Combination of low dose of genistein and daidzein has synergistic preventive effects on isogenic human prostate cancer cells when compared with individual soy isoflavone

The reduced incidence of prostate cancer (PCa) in Asia countries has been attributed to high soy diets, and major soy isoflavones, in particular daidzein and genistein, are thought to be the source of the beneficial and anti-cancer effects of soy foods. However, attention has been drawn to the safety of using high levels of soy isoflavones in humans, which is especially the concern for consumers taking regular soy isoflavone dietary supplements. The main objective of this study is thus to identify a soy isoflavone combination with lower levels of daidzein and genistein to be a more efficacious and safer chemo-preventive agent for PCa. The anticancer effects of daidzein and genistein, and their combinations on early-stage androgen-dependent PCa cells (LNCaP) and bone metastatic LNCaP-derivative PCa cells (C4-2B) were compared. Cells were treated with varying concentrations of daidzein, genistein (25–200 μM) or their combinations (25 or 50 μM) and cell proliferation, apoptosis, cell cycles and cellular uptakes of the isoflavones were measured after 48 h. Daidzein and genistein showed a synergistic effect on inhibiting cell proliferation and inducing apoptosis of both PCa cells. Twenty-five μM daidzein/50 μM genistein and 50 μM daidzein/50 μM genistein significantly increased the apoptotic effects on C4-2B cells although they did not show any effect when used individually. Except 50 μM daidzein/50 μM genistein, all other combinations had no impacts on cell cycles. For treatment with soy isoflavone combination, genistein was always better taken up than daidzein by both LNCaP and C4-2B cells.     

基于异黄酮类标志物的餐厨废弃油脂掺伪食用植物油鉴别

通过分析废弃油脂来源及流通,以大豆油异黄酮类标志物为切入点,利用磁固相萃取液相色谱串联质谱分析法探究了异黄酮类标志物的热稳定性以及大豆异黄酮在食用植物油和餐厨废弃油脂中的分布。模拟反复加热实验结果表明,标志物经过14h持续加热,依然可以检出,热稳定性相对良好。对芝麻油、菜籽油、茶籽油、花生油、亚麻籽油、大豆油及餐厨废弃油脂中的异黄酮标志物含量测定分析后作聚类分析图,结果表明通过聚类分析可以将六种食用植物油以及废弃油脂区分开。大豆油中同时含有四种大豆异黄酮,而餐厨废弃油脂中含有黄豆苷元和染料木素,以及少量染料木苷,不含黄豆苷。其他食用植物油不含有或者不同时含有大豆异黄酮类化合物,利用含量关系可以将餐厨废弃油脂与其他食用油区分开。因此,黄豆苷元、染料木素可以作为餐厨废弃油脂标志物。以芝麻油为例,掺伪5%餐厨废弃油脂的芝麻油的色谱图中可明显观察到黄豆苷元、染料木素特征峰,表明该方法可靠有效,可以为餐厨废弃油脂检测和市场监管提供参考依据。     

Determination of the absolute configurations of 4-mercapto-2-alkanones using the <Superscript>1</Superscript>H NMR anisotropy method and enzyme-catalyzed kinetic resolution of the corresponding 4-acetylthio-2-alkanones

To determine the capillary gas chromatographic (GC) elution order of the enantiomers of 4-mercapto-2-alkanones, racemic 4-acetylthio-2-alkanones were synthesized and hydrolyzed by lipases to get enantio-enriched 4-mercapto-2-alkanones that were resolved by capillary gas chromatography using a chiral stationary phase. The obtained enantio-enriched 4-mercapto-2-alkanones were esterified with (S)-2-methoxy-2-(1-naphthyl)propionic acid ((S)-MαNP acid), and the HPLC analysis of MαNP thioesters revealed the same isomeric ratios as the chiral GC analysis of the corresponding thiol enantiomers. The diastereoisomeric thioesters obtained were resolved by HPLC, and the absolute configurations were determined on the basis of ¹H NMR anisotropy effects. On the basis of these results, the GC elution order of the enantiomers of 4-mercapto-2-alkanones and of the corresponding 4-acetylthio-2-alkanones could be determined. The combination of enzymatic resolution and MαNP thioesterification proved to be a useful tool to determine the absolute configuration of secondary thiols.     

