葛根芩连汤血清药化学研究

上海交通大学硕士学位论文葛根芩连汤血清药化学研究姓名：金慧申请学位级别：硕士专业：药物分析学指导教师：闫超20081201葛根芩连汤血清药化学研究摘要中药复方葛根芩连汤源于《伤寒论》，处方由葛根、黄芩、黄连和甘草四味中药组成，具有解表清里、升清止利之效。

传统中药复方研究主要针对单味及复方给药后一个或数个有效成分进行药代动力学研究，但这无法反映复方整体的体内动态过程。

基于血清中含有的成分才是中药体内的直接作用物质，本研究采用血清药化学的研究思路，以口服葛根芩连汤后的大鼠血浆为研究对象，将多种现代技术综合应用，从血清中分离、鉴定移行成分，研究其与传统疗效的相关性，并构建血浆药代指纹图谱，阐明体内直接作用物质的代谢及体内的动态过程。

具体内容分为以下四个部分：1. 以大鼠口服葛根芩连汤后的血浆为研究对象，建立葛根芩连汤入血成分的分析方法：生物样本中主要干扰分析测定的组分是蛋白质。

本研究通过实验筛选出甲醇沉淀血浆中蛋白的前处理方法，样品经氮气吹干，用甲醇复溶后以12000 r/min的转速离心后得到的样品直接用于HPLC分析。

通过优化波长和流动相的pH等HPLC的条件，确定在270 nm的波长下，用乙腈和20 mM的乙酸铵溶液对大鼠血浆样品进行梯度洗脱。

2. 葛根芩连汤入血成分的归属：比较空白血浆加葛根芩连汤提取物、大鼠口服复方及各单药后的含药血浆色谱图，对复方给药后的入血成分进行归属。

借鉴指纹图谱的研究思路，以保留时间为依据，对复方含药血浆中的31个成分进行归属，其中15个来源于中药，13个为体内代谢产物，3个为进入体内后产生的新物质。

3. 建立了液相色谱-二极管阵列-质谱联用（HPLC-DAD-MS）对大鼠口服葛根芩连汤后的血浆样品分析鉴定的方法：首先根据葛根素、黄芩苷、小檗碱、巴马汀和药根碱这五个标准品来优化质谱条件，在质谱正负离子模式下分别进样分析，根据保留时间、紫外最大吸收波长和正负离子模式下的荷质比，经与标准品或是参考文献比对，初步鉴定或推测出大鼠口服葛根芩连汤后血浆中的14种原型药物成分，分别为葛根素、3’-甲氧基葛根素、8-C-芹糖基（1→6）葡萄糖-大豆苷元、大豆苷元、黄芩苷、汉黄芩苷、芹糖基-7-O-β-D-葡萄糖酐酸、木蝴蝶素A-7-O-β-D-葡萄糖苷酸、药根碱、黄连碱、小檗碱、巴马汀、甘草酸、甘草苷。

黄酮类化合物在体内容易发生首过效应，葡萄糖醛酸化是血浆中主要的代谢途径，本研究推测出4种代谢产物，分别为大豆苷元-7-O-β-D-葡萄糖苷酸、大豆苷元4’-O-硫酸酯、黄芩素-6-O-β-葡萄吡喃糖醛酸苷、6-O-甲基-黄芩素-7-O-硫酸酯，这些成分的鉴定有助于阐明葛根芩连汤的药效物质基础。

4. 构建血浆药代指纹图谱，并首次将化学计量学用于葛根芩连汤入血成分的经时变化过程的研究：首先对含药血浆的色谱方法进行方法学考察，构建血浆药代指纹图谱，结果表明其稳定性好，重复性高，适合药代动力学的研究。

进而连续考察了大鼠口服葛根芩连汤后48 h内，12种主要成分在体内的动态变化过程。

其中葛根的主要成分葛根素代谢较快，达峰时间为1 h，给药后10 h基本代谢完毕；甘草中有部分化合物的达峰时间1-1.5 h；而黄芩中的主要成分黄芩苷则出现双峰现象，达峰时间分别为2、10 h，有部分化合物因其结构与黄芩苷相似，与体内的肝肠循环有关而呈现不规则的多峰现象。

