中药多成分药代动力学的新方法和策略研究
发布时间：2018-08-28 08:25
【摘要】：中药多成分药代动力学(简称多成分药代)是中医药理论和作用机制阐明的重要研究内容。但是由于中药成分的复杂和多样性,以及中药成分与生物体系相互作用的复杂性和不确定性,中药多成分药代动力学的研究一直面临着巨大挑战:(1)中药化学成分复杂、体内代谢多样化,使得体内成分的结构鉴定困难;(2)药代动力学研究的目标化合物不明确、在中药及生物样本浓度低、内源性基质和组分之间干扰大、对照品缺乏,导致中药多成分检测和定量方法的建立困难;(3)药效指标不统一、有效成分不明确,导致中药药代动力学研究结果的综合评价和应用不充分。因此需要运用先进的分析技术,能够快速、准确、全面的定性分析中药体内多成分,灵敏、快速、准确、宽动态范围的定量分析中药体内多成分,建立符合中医治病整体观的更深层次和更广视角的新策略,为符合中医理论的中药多成分药代动力学研究提供技术支持和科学数据。本论文针对中药药代动力学研究存在的一些瓶颈问题,分别选择了巫山淫羊藿、小续命汤有效成分组和二仙汤为研究对象,探索性地开展了中药多成分药代动力学研究所需分析新技术和新策略的研究工作。论文第一部分针对中药多成分药代研究缺乏体内成分及其代谢物对照品的瓶颈问题,探索性地基于体内主要成分代谢途径的全面诠释,建立多成分药代指标成分的研究策略。该策略首先采用高效液相色谱-高分辨质谱/质谱联用方法(HPLC-HRMSn)和质谱树状图相似度过滤(MTSF)技术对淫羊藿5个主要异戊烯基黄酮成分在大鼠体内的代谢产物进行了分析和鉴定,基于体内代谢物的鉴定结果推断5种主要异戊烯基黄酮的代谢途径,将血浆中主要暴露的14个原型成分和葡萄糖醛酸代谢物选为潜在的药代指标成分,作为测定对象,采用高效液相色谱串联质谱分析技术(HPLC-MS/MS),初步研究了 5种主要异戊烯基黄酮灌胃给予大鼠后,这14个体内成分在血浆中的主要药动学参数特征;由于葡萄糖醛酸结合型代谢物没有对照品,因此采用酶水解的方法将血浆样本中的这14个体内成分转化为有对照品、且存在代谢转化关系的10个潜在药代指标成分作为药代动力学的研究对象,建立了 HPLC-MS/MS分析方法:在色谱分离方面,通过优化两种流动相的比例和混合时间,在二元液相泵上实现了四元流动相的分离效果,消除了朝藿定A-C/淫羊藿苷的cross-talk和淫羊藿素的基质效应;在质谱检测方面,采用分段多反应监测模式,缩短了质谱的duty cycle,从而增加了方法的灵敏度。所建立的方法经过了方法验证,能够满足生物样品分析,并被应用于研究5种主要异戊烯基黄酮和巫山淫羊藿灌胃给予大鼠后,10个潜在的药代指标成分在血浆中的主要药动学参数特征;采用Kendall's tau-b等级相关分析法对酶水解前后血浆中测得的指标成分的浓度随给药时间的变化进行相关性分析。实验结果表明淫羊藿素的葡萄糖醛酸结合型代谢物是巫山淫羊藿灌胃给药后,异戊烯基黄酮在大鼠体内的主要存在形式。另外,酶水解血浆中的总淫羊藿素水平能够同时反映异戊烯基黄酮在体内的暴露量和动态变化。因此,具有高暴露量和较好相关性(r0.5)的淫羊藿素可以作为巫山淫羊藿异戊烯基黄酮的药代指标成分。除此之外,我们还利用淫羊藿素的药代特征建立数学模型,用于预测大鼠体内巫山淫羊藿的暴露量。结果显示预测值与实验值有较好的一致性,进一步证实酶水解血浆中的淫羊藿素可以作为巫山淫羊藿中异戊烯基黄酮的药代指标成分。论文第二部分研究了中药复方小续命汤抗脑缺血的有效成分组(AF-XXMD)的多成分药代。采用HPLC-HRMSn方法和MTSF技术分析和鉴定AF-XXMD中68种化学成分,对大鼠血浆和脑内高暴露水平的化学成分,采用反向分子对接的方法,预测了药理活性,选择大鼠血浆和脑组织21个高暴露且预测有活性的化合物作为药代指标成分,建立高分离度快速液相色谱串联质谱法(RRLC-MS/MS)。