Biological Fingerprinting Analysis of Interaction Between Taxoids in Taxus and Microtubule Protein by Microdialysis Coupled with High-performance Liquid Chromatography/Mass Spectrometry for Screening Antimicrotubule Agents

Some natural products, such as traditional Chinese medicines（TCMs）, contain compounds with anticancer activity and have attracted a great interest in recent years as alternative anticancer therapies. A quick and convenient assay for screening antimicrotubule compounds in which in vitro microdialysis/high-performance liquid chromatography （HPLC） is used to monitor the binding of the compounds extracted from TCM Taxus cuspidata Siebold ＆ Zucc（Taxus） to microtubules is reported. It was observed that the extract of Taxus contains at least five compounds which have affinity interaction with microtubules by biological fingerprinting analysis, and they were identified as the taxoids of taxol, baccatin III, 10-deacetylbaccatin Ⅲ（10-DAB）, cephalomannine and 7-epi-10-deacetyltaxol （7-epi-10-DAT） based on the comparison of their high-performance liquid chromatographic/mass spectrometric and UV spectra with those of the standard samples, both assembly-promoting and disassembly-inhibiting characteristics of those compounds were evaluated. It was observed that baccatin Ⅲ and 10-DAB bound to microtubules and the binding degrees were influenced by GTP. Competitive binding behavior of taxol with other four taxoids to microtubules was also investigated.

Quantification of six bioactive compounds in Zhenqi Fuzheng preparation by high-performance liquid chromatography coupled with diode array detector and evaporative light scattering detector

A simple and accurate high-performance liquid chromatography（HPLC）coupled with diode array detector（DAD）and evaporative light scattering detector（ELSD）was established for the determination of six bioactive compounds in Zhenqi Fuzheng preparation（ZFP）.The monitoring wavelengths were 254,275 and 328 nm.Under the optimum conditions,good separation was achieved,and the assay was fully validated in respect of precision,repeatability and accuracy.The proposed method was successfully applied to quantify the six ingredients in 31 batches of ZFP samples and evaluate the variation by hierarchical cluster analysis（HCA）,which demonstrated significant variations on the content of these compounds in the samples from different manufacturers with different preparation procedures.The developed HPLC method can be used as a valid analytical method to evaluate the intrinsic quality of this preparation.

Extraction and RP-HPLC determination of taxol in rat plasma, cell culture and quality control samples

A rapid, sensitive, selective and validated reverse phase high-performance liquid chromatography （RP-HPLC） method for the estimation of paclitaxel in micro-sample of rat plasma and in culture of cancer cells was per- formed in this study. The mobile phase consisted of an （80： 20： 0.1, v/v/v）. Column elution at a flow rate of 1 optimized mixture of methanol：water： trifluroacetic acid mL/minute with UV detection at 225 nm at room tern- perature was used. The RP-HPLC method was successfully applied for the determination of paclitaxel in plasma samples and in culture of cancer cells with nano-quantity of estimation. The validation studies were performed in accordance with the International Conference on Harmonization （ICH） guidelines. The intra- and inter-day pre- cision showed that the coefficients of variation ranged from 1.07% to 4.27% at different levels of concentrations. To the best of our knowledge, this study also reported for the first time the optimization of different solvents for effective extraction of paclitaxel wherein tert.-butyl methyl ether （TBME）： diethyl ether （DEE） in 50：50 v/ v composition was found most efficient with extraction efficiency ranging between 77.99% and 91.74% and be- tween 76.14 and 93.66% in the plasma and cell culture, respectively. This proposed method was successfully ap- plied to study the pharmacokinetics of paclitaxel and the influence of verapamil and all-trans retinoic acid （atRA） on paclitaxel pharmacokinetics in rat models. This proposed method might emerge as a valuable aid in the labo- ratory monitoring of paclitaxel in a variety of in vitro as well as in vivo scenarios.