将不同时间采血点的色谱数据导入SIMCA-P软件进行主成分分析（PCA），通过PCA得分图可以清楚地区分不同采血时间色谱图的差异，通过偏最小二乘法（PLS）可以更进一步发现引起葛根芩连汤入血成分的经时变化的主要因素，从而有助于揭示葛根芩连汤的时效关系。

本文通过液相色谱、二极管阵列和质谱联用等方法，对葛根芩连汤进行血清药化学研究，对口服葛根芩连汤后大鼠血浆中的化学成分进行分离、归属和鉴定，并构建血浆药代指纹图谱，阐明了某些物质在体内的代谢及其动态变化过程，为中药药效物质基础研究提供新的思路和方法。

关键词：葛根芩连汤，中药血清药化学，高效液相色谱-二极管阵列-质谱联用，成分鉴定，药代指纹图谱STUDY ON SERUM PHARMACOCHEMISTRY OFGEGEN QINLIAN DECOCTIONABSTRACTGegen Qinlian Decoction (GQD) was one of the most widely used traditional Chinese medicines，composed of Radix Puerariae，Radix Scutellariae，Rhizoma Coptis and Glycyrrhiza. It has been used to treat for antipyretic， anti-diarrhea，anti-inflammatory and relieving muscles. There were many compounds in GQD including flavones，alkaloids and triterpenes. In conventional studies, a few mark compounds had been used to control the quality or investigate the pharmacokinetic of GQD. However，these studies were not able to reflect the decoction’s overall efficacy，because the multiple constituents may work synergistically，which was not combination of each herb simply. Based on the hypothesis that only the constituents migrating to blood after oral administration of TCM have the probability to be effective constituents，pharmacochemistry strategy was applied to study the effective compounds in rat plasma after oral administration of GQD.1. A chromatographic method was established on rat plasma after oral administration of GQD. The chromatographic separation was carried out on Globalsil C18 ( 250 mm × 4.6 mm ID，5 µm ) column with a binary mobile phase system consisting of 20 mmol·L-1 ammonium acetate and acetonitrile with gradient elution at 270 nm. Precipitation with methanol was chosen as a sample preparation method，because it not only removed a lot of endogenous impurities but also extracted most of the target compounds. 0.1 mol·L-1 ammonium acetate ( pH 4.0 adjusted by acetic acid ) was added during the sample preparation so as to improve the extraction recovery rate.2. Constituents migrating to blood after oral administration of GQD were ascribed. Learning from fingerprint strategy，rat plasma samples after oral administration of GQD and individual herb were compared. Accordingto the retention behavior，31 compounds in rat plasma were ascribed，including 15 of them came from herb，13 of them were metabolites，and 3 of them were the new substances in plasma.3. Constituents migrating to blood after oral administration of GQD were identified by HPLC-DAD-MS. Due to the advantages of sensitivity and specificity， LC-MS (ESI/APCI) was applied to determine the compounds in plasma after oral administration of GQD. According to the retention time， UV max and m/z in positive or negative ion mode，18 compounds were identified or tentatively elucidated comparing with standard compounds or literature. There were 14 compounds came from herb，including puerarin， 3’-methoxy puerarin，daidzein 8-C-apiosy (1→6) glucoside， daidzein， baicalin，oroxylin