中药的毛细管电泳指纹图谱研究

中药是我国医药中的瑰宝,是中国入世后最有竞争力的品种之一。虽然建国以来,对中药的内在质量标准的研究受到高度重视,但不少中药的鉴定方法仍处于避难就易、不足以真实反映其内在质量的状态。缺乏能真实反映中药内在质量的质量标准,成了中药现代化与出口的瓶颈。中药成分非常复杂,虽然用现代分析手段,可以搞清其某些成分,但由于中药的药效往往是多组分的协同作用,少数成分不足以表征中药的总体药效,因而出现了用指纹图谱总体评价中药质量的方法[1]。指纹图谱是当前中药质量研究的热门课题。　　指纹图谱是指某(或某产地)中药材或中成药…     

逍遥丸的毛细管电泳指纹图谱的建立

采用毛细管区带电泳法建立了逍遥丸(Xiaoyao Pill, XYP)的毛细管电泳指纹图谱(CEFP)。运用正方形优化法,以色谱指纹图谱分离量指数(RF)为优化的目标函数,对建立指纹图谱的实验条件进行了优化,确定了最佳背景电解质(BGE)溶液50 mmol/L硼砂-50 mmol/L磷酸氢二钠-150 mmol/L磷酸二氢钠-50 mmol/L碳酸氢钠(1:1:1:5, v/v/v/v; pH 7.40)、紫外检测波长228 nm、运行电压12 kV、重力进样25 s (高度14 cm)的分离检测条件。采用未涂层石英毛细管(70 cm×75 μm,有效分离长度57 cm)分离,以咖啡酸色谱峰为参照,确定13批逍遥丸样品的21个共有指纹峰。通过聚类分析确定用其中10批样品生成对照CEFP,以此为标准用系统指纹定量法鉴别13批逍遥丸的质量,结果显示: S3号样品的化学成分数量和分布比例不合格,S10和S12号样品含量明显偏高,其余批次质量均合格。所建立的正方形优化法操作简便,适用于中药的毛细管区带电泳BGE的选择;所建立的逍遥丸CEFP具有较好的精密度和重现性,可以为逍遥丸的质量控制提供新的参考。     

大青叶的毛细管电泳指纹图谱研究

采用高效毛细管电泳法,紫外检测波长228nm,电压12．5kV,以硼酸(100mmol／L)：硼砂(50mmol／L)=11：10(调pH11)为背景电解质进行指纹图谱研究。建立了大青叶药材的电泳指纹图谱(CEFP)。以胞苷峰为参照物峰,确定了18个共有峰,测定了10个产地大青叶的CEFP与共有模式间具有良好的相似性,用色谱指纹图谱指数对其进行评价。所建立的CEFP具有较好的重现性,可用于大青叶药材的质量控制。     

黄柏的非水毛细管电泳指纹图谱研究

采用非水毛细管电泳(NACE)法,以pH5.8的醋酸盐-甲醇为背景电解质溶液,运行电压25kV,温度20℃,检测波长210nm,正极压力(5kPa×10s)进样,建立了黄柏药材的NACE指纹图谱。以氯化两面针碱为内标,确定黄柏的NACE指纹峰为13个。测定了10个产地黄柏的NACE指纹图谱,与对照NACE指纹图谱的相似度为0.718~0.982。本方法具有较好的精密度,各指纹峰相对迁移时间的相对标准偏差(RSD)<1.5%,相对峰面积的RSD≤5%。方法可用于黄柏药材的质量控制。     

新疆紫草的毛细管电泳指纹图谱研究

建立了基于毛细管电泳技术的新疆紫草指纹图谱的质量控制方法.优化后的电泳条件:分离柱为50μm×40 cm未涂层毛细管柱,运行缓冲液为pH 8.0、100 mmol/L硼酸盐缓冲液(含25 mmol/L SDS及20%(V/V)无水乙醇),进样量0.5 psi×5 s,分离电压25 kV,检测波长214 nm.应用此条件,35 min内可实现新疆紫草有效成分的高效分离.在方法学验证的基础上,建立了新疆紫草指纹图谱.以新疆紫草对照药材指纹图谱为对照图谱,通过特征指纹峰、SFˊ相似度评价、聚类分析对不同购买地的新疆紫草进行质量评价和鉴别.此研究结果与其它中药鉴定方法对照结果一致.本方法准确、可靠、用时短,且具有良好的重现性,为新疆紫草的质量控制与评价提供了一种新的快速有效的鉴别方法.     

