoflavones; Kinetics; Matrines; Models, Chemical; Quinolizines; Silicon Dioxide; Trypsin; Trypsin Inhibitors

Dietary supplements high in isolated isoflavones are commercially available for human consumption primarily to alleviate menopausal symptoms in women. The isoflavone composition, quantity and importantly their estrogenic potency are poorly standardised and can vary considerably between different products. The aim of this study was to analyse the isoflavone composition of 11 dietary supplements based on soy or red clover using the HPLC/MS/MS technique. Furthermore, we investigated the transactivational potential of the supplements on the estrogen receptors (ER), ERα and ERβ, performing luciferase reporter gene assays. As expected, we found that the isoflavone composition varies between different products. The measured total isoflavone contents in various supplements were mostly comparable to those claimed by the manufacturers in their product information. However expressing the isoflavone content as isoflavone aglycone equivalents, soy-based supplements had a clearly lower quantity compared to the manufacturer information. All supplements transactivated more or less ERα and ERβ with a preference for ERβ. The transactivational efficiency exceeded partly the maximal 17β-estradiol induced ER activation. While the different soy-based supplements revealed similar transactivation potential to both ERs, red clover-based supplements differed considerably. We conclude that different commercial dietary supplements based on soy or red clover vary in their isoflavone composition and quantity. They are estrogenically active, although especially the red clover-based supplements show considerable differences in their estrogenic potential to ERα and ERβ. Thus, different isoflavone-rich products cannot be necessarily compared regarding possible biological effects.

Topics: Capsules; Dietary Supplements; Estrogen Receptor alpha; Estrogen Receptor beta; Food Inspection; Food Labeling; Genes, Reporter; Genistein; Germany; Glycine max; Glycosides; HEK293 Cells; Humans; Isoflavones; Phytoestrogens; Recombinant Proteins; Response Elements; Transcriptional Activation; Trifolium

The objective of this study was to evaluate the changes in oligosaccharides and isoflavone aglycone content in soymilk during fermentation with commercial kefir culture. Soymilk was fermented with kefir culture at 25 °C for 30 h. The counts of lactic acid bacteria, Lactococcus lactis, Leuconostoc sp and yeasts; measurements of pH, acidity, α-galactosidase and β-glucosidase activity, sugar and isoflavone contents were performed at the intervals of time. In the fermented soymilk, the lactic acid bacteria counts increased from 7.6 log to 9.1 CFU g(-1), pH reached to 4.9 and lactic acid reached 0.34 g 100  g(- 1). The α-galactosidase was produced (0.016 AU g(-1)) with 100% raffinose and 92% stachyose hydrolysis being observed after the depletion of galactose, glucose and sucrose. Kefir culture produced β-glucosidase (0.0164 AU g(-1)), resulting in 100% bioconversion of glycitin and daidzin and 89% bioconversion of genistin into the corresponding aglycones. The fermented soymilk presented 1.67 μmol g(-1) of daidzein, 0.28 μmol g(-1) of glicitein and 1.67 μmol g (-1) of genistein.

Topics: alpha-Galactosidase; beta-Glucans; beta-Glucosidase; Cell Survival; Colony Count, Microbial; Cultured Milk Products; Fermentation; Food Handling; Food Microbiology; Genistein; Hydrogen-Ion Concentration; Hydrolysis; Isoflavones; Lactococcus lactis; Leuconostoc; Levilactobacillus brevis; Oligosaccharides; Raffinose; Saccharomyces cerevisiae; Soy Milk

Despite considerable interest in the physiologic effects of isoflavones, the in vivo bioavailability of the most common isoflavone forms, malonylglucoside conjugates, has not been determined. Differences in the bioavailability of malonylglucosides compared with the nonconjugated β-glucoside forms may explain the inconsistent findings regarding the physiologic effects of isoflavones. Therefore, our objective was to determine the effect of malonyl- conjugation on isoflavone bioavailability in an animal model. Malonylgenistin and malonyldaidzin, and their corresponding nonconjugated glucosides, were extracted from soy grits and purified using liquid chromatography. Purity of the isolated forms was confirmed by nuclear magnetic resonance analysis. Male rats were gavaged with malonylgenistin, genistin, malonyldaidzin, or daidzin at a dose of 100 μmol/kg body weight. Blood and urine samples were collected at time intervals ranging from 0 to 48 h. Isoflavone metabolites in plasma and urine were determined using stable isotope dilution-liquid chromatography/mass spectrometry. Comparisons of pharmacokinetic variables were made between nonconjugated and conjugated glucosides and over time of plasma collection. The areas under the time-concentration curve of the metabolites in the plasma obtained after the administration of nonconjugated β-glucosides were 1 to 6 times higher than those of their respective malonylglucosides (P ≤ 0.05). Additionally, maximum plasma concentration and urinary excretion of isoflavone metabolites were significantly higher (1-9 times; P ≤ 0.05) after the administration of nonconjugated β-glucosides. To our knowledge, these results demonstrated, for the first time, that nonconjugated β-glucosides are relatively more bioavailable than their respective malonylglucosides. These differences in the bioavailability of conjugated and nonconjugated β-glucosides should be considered in future studies focused on the bioactivity of isoflavones.

Topics: Administration, Oral; Animal Feed; Animals; Deuterium; Glucosides; Glycine max; Isoflavones; Male; Nuclear Magnetic Resonance, Biomolecular; Rats; Rats, Wistar

This study compares conversion of three major soy isoflavone glucosides and their aglycones in a series of in vitro intestinal models.. In an in vitro human digestion model isoflavone glucosides were not deconjugated, whereas studies in a Caco-2 transwell model confirmed that deconjugation is essential to facilitate transport across the intestinal barrier. Deconjugation was shown upon incubation of the isoflavone glucosides with rat as well as human intestinal S9. In incubations with rat intestinal S9 lactase phlorizin hydrolase, glucocerebrosidase, and cytosolic broad-specific β-glucosidase all contribute significantly to deconjugation, whereas in incubations with human intestinal S9 deconjugation appeared to occur mainly through the activity of broad-specific β-glucosidase. Species differences in glucuronidation and sulfation were limited and generally within an order of magnitude with 7-O-glucuronides being the major metabolites for all three isoflavone aglycones and the glucuronidation during first pass metabolism being more efficient in rats than in humans. Comparison of the catalytic efficiencies reveals that deconjugation is less efficient than conjugation confirming that aglycones are unlikely to enter the systemic circulation.. Altogether, the data point at possible differences in the characteristics for intestinal conversion of the major soy isoflavones between rat and human, especially with respect to their deconjugation.

Topics: Animals; Biological Availability; Biological Transport; Caco-2 Cells; Dietary Supplements; Digestion; Glucosides; Glycine max; Humans; In Vitro Techniques; Intestinal Mucosa; Isoflavones; Liver; Rats

In view of the important role of isoflavonoids and their related glycoconjugates in human health, there is considerable interest in their enzymatic conversion. SBGL, a novel β-glucosidase isolated from Novosphingobium sp. GX9, was expressed in Escherichia coli and found to have high activity for hydrolysis of daidzin and genistin. SBGL showed very low K m values for daidzin and genistin, and the k cat/K m values for these substrates were 33,300 and 19,200 s(-1) mM(-1), respectively. The SBGL glucosidase could also transglycosylate the flavanol (+)-catechin at saturating acceptor concentrations, which has not previously been reported for a β-glucosidase and is difficult to achieve synthetically.

Topics: Bacterial Proteins; beta-Glucosidase; Catechin; Cloning, Molecular; Escherichia coli; Gene Expression; Glycosylation; Hydrolysis; Isoflavones; Kinetics; Molecular Sequence Data; Sequence Analysis, DNA; Sphingomonadaceae

In this paper, the metabolites of four soy isoflavones, daidzein, daidzin, genistein, and genistin, on perfused rat intestine-liver model were investigated by high-performance liquid chromatography coupled with high-resolution mass spectrometer/tandem mass spectrometer. Totally 16 metabolites were detected and identified based on accurate mass, fragmentation patterns, and multiple-stage mass data (MS(n)). The metabolic site of dadzein-7-methyl ether (D-7-M) was further confirmed by nuclear magnetic resonance. Methylation, glucuronide conjugation, and sulfate conjugation were the primary metabolic processes. Among them, six metabolites, daidzin-4',7-diglucoside, genistein-4'-glucoside, D-7-M, dadzein-4',7-dimethyl ether, genistein-4'-methyl ether, and genistein-7-methyl ether were detected in rats for the first time and not reported in humans. The metabolic pathways of daidzein, daidzin genistein, and genistin in rats were postulated. The biological effects of these metabolites are worthy of further investigation.