Determination of Four Pesticides in Soil by Homogeneous Ionic Liquid-based Microextraction Coupled with High-performance Liquid Chromatography

Homogeneous ionic liquid microextraction was developed for the simultaneceus extraction of dimetho- morph, mefenacet, isoprothiolane and oxadiazon from soil. 1-Butyl-3-methylimidazolium tetrafluoroborate was used as extraction solvent, and ammonium hexafluorophosphate was used as ion-pairing agent. High-performance liquid chromatography（HPLC） was employed for separation and determination of the analytes. The calibration curves show good linear relationship（r〉0.9988）. The recoveries are between 74.2% and 97.9% with relative standard devia- tions（RSDs） lower than 5.97%. The present method is free of volatile organic solvents, and expenditures of sample, extraction time and solvent are lower, compared with ultrasonic and Soxhlet extraction. There was no obvious diffe- rence in the extraction recoveries of pesticides obtained by the three extraction methods.

HPLC Fingerprint Chromatogram of Tetracera asiatica Produced in Guangxi

[Objectives] To establish fingerprint chromatogram of Tetracera asiatica and provide basis for controlling and assessing the quality of Tetracera asiatica.[Methods] The high-performance liquid chromatography( HPLC) method was used. Phenomenex C18( 5 μm,4. 6 ×250 mm),acetonitrile-0. 1% phosphoric acid water,gradient elution,flow rate 1 m L/min,detection wavelength 214 nm,and column temperature 25 ℃. The fingerprint chromatogram of 10 batches of samples of Tetracera asiatica was determined for similarity comparison. [Results] It established HPLC fingerprint chromatogram of Tetracera asiatica produced in Guangxi,determined a total of 13 common fingerprint peaks. The similarity of 10 batches of Tetracera asiatica was > 0. 9.[Conclusions] The method is rapid and accurate and can provide references for assessing the quality of Tetracera asiatica.

Quality Control of Zhuang Medicine Xiaoyan Zhiyang Lotion

[Objectives]To establish the TLC identification methods of Llicis Rotundae Cortex and Mahoniae Caulis and the determination method of syringin content in Zhuang Medicine Xiaoyan Zhiyang Lotion.[Methods]Thin layer chromatography(TLC)was used to qualitatively identify Llicis Rotundae Cortex and Mahoniae Caulis in Zhuang Medicine Xiaoyan Zhiyang Lotion.The quantitative analysis of syringin in Zhuang Medicine Xiaoyan Zhiyang Lotion was carried out by high-performance liquid chromatography(HPLC).Chromatographic conditions are as follows:Luna 5μm C_(18)((2)100 A(250 mm×4.6 mm,5μm)column was adopted;acetonitrile(10%)-water(90%)was as the mobile phase,and the flow rate was 1.0 mL/min;the detection wavelength was 265 nm;the sample size was 5μL;the column temperature was 30℃.[Results]Syringin had a good linear relationship with its peak area in the range of 0.190-2.856μg(R^(2)=0.9995).The RSD values of precision,stability and repeatability experiments were all lower than 2.5%,and the average sampling recovery rate(n=6)was 100.37%(RSD=-0.88%).[Conclusions]The method had the advantages of simple operation,strong specificity,good repeatability and stability,and can be used for the quality control of Zhuang Medicine Xiaoyan Zhiyang Lotion.