大豆异黄酮的提取及其生物学效能的研究

研究大豆异黄酮化合物对各试验组动物血脂、血粘度、凝血时间、血小板聚集率、脂质过氧化物及载脂蛋白水平的作用。采用多种溶剂浸提、分离、纯化法从大豆中提取大豆异黄酮 (SI)的 4个单位Daidzin、Genistin、Genistein、Daidzein ,并经薄层色谱和高效液相色谱与标准品比较鉴定后 ,分别作用于高血脂动物实验中。结果发现大豆异黄酮与高脂试验组相比具有明显地降低高脂血症小白鼠的血清TC ,TG ,LDL -C ,升高HDL -C(P <0 .0 1)的作用 ,血桨粘度、PtA和HCT降低 (P <0 .0 5 ) ,CgT延长 ,血SOD增加 ,LPO减少 (P <0 .0 1)。试验证明大豆异黄酮具有确切地调整血脂及抗脂质过氧化的作用 ,具有临床应用价值。     

Antioxidant properties of aqueous and methanol soy extracts in minced trout muscle

Antioxidant capacity of freeze-dried aqueous and methanolic extracts of soy protein isolate (SPI) and soybean meal (SBM) were assessed by 2, 2′-Diphenyl-1-picrylhydrazyl (DPPH) assay and total phenolic content (TPC). Caffeic and chlorogenic acids in liquid aqueous extracts, and genistein and daidzein in liquid methanolic extracts were measured by HPLC. Freeze-dried extracts were applied (1000 and 4000 μg/g) to trout mince and TBARS formation monitored during 14 days refrigerated storage. SBM aqueous extracts-treated mince (4000 μg/g) had significantly lower (P < 0.05) TBARS than all other treatments. SPI methanolic extract had the highest TPC (100 mg chlorogenic acid equivalents/100 g extract) and genistein and daidzein contents. SPI aqueous extracts had the highest chlorogenic acid (280 μg/g) while SBM aqueous extracts had the highest caffeic acids content (484 μg/g). SPI had the best in vivo antioxidant profile. SBM extracts were most effective at retarding lipid oxidation in refrigerated trout mince.     

Distribution of isoflavones and coumestrol in neglected tropical and subtropical legumes

BACKGROUND: Isoflavones and coumestrol from dietary legumes are plant constituents showing multiple beneficial effects on humans. Owing to their ability to bind with mammalian estrogenic receptors and thereby intervention in several kinds of hormone‐related cancers, they have received much attention. Soybean (Glycine max) is currently the major source of isoflavonoids in human diet. However, dozens of tropical and subtropical leguminous species remain unexplored for their isoflavonoids content. RESULTS: We have analyzed 55 extracts from 41 tropical and subtropical legume species used either in human or animal diet by high‐performance liquid chromatography for the content of soy isoflavones, biochanin A, daidzein, daidzin, formononetin, genistein, genistin, sissotrin, ononin and the coumestan coumestrol. Genistein and biochanin A were the most abundant compounds. The highest content of genistein was found in aerial parts of Andira macrothyrsa, seeds of Pachyrhizus tuberosus and aerial parts of Calopogonium mucunoides (598, 250 and 184 µg g^?1, respectively) and biochanin A in aerial parts of Cratylia argentea, C. mucunoides and flowers of A. macrothyrsa (76, 53 and 40 µg g^?1, respectively). CONCLUSION: None of the samples tested was richer overall source of soy isoflavones and coumestrol than soybean; nevertheless several species (C. mucunoides or A. macrothyrsa) may serve as a promising source of individual compounds. © 2012 Society of Chemical Industry     

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

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

Urinary isoflavone excretion in Korean adults: comparisons of fermented soybean paste and unfermented soy flour

