高效液相色谱-串联质谱法同时测定葛根中葛根素和大豆苷元

提出了高效液相色谱-串联质谱法测定葛根中葛根素和大豆苷元的含量。样品经乙醇提取,所得提取液用乙醇定容至100mL后经Waters Xterra MS C18色谱柱(150mm×3.9mm,5μm)分离,用乙腈与50mmol.L-1甲酸溶液(40+60)的混合液洗脱,采用电喷雾正离子电离多反应监测模式。葛根素和大豆苷元的质量浓度分别在0.050～0.50mg.L-1和5.0～50mg.L-1之间与峰面积呈线性关系,检出限(3S/N)均为5μg.L-1。在0.1,1.0,10.0mg.L-1 3个浓度水平进行加标回收试验,葛根素和大豆苷元的回收率分别为96.6%和97.4%。     

加压毛细管电色谱测定葛根中三种异黄酮类化合物

建立了加压毛细管电色谱(pCEC)检测葛根中异黄酮类化合物葛根素、大豆苷、大豆苷元的方法。采用C18毛细管色谱柱,以NaH2PO4缓冲盐水溶液和甲醇为流动相,优化流动相比例、流动相流速、NaH2PO4缓冲盐水溶液浓度和pH、分离电压等色谱条件。结果表明,在流动相为17.5 mmol/L NaH2PO4缓冲盐水溶液(pH 4.0):甲醇=55:45(V/V),分离电压3 kV,流动相流速80μL/min,检测波长250 nm的条件下,葛根素、大豆苷、大豆苷元质量浓度在200～1000μg/mL范围内线性关系良好,相关系数在0.9960～0.9982之间,平均回收率在98.6%～100.9%之间,RSD为3.1%～3.5%之间。该方法已用于葛根中异黄酮类物质的分离检测。     

高效液相色谱法同时测定葛枳胶囊中5种有效成分的含量

提出了高效液相色谱法同时测定葛枳胶囊中5种有效成分(葛根素、大豆苷、甘草苷、大豆苷元和甘草酸)的含量。样品经甲醇-水(50+50)溶液超声提取30min,采用Diamonsil C18色谱柱(200mm×4.6mm,5μm)为分离柱,以不同体积比混合的乙腈和磷酸(0.1+99.9)混合液为流动相进行梯度洗脱,甘草苷的检测波长为276nm,其余4种成分均为250nm。5种有效成分分别在一定的质量浓度范围内与其峰面积呈线性关系,方法的检出限(3S/N)在0.050～0.338mg·L-1之间。方法的回收率在97.1%～104%之间,测定值的相对标准偏差(n=6)在1.0%～2.7%之间。     

大豆苷元磺化物的合成、晶体结构及活性研究

对大豆苷元进行结构修饰和改性,利用磺化反应合成出强水溶性异黄酮新化合物大豆苷元磺酸钠,采用IR,MS和单晶X射线衍射法对其进行了表征和晶体结构测定,大豆苷元磺酸钠(C₃₀H₃₆Na₂O₂₃S₂)属三斜晶系,空间群P1,a=0.6948(14)nm,b=1.3277(3)nm,c=2.0401(4)nm,α=105.16(3)°,β=90.75(3)°,γ=92.73(3)°,V=1.8138(6)nm³,Z=2,μ=0.266mm^-1,F(000)=908,大豆苷元磺酸钠的晶体结构中包含大豆苷元磺化物异构体4′,7-二羟基异黄酮-3′-磺酸钠和4′,7-二羟基异黄酮-5′-磺酸钠及9分子水,配位水氧原子与钠离子配位在晶体内部形成一维聚合钠离子直链,该钠离子链将两种大豆苷元磺酸钠异构体及分子水联结在一起,形成空间网状结构,对于大豆苷元磺酸钠晶体结构的形成和稳定起了重要的作用,生理活性实验结果表明,大豆苷元磺酸钠的抗缺氧缺血作用明显优于大豆苷元.     