Topics: Animals; Chromatography, High Pressure Liquid; Disease Models, Animal; Genistein; Humans; Isoflavones; Male; Molecular Structure; Rats

A novel isoflavone 7- O -glucosyltransferase PlUGT1 was isolated from Pueraria lobata . PlUGT1 could convert daidzein to daidzin, genistein to genistin as well as formononetin to ononin. Pueraria lobata roots are traditionally consumed as a rich source of isoflavone glycosides that have various human health benefits. However, to date, the genes encoding isoflavone UDP-glycosyltransferases (UGTs) have only been isolated from the roots of soybean seedlings (GmIF7GT), soybean seeds (UGT73F2) and Glycyrrhiza echinata cell suspension cultures (GeIF7GT). To investigate the isoflavone metabolism in P. lobata, 40 types of partial UGT cDNAs were isolated from P. lobata, and seven full-length UGT candidates with preferential expression in roots were identified. Functional assays in yeast (Saccharomyces cerevisiae) revealed that one of these UGT candidates, designated PlUGT1 (official UGT designation UGT88E12), efficiently glycosylated isoflavone aglycones at the 7-hydroxy group. Recombinant PlUGT1 purified from Escherichia coli cells was characterized and shown to be relatively specific for isoflavone aglycones, while flavonoid substrates were poorly accepted. The biochemical results suggested that PlUGT1 was an isoflavone 7-O-glucosyltransferase. The deduced amino acid sequence of PlUGT1 shared only 26 % identity with GeIF7GT, 27 % with UGT73F2 and 63 % with GmIF7GT. The PlUGT1 gene was highly expressed in P. lobata roots relative to other organs and strongly induced by methyl jasmonate signal in P. lobata cell suspension culture. The transcript abundance of PlUGT1 was correlated with the accumulation pattern of isoflavone glycosides such as daidzin in P. lobata plants or in cell suspension culture. The biochemical properties and gene expression profile supported the idea that PlUGT1 could play a role in isoflavone glycosylation in P. lobata.

Topics: Acetates; Cloning, Molecular; Cyclopentanes; Glucosides; Glucosyltransferases; Isoflavones; Molecular Sequence Data; Oxylipins; Phylogeny; Plant Proteins; Plant Roots; Pueraria

Genistin and daidzein exhibit a protective effect on DNA damage and inhibit cell proliferation. Glycosylation and malonylation of the compounds increase water solubility and stability. Constructed pET15b-GmIF7GT and pET28a-GmIF7MAT were used for the transformation of Escherichia coli and bioconversion of genistein and daidzein. To increase the availability of malonyl-CoA, a critical precursor of GmIF7MAT, genes for the acyl-CoA carboxylase α and β subunits (nfa9890 and nfa9940), biotin ligase (nfa9950), and acetyl-CoA synthetase (nfa3550) from Nocardia farcinia were also introduced. Thus, the isoflavonoids were glycosylated at position 7 by 7-O-glycosyltranferase and were further malonylated at position 6(″) of glucose by malonyl-CoA: isoflavone 7-O-glucoside-6(″)-O-malonyltransferase both from Glycine max. Engineered E. coli produced 175.7 µM (75.90 mg/L) of genistin and 14.2 µM (7.37 mg/L) genistin 6''-O-malonate. Similar conditions produced 162.2 µM (67.65 mg/L) daidzin and 12.4 µM (6.23 mg/L) daidzin 6''-O-malonate when 200 µM of each substrate was supplemented in the culture. Based on our findings, we speculate that isoflavonoids and their glycosides may prove useful as anticancer drugs with added advantage of increased solubility, stability and bioavailability.

Topics: Angiogenesis Inhibitors; Antineoplastic Agents; Escherichia coli; Genetic Engineering; Glucosides; Glycosylation; Isoflavones; Malonates; Malonyl Coenzyme A; Uridine Diphosphate Glucose

Retinoids have been used as therapeutics for diverse skin diseases, but their side effects limit clinical usage. Here, we report that extracts of two soybeans, Glycine max and Rhynchosia nulubilis, and their ethyl acetate fractions increased the transcriptional activity of retinoic acid receptors (RARs), and that daidzin and genistin were the major constituents of the active fractions. Daidzin and its aglycone, daidzein, induced transcriptional activity of RAR and RARγ. FRET analysis demonstrated that daidzein, but not daidzin, bound both RAR and RARγ with EC50 values of 28μM and 40μM, respectively. Daidzein increased expression of mRNA of RARγ through direct binding of RAR and recruitment of p300 to the RARγ2 promoter. Further, mRNA and gelatinolytic activity of matrix metalloproteinase-9 were decreased by daidzein in HaCaT cells. Together, these results indicate that daidzein functions as a ligand of RAR that could be a candidate therapeutic for skin diseases.

Topics: Binding Sites; Cell Line; E1A-Associated p300 Protein; Gene Expression; Glycine max; Growth Inhibitors; Humans; Isoflavones; Keratinocytes; Ligands; Matrix Metalloproteinase 9; Molecular Docking Simulation; Plant Extracts; Promoter Regions, Genetic; Protein Binding; Receptors, Retinoic Acid; Retinoic Acid Receptor gamma; RNA, Messenger; Transcriptional Activation

Paenibacillus xylanilyticus KJ-03 isolated from konjac field, showed β-glucosidase activity on tryptic soy agar plate supplemented with 0.1 % esculin and 0.25 % ferric ammonium citrate. A genome library was constructed to obtain the β-glucosidase gene and a recombinant clone, pGlc2-3 was selected. The 2,247 bp gene encoding KJ-03 β-glucosidase consisted of 749 amino acids. The deduced amino acids of BglA were 61 % homologous with that of the β-glucosidase from Bacillus cereus AH1272, which belongs to the glycoside hydrolase family 3. His-tagged β-glucosidase was purified by using His-Trap column and characterized. KJ-03 β-glucosidase was showed as a single band with about 82 kDa on SDS-PAGE. The purified enzyme has optimal activity at 20 °C and pH 7.0 using p-NPβG and 72 % of the maximal activity was still remaining at 10 °C. The β-glucosidase has optimal activity at low temperatures indicating that it is a cold-active enzyme. The substrate specificity showed that the purified enzyme hydrolyzed aryl β-glucoside substrates and isoflavones such as daidzin and genistin.

Topics: Amino Acid Sequence; Bacterial Proteins; beta-Glucosidase; Enzyme Stability; Isoflavones; Kinetics; Molecular Sequence Data; Paenibacillus; Recombinant Proteins; Sequence Alignment; Substrate Specificity

Isoflavone profiles of a fermented soy food, cheonggukjang, were modified using almond powder. Isoflavones were analyzed by high performance liquid chromatography (HPLC) with an ultraviolet detector. Malonyl derivatives of isoflavones decreased and aglycones of isoflavones increased in samples with almond powder for 48 h. As added, almond powder increased from 0%, 5%, and 10% (w/w), amounts of aglycones increased to 21.11%, 26.63%, and 32.45% for 48 h, respectively. β-Glucosidase activity in 5% and 10% almond added samples was significantly higher than samples without addition of almond (P < 0.05). The content of succinyl daidzin and succinyl genistin, new metabolites from isoflavones, in almond-added cheonggukjang was significantly lower than control samples, implying that β-glucosidase activity from almond affected negatively the formation of succinyl derivatives (P < 0.05). Principal component analysis (PCA) for isoflavone distribution showed that first principal component (PC1) and second principal component (PC2) expressed 64.78% and 22.26% of the data variability, respectively. Biotransformation of isoflavones in any fermented soy foods can be achieved using natural products containing high β-glucosidase activity such as almond.. The results of this study can help to modify the structural transformation of phytochemicals in any fermented soy foods using natural products. Adjusting the content of almond powder can achieve wanted profiles, for example, high aglycones content. Also, content of metabolites such as succinyl derivatives can be controlled using proper amounts of almond and fermentation time.