柏子养心丸的毛细管电泳指纹图谱

建立了柏子养心丸(Baizi Yangxin Wan, BZYXW)毛细管区带电泳指纹图谱(CEFP)。采用三棱柱优化法优化背景电解质(BGE),以色谱指纹图谱分离量指数(RF)为实验条件优化的目标函数,最终确定BGE为50 mmol/L硼砂-50 mmol/L磷酸氢二钠-200 mmol/L硼酸-150 mmol/L碳酸氢钠(体积比为7:7:1:1,含4%乙腈,pH 9.70)。在其他优化的毛细管电泳条件为紫外检测波长228 nm,运行电压12 kV,重力进样25 s (高度14 cm)条件下,采用未涂层石英毛细管(70 cm(有效分离长度57 cm)×75 μm)分离,以阿魏酸峰为参照,确定了17个共有指纹峰。对样品聚类分析后用其中9批生成对照CEFP(RCEFP),以其为标准,用系统指纹定量法鉴别出12批BZYXW质量为: 3批好,1批良好,3批中,1批一般,4批劣。三棱柱优化法为BGE选择提供了重要参考,建立的BZYXW-CEFP精密度好、重现性高,可用于BZYXW的质量控制。     

不同产地金银花药材的HPCE指纹图谱分析

以40mmoL/L硼砂溶液为缓冲液,利用高效毛细管电泳(HPCE)法,建立不同产地金银花的HPCE指纹图谱并测定其中绿原酸的含量。工作电压为17kV,温度为25℃,压力进样时间为6s。不同产地金银花药材中绿原酸的含量为3.2%4.4%。通过对HPCE指纹图谱共有峰数据进行判别分析和聚类分析,可以将不同产地的金银花进行判别分类。HPCE指纹图谱可以用于较全面地考察金银花药材的质量。     

连翘的毛细管电泳指纹图谱研究

采用毛细管区带电泳法,以75 mmol/L硼砂溶液(用0.1 mmol/L氢氧化钠溶液调pH 9.7)为背景电解质,运行电压14 kV,检测波长228 nm,重力进样15 s(高度9 cm),建立了连翘药材的毛细管电泳指纹图谱(CEFP)。将10个不同产地的连翘药材进行比较,按各电泳峰共有率fi≥70%作为选择电泳指纹峰的依据,确定连翘的毛细管电泳指纹峰为29个。在方法的精密度和重复性试验中,各指纹峰的相对迁移时间的相对标准偏差(RSD)不大于5%,相对峰面积的RSD约为2%~15%。10个产地的连翘药材的CEFP与其对照CEFP的相似度均不低于0.94。同时,采用指纹图谱信息量指数I和相对信息量指数Ir评价了10个产地连翘药材的质量差异。     

基于毛细管电泳的黄芪抗氧化生物指纹图谱研究

根据评价抗氧化剂清除自由基能力的间接化学发光法,结合具有高效分离能力的毛细管电泳技术,建立了能表征中药抗氧化活性的生物指纹图谱。选取中药黄芪为研究对象,以黄芪甲苷为参照物峰,确定了6个共有峰,并首次测得黄芪甲苷对羟自由基的清除率。各共有峰相对迁移时间的RSD<1.5%,相对自由基清除率的RSD<8.4%,表明方法的精密度和重复性较好。7个产地的黄芪药材与道地药材具有良好的抗氧化活性相似度,相似度>96.5%。抗氧化生物指纹图谱对天然产物的质量控制与临床研究具有指导意义。     

毛细管电泳指纹图谱应用于叶下珠药材中黄酮的检测

建立了叶下珠药材中黄酮的毛细管电泳指纹图谱。以硼砂和十二烷基磺酸钠(pH9.0)为背景电解质溶液,运行电压15kV,紫外检测波长245nm,流体静压力进样15s(高度12cm),对不同产地叶下珠药材进样检测。按电泳峰共有率fi≥70%为依据,确定10个不同产地叶下珠药材中黄酮的毛细管电泳指纹峰为8个,各产地叶下珠的毛细管电泳指纹图谱与标准毛细管电泳指纹图谱的相似度较好。在制备供试液后不同时间进样测定,各指纹峰的相对迁移时间的相对标准偏差小于5%,相对峰面积的相对标准偏差在3.0%～7.8%之间,结果表明样品在48h内稳定。     

Simultaneous determination of iridoids, phenolic acids, flavonoids, and saponins in Flos Lonicerae and Flos Lonicerae Japonicae by HPLC-DAD-ELSD coupled with principal component analysis

A method, HPLC coupled with diode-array and evaporative light scattering detectors (HPLC-DAD-ELSD), was newly developed to evaluate the quality of Flos Lonicerae (FL) and Flos Lonicerae Japonicae (FLJ), through a simultaneous determination of multiple types of bioactive components. By employing DAD, the detection wavelengths were set at 240 nm for the determination of iridoids, 330 nm for phenolic acids, and 360 nm for flavonoids, respectively. While ELSD, connected in series after DAD, was applied to the determination of saponins. This assay was fully validated with respect to precision, repeatability, and accuracy. Moreover, principal component analysis (PCA) was used for the similarity evaluation of different samples, and it was proven straightforward and reliable to differentiate FL and FLJ samples from different origins. For PCA, two principal components have been extracted. Principal component 1 (PC1) influences the separation between different sample sets, capturing 54.598% variance, while principal component 2 (PC2) affects differentiation within sample sets, capturing 12.579% variance. In conclusion, simultaneous quantification of bioactive components by HPLC-DAD-ELSD coupled with PCA would be a well-acceptable strategy to differentiate the sources and to comprehensively control the quality of the medicinal plants FL and FLJ.     

