HPLC法测定豆制品中游离异黄酮的含量

利用高效液相色谱法（HPLC）对豆制品中的大豆黄素和染料木素的含量进行测定,从而研究豆制品加工中的游离大豆异黄酮分布情况。     

The effect of low pH on the glycitein-BSA conjugate interaction with specific antiserum: competitive inhibition study using surface plasmon resonance technique

Competitive inhibition serological assay for detection of the phytoestrogen glycitein (Glyc) was developed using surface plasmon resonance (SPR) technique with protein conjugates and polyclonal antibodies initially designed for the enzyme-linked immunosorbent assays (ELISA). The efficiency of the approach to the quantification of the soy isoflavone glycitein in water was investigated using the competitive reaction of analyte (free Glyc)and immobilized Glyc-BSA-conjugate with polyclonal antibodies. It was shown that the efficiency to detect Glyc drastically depends on the pH level of the probe solution. With the decrease in pH from 7.4 to 4.0, (i) the affinity of the specific reaction increases and (ii) the level of unspecific sorption becomes saturated. Non-specific adsorption to a SPR sensor surface obscures the specific component and shaded specific response at higher pH (6.0-7.4) when used serum for the quantification of specific analytes. The standard curves obtained in acidic solutions (pH 4-5) indicate that the linear part of the dependence completely covers the range between detection limit (0.1 μg/ml) and Glyc solubility in water (0.9 μg/ml). The difference in SPR- and ELISA-based analytical protocols as well as the requirements for increasing the efficiency in quantitative SPR analysis using purified antibodies is discussed.     

荧光光谱法研究黄豆黄素与牛血清白蛋白的相互作用

用荧光光谱法在pH 7.4的PBS中研究了黄豆黄素(Glycitein,GL)与牛血清白蛋白(Bovine serum albumin,BSA)分子之间的相互作用方式及机理.结果表明GL可静态猝灭BSA的内源荧光.310K和315K的结合位点数与表观结合常数分别为1.30,8.09×104L·mol-1和1.73,5....     

Hyphenated technique for the extraction and determination of isoflavones in algae: Ultrasound-assisted supercritical fluid extraction followed by fast chromatography with tandem mass spectrometry

New hyphenated technique for the extraction and determination of isoflavones in sea and freshwater algae and cyanobacteria was developed. The method consists of sonication sample pretreatment, extraction by supercritical CO₂ modified by 3% (v/v) of MeOH/H₂O mixture (9:1, v/v) at 35 MPa and 40 °C for 60 min, fast chromatography analysis by the means of Agilent 1200 Series Rapid Resolution and MS/MS determination. Agilent 1200 Series RRLC was used with Zorbax SB-CN chromatographic column (100 mm × 2.1 mm, particle size 3.5 μm), 3 μl injection volume, mobile phase consisting of 0.2% (v/v) acetic acid in water (solvent A) and acetonitrile (solvent B) and used with linear gradient (30% B at 0 min, from 0 min to 3 min up to 50% B, from 3 to 6 min up to 80% B and from 6 to 10 min down to 30% B). The flow-rate was 0.4 mL/min, column oven temperature 35 °C. MS detector Agilent Technologies 6460 Triple quadrupole LC/MS with Agilent Jet Stream was used in a negative ESI mode under following conditions: gas temperature 350 °C, gas flow 13 L/min, nebulizer gas pressure 50 psi, sheath gas temperature 400 °C, sheath gas flow 12 L/min, capillary voltage was 4 kV. Samples were analysed in the multiple reaction monitoring (MRM) mode. Eight isoflavone compounds were found for the first time in seven real samples of sea algae and in three control samples of freshwater algae and cyanobacteria. Usual optimisation study of extraction parameters was performed. Pressure and temperature optima for algae matrix are different from those obtained sooner for other matrices for most of the analytes, but the results of modifier optimisation study are in good accordance with those obtained sooner for spiked samples and red clover matrix. It seems that matrix has very small or no effect on the modifier selection. Two different approaches of sonication pretreatment were tested: sonication bath and the thorn instrument. In longer extraction time experiments, thorn sonication was more efficient and recovery of following supercritical fluid extraction was higher.     

The synthesis of [2,3,4-C3]glycitein

Glycitein is one of the soy isoflavones which have attracted considerable interest in recent years due to their possible beneficial effects on human health. However, glycitein has been much less studied than other members of this family due to the lack of good methods for its synthesis. Herein we report a short high yielding synthesis of a multiply ¹³C-labelled glycitein derivative, [2,3,4-¹³C₃]glycitein, which has been employed as an internal standard in LC–MS analysis. A key feature is a rapid and efficient synthesis of 2,4-dihydroxy-5-methoxy-[1′,2′-¹³C₂]acetophenone via acetylation of isovanillin with [¹³C₂]acetyl chloride followed by a Baeyer–Villiger reaction, selective hydrolysis and finally a BF₃ catalysed Fries rearrangement. An aldol reaction using 4-benzyloxy-[carbonyl-¹³C]benzaldehyde gave a chalcone and then thallium(III) mediated oxidative rearrangement, deprotection and cyclisation provided the [2,3,4-¹³C₃]glycitein. The overall yield for the 8 step reaction sequence, based on [¹³C₂]acetyl chloride, was 57%.     

黄豆黄素与黄豆黄苷吸收光谱和荧光光谱的比较研究

研究了黄豆黄素和黄豆黄苷在不同pH条件下的吸收光谱和荧光光谱, 从分子结构的角度解释了二者呈现不同光谱特征的原因. 黄豆黄素分子基本无荧光. 在弱碱性时, 黄豆黄素分子发生7-OH质子的电离, 导致吸收光谱中320 nm的吸收峰红移至348 nm. 采用pH-光度法测得7-OH质子的电离常数pK_a1=7.08±0.04. 黄豆黄素一价阴离子呈现较强荧光, 最大激发和发射波长λ_ex/λ_em分别为334 nm/464 nm, 荧光量子产率为0.049. 在碱性溶液中, 黄豆黄素4'-OH质子电离, 导致吸收光谱中254 nm的吸收峰红移至260 nm, 电离常数pK_a2=9.96±0.01. 黄豆黄苷分子基本无荧光. 在碱性条件下, 黄豆黄苷分子的4'-OH质子发生电离, 导致吸收光谱中256 nm的吸收峰红移至 280 nm, 电离常数pK_a=9.81±0.03. 黄豆黄苷阴离子基本无荧光, 但在热碱性条件下发生γ-吡喃酮环裂解反应而产生较强荧光, λ_ex/λ_em为288 nm/388 nm, 裂解产物的荧光量子产率为0.056. 虽然, 黄豆黄苷与黄豆黄素是苷与苷元的关系, 但黄豆黄苷不能在热碱性条件下通过糖苷水解转变为黄豆黄素, 二者的荧光增强机理存在本质不同.