人尿中异黄酮的高效液相色谱分析

建立了测定人尿中异黄酮组分(大豆苷原、黄豆黄素、雌马酚、染料木黄酮)含量的反相高效液相色谱法。在尿样中加入黄酮作为内标,异黄酮经酶解后在pH=7.0中性条件下用乙酸乙酯(1∶1)提取,然后用0.02%TFA-M eOH-ACN三元梯度洗脱的方法分离异黄酮。在该条件下,大豆苷原、黄豆黄素、雌马酚、染料木黄酮的检出限分别为12.9 nmol/L、13.9 nmol/L、71.6 nmol/L和11.8 nmol/L;回收率均在89%以上。本方法具有测试步骤简单、准确度高、重现性好等优点,适合大批量样品测定。     

Determination of isoflavones in red clover and related species by high-performance liquid chromatography combined with ultraviolet and mass spectrometric detection

High-performance liquid chromatography-UV-electrospray ionization-mass spectrometric detector (HPLC-UV-ESI-MSD) method for determination of isoflavones in red clover (Trifolium pratense L.) and related species has been developed. The separated isoflavones including aglycones, glycosides and glycoside malonates, were individually analyzed and identified by their molecular ions and characteristic fragment ion peaks using LC-MSD under MS and MS-MS mode, and in comparison with the standard isoflavones. A total of 31 isoflavones were detected in red clover. Several isoflavones were also identified for the first time in related species, T. repense L. (white clover), T. hybridum L. (alsike clover) and T. campestre Schreber (hop trefoil). Based on reversed phase HPLC, all 10 isoflavone aglycones, daidzein, formononetin, genistein, pseudobaptigenin, glycitein, calycosin, prunetin, biochanin A, irilone and pratensein in acidic hydrolyzed extracts were successfully separated within 40 min and quantified individually by UV and MS detectors. For the 10 target compounds, the investigated concentrations ranged from approximately 24 to approximately 12500 ng/ml for UV detection and approximately 6 to approximately 3125 ng/ml for MS detection, and good linearities (r2 > 0.999 for UV and r2 > 0.99 for MS) for standard curves were achieved for each isoflavone. The accuracy and repeatability (n = 10) were within 15% for these 10 compounds. This is the first method reported that enables the simultaneous quantitation of all 10 isoflavone aglycones in red clover and related species.     

Comparison of analytical methods for the quality control of a new formulation containing soy extract and melatonin

Three analytical methods have been developed and compared for the quality control of a new formulation (Soymen GN(R) capsules) containing soy extract and melatonin for the treatment of menopausal symptoms. The first method is based on MEKC with diode-array detection, using a mixture of basic carbonate buffer (95%) and methanol (5%), containing 55 mM SDS, as the BGE. The second method is an HPLC method with UV detection at 260 nm. The third method is an HPLC method coupled to amperometric detection which is carried out at an oxidation potential of +0.8 V. In both HPLC systems, the chromatographic separation is obtained on an RP C18 column using a mixture of ACN and an acidic phosphate buffer (25:75 v/v) as the mobile phase. A feasible pretreatment procedure with a methanol/water mixture has been implemented to achieve the quantitative extraction of the main soy isoflavones and of melatonin from the capsules. The results obtained with the three methods are in good agreement with each other and satisfactory in terms of linearity (r(2) >0.9996), precision (RSD <5.4%) and accuracy (recovery >97%). Thus, each of the three analytical methods seems to be suitable for the simultaneous analysis of the main soy isoflavones and melatonin in the new commercial formulation.     