反相高效液相色谱法测定葛根素和大豆苷元

利用反相高效液相色谱法,配以二极管阵列检测器测定了心可舒片中的葛根素和大豆苷元,建立了一种心可舒片的质量控制方法。色谱柱为C18柱,流动相为乙腈和0．02mol／L磷酸二氢钾,柱温为25℃,流速为0．8mL／min,梯度洗脱,检测波长为250nm。葛根素和大豆苷元的平均回收率分别为97．3％和101．4％：RSD为0．6％和1．9％。     

超高效液相色谱法分析淡豆豉配方颗粒中3种异黄酮

建立超高效液相色谱法同时测定淡豆豉配方颗粒中3种异黄酮(大豆苷元、黄豆黄素、染料木素)的含量。选择Eclipse Plus C₁₈ RRHD色谱柱(100 mm×2.1 mm,1.8μm)，流动相为乙腈-0.1%乙酸溶液，梯度洗脱，流量为0.3mL/min，柱温为30℃，检测波长为260 nm。大豆苷元、黄豆黄素、染料木素的质量浓度分别在18.41～184.14、0.48～4.82、30.65～306.46μg/mL范围内线性关系良好(r≥0.999 9)。样品测定结果的相对标准偏差分别为1.80%、2.20%、1.71%(n=6)，平均加标回收率分别为96.49%、100.93%、98.53%。该方法快速，准确、可靠，可为淡豆豉配方颗粒的质量控制提供参考。     

大豆苷元与氨基修饰β-环糊精包合物的制备、表征及水溶性

采用饱和水溶液法制备了大豆苷元分别与两种氨基修饰β-环糊精(ACD) 即单-6-氨基-β-环糊精(NCD) 和单-6-乙二胺基-β-环糊精(ENCD) 的固体包合物,并获得最佳包合条件:大豆苷元与环糊精投料比为3∶1(n/n) ,搅拌时间为72 h,分别获得83%和67%的产率.利用X-射线粉末衍射和热重分析对其进行了表征,证实了两种包合物的形成.利用Job′s曲线法确定了主客体的包合比为1∶1,并利用荧光光谱滴定分析测得其包合稳定常数KS分别为899.2和203.8 L/mol.水溶性实验表明,通过与NCD和ENCD形成包合物,25℃下大豆苷元在水中的溶解度由原来的8.31 μg/mL增至15.2和13.2 mg/mL,分别提高了约1800和1500倍.     

反相高效液相色谱法制备纯化大豆异黄酮糖苷

利用制备高效液相色谱法从大豆总异黄酮提取物中制备出了3种大豆异黄酮糖苷。在Nova-Pak HR C18色谱柱(100 mm×25 mm i.d.,6 μm)上,以甲醇-体积分数为0.1%的乙酸水溶液(体积比为23∶77)为流动相,流速为20 mL/min,采用 等度洗脱方式,制备了3种大豆异黄酮糖苷,经质谱分析,确认它们分别为大豆苷、黄豆苷和染料木苷。高效液相色谱分析 表明,所制备的3种化合物的纯度均达到了99%以上。     

大豆苷元与牛血清白蛋白的相互作用研究

采用荧光和紫外-可见吸收光谱,研究了大豆苷元与牛血清白蛋白(BSA)的相互作用.结果表明大豆苷元对BSA有较强的荧光猝灭作用;根据Stern-Volmer方程得到大豆苷元与BSA之间的结合常数KA为0.385×105 (30℃)、0.405×105(40℃)和0.431×105(50℃).根据F(o)rster非辐射能量转移理论,求出了大豆苷元与BSA之间的结合距离为2.34 nm(30℃)、2.48 nm(40℃)和2.71 nm(50℃).热力学数据表明大豆苷元与BSA之间的作用力主要为疏水作用力,同时用同步荧光光谱探讨了大豆苷元对BSA构象的影响.     