Topics: Bacillus subtilis; beta-Glucosidase; Chromatography, High Pressure Liquid; Diet; Fermentation; Functional Food; Glucosides; Isoflavones; Nuts; Plant Proteins; Principal Component Analysis; Prunus; Republic of Korea; Soy Foods; Time Factors

The recombinant β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus was purified with a specific activity of 330 U/mg for genistin by His-trap chromatography. The specific activity of the purified enzyme followed the order genistin > daidzin > glycitin> malonyl glycitin > malonyl daidzin > malonyl genistin. The hydrolytic activity for genistin was highest at pH 6.0 and 95 °C with a half-life of 59 h, a K(m) of 0.5 mM, and a k(cat) of 6050 1/s. The enzyme completely hydrolyzed 1.0 mM genistin, daidzin, and glycitin within 100, 140, and 180 min, respectively. The soybean flour extract at 7.5% (w/v) contained 1.0 mM genistin, 0.9 mM daidzin, and 0.3 mM glycitin. Genistin, daidzin, and glycitin in the soybean flour extract were completely hydrolyzed after 60, 75, and 120 min, respectively. Of the reported β-glucosidases, P. furiosusβ-glucosidase exhibited the highest thermostability, k(cat), k(cat)/K(m), yield, and productivity for hydrolyzing genistin. These results suggest that this enzyme may be useful for the industrial hydrolysis of isoflavone glycosides.

Topics: beta-Glucosidase; Enzyme Stability; Glycine max; Glycosides; Hot Temperature; Hydrolysis; Isoflavones; Kinetics; Pyrococcus furiosus; Recombinant Proteins; Substrate Specificity

In order to produce isoflavone aglycosides effectively, a process of isoflavone hydrolysis by Bacillus subtilis natto NTU-18 (BCRC 80390) was established. This process integrates the three stages for the production of isoflavone aglycosides in one single fermenter, including the growth of B. subtilis natto, production of β-glucosidase, deglycosylation of fed isoflavone glycosides. After 8 h of batch culture of B. subtilis natto NTU-18 in 2 L of soy medium, a total of 3 L of soy isoflavone glucoside solution containing 3.0 mg/mL of daidzin and 1.0 mg/mL of genistin was fed continuously over 34 h. The percentage deglycosylation of daidzin and genistin was 97.7% and 94.6%, respectively. The concentration of daidzein and genistein in the broth reached 1,066.8 μg/mL (4.2 mM) and 351 μg/mL (1.3 mM), respectively, and no residual daidzin or genistin was detected. The productivity of the bioconversion of daidzein and genistein over the 42 h of culture was 25.6 mg/L/h and 8.5 mg/L/h, respectively. This showed that this is an efficient bioconversion process for selective estrogen receptor modulator production.

Topics: Bacillus subtilis; Biotransformation; Culture Media; Fermentation; Isoflavones; Time Factors

Previous laboratory and animal studies reported that soy isoflavones were major bioactive compounds in soy to exert chemoprotection of prostate cancer. However, these studies cannot reflect the realistic effects that soy may induce through diets, and little is known about the bioavailability of isoflavones from whole soy food and their bioactivities after cooking and digestion. In this study, cooking and in vitro digestion were used to prepare soy extracts and the effects of cooking and digestion on the isoflavone contents and bioactivities of the whole soy extracts were examined. The cooking procedure generally increased the amount of daidzin, genistin and daidzein, but decreased that of genistein. Digestion process significantly lowered contents of daidzin and genistin in 60min cooked sample, while increased the contents of daidzin and daidzein and decreased the content of genistein in the uncooked sample. Antioxidant activities of soy extracts increased after cooking and in vitro digestion, while no consistent increase of the four soy isoflavones was determined. The apoptotic effects of soy extracts on both LNCaP and C4-2B cells were generally in a dose-dependent manner. Compared to purified single isoflavones, cooked and digested soy were more effective on induction of prostate cancer cell apoptosis, which indicated synergistic interactions between various bioactive compounds in the whole soy.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cooking; Digestion; Glycine max; Humans; Isoflavones; Male; Models, Biological; Plant Extracts; Prostatic Neoplasms

Phytooestrogens are known to cause anti-cancer effects on mamma carcinoma cells. In this study, the effects of the lignan secoisolariciresinol and the isoflavone glycosides and aglycones genistein, genistin, daidzein and daidzin were tested on MCF-7 and BT20 cells in vitro.. First, the cellular expression of hormone receptors was examined by immunohistochemical procedures. The effects of the phytooestrogens on the cells were detected by using three different assays measuring cell letality, viability and proliferation. The phytooestrogens were tested in concentrations of 1, 5, 10 and 50 μg/mL, respectively. 17β-oestradiol and tamoxifen were used as controls, respectively, in the same concentrations as the phytooestrogens.. The immunohistochemistry showed evidence of oestrogen- and progesterone receptors at the MCF-7 cell line, whereas no expression could be seen at the BT20 cells. Among the phytooestrogens, genistein and secoisolariciresinol showed various anti-cancerogenic effects on both cell lines, respectively, but only in the highest concentration. Regarding the controls, tamoxifen showed a stronger antivital and anti-proliferative effect on BT20 than on MCF-7. Oestradiol caused sporadic anti-cancer effects on both cell lines, respectively, at its highest concentration.. Genistein and Secoisolariciresinol have anti-cancer properties on MCF-7 and BT20 in vitro. There are differences in the effects of isoflavones depending on the glycolysation status. The role of the oestrogen receptors in the mechanisms of action of both the phytooestrogens and controls is of less importance. Further investigations have to be carried out, especially concerning the mechanisms of action. Phytooestrogens may be potential substances in the therapy of mamma carcinomas.

Topics: Breast Neoplasms; Butylene Glycols; Carcinoma; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Genistein; Humans; Immunohistochemistry; Isoflavones; L-Lactate Dehydrogenase; Lignans; Phytoestrogens; Receptors, Estrogen; Receptors, Progesterone

The purpose of this research was to design, test, and optimize a continuous microwave extraction method using temperature and residence time during and after microwave exposure as optimizing parameters for extraction of major isoflavones (genistin, genistein, daidzin, and daidzein) from soy flour. The extraction yield of four isoflavones at different heating temperatures (55 and 73 degrees C) and extraction times (0, 4, 8, 12, and 16 min) were investigated and compared with yields provided by a conventional solvent extraction method. The microwave prototype consisted of multiple, commercially available, batch-type, house-hold microwave units placed on top of each other in series to impart a continuous operation. The optimum parameters for microwave-assisted extraction of isoflavones were 73 degrees C for 8 min using a 3:1 ethanol-to soy flour ratio. At these parameters, the total yield of isoflavones extracted doubled, while the amount of oil extracted was 12%. Continuous microwave-assisted solvent extraction is a viable method for extraction of soybean isoflavones at relatively short residence times and high throughput.

