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.     

Determination of some phenolic compounds in flax seed and nettle roots by HPLC with coulometric electrode array detection

A method for determination of secoisolariciresinol, homovanillyl alcohol, p-coumaric acid, and ferulic acid in flax seed and nettle roots was developed. At first the naturally occurring esters and glycosides were cleaved by alkaline methanolysis (sodium methoxide) followed by enzymatic hydrolysis (cellulase) to get the aglycones. Then the compounds were isocratically separated (60 mM sodium acetate/ethanol/acetonitrile/glacial acetic acid =81:9:9:1, v:v:v:v, flow rate: 0.8 ml/min.) on a Spherisorb S-ODS 1 column (250 mm x 4.6 mm, i.d, particle size: 5 µm) using 7-hydroxy-4-methylcoumarin as internal standard and detected at eight electrodes in a coulometric electrode array detector. The potentials of the 1st to the 5th electrode were set to +150 to +350 mV ( in increments of 50 mV), the potentials of the 6th to the 8th electrode were adapted from +550 to +750 mV (in increments of 100 mV) against modified palladium reference electrodes. In six flax seed samples secoisolariciresinol was found in the range from 5,000 to 10,000 µg/g and the content of p-coumaric and ferulic acid varied between 100 and 450 µg/g. In nettle roots the content of the lignan is about 10 times lower than in flax seed.     

Phenolic compounds in tomatoes. Natural variations and effect of gamma-irradiation

A method for determination of p-hydroxybenzaldehyde, p-coumaric acid, ferulic acid, rutin and naringenin in tomatoes based on HPLC with coulometric electrode array detection was developed. After lyophilisation and extraction the compounds were separated isocratically (acetonitrile/buffer: 14:86, v:v, or acetonitrile/buffer: 27:73, v:v, flow rate: 0.8 ml/min) on reversed phase columns (LiChrospher 60 RP select B or Spherisorb ODS2) and detected at 16 respectively 10 working electrodes set at potentials between +50 and +750 mV against palladium reference electrodes. The natural content of these components showed a strong variation between different varieties, harvests and degrees of maturity. Gamma-irradiation reduced the concentration of the phenolic compounds markedly in all tomato samples investigated, however, this change was smaller than the naturally occurring differences. The irradiation products vanillin and eriodictyol, identified in standard solutions, could not be found in irradiated tomatoes.     

高效液相色谱法检测大豆粕大豆黄素及染料木素含量

该文介绍高效液相色谱法检测大豆粕提取的大豆异黄酮,液相操作条件为:甲醇-水-乙酸梯度洗脱,检测波长260 nm。大豆黄素,染料木素在0.1mg-0.5 mg/ml范围内与峰面积有很好线性关系(r=0.998-0.999)。平均回收率为98.1％-98.4％,6次进样相对标准差(RSD)=1.41％,1.27％。该法测定样品,快速准确,重复性好。     

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.     

UPLC法测定大豆制品及相关制剂中大豆异黄酮含量

目的：建立同时测定大豆制品及相关制剂中4种大豆异黄酮类化合物大豆苷、染料木苷、大豆苷元、染料木素含量的超高效液相色谱法。方法：色谱柱：Acquity UPLC BEH C18柱(50mm×2.1mm,1.7μm),流动相：A为体积分数0.2%甲酸溶液,B为乙腈,梯度洗脱;流速：0.4mL/min;检测波长：254nm,柱温：30℃。结果：线性范围：大豆苷0.625～40.0mg/L(r=1.000),染料木苷0.625～40.0mg/L(r=0.9999),大豆苷元0.04～2.56mg/L(r=0.9997),染料木素0.04～2.56mg/L(r=0.9996)。大豆和Natrol? soy isoflavones中大豆苷、染料木苷、大豆苷元、染料木素平均回收率为98.5%～99.8%。结论：本方法简便、快速、灵敏、准确,适用于大豆制品及相关制剂中大豆异黄酮含量测定。     

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.     

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.     

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.     

