Identification and determination of aesculin and aesculetin in ash barks by capillary zone electrophoresis

Summary A capillary zone electrophoresis method for identification and determination of aesculin and aesculetin has been established using borate-phosphate buffer containing 30% ethanol with on-column UV detection. A detailed investigation of the influence of changes in borate concentration, pH, applied voltage, temperature and organic modifier was then carried out. For both aesculin and aesculetin, a linear plot of migration time (MT) against borate concentration was obtained, and ln[measured peak area (MA)] and lnMT both gave linear plots against ln(applied voltage) with correlation coefficient r>0.999, which also resulted in a linear correlation between MA and MT (r≥0.9998) under varied voltage. Ethanol as organic modifier to the background electrolytes helped in separating aesculin and aesculetin from other components in ash barks. The reproducibility with relative standard deviation in MT and in normalized peak area(NA) and linearity based on NA against concentration were evaluated. Finally, the method was successfully applied to monitor the quality of different ash barks and to compare the effect of sample preparation on content of bioactive components in ash bark. Results indicate that CZE promises to be applicable to quality control of traditional Chinese medicines containing aesculin and aesculetin.     

Isolation and purification of coumarin compounds from Cortex fraxinus by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was successfully used for the isolation and purification of coumarin compounds from Cortex fraxinus, the Chinese herbal drug. n-Butanol-methanol-0.5% acetic acid (5:1.5:5, v/v) was used as the two-phase solvent system. 14.3 mg of fraxin, 26.5 mg of aesculin, 5.8 mg of fraxetin and 32.4 mg of aesculetin with the purity of 97.6, 99.5, 97.2 and 98.7%, respectively were obtained from 150 mg of crude extracts of C. fraxinus in a single run. The structures of the isolated compounds were identified by 1H NMR and 13C NMR.     

Comparative pharmacokinetic study of the main components of cortex fraxini after oral administration in normal and hyperuricemic rats

Cortex Fraxini is an important traditional Chinese herbal medicine used for the treatment of gout and hyperuricemia. An efficient and rapid ultra‐performance liquid chromatography mass spectrometry method was developed and validated for simultaneous quantitation of six coumarins (aesculin, fraxin, aesculetin, fraxetin, sopoletin and 7‐hydroxycoumarin) in normal and hyperuricemic rats plasma after oral administration of Cortex Fraxini. The method could successfully be applied for pharmacokinetics studies. The pharmacokinetic behavior of six coumarins in normal and hyperuricemia rats plasma was determined. Results showed that, for some of analytes, the pharmacokinetic parameters (AUC_0–t , AUC_0–∞, C _max, T _max and CL ) were significantly different between normal and hyperuricemic rats. The different pharmacokinetic parameters might result from renal impairment or a change of metabolic enzymes in the pathological state. The pharmacokinetic study in pathological state could provide more useful information to guide the clinical use of traditional Chinese herbal medicine.     

Analysis of aesculin and aesculotin in Cortex fraxini by capillary zone electrophoresis

A simple method for quantitative analysis of aesculin and aesculetin in Cortex fraxini was developed using capillary zone electrophoresis (CZE). Berberin was employed as an internal standard, The running buffer was 6 mM Na(2)B(4)O(7) and 10 mM NaH(2)PO(4) (pH 6.70). The linear calibration range was 32-256 mug ml(-1) (r=0.9996) for aesculin and 23-230 mug ml(-1) (r=0.9993) for aesculetin. The contents of aeculin and aesculetin in C. fraxini were easily determined within 12 min the pKa values of aesculin and asculetin determined by CZE were 6.56 and 5.62, respectively. A simple method for estimation of the temperature inside the capillary during running was also proposed.     

定量测定秦皮中秦皮甲素的二阶导数示差脉冲极谱法

建立了中草药秦皮中秦皮甲素的二阶导数示差脉冲极谱定量分析方法。秦皮甲素在乙醇－2mol/L氯化锂－水（体积比2：1：5）的底液中,于－1．703V（vs Ag/AgCl)处出现一良好的二阶导数示差脉冲极谱峰,其峰幅值与秦皮甲素在0．2－1．6mmol/L范围内呈非常显著的线性关系（P＜0．01）,检测下限脒50mmol/L。该法简便、快速、灵敏,结果准确。     