Topics: Biotechnology; Equipment Design; Genistein; Glycine max; Isoflavones; Microwaves; Plant Oils; Temperature; Time Factors

Several dietary intervention studies examining the health effect of soy isoflavones allude to the importance of equol in establishing the cardiovascular response to soy protein. Although, the specific mechanism by which this action occurs has not been established. The aim of this study was to investigate the inhibitory effect of soy-isoflavones and the metabolite of daidzein, equol, on agonist-induced platelet responses dependent on thromboxane A(2) (TxA(2)) receptor.. Competitive radioligand binding assay was used to screen for affinity of these compounds to the TxA(2) receptor. The effect of equol on platelet activation, evaluate through of release of the ATP, by analogs of TxA(2) was analyzed. The effect of equol on platelet aggregation was investigated with ADP, U46619 (a TxA(2) mimic) and the calcium ionophore A23187.. The data showed that aglycone isoflavones and equol bind to TxA(2) receptor in the micromol/L range, whereas their glucoside derivates had very low binding activity for this receptor. Under equilibrium conditions, the following order of the relative affinity in inhibiting [(3)H]-SQ29585 binding was: equol>genistein>daidzein>glycitein>>genistin, daidzin, glycitin. Equol interaction was reversible and competitive for labeled-SQ29548 with not apparent decrease in the number of TxA(2) binding sites. In addition, from platelet activation studies, equol effectively inhibited ATP secretion elicited by the TxA(2) analog U46619. On the other hand, equol inhibited the platelet aggregation induced by U46619 and A23187, while it failed to inhibit that induced by ADP.. The aglycone isoflavones from soy, and particularly equol, have been found to have biological effects attributable to thromboxane A(2) receptor antagonism. These findings may help elucidate how dietary isoflavone modulate platelet function and explain why soy-rich foods are claimed to have beneficial effects in the prevention of thrombotic events.

Topics: Binding, Competitive; Bridged Bicyclo Compounds, Heterocyclic; Calcium; Equol; Fatty Acids, Unsaturated; Genistein; Humans; Hydrazines; Isoflavones; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Receptors, Thromboxane A2, Prostaglandin H2

This paper highlights, for the first time, the changes in the phenolics fraction (anthocyanins, flavonoids and stilbenes) and tocopherols of unpeeled Pistacia vera L. var. bianca with ripening, and the effect of the sun-drying process. The total polyphenol levels in pistachios, measured as mg of Gallic Acid Equivalent (GAE), were: 201 +/- 10.1, 349 +/- 18.3 and 184.7 +/- 6.2 mg GAE/100 g DM in unripe, ripe and dried ripe samples, respectively. Most phenolics in ripe pistachios were found to be anthocyanins. They increased with ripening, while the sun drying process caused a susbtantial loss. Flavonoids found in all pistachio samples were daidzein, genistein, daidzin, quercetin, eriodictyol, luteolin, genistin and naringenin, which decreased both with ripening and drying. Before the drying process both unripe and ripe pistachios showed a higher content of trans-resveratrol than dried ripe samples. gamma-Tocopherol was the major vitamin E isomer found in pistachios. The total content (of alpha- and gamma-tocopherols) decreased, both during ripening and during the drying process. These results suggested that unpeeled pistachios can be considered an important source of phenolics, particularly of anthocyanins. Moreover, in order to preserve these healthy characteristics, new and more efficient drying processes should be adopted.

Topics: Anthocyanins; Desiccation; Flavanones; Flavonoids; Isoflavones; Phenols; Pistacia; Polyphenols; Stilbenes; Sunlight; Tocopherols

To investigate the isoflavones from the vines of Pueraria lobata.. The compounds were isolated by column chromatography over silica gel and RP-C18, and purified by Sephadex LH-20 column chromatography and preparative TLC. The structures were elucidated on the basis of physico-chemical properties and spectral data.. Twelve compounds were isolated and identified as: 3'-methoxydaidzein (1), formononetin (2), genistein (3), daidzein (4), daidzin (5), genistin (6), ononin (7), 5-hydroxyl ononin (8), calycosin (9), 6"-O-acetyl genistein (10), 6"-O-acetyl daidzin (11), puerarin (12).. For the first time, compounds 9-11 were isolated from the genus Pueraria plant, and compounds 1, 3, 6-8 were obtained from the vines of this plant.

Topics: Genistein; Glucosides; Isoflavones; Magnetic Resonance Spectroscopy; Plant Stems; Pueraria

Soymilk was fermented with five isolates of probiotic lactic acid bacteria and in combination with probiotic yeast Saccharomyces boulardii. Nutritional profile like fat, protein, ash, pH, acidity, polyphenol, and protein hydrolysis were analyzed. Polyphenol content decreased from 265.88 to 119 microg/ml with different cultures. Protein hydrolysis ranged from 2.46 to 2.83 mmol l(-1) with different cultures. The antioxidant activity was assessed using different methods like 1, 1-diphenyl-2-picrylhydrazyl free radical-scavenging assay, inhibition of ascorbate autoxidation, and measurement of reducing activity. The activities varied with the starters used but, nevertheless, were significantly higher than those found in unfermented soymilk. Bioconversion of the isoflavone glucosides (daidzin + genistin) into their corresponding bioactive aglycones (daidzein + genistein) was observed during soymilk fermentation. Total glucosides in soyamilk were 26.35 mg/100 ml. In contrast, aglycones genistein and daidzein were quantitatively lesser accounting 2.91 mg/100 ml (genistein 1.17 mg/100 ml and daidzein 1.19 mg/100 ml). Soymilk fermented with probiotic cultures resulted in the reduction of glycosides ranging from 0.40 mg to 1.36 mg/100 ml and increase in aglycones ranging from 6.32 mg to 13.66 mg/100 ml.

Topics: Antioxidants; Ascorbate Oxidase; Chromatography, High Pressure Liquid; Fats; Fermentation; Flavonoids; Genistein; Hydrogen-Ion Concentration; Isoflavones; Lactobacillus; Nutritive Value; Phenols; Polyphenols; Probiotics; Saccharomyces; Soy Milk; Soybean Proteins

Effects of riboflavin photosensitization on the distribution of isoflavones in commercially available soymilk were analyzed using high-performance liquid chromatography (HPLC). Total isoflavones (TI) in soymilk with riboflavin (1000 ppm, w/v) under light were significantly different from those stored in the dark for 24 h (P < 0.05), while TI in samples with 0 and 1000 ppm added riboflavin were not significant from each other in dark conditions (P > 0.05). To test the effects of the concentration of riboflavin on TI, soymilk was mixed with riboflavin to make 0, 100, 250, and 500 ppm (w/v) and stored under light at 25 degrees C for 24 h. TI in soymilk with 100 ppm riboflavin under light significantly decreased by 13.5% for 24 h (P < 0.05) compared to control samples and were not significantly different from those with 250 or 500 ppm samples (P > 0.05). Daidzin and genistin were predominant isoflavones in soymilk, and the rate of photo degradation of genistin was higher than that of daidzin for 24 h in soymilk under riboflavin photosensitization.

Topics: Chromatography, High Pressure Liquid; Food Handling; Isoflavones; Light; Riboflavin; Soy Milk

There is evidence that metabolic activation can increase the estrogenic activity of the phytoestrogen-rich herb in tests with HepG2 cells. Variation in both plant genetics and harvest season may also influence estrogenic activity of the plant materials. We evaluated the influence of in vitro metabolic activation by S9 mixture on the estrogenic activity of tuberous samples of different cultivars of the phytoestrogen-rich herb, Pueraria mirifica, harvested in different seasons.. Plant extracts were derived from the tubers of five plant cultivars collected during summer, rainy season and winter and administered to MCF-7 cultures, an ERalpha-positive human mammary adenocarcinoma cell line for 3 days at dosages of 0.1, 1, 10, 100 and 1000microg/ml. These data were compared with the major plant isoflavonoids puerarin, daidzin, genistin, daidzein and genistein and with 17beta-estradiol, at concentrations of 10(-12) to 10(-6)M. The test system was done in the absence and presence of the S9 mixture.. The major plant isoflavonoids and the plant extracts exhibited variable degrees of estrogenic activities as evaluated by altered proliferation of the MCF-7 cell line which were significantly enhanced in the presence of the S9 mixture.. Metabolic activation of plant isoflavonoids at least in vitro by S9 mixture plays a significant role in amplification of the estrogenic activity of the phytoestrogen-rich plant. In addition, the estrogenic activities of the plant samples were potentially influenced by both seasonal changes and plant genetics.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Estradiol; Female; Genistein; Humans; Isoflavones; Phytoestrogens; Plant Extracts; Pueraria; Seasons

Complete separation of aglycones and glucosides of selected isoflavones (genistin, genistein, daidzin, daidzein, glycitin, glycitein, ononin, sissotrin, formononetin, and biochanin A) was possible in 1.5 min using an ultrahigh-pressure liquid chromatography (U-HPLC) on a different particular chemically modified stationary phases with a particle size under 2 microm. In addition, selected separation conditions for simultaneous determination of isoflavones together with a group of phenolic acids (gallic, protocatechuic, p-hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, ferulic, and sinapic acid) allowed separation of all 19 compounds in 1.9 min. Separations were conducted on a non-polar reversed phase (C(18)) and also on more polar phases with cyanopropyl or phenyl groups using a gradient elution with a mobile phase consisting of 0.3% aqueous acetic acid and methanol. Chromatographic peaks were characterised using parameters such as resolution, symmetry, selectivity, etc. Individual substances were identified and quantified using UV-vis diode array detector at wavelength 270 nm. Limits of detection (3S/N) were in the range 200-400 pg ml(-1). Proposed U-HPLC technique was used for separation of isoflavones and phenolic acids in samples of plant materials (Trifolium pratense, Glycine max, Pisum sativum and Ononis spinosa) after acid hydrolysis of the samples and modified Soxhlet extraction.