酱油中异黄酮物质的分离与鉴定

本采用真空肖缩干燥酱油、乙酸乙酯热回流抽取酱油中黄酮物质,并使用薄层层析法进行纯化,分离。     

热烫处理对大豆中异黄酮苷原稳定性影响的研究

研究了热烫处理下大豆中黄豆苷原和染料木黄酮等2种异黄酮苷原的热稳定性,并对其进行降解动力学分析。结果表明,染料木黄酮在热处理的前13min内损失33.73%,遵循一级反应动力学关系;而黄豆苷原的热降解则在9～13min和45～60min内遵循一级反应动力学关系,损失率分别为22.56%和9.08%。     

Bioavailability of soy isoflavones in rats Part I: application of accurate methodology for studying the effects of gender and source of isoflavones

There are limited and controversial reports about the effects of gender and source of isoflavones on their bioavailability. Moreover, several previous studies have not used appropriate methodology to determine the bioavailability of soy isoflavones, which requires comparing the area under the plasma concentration-time curve after both oral and intravenous injection (IV) administration. Therefore, the present study was conducted to determine the bioavailability of isoflavones from different sources following both oral and IV administration in male and female rats. Three sources of isoflavones; Novasoy (a commercial supplement), a mixture of synthetic aglycones (daidzein, genistein and glycitein) and a mixture of synthetic glucosides (daidzin, genistin and glycitin) were tested. Following administration, blood samples were collected at several time points (0, 10, 30 min and 1, 2, 8, 24, 48 h post oral gavage and 0, 10, 30, 45 min and 1, 2, 3, 4, 8 h post-IV dosing) and plasma isoflavones were measured by LC/MS. Bioavailability values for daidzein, genistein and glycitein were significantly (p <0.05) higher (up to sevenfold) in Novasoy and the glucoside forms of isoflavones compared with those of the aglycone forms. Moreover, significant (p <0.05) gender differences in the bioavailability of 7-hydroxyl-3-(4'-hydroxyphenyl)-chroman (a metabolite of daidzein), glycitein and daidzein were observed for Novasoy, with higher values in male rats. In summary, the source of isoflavones and the sex of rats had significant effects on isoflavone bioavailability.     

Direct and indirect photolysis of the phytoestrogens genistein and daidzein

Genistein and daidzein are two estrogenic compounds derived from plants, especially legumes. This research begins to explore their environmental fate, focusing on direct and indirect photolysis. UV-visible spectra for both compounds at varying pH values were taken, the pK(a) values for both compounds were measured, and UV-visible spectra for each protonation state were determined. The loss of both compounds in deionized water was observed upon exposure to natural sunlight, and the quantum yields were determined for each protonation state. In Mississippi River water, direct photolysis does not account for all of the loss of genistein and daidzein. The mechanism of indirect photolysis was probed using quenchers and sensitizers, and results suggest that daidzein is transformed mainly via direct photolysis and singlet oxygenation, while genistein is transformed mainly via reaction with triplet-state natural organic matter. The parameters determined in this study will allow for estimation of the concentration of genistein and daidzein in sunlit surface waters, which will allow for assessment of any risks posed to aquatic wildlife.     

偏钒酸钠促进大豆芽异黄酮类物质黄豆苷元和染料木黄酮合成的研究

目的:分析比较偏钒酸钠对大豆芽合成异黄酮类物质-黄豆苷元和染料木黄酮的影响;方法:将大豆种子在不同浓度的偏钒酸钠水溶液(0、200、400、600、800、1000、1200、1400、1600mg/LNaVO3)于28℃萌发6d,第6d收获富钒大豆芽,洗净烘干至恒重,用95%乙醇提取大豆芽中的异黄酮类物质-黄豆苷元和染料木黄素,黄豆苷元和染料木黄素含量由高效液相色谱法测定;结果:分析测定结果表明,当大豆种子在浓度为200～600mg/L偏钒酸钠溶液中于28℃萌发6d,收获的大豆芽中的异黄酮类物质黄豆苷元和染料木黄酮的含量高于同等条件下其他浓度偏钒酸钠萌发液中萌发的大豆芽;结论:综合大豆芽的生长情况及大豆芽中异黄酮类物质-黄豆苷元和染料木黄酮含量等因素,在本实验条件下,萌发富钒大豆芽的适宜偏钒酸钠酸浓度为200～600mg/L,适宜的培育时间为96～120h,适宜温度为28℃。     

Apoptotic effects of cooked and in vitro digested soy on human prostate cancer cells

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.     