Effects of ultrasound on growth, bioconversion of isoflavones and probiotic properties of parent and subsequent passages of Lactobacillus fermentum BT 8633 in biotin-supplemented soymilk

This study aimed to evaluate the effects of ultrasound on Lactobacillus fermentum BT 8633 in parent and subsequent passages based on their growth and isoflavone bioconversion activities in biotin-supplemented soymilk. The treated cells were also assessed for impact of ultrasound on probiotic properties. The growth of ultrasonicated parent cells increased (P < 0.05) by 3.23-9.14% compared to that of the control during fermentation in biotin-soymilk. This was also associated with enhanced intracellular and extracellular (8.4-17.0% and 16.7-49.2%, respectively; P < 0.05) β-glucosidase specific activity, leading to increased bioconversion of isoflavones glucosides to aglycones during fermentation in biotin-soymilk compared to that of the control (P < 0.05). Such traits may be credited to the reversible permeabilized membrane of ultrasonicated parent cells that have facilitated the transport of molecules across the membrane. The growing characteristics of first, second and third passage of treated cells in biotin-soymilk were similar (P > 0.05) to that of the control, where their growth, enzyme and isoflavone bioconversion activities (P > 0.05) were comparable. This may be attributed to the temporary permeabilization in the membrane of treated cells. Ultrasound affected probiotic properties of parent L. fermentum, by reducing tolerance ability towards acid (pH 2) and bile; lowering inhibitory activities against selected pathogens and reducing adhesion ability compared to that of the control (P < 0.05). The first, second and third passage of treated cells did not exhibit such traits, with the exception of their bile tolerance ability which was inherited to the first passage (P < 0.05). Our results suggested that ultrasound could be used to increase bioactivity of biotin-soymilk via fermentation by probiotic L. fermentum FTDC 8633 for the development of functional food.     

Optimization of matrix solid-phase dispersion extraction method for the analysis of isoflavones in Trifolium pratense

A method based on matrix solid-phase dispersion (MSPD) has been developed for the determination of 12 isoflavones in Trifolium pratense L. Dried leaf samples were blended with C₁₈, placed in small columns and isoflavones extracted with dichloromethane–methanol. Analyses were performed by high performance liquid chromatography with diode array detection (HPLC-DAD) with 2-methoxyflavone as internal standard. Several dispersants, eluents and clean-up steps were tested during the optimization of the process in order to obtain the best selectivity and yields. Mean recoveries ranged from 70% to 119%, with relative standard deviations <18%. The limits of detection were between 0.006 mg/l for biochanin A and 0.108 mg/l for daidzin. The performance of the optimized method in real samples was compared with a conventional method based in solid–liquid extraction (SLE).     

Availability of Isoflavones (Genistin,Genistein and Daidzein) in Soybean ( Glycine Max ) Curd Treated under High Pressure

The article describes the concentration of isoflavones (genistin, genistein and daidzein) in a Spanish commercial tofu treated under high-pressure. The tofu was subjected to pressure of 400 MPa, in cycles of 10 min each with a total of 4 cycles with the progression of 10, 20, 30 and 40 min, and a temperature of about 10-12 °C. The isoflavones were extracted from tofu with aqueous methanol solution (80:20). The content of isoflavones went up directly proportional to the pressure applied for the first 30 minutes. Afterwards, at 40 min, the values dropped close to the control. Significant differences were found in genistein, genistin and daidzein treated for 20 and 30 min in comparison to control values. Finally, we came to the conclusion that, the isoflavones (genistin, genistein and daidzein) of treated tofu were not destroyed at 400 Mpa for 30 min and a major extraction was observed when compared with the untreated tofu.     

Microwave assisted extraction of soy isoflavones

A fast and reliable analytical method using microwave assisted extraction has been developed. Several extraction solvents (methanol (MeOH) and ethanol (EtOH), 30-70% in water and water), temperatures (50-150 °C), extraction solvent volume, as well as the sample size (1.0-0.1 g) and extraction time (5-30 min) were studied for the optimization of the extraction protocol. The optimized extraction conditions for quantitative recoveries were: 0.5 g of sample, 50 °C, 20 min and 50% ethanol as extracting solvent. No degradation of the isoflavones was observed using the developed extraction protocol and a high reproducibility was achieved (>95%).     