BACKGROUND: This study compared the 24 h urinary excretion of isoflavone algycones after the consumption of fermented soybean paste or unfermented soy flour in a population of healthy Koreans (four males and five females). An equivalent amount (20 mg) of total isoflavone was consumed as either 52 g of soybean paste or 12.2 g of soy flour in a randomised, crossover trial consisting of two single‐time‐point feedings and having 3 day run‐in and washout periods between feedings. RESULTS: Analysis of starting materials indicated that daidzein (1.4%) and genistein (1.4%) constituted a smaller proportion of the total isoflavones in soy flour as compared with soybean paste (55.3% daidzein and 45.7% genistein). Free and glycosidic forms of isoflavones differed significantly between the two soy products. Levels of β‐glycosides as well as total glycosides (β‐glycosides, malonyl and acetyl forms) were two‐fold higher in soy flour as compared with soybean paste. Malonyl forms predominated in soy flour. CONCLUSIONS: The consumption of soybean paste resulted in significantly higher urinary excretion of both daidzein and genistein as compared with soy flour (P < 0.05 and P < 0.01 respectively). Copyright © 2007 Society of Chemical Industry     

HPLC with coulometric electrode array detection Determination of daidzein and genistein in soy based infant food, soy milk and soy based supplements

A HPLC method coupled with coulometric electrode array detection for determination of daidzein and genistein in soy milk, soy-based infant food and nutritional supplements is described. The samples were spiked with bisphenol A (internal standard), extracted and hydrolyzed in an acid ethanolic solution. After purification of the extract on a C-18 cartridge the compounds were separated on a C-18 reversed phase column (50?mM sodium acetate adapted to pH?4.8 with glacial acetic acid/methanol at 450/550, v/v). The compounds were detected at eight electrodes set on potentials between +390?mV and +810?mV (in increments of 60?mV) against modified palladium electrodes. The contents of daidzein and genistein were determined in several commercial samples.     

High levels of equol in organic skimmed Finnish cow milk

The isoflavonoids, equol, formononetin, daidzein, genistein, biochanin A, and O-demethylangolensin (O-DMA), were analyzed from commercial cartons of skimmed Finnish milk by HPLC-diode array detector (DAD)-FL. We found 411 +/- 65 ng/mL of equol and traces of formononetin and daidzein in organic skimmed milk whereas conventionally produced milk contained 62 +/- 16 ng/mL of equol and no formononetin or daidzein.     

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

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

Studies on flavonol degradation by peroxidase (donor: H2O2-oxidoreductase,EC 1.11.1.7): Part 2—Quercetin

The peroxidatic transformation of quercetin was studied at pH 5·5 under UV-spectrophotometric control. The reaction products obtained after 3 minutes' incubation of quercetin (dissolved in ethylene glycol monomethyl ether) with peroxidase (Boehringer, Mannheim)/H₂O₂ were fractionated on preparative scale by Sephadex LH-20 liquid chromatography using methanol as solvent. More than twenty compounds could be detected by analytical HPLC, from which the following substances were characterized by their spectroscopic data (UV; IR; ¹H- and, in part, ¹³C-NMR; EI- and, in part, FAB- and FD-MS): 2,4,6-trihydroxybenzoic acid (Q1); 3,4-dihydroxybenzoic acid (Q2); methyl 2,4,6-trihydroxyphenyl-glyoxylate (Q3); 2-(3,4-dihydroxyphenyl)-2-hydro-3,5,7-trihydroxy-3-methoxy-4H-1-benzopyran-4-one (‘quercetinchinone’) (Q5); 2,3-epoxy-2-(3,4-dihydroxyphenyl)-3-[4O-[2-(3-hydroxyphenyl)-3,5,7-]trihydroxy-4H-1-benzopyran-4-onyl]-5,7-dihydroxy-4H-1-benzopyran-4-one (2 isomers) (Q7) and a trimer (Q8) structurally related to Q7.     

