【作者】 高荣；

【导师】 张志荣；

【作者基本信息】 四川大学 ， 药剂学， 2007， 博士

【摘要】 目前中药的作用机理研究大多照搬化学药的研究模式，即少数有效成分的药物动力学研究和病理模型的药效动力学研究。但是既然中药大多为复方且成分非常复杂，那么少数有效成分的药物动力学研究就不能代表整个中药复方的药动学特征，而且这也不符合中医的“整体论”；另外，由于中医的证候非常复杂，很难选择某个或某几个相关性高、特异性强的实验室“金指标”，使中药药效动力学研究变得较为困难。代谢组学的兴起，为中药作用机理的研究带来难得的机遇。代谢组学是指对生物系统对病理生理刺激及基因改变的代谢应答的定量分析，是一种探索机体及其共生物的整体代谢机制的方法，它主要研究药物作用于机体后内源性代谢物的变化，更直接地反映生化过程和状态改变，又与中医药整体动态性原则具有很大的趋同性。为了揭示中药复方的药物动力学和药效动力学研究缺陷，了解代谢组学在中药复方作用机理研究上的优势和潜力，本课题选择银黄注射液和银黄口服液分别进行经典的药物动力学、药效动力学研究和代谢组学的初步研究。银黄注射液和银黄口服液由金银花提取物和黄芩提取物制备而成，具有清热、解毒、利咽之功效，用于急性扁桃体炎、上呼吸道感染及风热犯肺而致发热、咳嗽、咽痛等症。绿原酸和黄芩苷分别是金银花和黄芩的主要有效成分，其中绿原酸具有抗炎、保肝利胆、抗菌抗病毒等作用，黄芩苷具有抗炎、抗菌抗病毒、保护缺血再灌注损伤、保肝利胆等作用。1银黄注射液和口服液体内分析方法研究1．1血浆中同时测定绿原酸和黄芩苷的HPLC方法研究利用甲醇-乙腈（3∶1）的混合溶剂提取血浆中的绿原酸和黄芩苷；使用C₁₈色谱柱，流动相为0．2％磷酸水溶液（A相）—甲醇／乙腈（1∶1，B相），当B相由15％线性递增至54％时可使两成分得到满意的分离；检测波长绿原酸为327nm，黄芩苷为278nm；流速为1ml·min_-1；葛根素和芦丁分别作为绿原酸和黄芩苷的内标。绿原酸和黄芩苷的线性浓度范围分别在0．388-12．4μg·ml^-1和0．485-124μg·ml^-1内其相关系数均大于0．998；绿原酸和黄芩苷的日内、日间精密度的RSD值均分别小于7％和9％，准确度RE值的范围分别为-9．5％至．3％和-4．2％至1．8％；绿原酸和黄芩苷的检测限分别为0．194μg·ml^-1和0．122μg·ml^-1，定量限分别为0．388μg·ml^-1和0．485μg·ml^-1。1．2尿液中毛细管区带电泳代谢指纹图谱分析方法研究建立了基于毛细管区带电泳的大鼠尿样代谢指纹图谱的电泳方法：选择浓度为40mmol／L且pH值为8．2硼酸盐缓冲溶液作为毛细管电泳的背景电解质，电泳电压为24kV，毛细管柱温为25℃，进样量为3s×0．5psi．，检测波长为214nm。所得到的二维指纹图谱以ASCⅡ格式输出excel数据，然后载入“计算机辅助相似性评价系统”，校正指纹图谱中对应组分色谱峰的迁移时间后，求算出各指纹图谱全谱的相合系数（夹角余弦法）以比较各组内指纹图谱的相似度。对所建立的大鼠尿样的毛细管代谢指纹图谱方法进行了方法学考察，发现方法的重现性和稳定性良好；大鼠表现在尿液代谢指纹图谱上的个体差异较小。2银黄注射液和口服液的药动学—药效学研究2．1银黄注射液和口服液的药物动力学研究10只雄性Wistar大鼠，随机均分成两组，分别单次静注银黄注射液0．5 ml·kg^-1和单次灌胃银黄口服液20 ml·kg^-1，收集不同时间点的血液样品，离心获得血浆，用上述建立的HPLC方法分别测定不同时间点的绿原酸和黄芩苷的浓度，用DAS1．0药动学程序求算药动学参数并进行药动学模型拟合。结果表明大鼠单次静注银黄注射液后，血浆中能够同时检出绿原酸和黄芩苷，其中绿原酸的药-时曲线变化呈三室开放模型，黄芩苷的药-时曲线变化呈二室开放模型；大鼠单次灌胃银黄口服液后，血浆中检不出绿原酸，而黄芩苷的药-时曲线呈多峰现象，其t_max分别为0．5、3、5、12 h，其绝对生物利用度为15．2％。2．2银黄注射液和口服液的药效动力学研究30只Wistar大鼠，♀♂各半，随机均分为口服液组、注射液组和对照组，每组10只，♀♂各半。口服液组和注射液组连续给药4天，每天2次，每次分别灌胃银黄口服液1ml和静注银黄注射液0．2ml；对照组自由取食和饮水不做任何处理。三组每只大鼠分别在第3天以1％的角叉菜生理盐水溶液于双后爪足趾皮下分别注射0．1ml造炎症模型，分别于给药前、给药后不同时间点测量各鼠的右后爪体积，求算出肿胀率。结果表明，注射液组和口服液组的足跖肿胀率在造模后不同时间点均显著地低于对照组（P＜0．05），说明两组均具有抗炎疗效；但注射液组和口服液组相比，造模后不同时间点的肿胀率差异均无统计学意义（P＞0．2），说明两种剂型的抗炎疗效相当。3银黄口注射液和口服液的代谢组学初步研究30只Wistar大鼠，♀♂各半，随机均分为口服液组、注射液组和对照组，单独饲养于代谢笼中。口服液组和注射液组连续给生理盐水2天，每天两次，每次分别灌胃生理盐水1ml和静注生理盐水0．2ml；然后连续给药4天，每天2次，每次分别灌胃银黄口服液1ml和静注银黄注射液0．2ml；对照组不做任何处理。三组的每只大鼠分别在第5天以1％的角叉菜生理盐水溶液于双后爪足趾皮下注射0．1ml造炎症模型。收集各鼠不同阶段的尿液，用上述建立的毛细管区带电泳方法获得了150份尿样的代谢指纹图谱。对获得的代谢指纹图潜进行峰平移校准和数据自标度化，主成分分析得到PCA得分总图，图中显示的150个点除了个别游离外，基本上聚集成4大区域。对代谢指纹数据进行分组分类PCA分析和变量权重分析，结果如下：对照组正常尿样的昼夜和性别等生理性差异PCA分析没有显著性；灌胃操作的刺激所引起的代谢组的变化PCA分析没有显著性差异，而静脉注射的刺激所引起的代谢组的变化PCA分析则有显著性差异；灌胃给药前后PCA分析没有显著性差异，而静注给药前后PCA分析有显著性差异；灌胃给药后和静注给药后的PCA分析有显著性差异；三组造模前后PCA分析均有非常显著的差异，变量权重分析结果表明有多个特征峰的变化非常明显；三组造模后0-36 h的组内PCA分析均无显著性差异；造模后0-36 h两给药组和对照组之间PCA分析有显著性差异，但灌胃组和静注组之间PCA分析则没有显著性差异，表明两种给药方式均能产生明显的抗炎疗效，且两者的疗效相当。结论药物动力学结果显示，经不同的给药途径后，银黄注射液和口服液中绿原酸和黄芩苷在大鼠体内所表现的药物动力学特征相差很大，而药效动力学研究结果却显示其抗炎疗效相当，药物动力学研究结果和药效动力学结果有矛盾之处，表明用经典的药物动力学和药效动力学的方法研究中药复方制剂的作用机理是不可靠的。应用基于毛细管电泳的尿样代谢指纹图谱的代谢组学研究方式，成功地对不同的处理的代谢表型进行有效的区分，而生理差异则可以忽略，并找出了相应的生物标志物的特征峰，表明该方法是一种全面、快捷、可靠的研究中药作用机理的有效方式。更多还原