Rapid Determination of Coumarins in Plants by Capillary Electrophoresis

The goal of this study was to optimize the parameters for capillary electrophoresis to separate and determine aesculin, aesculetin, umbelliferone, and dihydrocoumarin in plant materials. A simple and rapid capillary zone electrophoresis method is reported for this separation that required less than nine minutes. A bare fused silica capillary was employed using 20 mM borax in 5% methanol at pH 10.1 as the background electrolyte with an applied voltage of 30 kV at 27°C. Good linearity and reproducibility were obtained with high correlation coefficients. The analytes were determined by molecular absorption at 194 and 206 nm. Coumarins were determined in Aesculus hippocastanum L., Cichorium intybus L., Melilotus officinalis L., and Juniperus communis L. “Pendula.” The concentrations of aesculin and aesculetin were 3.07 and 6.31 mg g^?1 in Aesculus hippocastanum. In Cichorium intybus, the aesculetin concentration was 2.42 mg g^?1. The dihydrocoumarin concentration was 0.54 mg g^?1 in Melilotus officinalis, and the concentration of umbelliferone was 0.58 mg g^?1 in Juniperus communis “Pendula.”     

Study of polyethylene glycol as a green solvent in the microwave-assisted extraction of flavone and coumarin compounds from medicinal plants

In this paper, the application of polyethylene glycol (PEG) aqueous solution as a green solvent in microwave-assisted extraction (MAE) was firstly developed for the extraction of flavone and coumarin compounds from medicinal plants. The PEG solutions were optimized by a mono-factor test, and the other conditions of MAE including the size of sample, liquid/solid ratio, extraction temperature and extraction time were optimized by means of an orthogonal design L(9) (3(4)). Subsequently, PEG-MAE, organic solvent-MAE, and conventional heating reflux extraction (HRE) were evaluated with nevadensin extraction from Lysionotus pauciflorus, aesculin and aesculetin extraction from Cortex fraxini. Furthermore, the mechanism of PEG-MAE was investigated, including microwave-absorptive property and viscosity of PEG solutions, the kinetic mechanism of PEG-MAE and different microstructures of those samples before and after extraction. Under optimized conditions, the extraction yields of nevadensin from L. pauciflorus, aesculin and aesculetin from C. fraxini were 98.7%, 97.7% and 95.9% in a one-step extraction, respectively. The recoveries of nevadensin, aesculin and aesculetin were in the range of 92.0-103% with relative standard derivation lower than 3.6% by the proposed procedure. Compared with organic solvent-MAE and conventional extraction procedures, the proposed methods were effective and alternative for the extraction of flavone and coumarin compounds from medicinal plants. On the basis of the results, PEG solution as a green solvent in the MAE of active compounds from medicinal plants showed a great promising prospect.     

三维荧光光谱法结合交替三线性分解算法同时分辨及定量测定秦皮中的秦皮甲素和秦皮乙素成分

用三维荧光分析结合交替三线性分解算法对秦皮中药成分进行定性定量分析,为直接测定秦皮甲素和秦皮乙素提供了一种快速简洁的方法。相关系数r分别为1．000和0．998。多次测定结果的相对误差分别为2．01％和2．21％。     

高效液相色谱-电感耦合等离子体质谱(ICP-MS)法测定中药材中无机汞、甲基汞、乙基汞

为准确测定中药材中不同形态的汞含量,通过选用人工胃液作为提取液,经水浴加热提取后,以甲醇-乙酸铵为流动相,采用C18反相色谱柱对样品溶液进行分离,最后经电感耦合等离子体质谱(ICP-MS)法测定3种汞形态化合物的含量。建立了中药材中无机汞、甲基汞、乙基汞的高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)法的测定方法,结果表明,3种汞形态化合物在0.5～5 ng/mL范围内均获得良好的线性关系,相关系数均大于0.999。通过加标回收的方法进行准确性评价,加标回收率为75.5%～118%,相对标准偏差RSD为2.4%～9.7%。测定的106批次中药材中,均未检出甲基汞、乙基汞。无机汞检出率为88.7%,合格率为100%。方法能够准确、高效地测定中药材中3种汞形态化合物的含量。106批次中药材中的汞存在形态主要是以无机汞为主。考察的106批次根及根茎类中药材中3种汞形态化合物的含量,积累了基础数据,为中药饮片的质量安全性监管提供技术支撑。     

能量色散X射线荧光光谱法测定中草药中的Cd元素

利用能量散射X射线荧光光谱（ED-XRF）法快速测定中草药中的Cd元素.方法对中草药进行简单预处理（粉碎）,使用X射线荧光光谱法建立中草药中Cd元素含量与相对强度的校正工作曲线,分析中草药样品中的Cd元素.Cd元素的检出限为0.083 mg/kg,定量限为0.207 mg/kg,定量限低于国家控制标准0.3 mg/kg.测量定量限以上的中草药样品时,其准确性与化学分析结果相当,重复性和稳定性等结果完全满足GB/T 4889-2008数据的统计处理标准.方法预处理时间不超过5 min,完成一个样品的检测时间不超过15 min,可应用于中草药生产企业中的重金属监控.     