微透析技术在植物生理生化研究中的应用

微透析是一种可以应用于生物体体内和体外的样品处理技术。可以与多种分析测试仪器耦合,对细胞外空间或其他水溶液环境中随时间、浓度发生变化的生理活性物质提供实时动态的监测信息。其突出优点是在不影响生物体正常生长的情况下可以准确地给出被分析物随时间的变化情况,而且可以对目标生物体进行各种处理,以得出被分析物受外界环境影响下的即时变化情况,而且微透析的样品在用高精密仪器分析时,无需进行样品前处理,可真正做到实时、在线、廉价的跟踪检测研究。阐述了微透析在植物体上的应用,微透析技术应用在动物体,主要取样是细胞间质,在植物体上可以检测植物质外体各种物质的变化,克服以往研究质外体的瓶颈,还包括了微透析技术与GC、HPLC、CE等分析仪器的耦合使用研究现状。

α-氨基丁酸为内标物高效液相法测定脑微透析液中氨基酸含量

目的研究以α 氨基丁酸 (AABA)为内标物 ,用高效液相色谱仪测定大鼠脑微透析液中的神经递质类氨基酸含量。方法以AABA为内标 ,用高效液相色谱仪测定大鼠脑微透析液中的神经递质类氨基酸含量 ,采用OPA柱前衍生 ,ODS C18色谱柱 ,荧光检测器 ,AABA作为内标 ,使用磷酸缓冲液和甲醇二元梯度洗脱方法 ,3 5min内完成。结果氨基酸与内标在给定条件下分离良好 ,每种氨基酸的回收率为 88.2 %— 10 2 .3 % ,线性回归相关系数平均为 0 .998± 0 .0 0 15 ,每种氨基酸的检测限为 1.0— 8.6ppm。 结论本方法准确、灵敏 。

高效液相色谱法测定补骨脂中补骨脂素和异补骨脂素的分析

建立高效液相色谱测定补骨脂中的补骨脂素(Psoralen)和异补骨脂素(Isopsoralen)的方法。色谱条件为Ecosil C18(200 mm×4.6 mm,5μm)色谱柱、水-甲醇溶液为流动相、等梯度洗脱、波长246 nm,流速1.0 m L/min,柱温30℃。采用这种方法,补骨脂素和异补骨脂素能达到基线分离的效果,线性关系良好,具有良好的稳定性和准确度。

Effect of single-use versus combined-use moschus and diazepam on expression of amino acid neurotransmitters in the rat corpus striatum

The present study analyzed expressional changes of excitatory neurotransmitters and inhibitory neurotransmitters in the rat corpus striatum after single-use and combined-use diazepam and Chinese herb moschus. The influence of moschus on the central nervous system was analyzed, in particular whether moschus increased penetration of other drugs into the brain. Reverse-phase high-performance liquid chromatography, which included pre-column derivation with orthophthaladehyde detection, showed varied increased levels of excitatory neurotransmitters, including aspartate and glutamate, and inhibitory neurotransmitters, including glycine and Y-aminobutyric acid, in the corpus striatum after treatment with moschus alone, diazepam alone, or a combination of both. Compared with the diazepam group, aspartate levels significantly decreased at 30 and 60-105 minutes after combined treatment with moschus, while glutamate significantly increased at 45 and 75-105 minutes, glycine levels significantly increased at 105 minutes, and γ-aminobutyric acid increased at 30 and 75-105 minutes. These findings suggested that moschus increased the inhibition effects of diazepam on the brain.

Effect of borneol, moschus, storax, and acorus tatarinowii on expression levels of four amino acid neurotransmitters in the rat corpus striatum

The present study collected cerebrospinal fluid samples from the corpus striatum in rats treated with borneol, moschus, storax, and acorus tatarinowii using brain microdialysis technology. Levels of excitatory neurotransmitters aspartic acid and glutamate, as well as inhibitory neurotransmitters glycine and ^-aminobutyric acid, were measured in samples using reversed-phase high-performance liquid chromatography, phosphate gradient elution, and fluorescence detection. Results showed that concentrations of all four amino acid neurotransmitters significantly increased in the corpus striatum following treatment with borneol or moschus, but effects due to borneol were more significant than moschus. Acorus tatarinowii treatment increased ^-aminobutyric acid expression, but decreased glutamate concentrations. Storax increased aspartic acid concentrations and decreased glycine expression. Results demonstrated that borneol and moschus exhibited significant effects on con amino acid neurotransmitter expression; storax exhibited excitatory effects and acorus tatarinowii resulted in inhibitory effects.