A-7-O-β-D-glucuronide，apigenin-7-O-β-D-glucuronide， wogonoside，jatrorrhizine， coptisine，palmatin， berberine， liquiritin， glycyrrhizin. Furthermore， Four metabolites were tentatively designated to be daidzein4'-O-sulfate，daidzein 7-O-β-D-glucuronide, 6-O-Methyl-baicalin-7-O-sulfate，baicalin -6-O-β- glucopyranuronoside.4. Peak area vs. time curves were studied after oral administration of GQD in rat. Based on the previous chromatographia conditions，repeatability and stability of the method were investigated，and they were suitable to apply to study the pharmacokinetic of GQD. The peak areas of 12 components in rat plasma were measured at the same time at 270nm. Drug peak area vs. time curves of compounds including components from GQD and metabolites were studied after oral administration of GQD in 48h. The results showed that components in Pueraria were absorbed well with rapid metabolism， while the components in Scutellaria were easy to present double-peak trends due to hepatoenteral circulation. Then， the chromatography data was imported to SIMCA-P to perform Principal Component Analysis (PCA)， which was helpful to reveal time-effect relation of GQD.In conclusion，the modern liquid chromatography coupled with DAD detection and MS identification was successfully used in the study on thepharmacochemistry of Gegen Qinlian Decoction. With the fingerprint strategy，18 compounds were identified and 12 peak area vs. time curves were studied， which provided a new method for analyzing the effective compounds in TCM.KEY WORDS: Gegen Qinlian Decoction (GQD)， Pharmacochemistry，HPLC-DAD-MS，Identification，Fingerprint in kinetics图片目录图1 不同检测波长下空白血浆加葛根芩连汤的HPLC图 (14)图2 不同pH的流动相对葛根芩连汤分离效果的影响 (15)图3 不同预处理方法得到的含药血浆的色谱图 (16)图4 甲醇和乙腈预处理空白血浆后的色谱图 (17)图5 比较不同条件处理血浆样品后黄芩苷的提取率 (19)图6 甲醇处理前后的葛根芩连水煎液及含药血浆的HPLC图（270nm） (22)图7 来源葛根血中移行成分的HPLC图 (23)图8 来源黄芩血中移行成分的HPLC图 (24)图9来源黄连血中移行成分的HPLC图 (24)图10来源甘草血中移行成分的HPLC图 (25)图11复方水煎液及其单味药HPLC图 (25)图12大鼠含药血浆的HPLC图（270nm） (31)图13来源于黄芩的化合物的MS和UV max (32)图14来源于葛根的化合物的MS和UV max (34)图15来源于黄连的化合物的MS和UV max (35)图16来源于甘草的化合物的MS和UV max (36)图17葛根芩连汤入血成分的HPLC图（270nm） (43)图18不同时间点的大鼠含药血浆的HPLC图（270nm） (45)图19 12种入血成分在大鼠体内随时间的变化趋势 (48)图 20大鼠含药血浆15个时间点按主成分1与主成分2分类图 (52)图21 正常大鼠灌胃葛根芩连汤0.17和2h血浆样本的PLS分析图（上）和载荷图（下） (53)图22正常大鼠灌胃葛根芩连汤0.17和8h血浆样本的PLS分析图（上）和载荷图（下） (54)图23正常大鼠灌胃葛根芩连汤0.17和48h血浆样本的PLS分析图（上）和载荷图（下） (55)表格目录表1 梯度洗脱条件 (13)表2 不同处理方法所得的样品 (18)表3葛根芩连入血成分色谱图初步归属结果 (26)表4 MS质谱参数 (29)表5 18种葛根芩连汤入血成分HPLC-DAD-MS的数据及结构推测 (37)表6连续进样重复性试验 (44)表7分析方法重复性试验 (44)表8稳定性试验 (45)表9 12种成分对应的峰号 (46)表10不同时间点的大鼠含药血浆的相关性 (51)符号说明GQD Gegen Qinlian Decoction 葛根芩连汤HPLC High Performance Liquid Chromatography 高效液相色谱Spectrometry 质谱MS MassIonization 电喷雾电离ESI ElectrosprayAPCI Atmospheric Pressure Chemical Ionization 大气压化学电离mode 选择离子模式ionSIM Selecteddetector 二极管阵列检测器DAD DiodearrayPCA Principle component analysis 主成分分析squares 偏最小二乘法leastPLS PartialRSD Relative standard deviation 相对标准偏差上海交通大学学位论文原创性声明本人郑重声明：所呈交的学位论文，是本人在导师的指导下，独立进行研究工作所取得的成果。