铂纳米颗粒-甘氨酸功能化石墨烯电化学传感器的制备及在大豆苷元分析中的应用

本文以甘氨酸为还原剂和功能化试剂，合成了铂纳米颗粒-甘氨酸功能化还原氧化石墨烯材料(Pt-G-rGO)。通过场发射透射电镜观察了材料的形貌，使用红外光谱和XRD图谱对其结构和化学组成进行分析。在不加H₂PtCl₆的情况下，采用同一方法制备了甘氨酸功能化石墨烯材料(G-rGO/GCE)。将这两种材料分别修饰于玻碳电极(GCE)表面，制备了电化学传感器(Pt-G-rGO/GCE和G-rGO/GCE)。与GCE和G-rGO/GCE相比，Pt-G-rGO显示了更快的电子传递速率，并对大豆苷元具有更灵敏的响应。考察了溶液pH及扫速对响应信号的影响，依据动力学参数，分析了大豆苷元的电化学反应。建立大豆苷元的测定方法，应用于市售药品含量分析。     

Simultaneous analysis of nine active components in Gegen Qinlian preparations by high-performance liquid chromatography with diode array detection

HPLC with diode array detection (HPLC/DAD) was employed to determine the quantities of puerarin, daidzin, daidzein, berberine, palmatine, coptisine, baicalin, baicalein, and glycyrrhizin in Gegen Qinlian preparations of three different pharmaceutical forms including decoction, dispensing granule and pill. The calibration curves for the nine bioactive components were linear in the given concentration ranges. The precision of the method was in the range of 0.2 - 5.0% (RSD), and the recoveries of this method were between 96.5 and 104.1%. The proposed method was applicable to analyze Gegen Qinlian preparations.     

Simultaneous Separation and Determination of Four Bioactive Constituents in Traditional Chinese Medicinal Tablet Xinkeshu by HPLC-DAD

Introduction In recent years there has been a renaissance of inter-est in use of traditional Chinese medicinal preparation (TCMP) because of the realization that traditional Chi-nese medicine can act in a synergistic manner in the human body, and can provide unique therapeutic prop-erties with minimal or no undesirable side-effects.1 The modernization research on TCMP is in process in China, and the first step is to establish simple and reliable ana-lytical technologies and methodologies f…     

A Novel Medium Poly(vinyl acetate-triallyl isocyanurate-divinylbenzene) Coupled with Oligo-β-Cyclodextrin for the Isolation of Puerarin from Pueraria Flavones

Poly(vinyl acetate-triallyl isocyanurate-divinylbenzene) matrix coupled with oligo-β-cyclodextrin was synthesized to separate puerarin. The separation of three isoflavone homologues, puerarin, daidzin and daidzein, was achieved by increasing the proportion of methanol from 40 to 85%. The interaction mechanism between the crosslinkers and puerarin was investigated through molecular dynamics simulation and the mixed-mode character was demonstrated by computational and conformational methods. The research confirms that in order to enhance the recognition, the addition of triallyl isocyanurate is feasible. Meanwhile, an alternative matrix for the separation of puerarin was provided, with the advantages of moderate mechanical rigidity, ease of modification and low cost.     

Identification of isoflavonoids in several kudzu samples by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Pueraria lobata is a rich source of isoflavonoids. The detection and identification of isoflavonoid components from Pueraria radix (RP), callus and cell cultures, is very important for the safest and most effective use of kudzu as a medicinal plant, and for the studies on quantitative analysis and secondary metabolism of isoflavonoids in vitro cultures. Liquid chromatography is coupled with negative and positive electrospray ionization (ESI) tandem mass spectrometry (MS-MS), and photodiode array detection is used to characterize and detect isoflavonoids in root, callus, and cell samples of P. lobata. Characteristic product ions of aglycones, O-glucosides, and C-glucosides were obtained from the full-scan ESI-MS chromatography of the major peaks and the MS-MS spectra of the protonated ions. Five major components of puerarin, daidzin-6"-O-acetylester, genistin-6"-O-malonylester, biochanin A-7-O-glucoside-6"-O-malonylester, and daidzein are detected and identified from the methanolic extract of P. lobata callus cultures. The major isoflavonoid components of P. lobata cell suspension cultures are identified as puerarin, daidzin, daidzin-6"-O-acetylester, genistin-6"-O-malonylester, biochanin A-7-O-glucoside-6"-O-malonylester, genistein-8-C-glucoside-6"-O-malonylester, and daidzein, on the basis of ESI-MS and MS-MS spectra analysis. Likewise, puerarin, daidzin, genistein-6"-O-malonylester, 3'-methoxypuerarin, and daidzein are detected and identified from RP. Of those isoflavonoid components detected, daidzin-6"-O-acetylester is a new isoflavonoid glucoside and is for the first time detected from P. lobata cultures in vitro.     