Ultrasound-assisted extraction of isoflavones from soy beverages blended with fruit juices

A new method for the fast determination of isoflavones from soy beverages blended with fruit juices without the need of freeze-drying the sample was developed. During the method development, several parameters were studied: solvent (methanol and ethanol), sample:solvent ratio (5:1 to 0.2:1), temperature (10-60 °C) and extraction time (5-30 min). The most important parameter for the extraction of isoflavones from soy drinks was the sample:solvent ratio. The optimized method consists of extracting the sample with ethanol with a sample:solvent ratio of 0.2:1 on an ultrasound bath at 45 °C during 20 min. Also, samples were freeze-dried, extracted using conventional method and compared with the optimized method and no significant difference was observed on total and individual isoflavone concentration. The most representative samples from the Spanish market, with a wide variation of isoflavone concentration were analyzed using the optimized method. Differences between manufacturers reached an almost 10 times fold variation. Overall isoflavone concentration ranged from 6.7 to 58.2 mg L⁻¹.     

Ultrasound‐assisted solid‐phase extraction coupled with photodiode‐array and fluorescence detection for chemotaxonomy of isoflavone phytoestrogens in Trifolium L. (Clover) species

Detailed chemotaxonomic studies were undertaken to establish the qualitative profile and real amounts of the pharmacologically active isoflavone aglycones genistein, daidzein, formononetin, and biochanin A in aerial parts of thirteen Trifolium L. (clover) species, native to Poland. A newly elaborated micropreparative technique – SPE – on BakerBond octadecyl, cyclohexyl, and phenyl cartridges was used in combination with ultrasound‐assisted extraction for isolation of isoflavone aglycones from hydrolyzed samples. The effectiveness of all three SPE sorbents in the purification of plant extracts was compared and very high recoveries (>96%) were documented for four isoflavones. Classical photodiode‐array and very sensitive fluorescence detection, coupled with reversed‐phase high‐performance liquid chromatography (RP‐HPLC), were employed to obtain the most reliable qualitative and quantitative results. Chemotaxonomic differences combined with flower color variability were demonstrated within thirteen clover species. Concentration levels of particular isoflavones in ten Trifolium species possessing flowers with white, pink, or purple‐red corolla ranged from ∼︁3 to ∼︁3300 μg/g dry weight, while in three yellow flowering clovers (T. aureum, T. dubium, and T. campestre) isoflavone compounds have not been detected at all. RSD values, determined for intra‐ and inter‐day precision of the quantitative results, were not higher than 6.2% and 7.1%, respectively.     

Simultaneous Quantification of Puerarin and Daidzein in Rat Plasma by High-Performance Liquid Chromatography with Post-Column Modification and Fluorescence Detection

A sensitive HPLC method based on post-column modification and fluorescence detection has been developed for determination of puerarin and daidzein in rat plasma. Chromatographic separation was performed on a C₈ column with a linear gradient prepared from 0.5% aqueous acetic acid and 0.5% acetic acid in acetonitrile, delivered at a flow rate of 0.8 mL min⁻¹. Naringin was used as the internal standard. It was necessary to use acetic acid in the mobile phase to achieve good separation, but this led to fluorescence signal suppression, because puerarin and daidzein have native fluorescence at pH 8.0–9.0. To enhance the sensitivity, post-column modification with alkaline buffer was adopted. After this modification, detection sensitivity for puerarin and daizein increased more than 500-fold and 600-fold, respectively, compared with direct fluorescence detection. Signal-to-noise ratios for detection for puerarin were more than 150 times better than for UV detection after use of the same method of sample preparation. This sensitive analytical method was successfully used to determine pharmacokinetic data for puerarin and daidzein in rat plasma after oral administration of a single dose of Puerariae radix extract containing puerarin (approx. 8.4 mg) and daizein (approx. 5.9 mg) to male SD rats.