Topics: Caffeic Acids; Chromatography, High Pressure Liquid; Coumaric Acids; Gallic Acid; Genistein; Glycine max; Hydroxybenzoates; Isoflavones; Molecular Structure; Pisum sativum; Plant Extracts; Propionates; Trifolium; Vanillic Acid

Pueraria mirifica tubers collected from 28 out of 76 provinces of Thailand and Pueraria lobata tubers collected from Guangzhou province, China were submitted to HPLC analysis with the established gradient system comprising 1.5% acetic acid and acetonitrile. Five major isoflavonoids, including puerarin, daidzin, genistin, daidzein and genistein, were adopted as authentic standards. P. mirifica tubers showed intra- as well as inter-provincial differences in isoflavonoid and total isoflavonoid contents. The difference in both cases should be mostly influenced by genetic and environmental factors. In comparison with P. lobata, P. mirifica population exhibited differences only with a lower amount of daidzein.

Topics: Calibration; China; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Genistein; Isoflavones; Molecular Structure; Phytoestrogens; Plant Tubers; Pueraria; Quality Control; Seasons; Thailand

Pueraria mirifica is a tuberous plant enriched with active phytoestrogens. There is no established information about the factors influencing isoflavonoid storage in the tubers. We investigated the tuberous storage of the major isoflavonoids of 1-year-old plants. Four cultivars of P. mirifica were cultivated in the same field trial during the same period to establish a unique plant age and differentiation under the same environment and soil conditions. The tubers collected from the 1-year-old plants in the summer, rainy season and winter were submitted to an HPLC analysis with a gradient system comprising 0.1% acetic acid and acetonitrile. Five major isoflavonoids, puerarin, daidzin, genistin, daidzein and genistein, were adopted as standards. P. mirifica tubers of different cultivars collected in the same season exhibited significant differences in individual and total isoflavonoid contents, showing chemovariety. P. mirifica tubers of the same cultivar collected from different seasons also exhibited significant differences in individual and total isoflavonoid contents, showing the influence of season. In conclusion, the tuberous storage of major isoflavonoids in 1-year-cultivated plants was greatly diverse and was strongly influenced by the season and plant genetics.

Topics: Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Genistein; Isoflavones; Models, Biological; Molecular Structure; Phytoestrogens; Plant Tubers; Pueraria; Rain; Seasons; Time Factors

Hydrolysis of isoflavone glycosides by Bacillus subtilis natto NTU-18 in black soymilk is reported. At the concentration of 3-5% (w/v), black soymilk in flask cultures, the isoflavones, daidzin, and genistin were highly deglycosylated within 24 h. Deglycosylation of isoflavones was further carried out in a 7-l fermenter with 5% black soymilk. During the fermentation, viable cells increased from 10(3) to 10(9) CFU ml(-1) in 15 h, and the activity of beta-glucosidase appeared at 8 h after inoculation and reached a maximum (3.3 U/ml) at 12 h, then decreased rapidly. Deglycosylation of isoflavone glycosides was observed at the same period, the deglycosylation rate of daidzin and genistin at 24 h was 100 and 75%, respectively. It is significantly higher than the previous reports of fermentation with lactic acid bacteria. In accordance with the deglycosylation of isoflavone glycosides, the estrogenic activity of the 24 h fermented black soymilk for ERbeta estrogen receptor increased to threefold; meanwhile, the fermented broth activated ERalpha estrogen receptor to a less extent than ERbeta. These results suggest that this fermentation effectively hydrolyzed the glycosides from isoflavone in black soymilk and the fermented black soymilk has the potential to be applied to selective estrogen receptor modulator products.

Topics: Bacillus subtilis; beta-Glucosidase; Biotechnology; Chromatography, High Pressure Liquid; Estrogens; Fermentation; Glycine max; Glycosides; Glycosylation; Hydrolysis; Isoflavones; Soy Milk; Time Factors

A mini-hydroponic growing system was employed for seedlings of kudzu vine (Pueraria montana) and contents of isoflavones (daidzein, genistein, daidzin, genistin, and puerarin) from shoot and root parts of seedlings were analyzed quantitatively. In addition, exogenous cork pieces, polymeric adsorbent, XAD-4, and universal elicitor, methyl jasmonate (MeJA), were used to regulate the production of these isoflavones. It was shown that cork pieces up-regulate the production of daidzein and genistein up to seven- and eight-fold greater than the levels obtained for control roots. In contrast, levels of glucosyl conjugates, daidzin and genistin, decrease up to five- and eight-fold, respectively. Cork treatment also induces the excretion of the root isoflavone constituents into the growth medium. Minimal levels of isoflavones are absorbed by the cork pieces. XAD-4 stimulates the production of glucosyl conjugates, daidzin and genistin, in root parts about 1.5-fold greater than that obtained in control roots. These are the highest amounts of daidzin and genistin that are observed (5.101 and 6.759 mg g(-1) dry weight, respectively). In contrast to these two adsorbents, MeJA increases the accumulation of isoflavones in shoot rather than in root parts of seedlings, about three- to four-fold over control levels, with the exception of genistein. These studies reveal new observations on the regulation of isoflavone production in hydroponically grown Pueraria montana plants by two adsorbents (cork pieces and XAD-4) and MeJA elicitor.

Topics: Acetates; Cyclopentanes; Flavonoids; Genistein; Glycoconjugates; Hydroponics; Isoflavones; Oxylipins; Plant Roots; Plant Shoots; Polystyrenes; Polyvinyls; Pueraria; Seedlings; Wood

To examine the effect of daidzin, genistin, and glycitin on the osteogenic and adipogenic differentiation of bone marrow stromal cells (MSC) and the adipogenic transdifferentiation of osteoblasts.. MTT test, alkaline phosphatase (ALP) activity measurement, Oil Red O stain and measurement were employed.. Daidzin, genistin, and glycitin 1*10(-8), 5*10(-7), 1*10(-6), 5*10(-6), and 1*10(-5) mol/L all promoted the proliferation of primary mouse bone MSC and osteoblasts. Daidzin 5*10(-7) mol/L and genistin 1*10(-6) mol/L promoted the osteogenesis of MSC. Genistin 1*10(-8), 5*10(-7), 1*10(-6), 5*10(-6), and 1*10(-5) mol/L and glycitin 1*10(-8), 1*10(-6), and 1*10(-5) mol/L inhibited the adipogenesis of MSC. Daidzin, genistin, and glycitin 1*10(-8), 5*10(-7), 1*10(-6), 5*10(-6), and 1*10(-5) mol/L all inhibited the adipocytic transdifferentiation of osteoblasts.. Daidzin, genistin, and glycitin may modulate differentiation of MSC to cause a lineage shift toward the osteoblast and away from the adipocytes, and could inhibit adipocytic transdifferen-tiation of osteoblasts. They could also be helpful in preventing the development of osteonecrosis.