【Abstract】 The mechanism study on traditional Chinese medicine （TCM） usually imitates the model of study on pharmaceutical chemicals, which involves the pharmacokinetic study of major active components and pharmacodynamic study on pathological model. Since most TCMs are compound recipes which contain complex components, the pharmacokinetic study on minor active components does not represent the pharmacokinetic characteristics of the whole TCM combination formula, and mismatches the "holism" of TCM. In addition, due to the very complex syndrome of TCM, it is difficult to choose one or several laboratory "golden standards" of high correlation and good specificity, which makes the study on pharmacodynamics of TCM truly difficult. The emergence of metabonomics will spell god-given chance element for the study on mechanism of action of TCM. Metabonomics, defined as the quantitative measurement of the multi-parametric metabolic response of living systems to pathophysiological stimuli or genetic modifications, is a branch of science concerned with the exploration on global metabolic mechanism of organism and its coinosite. It mainly investigates the change of endogenous metabolism response to drug, and reflects the biochemical process and condition modification and fits the global and dynamic principle of TCM. In the present study, a comparative study on classical pharmacokinetics-pharmacodynamics and metabonomics in Yinhuang injection and Yinhuang oral solution was undertaken to reveal the defects of pharmacokinetics and pharmacodynamics and to evaluate the potential of metabonomics on TCM combination formulas.Yinhuang injection and Yinhuang oral solution, prepared with extracts from Flos Lonicerae and Radix Scutellariae, are widely used to treat acute tonsillitis, upper respiratory infection, febris, tussis, pharyngalgia, etc., with such therapeutic effects as relieving fever, deintoxication and relieving sore-throat. Chlorogenic acid （ChA） is the major active component of Flos Lonicerae, having therapeutic effects of anti-inflammatory, conservancy of hepar, cholagogue, antibiosis and antivirus, etc.; Baicalin （Bc） is the major active component of Radix Scutellariae, having therapeutic effects of anti-inflammatory, antibiosis and antivirus, conservancy of ischemical reperfusion injury, conservancy of hepar, cholagogue, etc.1 The study on analytical method in vivo of Yinhuang injection and Yinhuang oral solution1.1 The study on HPLC analytical method for simultaneous determination of ChA and Bc in plasmaExtracted from the plasma samples with methanol-acetonitrile （3:1）, the two compounds were successfully separated using a C₁₈ column with a gradient elution composed of 15% and 54% methanol-acetonitrile （1:1） in 0.2% phosphoric acid water solution. The flow-rate was set at 1 ml·min^-1 and the eluents were detected at 327 nm for ChA, 278 nm for Bc. Puerarin and rutin were used as the internal standards for chlorogenic acid and baicalin, respectively. The method was linear over the range of 0.388 - 12.4μg·ml^-1, 0.485 - 124μg·ml^-1 for chlorogenic acid and baicalin, respectively. The correlation coefficient for each analyte was above 0.998. The intra-day and inter-day precisions were better than 7% and 9%, with the relative error ranging from -9.5% to 7.3% and from -4.2% to 1.8%. The limit of detection and the limit of quantification for ChA and Bc in plasma were 0.194μg·ml^-1, 0.122μg·ml^-1, 0.388μg·ml^-1, and 0.485μg·ml^-1, respectively. 