Determination of Major Isoflavones in Soy Drinks by Solid-Phase Micro Extraction Coupled to Liquid Chromatography

A simple method for determination of major isoflavones (daidzein, genestein, genistin) and equol, a daidzein metabolite, has been developed using solid-phase micro extraction coupled to liquid chromatography with diode array UV detection. All the aspects influencing adsorption (fibre coating, extraction time, temperature, pH and salt addition) and desorption of the analytes (desorption and injection time and minimize carryover) were carefully investigated. The limits of detection obtained using a polydimethylsiloxane /divinylbenzene fiber coating were in the range 4 (genistein) to 38 (genistin) nM. The method proved to be useful for determination of analytes in soy-based products. Daidzein, genistein, and genistin were found in all samples (three soy milks and two blended rice/soy beverages) at concentrations ranging from 0.8?±?0.2 (daidzein) to 180.4?±?12.1 (genistin) μM, while glycitein and equol were found in three and in one of the drinks at levels between 1.7?±?0.1 and 5.0?±?0.2 μM and at 2.7?±?0.2 μM, respectively. The procedure was used to define the binding constants of isoflavones with serum albumin bovine that were found from 2.5?×?10⁴ (daidzein) to 2.9?×?10⁵ (genistein) L mol^?1. The method proposed restricts the use of organic solvents and can be used widely in most laboratories with standard equipment. In addition, it is suitable for easily studying the isoflavone interaction with the main constituents in plants, e.g., proteins and lipids, to evaluate their bioavailability and to clarify the controversial question on benefits for or damage to human health resulting from soy intake.     

Determination of isoflavones in commercial soy products by HPLC and coulometric electrode array detection

The concentrations of the isoflavones daidzein, genistein and glycitein were determined in a large number of commercially-available soy products by high-performance liquid chromatography with coulometric electrode array detection using estriol as internal standard.During extraction, the naturally occurring glycosides were converted into their respective aglycones by acid hydrolysis. The analytes were separated on a C-18 reversed phase column, eluted with methanol/acetonitrile/50 mM sodium acetate pH 4.8 (40/5/55, v/v/v), and detected by a coulometric electrode array detector using twelve electrodes set to potentials between +250 and +800 mV (in increments of 50 mV) against palladium reference electrodes.The isoflavones could be determined over a wide concentration range (0.8–1135.0 mg/kg for daidzein, 1.9–1442.5 mg/kg for genistein and 0.5–154.6 mg/kg for glycitein). The recovery of the isoflavones in the different matrices was determined by the standard addition method and varied between 40.9–94.4%. The detection limits (S/N=3) depend on the matrix of the soy product and were found for daidzein between 0.3–1.6 mg/kg, for genistein between 0.3–1.7 mg/kg and for glycitein between 0.5–2.3 mg/kg.     

Effects of riboflavin photosensitization on daidzein and its photosensitized derivatives

Photosensitized compounds from daidzein were studied in a riboflavin model system under visible light irradiation by high-performance liquid chromatography (HPLC). As the period of light irradiation increased, concentration of daidzein decreased significantly (P < 0.05) and new peaks of daidzein derivatives were observed and changed during photosensitization. Three new peaks from photosensitized daidzein were tentatively identified as 7-, 3', 4'-trihydroxyisoflavone (or 3'-hydroxydaidzein) and 2 dimmers of daidzein by a combination of HPLC-mass spectrometry (MS) and retention times of standard compounds by HPLC. Addition of sodium azide and removal of headspace oxygen treatment affected the formation of newly formed peaks. The type I pathway of riboflavin photosensitization played more important roles than type II pathways on the formation of daidzein derivatives. Practical Application: Isoflavones are important phytochemicals found in soy foods. Generally, many foods containing soy ingredients are displayed under visible light irradiation. Also, riboflavin can be found in many foods containing vegetables. The results of this study can be used to understand the stability and changes of isoflavone aglycones in soy and soy-based foods under visible light irradiation.     

Enhanced technological and permeation properties of a microencapsulated soy isoflavones extract

Functional extracts from soybean have numerous health benefits deriving from their content in isoflavones, especially daidzein and genistein, natural antioxidant and estrogen-like (ES-like) molecules. However, soybeans denaturated dried extracts possess critical properties for industrial applications such as poor water-solubility and inadequate storage stability. The present research was aimed at developing a new sodium-carboxymethylcellulose (NaCMC) matrix by spray-drying to microencapsulate soy extracts obtaining enhanced technological and permeation properties. Microparticles (NaCMC-Iso) loaded with marketed soybeans extract (Iso), enriched in daidzein and genistein (about 90% w/w), were produced. Different liquid feeds, based on H₂O/EtOH/acetone co-solvent systems, and various process parameters were tested to obtain a free-flowing and manageable powder with satisfying yield. The engineered NaCMC-Iso powders showed satisfying loading efficiency, high flow properties, good stability and enhanced technological properties. Interestingly, the in vitro dissolution and permeation rates were dramatically improved suggesting a higher bioavailability after the oral administration. The made-up powder appears a newsworthy ingredient of easier manipulation for enclosing in dietary supplements and nutraceutical products.     