Topics: Adipocytes; Animals; Bone Marrow Cells; Cell Differentiation; Isoflavones; Mice; Osteoblasts; Osteogenesis; Phytoestrogens; Stromal Cells

A method for simultaneous detection and quantification is presented to determine the presence of isoflavones and bisphenol A in a biological sample. A coulometric array detector was used with reversed-phase high-performance liquid chromatography (HPLC). Daidzein (1), glycitein (2), genistein (3) and their glucoside conjugates, daidzin (4), glycitin (5) and genistin (6), were measured as phytochemicals. Also assayed here was equol (7), a metabolite from compound 1, and bisphenol A (8), an industrial chemical that acts as an endocrine disrupter. All chemicals were simultaneously detected by using a 600-mV single detection voltage with high efficacy. A mixture of 1, 3 and 8 was orally administered to rats, and the levels of these three chemicals in the serum were clearly increased after a 4 kU beta-glucuronidase treatment. The levels of compounds 1 and 3 in the serum were detected at 1665 and 2040 ng/ml, while 8 was at a low level of 417 ng/ml. Compound 7 in the serum was not detected until after enzymatic hydrolysis (72 ng/ml). These results suggest that this analytical method would be useful for metabolic and pharmacokinetic studies on isoflavones and bisphenol A.

Topics: Administration, Oral; Animals; Benzhydryl Compounds; Calibration; Chromatography, High Pressure Liquid; Colorimetry; Genistein; Glucuronidase; Isoflavones; Male; Phenols; Rats; Rats, Sprague-Dawley; Sensitivity and Specificity

The estrogenic isoflavones of soybeans and their glycosides are products of the shikimate pathway, the target pathway of glyphosate. This study tested the hypothesis that nonphytotoxic levels of glyphosate and other herbicides known to affect phenolic compound biosynthesis might influence levels of these nutraceutical compounds in glyphosate-resistant soybeans. The effects of glyphosate and other herbicides were determined on estrogenic isoflavones and shikimate in glyphosate-resistant soybeans from identical experiments conducted on different cultivars in Mississippi and Missouri. Four commonly used herbicide treatments were compared to a hand-weeded control. The herbicide treatments were (1) glyphosate at 1260 g/ha at 3 weeks after planting (WAP), followed by glyphosate at 840 g/ha at 6 WAP; (2) sulfentrazone at 168 g/ha plus chlorimuron at 34 g/ha applied preemergence (PRE), followed by glyphosate at 1260 g/ha at 6 WAP; (3) sulfentrazone at 168 g/ha plus chlorimuron at 34 g/ha applied PRE, followed by glyphosate at 1260 g/ha at full bloom; and (4) sulfentrazone at 168 g/ha plus chlorimuron at 34 g/ha applied PRE, followed by acifluorfen at 280 g/ha plus bentazon at 560 g/ha plus clethodim at 140 g/ha at 6 WAP. Soybeans were harvested at maturity, and seeds were analyzed for daidzein, daidzin, genistein, genistin, glycitin, glycitein, shikimate, glyphosate, and the glyphosate degradation product, aminomethylphosphonic acid (AMPA). There were no remarkable effects of any treatment on the contents of any of the biosynthetic compounds in soybean seed from either test site, indicating that early and later season applications of glyphosate have no effects on phytoestrogen levels in glyphosate-resistant soybeans. Glyphosate and AMPA residues were higher in seeds from treatment 3 than from the other two treatments in which glyphosate was used earlier. Intermediate levels were found in treatments 1 and 2. Low levels of glyphosate and AMPA were found in treatment 4 and a hand-weeded control, apparently due to herbicide drift.

Topics: Drug Resistance; Genistein; Glycine; Glycine max; Glyphosate; Herbicides; Isoflavones; Isoxazoles; Organophosphonates; Seeds; Shikimic Acid; Tetrazoles

Recently, dietary phyto-oestrogens (PO) have been suggested as possible alternatives to oestrogen therapy (hormone replacement therapy) as a means of preventing bone loss associated with ovarian hormone deficiency. While PO, which exhibit oestrogen-like activity, act directly on bone cells, their protective effect on bone may be partly due to their ability to enhance Ca absorption. Therefore, the aim of the present study was to investigate the effect of 17beta-oestradiol and two commonly consumed soyabean PO (genistein and daidzein) on Ca absorption in the human Caco-2 intestinal-like cell model. Caco-2 cells were seeded onto permeable filter supports and allowed to differentiate into monolayers. On day 21, the Caco-2 monolayers (n 8-18 per treatment), grown in oestrogen-replete or -deplete media, were then exposed to 10 nm-17beta-oestradiol, 1 nm-1,25-dihydroxycholecalciferol, or 50 micro m-genistein or -daidzein for 24 h. After exposure, transepithelial and transcellular transport of 45Ca and fluorescein transport (a marker of paracellular diffusion) were measured. As expected, 1,25-dihydroxycholecalciferol stimulated Ca absorption in Caco-2 cells, by up-regulating transcellular transport, whereas 17beta-oestradiol had no effect on Ca absorption. Unexpectedly, both PO decreased Ca absorption (by about 17-19 % compared with control, P<0.05), by reducing transcellular Ca transport in Caco-2 cells grown in oestrogen-replete media. This inhibitory effect disappeared when monolayers were grown in oestrogen-deplete media. In conclusion, PO at high luminal concentrations either had no effect or reduced Ca absorption in Caco-2 cells, dependent on oestrogen status. The effect of lower concentrations of these compounds needs to be investigated.

Topics: Analysis of Variance; Caco-2 Cells; Calbindins; Calcium; Depression, Chemical; Estradiol; Genistein; Glucosephosphate Dehydrogenase; Humans; Intestinal Absorption; Intestinal Mucosa; Isoflavones; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; S100 Calcium Binding Protein G

To study the methods of determining the amount of Isoflavones in red clover extractive.. RP-HPLC is employed to determine the Isoflavones, with a C18 RP Column, a moble phase of MeOH-CH3CN-0.1%H3PO4 and a detection wavelength of 260 nm.. After being water-solved, the four groups of flavoes elements satisfactorily separated, the amount of feeding in a range of 0.024-0.336 microg which has a good linear relationship with the peaks, and the total isoflavones determining results repetition, RSD=10.1%.. A simple, reliable and effective quality-control method is given.

Topics: Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Hydrolysis; Isoflavones; Plants, Medicinal; Quality Control; Trifolium

The soy isoflavones daidzin, glycitin, and genistin were purified from defatted soy flour using preparative-scale reverse-phase HPLC. The stabilities of the three isoflavones at different heating temperatures were investigated. Daidzin, glycitin, and genistin were lost at a rate of 26, 27, and 27% of their original concentration, respectively, after 3 min at 185 degrees C. At 215 degrees C, decreases of daidzin, glycitin, and genistin were 65, 98, and 74% after 3 min and 91, 99, and 94% after 15 min, respectively. The order of the thermal stabilities, from lowest to highest, was glycitin, genistin, and daidzin. Acetyl daidzin and acetyl genistin, daidzein, glycitein, and genistein were produced during heating at temperatures above 135 degrees C. The rate of binding of an acetyl group to form acetyl daidzin and acetyl genistin from daidzin and genistin was higher than the rate of loss of a glucoside group to form daidzein and genistein. However, acetyl daidzin and acetyl genistin decreased sharply at temperatures above 200 degrees C, while daidzein, glycitein, and genistein were relatively stable over 30 min. The stability of daidzein was higher than that of glycitein or genistein.

Topics: Acetylation; Chromatography, High Pressure Liquid; Genistein; Glycine max; Hot Temperature; Isoflavones; Kinetics

Peroxynitrite, a potent oxidant formed in vivo from the reaction of nitric oxide with superoxide, can mediate low-density liprotein (LDL) oxidation which is thought to increase the risk of atherosclerosis. This study investigates the inhibitory effect of the isoflavones, genistein and daidzein, together with their glycosidic forms, genistin and daidzin, on the peroxynitrite-mediated LDL oxidation and nitration of tyrosine. Genistein and daidzein were observed to dose-dependently inhibit peroxynitrite-mediated LDL oxidation, while their glucoside conjugates showed less activity. Moreover, all the isoflavones used in this study were found to be potent peroxynitrite scavengers, preventing the nitration of tyrosine. The ability of the isoflavones at 50 microM to decrease the tyrosine nitration induced by peroxynitrite (1 mM) was in the ratios of genistein (49%), daidzein (40%), daidzin (41%) and genistin (42%) when compared to the control (tyrosine incubated only with peroxynitrite). These results suggest that an intake of isoflavones could contribute to protecting against cardiovascular diseases and chronic inflammatory diseases.