Simultaneous determination of six isoflavonoids in rat plasma after administration of total flavonoid from Gegen by ultra-HPLC-MS/MS

A simple, specific, and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of 3'-hydroxypuerarin, 6'-O-xylosylpuerarin, mirificin, puerarin, 3'-methoxypuerarin and daidzin in rat plasma. After the addition of methanol containing 0.1% formic acid and 10% ascorbic acid, the analytes and rutoside were obtained by protein precipitation, then separated on a Thermo Syncronis C18 column (2.1 mm × 10 cm, 1.7 μm) by gradient elution and monitored using an electrospray ionization interface operating in positive ion and selective reaction monitoring acquisition mode. The calibration curves of these analytes showed good linearity (r > 0.99) within the test ranges. The lower limit of quantification was 0.0200 μg/mL for 3'-hydroxypuerarin, 0.0101 μg/mL for 6'-O-xylosylpuerarin, 0.0100 μg/mL for mirificin and puerarin, 0.0098 μg/mL for 3'-methoxypuerarin, and 0.0090 μg/mL for daidzin. The intraday and interday precision and accuracy were all within 15%. The extraction recoveries were from 74.0 to 95.8%. The validated method was successfully applied to pharmacokinetic studies of the six isoflavonoids in rat plasma after intravenous administration of total flavonoids from Gegen.     

Separation and determination of isoflavonoids in several kudzu samples by high-performance capillary electrophoresis (HPCE)

Pueraria lobata is a rich source of isoflavonoids. The detection and identification of isoflavonoid components from root, stem, leaf, callus and cell samples, is very important for the best, safest and most efficacious use of kudzu as a medicinal plant, and for the studies on quantitative analysis in the secondary metabolism of isoflavonoids. In this paper, a simple, rapid and precise high-performance capillary electrophoresis (HPCE) method with diode array detection (DAD) has been developed for separation and determination of isoflavonoids in several kudzu samples. The isoflavonoids could be well separated within 15 min in a 40 cm length capillary at a separation voltage of 15kV in a 30 mmol L(-1) borax buffer (pH9.29), and this proposed method demonstrated excellent reproducibility and accuracy with relative standard deviations of less than 5% for isoflavonoid content (n = 5) of different kudzu samples. The relationship between peak areas and isoflavone concentrations, in a specified working range with linear response, was determined by first-order polynomial regression over the range 0.05-0.5 mg mL(-1) for puerarin and 2.5-50 microg mL(-1) for 3'-methoxypuerarin, daidzin and daidzein, respectively, and quantitative evaluation of those four main isoflavonoid components was determined by ultraviolet absorption at lambda = 192 nm. The differences were also illustrated by comparison of the determination of isoflavonoid components from kudzu root, stem, leaf samples and plant tissue cultures in vitro.     