Relationship between morphospecies and microcystin-producing genotypes of Microcystis species in Chinese freshwaters

Twenty water bodies in China were sampled,and 186 strains of different Microcystis species were isolated,from which eight morpho species were identified and 43 stains containing the mcyB gene were detected.Phylogenetic analysis based on the mcyB gene indicated that the microcystin(MC)-producing Microcystis in China could be divided into two groups(ⅠandⅡ)and showed significant differences between the two groups.The maximum sequence similarity was 69.1%.Microcystins(MCs)were measured by high-performance liquid chromatography(HPLC)analysis,and no microcystin-RR(MC-RR)was detected in some strains belonging to GroupⅡ.Compared to other regions of the world,the proportion of Chinese MC-producing was different,and the regional differences were more obvious.A whole-cell polymerase chain reactio(PCR)assay was conducted to analyze the proportion of the mcyB gene in the laboratory cultured and field cultured Microcystis.The proportion of four morphospecies(M.vividis,M.ichthyoblabe,M.novacekii,and M.aeruginosa)that contained the mcyB gene exceeded 50%in the field cultured sample s.Compared with former studies,M.aeruginosa was the mo st likely morphotype that can produce MCs in the world.This study provided new insight of Microcystis hazard assessment and field monitoring.

Separation and Detection of Ceramides by HPLC Followed by Evaporative Light-Scattering Detection and Thin Layer Chromatography

Ceramides are important signaling molecules involved in a variety of cellular processes, including cell growth, differentiation, and apoptosis. Currently, different methods are used for ceramide analysis, some of which are insensitive or cumbersome. This paper described methods utilizing thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) followed by evaporative light scattering detection (ELSD) to detect ceramide directly in cell extracts without derivatization, which was proved to be efficient and reproducible. Five kinds of ceramides were used as standards. Both TLC and normal-phase HPLC/ELSD results indicate that yeast contains several kinds of ceramides.

Determination of Nine Phenolic Components in Leaves of Crataegus pinnatifida Bge.

[Objectives] To determine the nine phenolic components in the leaves of Crataegus pinnatifida Bge. [Methods] The reversedphase high-performance liquid chromatography( RP-HPLC) was applied. [Results] Nine phenolic components showed a good linear relationship in the range of 2-500 μg/m L with r in the range of 0. 999 5-0. 999 9. The recovery rate of spiked samples ranged from 93. 7% to110. 2%,and the relative standard deviation was in the range of 0. 69%-4. 58%. The leaves of 29 cultivars of C. pinnatifida Bge. were measured,and the average content of the nine phenolic components was as follows: isoquercitrin,hyperoside,procyanidin C1,procyanidin D1,epicatechin,procyanidin B2,chlorogenic acid,eucomic acid,and vitexin 2 "-O-rhamnoside. The contents of flavonoids and phenolic acids were high,up to 15 mg/g D. W,and the content of procyanidins was up to 6 mg/g D. W. [Conclusions]This method is easy and accurate in determination of phenolic components in the leaves of C. pinnatifida Bge.

Determination of Total Anthraquinone and Free Anthraquinone in Different Processed Products of Rhubarb