Topics: Genistein; Humans; Isoflavones; Lipoproteins, LDL; Nitrates; Oxidants; Oxidation-Reduction; Peroxynitrous Acid; Tyrosine

Hormonally active chemicals (HACs) that are capable of inducing adverse effects on wildlife as well as human beings are featured as "endocrine disruptors". Various animal studies conducted to clarify the characteristics of HACs, including the uterotrophic assay, are sufficiently sensitive to detect the effect of 17-beta-estradiol in micrograms per kilogram of body weight or lower. In such systems, a trace amount of HACs in the dietary pellets may interfere with the test results and thus can be a serious problem for the low-dose issue, which is now a major topic in the field of endocrine disruptor research. Here, the significance of the hormonal effects of phytoestrogen components in the NIH-07 diet is confirmed and a NIH-07-based open formula "phytoestrogen-low diet" (PLD) is proposed, which effectively reduces uterine weight as well as the uterine luminal epithelial labeling index in ovariectomized rats.

Topics: Animals; Diet; Estrogens, Non-Steroidal; Female; Genistein; Isoflavones; Organ Size; Ovariectomy; Phytoestrogens; Plant Preparations; Rats; Uterus

Soy isoflavonoids have attracted much attention because of their estrogenic activity. To study the intestinal absorption of the isoflavonoids, we investigated the cellular uptake and metabolism of genistein and daidzein and their glucosides, genistin and daidzin, by Caco-2 cell monolayers as a model of the human intestinal epithelium. When Caco-2 monolayers were incubated with genistein or daidzein at 10 micromol/L from the apical (mucosal) side, aglycone was lost from the apical solution for 2.0 h (P < 0.05) and the glucuronide/sulfates appeared at the level of 1-2 micromol/L. In the basolateral (serosal) solution, both intact aglycones and their glucuronide/sulfates increased (P < 0.05) with time and reached approximately 20 and 15% of the initial dose, respectively. The transport of their glucosides, genistin and daidzin, through Caco-2 monolayers was less than one tenth that of the aglycones. The cellular metabolism of genistein was compared with quercetin, kaempferol, luteolin and apigenin. Only genistein aglycone was transported intact to the basolateral solution. Transport was greater (P < 0.05) than that of flavonoid aglycones and was without an appreciable decrease of transepithelial resistance. Radical scavenging activity was not related to the susceptibility to conjugation of flavonoids/isoflavonoids. Affinity to the liposomal membrane was increased in the order of genistin = daidzin < daidzein < genistein << flavonoid aglycones. These results strongly suggest that isoflavone aglycones are taken up into enterocytes more efficiently than their glucosides because of their moderate lipophilicity. Furthermore, they are generally transported to the basolateral side in intact form in contrast to flavonoids, probably due to their unique isoflavonoid structure.

Topics: Binding, Competitive; Caco-2 Cells; Cell Extracts; Electric Impedance; Flavonoids; Free Radical Scavengers; Genistein; Humans; Intestinal Mucosa; Intestine, Small; Intracellular Membranes; Isoflavones; Liposomes

The effects of the soy isoflavone glycoside, daidzin, genistin, and glycitin on bone loss and lipid metabolism in ovariectomized (ovx) rats were compared with those of estrone. Thirty-six 11-week-old female Sprague-Dawley rats were assigned to six groups, sham-operated, ovx, ovx+glycitin, ovx+daidzin, ovx+genistin, and ovx+estrone and fed matched amounts of a commercial calcium-deficient diet for 4 weeks. Throughout this period, daidzin, genistin or glycitin (25, 50 or 100 mg/kg/d) was given orally using a stomach tube, or estrone (7.5 microg/kg/d) was administered subcutaneously. Daidzin, genistin and glycitin significantly prevented bone loss in ovx rats at a dose of 50 mg/kg/d, like estrone. At this dose glycitin and daidzin also prevented ovx-induced uterine atrophy and increases in body weight gain, abdominal fat, serum total cholesterol and triglyceride, and urinary excretion of pyridinoline and deoxypyridinoline with statistical significance, like estrone. On the other hand, genistin prevented ovx-induced uterine atrophy only at a dose of 100 mg/kg, but did not block any other change of ovx rats at a dose of 50 or 100 mg/kg. These findings indicate that daidzin, glycitin, and genistin are effective in preventing bone loss and the former two compounds are effective in reversing the unfavorable changes of lipid metabolism in this model. It is suggested that the preventive effect of daidzin or glycitin on bone loss in ovx rats is due to suppression of bone turnover, as in the case of estrone, but genistin has a different mechanism of action from the other compounds. Soy isoflavone glycosides may represent a potential alternative therapy in the treatment of bone loss and lipid metabolism abnormality in ovarian hormone-deficient women.

Topics: Adipose Tissue; Animals; Atrophy; Calcium; Eating; Female; Glycine max; Isoflavones; Lipid Metabolism; Ovariectomy; Phosphorus; Rats; Rats, Sprague-Dawley; Uterus; Weight Gain

Commercial processing wastes or by-products of crops were found to be sources of antimutagens and human tumor cell growth suppressors. We developed a microplate method to measure genomic DNA damage in Chinese hamster ovary cells with a modified single cell gel electrophoresis (SCGE) assay. This allowed us to measure the repression of 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage by very small amounts of complex mixtures, fractions or individual chemicals isolated from agricultural by-products. We previously demonstrated that PCC, an ethanol extract of a commercial soybean processing by-product, repressed induced genomic DNA damage in mammalian cells. PCC was separated into a series of chemically defined fractions and two fractions (PCC70 and PCC100) repressed mutagen-induced damage. Of the isoflavones isolated from soybean fraction PCC70, daidzein expressed antigenotoxic activity, however, genistin and genistein enhanced DNA damage. An antigenotoxic response also was observed with a fraction isolated from corn distillate solids (CDS40). We developed a microplate assay to measure the suppression of the growth rate of human cancer cells in which the cytostatic/cytotoxic status at each concentration of the test sample was quantitatively determined. Genistein, genistin, daidzein and daidzin isolated from soybean fraction PCC70 expressed a wide range of growth suppression of HT-29 human colon cancer cells. The biological assays were integrated with, and directed, the separation and analytical chemistry component of this project. Compounds were purified from biologically active fractions and the structure of individual chemicals was determined with analytical HPLC and LC-mass spectroscopy (LC-MS). This research may lead to the isolation of novel chemoprotectants from agronomic commercial processing products and by-products.

Topics: Animals; Antimutagenic Agents; Antineoplastic Agents; Cell Division; Chemical Fractionation; CHO Cells; Chromatography, High Pressure Liquid; Colonic Neoplasms; Cricetinae; DNA Damage; Drug Screening Assays, Antitumor; Genistein; Glycine max; HT29 Cells; Humans; Isoflavones; Mass Spectrometry; Mutagenicity Tests; Plant Extracts; Zea mays

High intake of phytoestrogens through soybeans and their products is thought to be associated with low incidences of prostate and / or breast cancer in Asian countries. Possible chemopreventive effects of genistin or daidzin on rat prostate carcinogenesis were therefore investigated. Male F344 rats were given 10 biweekly subcutaneous injections of 3,2'-dimethyl-4-aminobiphenyl (DMAB) and then either genistin or daidzin in the diet at a concentration of 0.1% for 40 weeks. Other groups of rats given DMAB were treated with genistin or daidzin together with a high dose of testosterone propionate (TP). Both genistin and daidzin reduced the numbers of ventral prostate carcinomas (P < 0.05), with a tendency for decrease in incidence. Invasive carcinomas which developed in the anterior prostate and seminal vesicles with TP were, however, not influenced by the two isoflavones. Thus, the present data suggest that genistin and daidzin possess anti-cancer effects at relatively early stages of prostate cancer development, providing experimental support for epidemiological findings.