Liquid chromatography-mass spectrometric analysis of puerarin and its metabolite in human urine

A specific LC-MS method was developed that allowed simultaneous determination of puerarin (PU) and its major metabolite, daidzein (DA), in human urine samples. PU and DA were separated on a packed capillary ODS column with on column concentration. Identification and quantification of the analytes were performed with ESI-Q-TOF mass spectroscopy in negative ionization mode. The method was validated, yielding calibration curves with correlation coefficients greater than 0.998. The LOQ for PU and DA from human urine samples was 0.1 and 0.05 nmol/mL, respectively. Assay accuracy and precision of quality control samples were within +/- 15%. Recoveries of PU and DA in spiked samples were in the range of 79.6-90.4 and 82.3-92.4%, respectively.     

Simultaneous determination of five isoflavones in rat plasma by LC‐MS/MS: Comparative pharmacokinetic characteristics of Puerariae lobatae radix in normal and type 2 diabetic rats

A rapid and sensitive liquid chromatography with tandem mass spectrometry method was developed and validated for simultaneous determination of puerarin, daidzin, daidzein, 3′‐hydroxy puerarin, and genistein in rat plasma after oral administration of Puerariae lobatae radix extract. The method of protein precipitation with acetonitrile was used for sample preparation. Chromatographic separation was achieved on a C₁₈ column with the mobile phases of acetonitrile/water containing 0.1% formic acid. The analytes were detected by mass spectrometer with an electrospray ionization source operating in the negative ion mode. The linearity, precision, accuracy, dilution reliability, recovery, matrix effects, and stability of the method were within acceptable ranges. The developed method was successfully used to compare the pharmacokinetic characteristics of five analytes in normal and type 2 diabetics rats after oral administration of Puerariae lobatae radix extract. Several pharmacokinetic alterations were observed and this might be caused by the pathological state of type 2 diabetes.     

Enhanced daidzin production from jasmonic and acetyl salicylic acid elicited hairy root cultures of Psoralea corylifolia L. (Fabaceae)

Daidzin (7-O-glucoside of daidzein) has several pharmacological benefits in herbal remedy, as antioxidant and shown antidipsotropic activity. Hairy root culture of Psoralea corylifolia L. was developed for biomass and enhanced daidzin production using signalling compounds such as jasmonic acid (JA) and acetyl salicylic acid (ASA). Best response of 2.8-fold daidzin (5.09% DW) with 1 μM JA treatment after second week and 7.3-fold (3.43% DW) with 10 μM JA elicitation after 10th week was obtained from hairy roots compared to untreated control. ASA at 10 μM promoted 1.7-fold increase in daidzin (1.49% DW) content after seventh week compared to control (0.83% DW). Addition of 25 μM ASA resulted in 1.44% DW daidzin (1.5-fold increase) with 0.91% DW in control after fifth week and 1.44% DW daidzin (2.3-fold increase) after eighth week when compared to untreated control (0.62% DW). Reduced biomass with increased daidzin content was facilitated by elicited hairy root cultures.     

Simultaneous separation and determination of seven isoflavones in Radix Puerariae by capillary electrophoresis with a dual cyclodextrin system

A simple, comprehensive and efficient capillary electrophoresis method using a dual cyclodextrin system was developed for the simultaneous determination of seven isoflavones (3′‐methoxypuerarin, puerarin, 3′‐hydroxypuerarin, ononin, daidzin, daidzein and genistin). Baseline separations of the seven isoflavones were achieved within 11 min with the running buffer consisting of 35 mm sodium tetraborate, 9.0 mm sulfobutylether‐β‐cyclodextrin and 30 mm α‐cyclodextrin at pH 9.34, and peaks were detected at 254 nm. Other separation parameters included the separation voltage for 15 kV and the working temperature for 25°C. Under the optimum conditions, good linearities were obtained with linear correlation coefficients of seven isoflavones of 0.9978–0.9992. The limits of detection and the limits of quantification were 0.7–2.9 and 2.5–9.5 μg/mL, respectively. Excellent precision and accuracy were obtained. The intraday and interday precision ranged from 0.7 to 2.0% and from 0.8 to 1.9%, respectively. The recoveries of seven analytes were from 97.7 to 103.1%. This method was successfully applied to determine the seven analytes in Radix Puerariae and its preparations.