Studies on flavonol degradation by peroxidase (donor: H2O2-oxidoreductase,EC 1.11.1.7): Part 1—Kaempferol

The peroxidatic degradation of kaempferol was studied at pH 5·5 under UV-spectrophotometric control. The reaction products obtained after incubation of kaempferol (dissolved in ethylene glycol monomethylether) with peroxidase (Boehringer, Mannheim)/H₂O₂ were fractionated in preparative scale by liquid chromatography on Sephadex LH-20. About twenty compounds were detected by analytical HPLC, from which four substances could be characterized by means of their spectroscopic data: 2,4,6-trihydroxybenzoic acid, 4-hydroxybenzoic acid, 2,2-dihydroxy-1(2,4,6-trihydroxyphenyl)-3(4-hydroxyphenyl)-1,3-propandione and 2-(4-methoxyphenyl)-5,7-dihydroxy-4H-1-benzopyran-4-one (acacetin).     

A new ultra-high pressure liquid chromatography method for the determination of total isoflavone aglycones after enzymatic hydrolysis: Application to analyze isoflavone levels in soybean cultivars

Since only isoflavone aglycones are considered to be bioactive, the determination of total aglycones that are released from conjugated isoflavones after hydrolytic treatment may facilitate an objective alternative for quantifying isoflavone contents in soy products. Given this major benefit, a new ultra-high pressure liquid chromatography (UV-UPLC?) method was developed for the fast and reliable determination of total aglycones in soybeans (daidzein, glycitein, and genistein) after enzymatic hydrolysis applying helix pomatia digestive juice. Capitalizing on the enhanced performance of UPLC?, aglycones were separated within 3 min only, with a total runtime of 8 min till the next injection. Thus, especially compared to HPLC protocols, UPLC? proved to be superior due to significantly shorter runtimes and accordingly increasing sample throughput. Additionally, regarding the performed validation (linearity, precision, recovery, selectivity, and robustness), the established method proved to be suitable for quantifying total aglycones in soybeans. Moreover, method applicability was demonstrated by analyzing 23 commercial soybean cultivars for their isoflavone contents. Cumulative aglycone levels ranged from 100 to 255 mg per 100 g, hence implying an average ratio of 52%, 41%, and 7% of total isoflavones for genistein, daidzein and glycitein, respectively. However, for some soybeans, other distinct aglycone distributions were observed as well.     

Production of soy yogurt enriched with glyceollins

Soy milk was prepared from regular soybean (M1), soybean germinated for 3 days at 25°C (M2), and soybean germinated under fungal infection (M3). Rhizopus microsporus var. oligosporus ATCC 22959 was used as the elicitor for glyceollin production. Each soy milk was fermented with Streptococcus infantarius 12 and Weissella sp. 4 (1:5, v/v) for 12 h at 37°C. Significant induction of glyceollins was confirmed only in M3 soy milk and glyceollins maintained stably during 12 h fermentation period. The concentration of glyceollins in M3 yogurt was 2,400.4±83 and 2,525.2±158 μg/g dry matter (d.m.) at 0 and 12 h, respectively. The amount of daidzein was significantly higher in M3 yogurt (635.1±21) than that of M1 (417±11) and M2 (545±17 μg/g d.m.) yogurt in 12 h (p<0.05). M2 yogurt had the highest amount of genistein (695.3±17) followed by M3 (634.5±26) and M1 (612.5±14 μg/g d.m.) yogurt. M3 soy yogurt also showed the highest content of total phenolic compounds (5.37 mg/g) and antioxidant activity. The results indicated that functional soy yogurt can be prepared from soybean enriched with glyceollins.