胶束萃取-高效液相色谱法同时测定葛根粉中5种异黄酮

目的：基于表面活性剂作为萃取剂,应用胶束萃取-反相高效液相色谱法测定葛根粉中葛根素、大豆苷、染料木苷、大豆苷元和染料木素5种异黄酮的含量。方法：以非离子表面活性剂Triton X-114为萃取剂,采用Phenomenex C18色谱柱,甲醇-体积分数0.25%冰醋酸溶液为流动相,梯度洗脱,利用紫外检测器在250nm波长处进行检测。考察色谱测定条件,优化胶束萃取和浊点预富集参数。结果：采用3g/100mL Triton X-114表面活性剂,液固比为100:1(mL/g),40℃超声萃取30min,所得的萃取率最大。在表面活性剂提取液中,加入一定量的氯化钠进行浊点预富集,可以提高萃取率和预富集因子。该方法的定性检测限范围是0.016～0.079μg/mL(RSN=3),平均回收率为93.7%～98.86%,预富集因子平均值可达到12.5。结论：该方法简便快速、灵敏可靠、绿色环保,适用于葛根粉中葛根素等5种异黄酮成分含量的测定和产品质量控制。     

去甲基化法制备黄豆苷元和染料木黄酮及抗氧化性分析

以KBr-H3PO4或KI-H3PO4为反应介质,对芒柄花黄素和鸡豆黄素进行去甲基化反应转化成黄豆苷元和染料木黄酮,并对产物的抗氧化性进行分析。结果表明：以KBr-H3PO4为反应介质,制备黄豆苷元最佳条件为反应时间5?h、反应温度120?℃、料液比1∶45（g/mL）、最佳饱和度75%,产率为89.42%;制备染料木黄酮最佳条件为反应时间5?h、反应温度120?℃、料液比1∶45（g/mL）、最佳饱和度100%,产率为85.98%;以KI-H3PO4为反应介质,制备黄豆苷元最佳条件为反应时间4?h、反应温度100?℃、料液比1∶15（g/mL）、最佳饱和度75%,产率可达87.03%;制备染料木黄酮最佳条件分别为反应时间5?h、反应温度100?℃和料液比1∶45（g/mL）、最佳饱和度75%,产率可达88.51%。与已有合成方法相比,本实验方法具有反应过程简单、反应条件温和、反应时间较短、产率较高等优点。所制备黄豆苷元和染料木黄酮具有一定的1,1-二苯基-2-三硝基苯肼自由基和羟自由基清除能力,以及较强的抗亚油酸氧化能力,表现出较好的抗氧化性。     

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.     

红车轴草提取物中异黄酮成分的分析

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

Effect of thermal processing on genistein,daidzein and glycitein content in soymilk

Soymilk was subjected to various heat treatments at 95, 121 and 140 °C for various lengths of time. The contents of the aglycones of isoflavone (daidzein, glycitein and genistein) of the soymilk were determined using C₁₈ reversed‐phase high‐performance liquid chromatography. Genistein showed greater stability to heat treatment than daidzein and glycitein. Both the daidzein and glycitein contents decreased rapidly during the early stage of heating, but on continued heating the rates of decrease were much slower. Heating may cause an increase or decrease in the genistein content of soymilk depending on the temperature and time used. Upon heating at 95 and 121 °C, there was an increase in the genistein content in the early stage of heating, possibly due the conversion of genistin to genistein. Heating at 140 °C for more than 15 s and prolonged heating at 95 and 121 °C, however, caused a slow decline in the genistein content. Copyright © 2006 Society of Chemical Industry