微波辅助提取-气相色谱法同时测定中草药中有机磷和氨基甲酸酯农药残留

建立了中草药中有机磷和氨基甲酸酯类农药同时检测的气相色谱分析新方法.中药材试样依据正交实验的优化条件,用正己烷-丙酮(1∶4,V∶V)混合提取剂进行微波辅助提取,经弗罗里硅土和中性氧化铝混合层析柱净化后,采用HP-5毛细管柱分离,氮磷检测器同时检测中草药中15种有机磷和6种氨基甲酸酯类农药残留量.21种农药在0.01～1.0 mg/L的浓度范围内线性良好,线性相关系数为0.9950～1.000,检出限为0.002～0.01 mg/L.在0.05、0.2、0.5 mg/kg三个添加水平的平均回收率分别为75.11%～128.57%、75.85%～120.71%和76.43%～117.25%,相对标准偏差分别为 3.10%～10.58%、5.27%～9.94%和4.03%～9.03%.方法用于中草药中有机磷和氨基甲酸酯类农药残留的同时检测,结果良好.     

王水-过氧化氢热浸提原子荧光光谱法快速测定原生中草药中的汞

采用王水-过氧化氢预氧化水浴热浸提处理样品,应用原子荧光光谱法快速检测原生中草药中痕量汞.其方法回收率在98.6%~105.4%之间,相对标准偏差在3.0%~7.4%之间,标准系列质量浓度在0.000~6.000μg/L范围内线性关系良好,相关系数为0.999 9,一元线性回归方程为：IF=301.512C＋1.235,方法检出限达到0.021 6μg/L.试验证实,方法具有简便、快速、灵敏度高、取样量少等优点,为原生中草药中痕量汞的检测提供了有效的分析方法.     

中草药活性成分的高通量筛选技术研究进展

随着高通量筛选技术的不断发展,该技术已经成为发现新药物的重要途径之一。高通量筛选技术已大量应用于筛选药物活性成分的领域中,但是其中大部分为从化合物库中筛选活性成分,仅有十几篇文献应用于中药活性成分的筛选,而中国传统中草药却是探索和发展新药物的丰富来源。本文通过综述国内外2008年到2017年的相关文献,阐述了分子和细胞水平上的高通量筛选技术在中草药活性成分的筛选及其应用进展,为今后中草药新药研发提供参考。     

Sensitive and Selective LC–ESI–MS Analysis of Aesculin in Rat Plasma

A rapid and sensitive liquid chromatography–electrospray ionization mass spectrometry method was developed for the determination of aesculin in rat plasma. The analyses were chromatographed on a Zorbax Extend-C18 analytical column (150 × 2.1 mm I.D., 5 µm) with 30:70 (v/v) methanol–0.1% formic acid as mobile phase. Detection was performed by triple-quadrupole tandem mass spectrometry in multi-reaction-monitoring mode with an electrospray ionization source. The method was validated for accuracy and precision, and linearity in the two matrices was good. The assay was linear in the range 12.5–1,800 ng mL^?1. The lower limit of quantification of aesculin (LLOQ) was 12.5 ng mL^?1. The recovery of aesculin and tinidazole (IS) were well above 85%. The within- and between-batch accuracy was 100–104% and 97–109%, respectively. There were no stability-related problems in the procedure for the analysis of aesculin. The method was successfully used in a preclinical study of the pharmacokinetics of aesculin in rats.     

Identification of the chemical constituents of Chinese medicine Yi‐Xin‐Shu capsule by molecular feature orientated precursor ion selection and tandem mass spectrometry structure elucidation

The incomplete identification of the chemical components of traditional Chinese medicinal formula has been one of the bottlenecks in the modernization of traditional Chinese medicine. Tandem mass spectrometry has been widely used for the identification of chemical substances. Current automatic tandem mass spectrometry acquisition, where precursor ions were selected according to their signal intensity, encounters a drawback in chemical substances identification when samples contain many overlapping signals. Compounds in minor or trace amounts could not be identified because most tandem mass spectrometry information was lost. Herein, a molecular feature orientated precursor ion selection and tandem mass spectrometry structure elucidation method for complex Chinese medicine chemical constituent analysis was developed. The precursor ions were selected according to their two‐dimensional characteristics of retention times and mass‐to‐charge ratio ranges from herbal compounds, so that all precursor ions from herbal compounds were included and more minor chemical constituents in Chinese medicine were identified. Compared to the conventional automatic tandem mass spectrometry setups, the approach is novel and can overcome the drawback for chemical substances identification. As an example, 276 compounds from the Chinese Medicine of Yi‐Xin‐Shu capsule were identified.     