[Objectives]The content of total anthraquinone and 5 free anthraquinones in different processed products of rhubarb were determined.[Methods]Using emodin as the reference,the concentration of 1%magnesium acetate methanol solution was used for color development,and the absorbance was measured at 516 nm wavelength;the mobile phase was methanol-0.1%phosphoric acid(76∶24)aqueous solution,the detection wavelength was set at 256 nm,the flow rate was 1.0 mL/min,the column temperature was 30℃,and the sample size was 10μL.[Results]The content of total anthraquinones in rhubarb was determined by UV spectrophotometry.The linear range was 0.98-23.52μg/mL,R2=0.9993,the average recoveries were 100.12%,99.66%and 99.15%,with RSDs of 1.58%,0.94%and 1.41%,respectively.The linear ranges of emodin,emodin,emodin,chrysophanol and emodin methyl ether were 0.0207-0.414(R2=0.9999),0.1721-3.442(R2=0.9996),0.0203-0.406(R2=0.9999),0.148-2.96(R2=0.9998)and 0.164-3.28(R2=0.9993),respectively.[Conclusions]The contents of total anthraquinones and free anthraquinones in different processed products of rhubarb were determined by UV spectrophotometry and HPLC.The method can be used for quality control of rhubarb and its different processed products.

脑缺血再灌注脑内纹状体区细胞外液谷胱甘肽的动态变化及丹参的影响

采用脑内微透析技术，应用高压液相色谱电化学检测方法（ＨＰＬＣ－ＥＤ），活体动态观察沙土鼠全脑缺血３０分钟，再灌注１２０分钟的细胞外液中的谷胱甘肽（Ｇｌｕｔａｔｈｉｏｎｅ，ＧＳＨ）的变化及丹参对它的影响。结果显示：全脑缺血后，细胞外液ＧＳＨ水平迅速升高（Ｐ＜０．０１），缺血３０分钟达高峰为缺血前的５。８２倍。再灌注后ＧＳＨ水平明显降低，于３０分钟趋于正常。脑缺血前３０分钟给予丹参注射液不影响细胞外液ＧＳＨ水平。表明脑缺血及再灌注期，ＧＳＨ反应性增高。ＧＳＨ作为内源性抗氧化剂及ＮＭＤＡ受体拮抗剂在脑缺血损伤中起重要作用。

基于微透析采样技术分析羟基喜树碱磁性脂质体在大鼠体内的药代动力学

目的:探究羟基喜树碱(HCPT)磁性脂质体在大鼠体内药代动力学特征,为该药物的制剂开发提供参考。方法:建立微透析样品中开环HCPT(C-HCPT),闭环HCPT(L-HCPT)的HPLC,SD大鼠尾静脉注射HCPT磁性脂质体和HCPT注射液,腹腔注射HCPT注射液(剂量以HCPT计均为10 mg·kg-1),于给药后采用微透析技术定时采样。微透析样品经各样本自身体内回收率校正,利用DAS 2.1.1软件计算相关药物动力学参数。结果:HCPT磁性脂质体尾静脉给药组,HCPT注射液尾静脉给药组和HCPT注射液腹腔给药组的药峰浓度(Cmax)分别为(2.789±0.158),(4.537±0.092),(0.340±0.066)mg·L-1,达峰时间(tmax)分别为(24±0),(6±0.127),(24±0.127)min,平均滞留时间(MRT0-∞)分别为(72.255±4.668),(20.325±4.288),(112.630±29.969)min,药时曲线下面积(AUC0-∞)分别为(216.870±3.271),(150.668±7.306),(34.883±10.245)mg·L-1·min;与HCPT注射液尾静脉给药组相比,HCPT磁性脂质体尾静脉给药组的tmax,MRT0-∞较大,差异有统计学意义;与HCPT注射液腹腔给药组相比,HCPT磁性脂质体尾静脉给药组的Cmax,AUC0-∞较大,差异有统计学意义。结论:HCPT磁性脂质体尾静脉注射能显著提高药物血药浓度、生物利用度及其在体内的滞留时间;且脂质体可保护HCPT活性必需基团内酯环不被破坏,从而保证HCPT的抗肿瘤活性。