1.2 The study on capillary zone eleetrophoresis （CZE） analytical method for metabolite fingerprints in rat urineThe optimum metabolite fingerprints based on CZE was obtained under following conditions: 40 mM sodium borate as the run buffer （pH was adjusted to pH 8.2 with 0.5 M hydrochloric acid）; 24 kV applied voltage, 25℃temperature; injection 3s×0.5 psi. Each CZE data was imported into Computer Aided Similarity Evaluation System as ASCⅡformat and treated as a two-dimensional matrix （time, absorbance）. After the peak alignments of the electropherograms, the congruence coefficient based on included angle cosine was calculated to evaluate the similar degree of intragroup fingerprints. The results showed that this method had good reproducibility and stability and the individual variation in fingerprints of rat urine was small.2 Comparative study on pharmaeokinetics and pharmaeodynamics of Yinhuang injection and Yinhuang oral solution2.1 Comparative study on pharmacokinetics of Yinhuang injection and Yinhuang oral solution10 male Wistar rats, randomly divided into two equal groups, were intravenously （i.v.） administrated with Yinhuang injection at a single dose of 0.5 ml·kg^-1 and intragastrically （i.g.） administrated with Yinhuang oral solution at a single dose of 20 ml·kg^-1, respectively. Collected blood salnples at different time points were centrifuged to obtain plasma and the concentration of ChA and Bc in plasma was determined simultaneously byHPLC described as above. Calculations of pharmacokinetic parameters and pharmacokinetic modeling were carried out using DAS 1.0 software. It was shown that the concentration of ChA and Bc in plasma could be determined simultaneously after i.v. administration of Yinhuang injection; the AUC change of ChA followed the three open - compartment model and that of Bc followed the two open - compartment model. It was shown that the ChA in plasma could not be determined via i.g. administration of Yinhuang oral solution and the AUC change of BC presented multiple - peak phenomenon, and the absolute bioavailability of Bc was about 15.2%.2.2 Comparative study on pharmacodynamics of Yinhuang injection and Yinhuang oral solution30 Wistar rats, 15 male and 15 female, were randomly assigned to 3 equal groups: the oral solution group, the injection group and the control group. Each rat in the injection group and the oral solution group was intravenously administrated with Yinhuang injection at 1 ml·kg^-1 and intragastrically administrated with Yinhuang oral solution at 5 ml·kg^-1, respectively, twice a day, for 4 days successively; the rats in the control group were allowed access to water ad libitum without any other treatment. On the third day, 0.1ml of the 1% carrageen normal saline solution was hypodermically injected into left and right back voix pedis of each rat in the three groups. The bulk of right back voix pedis at different time after administration of carrageen and the normal voix pedis were measured and the degree of tumefaction was calculated. It was shown that the tumefaction values of two dosage groups were significantly lower than that in the control group （P＜0.05） and the degree of tumefaction in the oral solution was equivalent to that in the injection group （P＞0.2）, which indicated that both of the two dosage groups have equivalent anti-inflammatory effect.3 Preliminary study on metabonomics of Yinhuang injection and Yinhuang oral solution30 Wistar rats, 15 male and 15 female, acclimated in individual metabolism cages, were randomly assigned to 3 equal groups: the oral solution group, the injection group and the control group. Each rat in the injection group and the oral solution