顺序注射氢化物发生-原子荧光光谱法同时测定中草药中的铅和锡

建立了一种顺序注射氢化物发生一原子荧光光谱法同时测定中草药中的铅和锡含量的方法,同时讨论了共存离子的干扰情况。结果表明,在最佳实验条件下,铅和锡的检出限分别为0．0253μg／L和0．0425μg／L,加标回收率为95．5％～103．1％,相对标准偏差小于5．2％,被测试样中共存的离子对铅和锡的测定没有干扰。该法操作方便、快速,用于中草药试样中铅和锡的同时测定,具有很好的可行性和实用性。     

Identification and determination of geniposide contained in Gardenia jasminoides and in two preparations of mixed traditional Chinese medicines

A high-performance liquid chromatographic method was applied to the determination of the geniposide concentration in Gardenia fruit and preparations of traditional Chinese medicine using a mobile phase of acetonitrile-methanol-5 mM monosodium phosphate (pH 4.6) (5:15:80, v/v/v). Intra-assay and inter-assay accuracy and precision of the analyses were < or = 10% in the range of 0.1 through 50 microg/ml. The presence of geniposide in the medicinal herb and its preparations was ascertained by retention time, spiking with an authentic standard, change of detection wavelength and change of the composition of the mobile phase. The concentration of geniposide in the fruit of Gardenia jasminoides Ellis var. grandiflora Nakai is higher than that in Gardenia jasminoides Ellis. The concentration of geniposide in the traditional Chinese herbal medicine preparations, Huang-Lian-Jiee-Dwu-Tang (66.27 +/- 1.98 mg/g) and In-Chern-Hau-Tang (68.54 +/- 2.62 mg/g) was less than in the herb Gardenia jasminoides Ellis (73.44 +/- 2.62 mg/g) itself.     

Determination of Sulfite in Botanical Medicine Using Headspace Thin-Film Microextraction and Surface Enhanced Raman Spectrometry

A facile method using headspace thin-film microextraction (HS-TFME) coupled with surface enhanced Raman spectrometry (SERS) has been developed for the determination of sulfite in traditional Chinese herbal medicine. The extraction substrate was synthesized by depositing urchin-like ZnO micron particles on glass sheets using chemical liquid phase deposition. Under the optimal conditions, the intensity of the SERS signal at 630–640?cm^?1 provided a good linear relationship with the concentration of sulfite from 25 to 400?mg/kg, and the linear correlation coefficient (R) was 0.996 with a detection limit of 6?mg/kg. The method was employed for the determination of sulfite in herbal medicines, and the results were confirmed by a traditional distillation-titration method. Therefore, this developed HS-TFME-SERS method may play an important role in the rapid, simple, and selective determination of sulfite residues in Chinese herbal medicine and become a potentially universal method for this analyte in various solid samples.     

Detecting aristolochic acids in herbal remedies by liquid chromatography/serial mass spectrometry

Targeted liquid chromatography/serial mass spectrometry (LC/MS/MS) analysis, using a quadrupole ion-trap mass spectrometer, permitted the detection of aristolochic acids I and II in crude 70% methanol extracts of multi-component herbal remedies without any clean-up or concentration stages. The best ionisation characteristics were obtained using atmospheric pressure chemical ionisation (APCI) and by including ammonium ions in the mobile phase. Limits of detection for aristolochic acids were influenced by the level of interference created by other components in the sample matrix. They were determined to be between 250 pg and 2.5 ng on-column within a matrix containing compounds extracted from 2 mg of herbal remedy. With a herbal remedy that only permitted the higher limit of detection, this sensitivity was sufficient to detect the aristolochic acids extracted from 0.1% dry weight of Aristolochia manshuriensis included in the preparation.     

中草药中微量锰的测定

以Ｍｎ＾２＋催化ＫＩＯ４氧化结晶紫为指示反应，用催化滴定法测定了菌陈、丹皮等六种中草药中的锰含量，并用原子吸收分光光度法作对照试验，结果满意。实验表明，该法兼具催化动力学分析法的高灵敏度和滴定分析的高准确度的特点，且仪器简单，操作方便。