基于微透析技术探讨安神定志灵对SHR大鼠前额叶NE、DA及其代谢产物含量的影响

目的:研究安神定志灵对SHR大鼠多动冲动行为及前额叶皮质单胺类神经递质及其代谢产物的影响。方法:依据随机数字表将SHR大鼠分为模型组,择思达组(0.045mg/kg),安神定志灵低、中、高剂量组(6.7,13.4,26.7g/kg),每组8只,另设WKY大鼠、SD大鼠各8只为正常组,灌胃给药28d。旷场实验观察大鼠运动距离及运动速度,微透析联合HPLC-ECD检测前额叶去甲肾上腺素(NE)、多巴胺(DA)和代谢产物3,4二羟基苯乙酸(DOPAC)、高香草酸(HVA)含量。结果:(1)给药前,模型组、择思达组及安神定志灵各剂量组运动距离及运动速度较正常组1(WKY)显著升高(P﹤0.05);给药第14、21、28天,择思达组和安神定志灵中剂量组上述指标较模型组显著降低(P﹤0.05)。(2)给药前,模型组、择思达组及安神定志灵各剂量组NE、DA、DOPAC、HVA含量较WKY组显著降低(P﹤0.05);模型组NE、HVA含量较正常组2(SD)显著降低(P﹤0.05);给药第14天,择思达组NE、DA含量较模型组显著升高(P﹤0.05);给药第28天,择思达组、安神定志灵中剂量组NE、DA含量较模型组显著升高(P﹤0.05),安神定志灵中剂量组DOPAC含量较模型组显著升高(P﹤0.05)。结论:安神定志灵能调控SHR多动、冲动行为,改善NE、DA缺乏状态。

Purines Change at Acupoints along the Pericardium Meridian in Healthy and Myocardial Ischemic Rats

Objective: To quantify the purine concentrations of the acupoints along the pericardium and nonpericardium meridians under healthy and myocardial ischemia conditions to investigate the relationship between acupoint purine change and body functional status in rats.Methods: A total of 70 rats underwent an operation for myocardial ischemia,while 40 of them survived.They were randomly assigned to the following 5 subgroups:Neiguan(PC 6),Quze(PC 3),Tianquan(PC 2),Quchi(LI 11),and Jianyu(LI 15).Simultaneously,another40 healthy rats were also randomized into the same 5 subgroups as the control group.The tissue ?uids at the acupoints were collected by microdialysis for 30 min.Subsequently,the concentration of adenosine triphosphate(ATP),adenosine diphosphate(ADP),adenosine monophosphate(AMP),and adenosine(ADO) were quanti?ed using the high-performance liquid chromatography method.Results: Compared with the healthy group,the ADO at PC 6(P=0.012),PC 3(P=0.038),PC 2(P=0.024),and LI 15(P=0.042) obviously increased in the model group,while no signi?cant difference was observed at LI 11(P=0.201).However,ATP,ADP,and AMP manifested no significant changes in these areas,except for ATP at LI 15(P=0.036).Conclusions: Myocardial ischemia could induce an increase in ADO at acupoints of the upper arm and shoulder area,suggesting that the body functional status could affect the responsiveness of acupoints.The status of these acupoints could be pathogenically activated by disease,and distribution following some speci?c courses.

Separation of mandelic acid and its derivatives with new immobilized cellulose chiral stationary phase

A new liquid chromatographic method has been developed for the chiral separation of the enantiomers of mandelic acid and their derivatives 2-chloromandelic acid, 4-hydroxymandelic acid, 4-methoxymandelic acid, and 3,4,5-trismethoxymandelic acid. The enantiomers were separated by a CHIRALPAKIC (250 mm×4.6 mm, 5 μm). Mandelic acid, 4-methoxymandelic acid, and 3,4,5-trismethoxymandelic acid were baseline resolved (resolution factor (RS )=2.21, RS =2.14, and RS =3.70, respectively). In contrast, the enantioselectivities between CHIRALPAKIC and 2-chloromandelic acid and 4-hydroxymandelic acid investigated were low. By comparing the chromatographs of mandelic acid enantiomers and mandelic acid spiked with (R)-mandelic acid, it was determined that the first effluent was (R)-mandelic acid.