Binding study of Flos Lonicerae Japonicae with bovine serum albumin using centrifugal ultrafiltration and liquid chromatography

The screening and analysis of bioactive components in traditional Chinese medicines (TCMs) is very important not only for the quality control of Chinese herbs but also for elucidating the therapeutic principles. This study developed a new method for screening and analyzing bioactive compounds from TCMs using centrifugal ultrafiltration coupled with high-performance liquid chromatography. The method was successfully applied in the binding study of Flos Lonicerae Japonicae with bovine serum albumin (BSA), and 11 compounds were found to be bound with the BSA. Eight of them were positively identified as chlorogenic acid, caffeic acid, rutin, quercetin-3-O-glucoside, luteolin-7-O-glucoside, lonicerin, 3, 5-di-O-caffeoyl quinic acid and 3,4-di-O-caffeoyl quinic acid. Another three compounds were tentatively identified as two isomers of chlorogenic acid and one isomer of di-O-caffeoyl quinic acid by comparing the UV data and MS data with the previous reports. Based on modern pharmacological study, these compounds are the major bioactive components in Lonicera japonica. Therefore, the proposed method could be a good approach to predicting the potential bioactivities of multiple compounds in TCMs simultaneously.     

Plasma metabolomics analysis of endogenous and exogenous metabolites in the rat after administration of Lonicerae Japonicae Flos

Lonicerae Japonicae Flos (LJF) is a typical herbal medicine and is used as a functional food. LJF, which has complex chemical compounds, has various biological effects. The global metabolomics, focusing on both the endogenous and exogenous metabolites, have not yet been investigated for LJF in normal healthy rats using LC–MS. In this study, plasma metabolomics was analyzed after the administration of LJF at different time intervals, and the exogenous metabolites were identified. Partial least squares discriminant analysis showed significant differences in chemical content in the dosed rats. Cholic acid, indoleacrylic acid, indolelactic acid, hippuric acid, N-acetyl-phenylalanine, and N-acetyl-serotonin significantly accumulated in the dosed rats. Lysophosphatidylethanolamine and lysophosphatidylcholine content, including plasmalogen, increased. There were 25 components of LJF, including 15 prototypes and 10 metabolites, that were identified. The 15 prototypes included phenolic acids, flavonoids, and iridoids, and their contents decreased with an increase in the administration time. Glucuronidation and sulfation of polyphenols were found for LJF. The exogenous glucuronide and sulfate metabolites—including dihydrocoumaric acid-sulfate, dihydrocaffeic acid-sulfate, dihydroferulic acid-sulfate, apigenin-glucuronide, apigenin-glucuronide-sulfate, isorhamnetin-glucuronide-sulfate, and others—were identified with a neutral loss of 176 and 80, respectively. The metabolic differences found in the study may serve as biomarkers of LJF consumption and promote the understanding of the mechanism of action of LJF.     

Rapid and simple method for screening of natural antioxidants from Chinese herb Flos Lonicerae Japonicae by DPPH-HPLC-DAD-TOF/MS

A rapid and simple method has been developed for the screening and identification of natural antioxidants of Flos Lonicerae Japonicae (FLJ), derived from the flower buds of Lonicera japonica. The hypothesis is that upon reaction with 1,1-diphenyl-2-picrylhydrazyl (DPPH), the peak areas (PAs) of compounds with potential antioxidant effects in the HPLC chromatograms will be significantly reduced or disappeared, and the identity confirmation could be achieved by HPLC-DAD-TOF/MS hyphenated technique. Using the proposed approach, about 14 compounds in the FLJ extract were found to possess a potential antioxidant activity. They were identified as chlorogenic acid (1), 1-O-caffeoylquinic acid (1-O-CQA, 2), caffeic acid (4), 4-O-CQA (5), rutin (7), isoquercitrin (8), luteolin-7-O-glucoside (9), lonicerin (10), 4,5-O-dicaffeoylquinic acid (4,5-O-diCQA, 11), 3,5-O-diCQA (12), 1,3-O-diCQA (13), 3,4-O-diCQA (14), 1,4-O-diCQA (16), and luteolin (17). In addition, the free radical scavenging capacities of the available identified compounds were also investigated by HPLC assay. The results indicated that the compounds with PAs significantly decreasing were natural antioxidants, whereas those with PAs not changing presented no activities, which accordingly indicated that this newly proposed method could be widely applied for rapid screening and identification of natural antioxidants from complex matrices including Chinese herbal medicines.     