Topics: Aminobiphenyl Compounds; Animals; Cell Division; Enzyme Inhibitors; Estrogens, Non-Steroidal; Isoflavones; Male; Neoplasm Invasiveness; Organ Size; Prostatic Neoplasms; Rats; Rats, Inbred F344; Testosterone; Tumor Cells, Cultured

Since topoisomerase poisons allow the enzyme to cut and covalently bind to DNA but abort the subsequent rejoining of the molecule after relieving the torsional stress. To study their action we have made use of a supercoiled form of the pRYG plasmid that bears a specific topoisomerase recognition and binding region. The conversion of the supercoiled circular double-stranded DNA to the linear and open circle forms in the presence of a topoisomerase II poison and a denaturation step by proteinase K-SDS is indicative of the efficiency of our test agents to stabilize the cleavable complex. Using this system, three glucosylated isoflavones (6'-methoxy-pseudobaptigenin-7-O-beta-glucoside, genistin, and daidzin) isolated from cytotoxic chloroform and ethyl acetate extracts of Retama sphaerocarpa Boissier, were found to have the ability to stabilize the cleavage complex human DNA topoisomerase II.

Topics: DNA; DNA Topoisomerases, Type II; Drugs, Chinese Herbal; Electrophoresis, Agar Gel; Enzyme Inhibitors; Estrogens, Non-Steroidal; Glycosylation; Humans; Isoflavones; Models, Chemical; Nucleic Acid Conformation; Plant Extracts; Topoisomerase II Inhibitors

Fecal bacteria from a healthy individual were screened for the specific bacteria involved in the metabolism of dietary isoflavonoids. Two strains of bacteria capable of producing primary and secondary metabolites from the natural isoflavone glycosides daidzin and genistin were detected. The metabolites were identified by comparison of their HPLC/mass, 1H NMR and UV spectra with those of standard and synthetic compounds. Both Escherichia coli HGH21 and the gram-positive strain HGH6 converted daidzin and genistin to the their respective aglycones daidzein and genistein. Under anoxic conditions, strain HGH6 further metabolized the isoflavones daidzein and genistein to dihydrodaidzein and dihydrogenistein, respectively. The reduction of a double bond between C-2 and C-3 to a single bond was isoflavonoid-specific by strain HGH6, which did not reduce a similar bond in the flavonoids apigenin and chrysin. Strain HGH6 did not further metabolize dihydrodaidzein and dihydrogenistein. This is the first study in which specific colonic bacteria that are involved in the metabolism of daidzin and genistin have been detected.

Topics: Anaerobiosis; beta-Glucosidase; Biotransformation; Chromatography, High Pressure Liquid; Escherichia coli; Feces; Genistein; Gram-Positive Bacteria; Humans; Intestines; Isoflavones; Magnetic Resonance Spectroscopy; Mass Spectrometry; Oxidation-Reduction; Spectrophotometry, Ultraviolet

Topics: Environmental Monitoring; Environmental Pollutants; Estradiol Congeners; Estrogens, Non-Steroidal; Glycine max; Humans; Isoflavones; Tamoxifen; Vitellogenins; Xenobiotics

Absorption of isoflavone aglycones and glucosides was compared in rats. Daidzein, genistein, daidzin and genistin were orally administered at a dose of 7.9 micromol/kg in 25 mM Na2CO3 and next their metabolite concentration in blood plasma was monitored for 30 min. After isoflavone glucosides administration, their metabolites appeared in plasma with a few minutes delay as compared to aglycones, which suggested that aglycones, but not glucosides, were absorbed already in the rat stomach. This observation was confirmed when absorption site was restricted solely to the stomach and absorption was shown to be independent of the vehicle pH used for administration.

Topics: Absorption; Animals; Gastric Mucosa; Genistein; Glucosides; Hydrogen-Ion Concentration; Isoflavones; Male; Rats; Rats, Wistar

The mechanisms by which soybean- and soybean isoflavone-enriched diets inhibit carcinogenesis are not known. We found that the isoflavones genistin and daidzin, and their respective aglucone forms daidzein and genistein, block 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin)-induced CYP1A1 enzyme activity. This inhibition is correlated with the capacity of the isoflavones to prevent CYP1A1-mediated covalent binding of benzo[a]pyrene (BaP) metabolites to DNA. We further evaluated daidzein and genistein, believed to be the active forms of the isoflavones, for the mechanism of the inhibitory process. Although daidzein and genistein appear structurally similar to known aromatic hydrocarbon receptor (AHR) agonists and antagonists, gel mobility shift assays indicated that the isoflavones do not inhibit dioxin-induced activation of the AHR or the accumulation of CYP1A1 mRNA, suggesting that the isoflavones do not act at the transcriptional level. We therefore evaluated the isoflavones for direct effects on the CYP1A1 enzyme. Daidzein and genistein non-competitive with the CYP1A1 substrate BaP for microsomal BaP hydroxylation, with apparent Ki values of 325 microM and 140 microM, respectively. The extent of CYP1A1 inhibition increases with time of preincubation at 37 degrees C, but not at 4 degrees C, in the presence of isoflavone plus NADPH; after 60 min preincubation the inhibition remains non-competitive, with apparent Ki values of 55 microM and 50 microM, respectively. Inhibition is neither prevented nor reversed by the thiol antioxidant dithiothreitol, nor by the iron chelator deferoxamine. Repeated washing of the microsomes does not reverse the inhibition. The dependency on NADPH, temperature and time for inhibition of CYP1A1 suggests that metabolism of either isoflavone or molecular oxygen to reactive species is required. Isoflavone-mediated inhibition of CYP1A1 activity may contribute to the mechanism by which these soybean isoflavones protect against carcinogenesis.

Topics: Animals; Anticarcinogenic Agents; Cell Division; Cytochrome P-450 CYP1A1; Enzyme Activation; Enzyme Induction; Enzyme Inhibitors; Genistein; Glycine max; Isoflavones; Liver Neoplasms, Experimental; Mice; Transcription, Genetic; Tumor Cells, Cultured

A method for determining flavonoids in human plasma is presented for application to pharmacokinetic studies of two flavonoids, rhoifolin and daidzin. Isocratic reversed-phase high-performance liquid chromatography (HPLC) was used with genistin as an internal standard and solid-phase extraction using a Sep-Pak C18 cartridge. The mobile phases were acetonitrile-0.1 M ammonium acetate solution (20:80, v/v) for rhoifolin and methanol-0.1 M ammonium acetate solution (33:67, v/v) for daidzin. The detection limits on-column were 2 ng for rhoifolin and 0.5 ng for daidzin.

Topics: Acetates; Acetonitriles; Chromatography, High Pressure Liquid; Disaccharides; Flavonoids; Glycosides; Humans; Isoflavones

The oestrogenic activity of genistin and daidzin was investigated in the B6D2F1 strain of mouse. Diethylstilboestrol and genistein were used as positive controls. The oestrogenic response to 1.5 mg genistin was equivalent to that of 1 mg genistein, giving a 1:1 molar relationship in oestrogenic activity between genistin and genistein. The oestrogenic response to 3.8 mg daidzin was equivalent to that of 1 mg genistein. Analysis of the blood obtained from the mice in this experiment failed to reveal any free genistein or daidzein in the plasma.

Topics: Animals; Chromans; Diet; Equol; Estrogens; Female; Fertility; Flavonoids; Isoflavones; Mice; Mice, Inbred Strains; Organ Size; Uterus

The soya-bean isoflavones genistein and daidzein have been reported to exhibit oestrogenic activity in mice, but there is a significant difference in the response of different mouse strains to these compounds. The CD-1 mouse was found not to respond to these oestrogenic compounds from soya-beans or to diethystilboestrol at levels previously reported to be effective in other strains.

Topics: Animals; Diethylstilbestrol; Estrogens, Non-Steroidal; Female; Flavonoids; Genistein; Glycine max; Isoflavones; Mice; Mice, Inbred Strains; Organ Size; Uterus