group i.v. administrated with normal saline solution at 1 ml·kg^-1 and i.g. administrated with normal saline solution at 5 ml·kg^-1, respectively, twice a day, for 2 days successively; then, rats were i.v. administrated with Yinhuang injection at 1 ml·kg^-1 and i.g. administrated with Yinhuang oral solution 5 ml·kg^-1, respectively, twice a day, for 4 days successively. The rats in the control group were allowed access to water ad libitum without any other treatment. On the fifth day, 0.1ml of the 1% carrageen normal saline solution was hypodermically injected into left and right back voix pedis of each rat in the three groups. At selected time intervals, urine samples from each rat were collected and 150 metabolite fingerprints were obtained by CZE method described as above.After peak alignments and data autoscaling, 150 fingerprints were subjected to Principal Components Analysis （PCA） to obtain PCA general scores plot, which showed that most of the scores were mapped in four different clusters except for several scores. The grouping and typing fingerprints were subjected to PCA and variance weight analysis to be shown as follows: The performance of PCA of fingerprints of normal urine sample revealed that the sexual and diurnal variation was not significant. The variation of PCA induced by the stimuli of i.g. was not significant while that induced by the stimuli of i.v. was significant. The variation of PCA was not significant in the group via i.g. administration of Yinhuang oral liquid and normal saline solution while significant in the i.v. group administrated with Yinhuang injection and normal saline solution. The variation of PCA was significant in i.g. administration of Yinhuang and i.v. administration of Yinhuang injection. The performance of PCA of fingerprints in three groups revealed significant discrimination where samples with inflammation lay apart from samples preinflammation, and the variance weight analysis was shown that multitude characteristic peaks varied significantly; while the variation of PCA of fingerprints in three groups was not significant during 0 - 36 h after causing inflammation. The variation of PCA during 0 - 36 h after causing inflammation was significant between two dosage groups and control group, while that was not significant between oral solution group and injection group, which indicated that both of the two dosage groups have equivalent therapeutic effect of anti-inflammation.ConclusionThe study on pharmacokinetics :indicated that pharmacokinetic characteristics of ChA and Bc in Yinhuang injection and Yinhuang oral solution varied greatly, while the study on pharmacodynamics indicated that the curative effect of Yinhuang oral solution equaled to that of Yinhuang injection. It is evident that the findings of pharmacokinetics and pharmacodynamics are inconsistent, which demonstrates that it is unreliable to study the mechanism of TCM combination formulas by traditional pharmacokinetic and pharmacodynamic methods.Application of CZE-based urine metabolite fingerprints could successfully discriminate the metabolite phenotypes of different treatment, overlook physiological variations and obtain characteristic peaks of biomarkers, which indicates that metabonomics is an all-round,, fast and reliable science branch on study of the mechanism of action of TCM combination formulas.更多还原