西他沙星差向异构体的毛细管电泳分离

以γ-环糊精和D-苯丙氨酸为手性选择剂,采用毛细管区带电泳成功地分离了新型抗菌素西他沙星差向异构体。考察了添加剂的种类、浓度和缓冲溶液的pH对毛细管电泳分离西他沙星的影响。分离电压为15 kV,选用60 cm(有效长度52.5 cm)×50 μm i.d.的石英毛细管,缓冲溶液组成为10 mmol/L KH2PO4-K2HPO4(pH 4.5),10 mmol/L CuSO4,20 mmol/L γ-环糊精和 10 mmol/L D-苯丙氨酸。实验结果表明,添加剂的种类和浓度是影响西他沙星手性分离的重要因素,只有当 D-苯丙氨酸、铜离子和γ-环糊精同时存在并达到一定浓度时,西他沙星差向异构体在毛细管电泳中才具有良好的分离效果。该方法可用于西他沙星差向异构体的定量分析。     

毛细管电泳样品电堆积富集过程的数值研究

毛细管电泳样品电堆积富集过程可以浓缩样品组分,从而提高检测灵敏度,是一种有效的样品富集技术。本文通过合理的简化和假设,把毛细管中电堆积富集过程中所涉及的主要变量根据电势分布方程、缓冲溶液的浓度方程和样品粒子的质量传输方程进行耦合求解,建立了一个一维的数学模型,并应用有限元的方法对该模型进行了求解。计算结果给出了毛细管中缓冲溶液浓度及电场强度的分布随时间变化的过程,以及富集过程中毛细管中的电势分布曲线;得到了样品粒子浓度在电堆积富集过程和富集之后的再次扩散过程中的分布曲线以及正、负样品粒子的分离过程;最后分析了不同缓冲溶液浓度比对样品富集效果的影响。该研究为样品电堆积富集技术的进一步完善提供了一种简单可行的理论研究方法。     

毛细管电泳法同时分析茶黄素类和儿茶素类化合物

为了建立一种快速、准确、简便的同时分析茶黄素类和儿茶素类化合物的毛细管电泳方法以满足茶黄素体外氧化制备过程监测和茶多酚酶促氧化动力学研究的需要,研究了毛细管电泳同时分析4种茶黄素类和6种儿茶素类化合物的最佳分析条件,并将建立的方法进行应用评价。结果表明:以含有200 mmol/L H3BO3(pH 7.7)、10 mmol/L KH2PO4、9 mmol/L β-环糊精和27.5%乙腈为电泳介质,在电压25 kV、柱温30 ℃下分离和200 nm波长处检测,可在8 min内将10种待测组分全部分离,且各组分的浓度与峰面积呈良好的线性关系,相关系数r为0.9907～0.9998,检测限为0.39～0.88 μg/mL,各组分的加标回收率为91.5%～113.5%,相对标准偏差小于5%。     

高效毛细管电泳I. 分析化学中的一个新的前沿领域

本文是关于高效毛细管电泳(HPCE)的详细综述的第一部分,概括了HPCE技术发展的历史、国内外研究现状,总结了HPCE的基本原理、主要操作方式以及HPCE所具有的特点,并讨论了该技术目前存在的问题及可能的解决方法。     

基于超高效液相色谱联用高分辨质谱自动解析方法的金银花不同加工方式分析

金银花干燥加工方式直接影响其药用价值及经济效益。 采用基于超高效液相色谱联用静电场轨道阱高分辨质谱的非靶向代谢组学技术结合化学计量学自动化数据分析策略,分析了不同加工方式对金银花中化学成分的影响。金银花样品经甲醇-水（8∶2,体积比）溶液超声提取30 min,两次离心后取上清液,以Waters Acquity UPLC HSS T3色谱柱（2.1×100 mm, 1.8 μm）分离,乙腈-水溶液（均含有体积分数0.1%的甲酸）进行梯度洗脱。高分辨质谱选择正离子扫描模式（ESI+）并采用FULL MS/DD-MS2（TOP4）模式完成数据采集。在自动化解析方法软件平台对仪器采集的原始信号直接解析,鉴定出绿原酸、芦丁和异槲皮苜等16种差异性化合物。在化学成分解析的基础上,利用层次聚类分析和主成分分析等方法评价了晒干、阴干、电热烘干、烘房烘干、热泵烘干和真空冷冻干燥这6种加工方式获得的金银花样品品质差异。真空冷冻干燥、烘房烘干和热泵干燥等加工方式能够获得外观特征优质的金银花。在真空冷冻干燥方式下,金银花样品中的抗氧化功能性成分能够得到更好的保存。