前处理方法通过添加抗坏血酸保护血浆中的黄芩苷和黄芩素不被氧化,通过脂质预处理柱除去磷脂,以消除血浆和脑组织中甘草酸的基质效应;色谱分离采用RRLC法,可实现3对同分异构体的基线分离;质谱检测采用分时间段的正负离子切换模式,并通过添加微量的甲酸铵(0.08mmol l-1)提高各化合物的质谱响应。对所建方法进行了方法验证,表明该方法符合生物样品分析方法的技术要求,满足大鼠脑组织和血浆中的药代动力学研究需要;采用正常大鼠与双侧颈总动脉结扎导致的慢性脑缺血模型大鼠,灌胃给予AF-XXMD,比较研究了 21个指标成分的血浆和靶器官脑的药动学特征和差异。研究结果显示在慢性脑缺血病理模型下AF-XXMD的药代特征发生明显的改变,包括血脑透过率、脑部动态变化、脑部暴露量和脑组织不同部位分布。慢性脑缺血大鼠灌胃给予AF-XXMD后最主要的化合物是黄酮和色原酮类化合物,而脑部最主要的化合物为色原酮和生物碱类化合物。AF-XXMD对慢性脑缺血的治疗效果与色原酮、黄酮和生物碱类化合物作用于多个靶点和多条代谢通路有关。论文第三部分以中药复方二仙汤为研究对象,整合中药复方中化合物的识别与鉴定、代谢组学、生物信息学的技术与方法,从中医药治病整体观角度,探索中药体内药效物质基础,包括来源于中药的外源性成分、被中药扰动的机体内源性成分,以及这些成分与药效的关系和其作用机制的研究思路和策略。首先,我们建立了快速识别与鉴定中药复方中未知化合物的新策略。这个新策略能够快速识别候选化合物的结构信息,并为结构鉴定提供有效的线索。这个策略主要包括以下四步:1、建立了 HPLC-HRMSn的中药成分的分析方法,获得高分辨质量数和多级质谱数据,利用MTSF技术,识别潜在化合物并获得其母核信息;2、基于保留时间和高分辨质量数,利用判别分析方法,对潜在化合物按类别进行分类;3、将MTSF技术和判别分析方法获得的潜在化合物的结构母核信息进行匹配,母核信息一致的潜在化合物作为候选化合物进行结构鉴定;4、基于同位素分布数据对鉴定的候选化合物结构进行确证。这个新策略能够在MTSF技术的基础上进一步排除约41%的不相关离子信息,同时还能够提供553个候选化合物的准确结构类型信息,最终鉴定出71个化合物。以鉴定的71个化合物为模板,基于MTSF技术,我们从二仙汤灌胃给药后的大鼠血浆、尿、粪、胆汁中鉴定出13个原型成分和150个代谢物。其次,我们建立了高通量不同官能团类型甾体激素的UHPLC-MS/MS定量分析方法,实现了一份生物样本一次分析中,同时进行羰基和酚羟基甾体激素的衍生化分析。所建立的方法仅需要500 μL血清样本,可在单次运行时间28 min内定量分析20个甾体激素类化合物。该方法首先采用液液萃取技术从生物样本中提取甾体激素,紧接着进行离线的吉拉德P衍生化,然后采用自动化的进样程序自动吸取两次样本:第一次将吉拉德P衍生化的羰基类甾体激素加载到色谱柱上;第二次使甾体激素在进样针中进行丹磺酰氯的在线衍生化反应,反应结束后再将丹磺酰氯衍生化的酚羟基类甾体激素加载到色谱柱上。与此同时,质谱切换阀与进样程序密切配合,以导入衍生化的甾体激素和去除多余的衍生化试剂,而后质谱对导入的两种类型的甾体激素衍生化产物按顺序分别进行分析。方法验证结果表明:线性范围为2～400 pg/mL,相关系数r值均高于0.988,最低定量限范围为2～4 pg/mL,各化合物精密度的相对标准偏差(RSD)值不大于20%,准确度在80～120%之间,完全能够满足生物样本中甾体激素的分析要求。再次,基于脂质组学开展了二仙汤对不同绝经阶段双侧去卵巢大鼠的调节作用研究,设置了假手术组、双侧去卵巢模型组、模型给予二仙汤组和模型给予尼尔雌醇组。将体重、血脂指标(胆固醇、高密度脂蛋白、低密度脂蛋白和甘油三酯)和肝脏油红O染色切片检查作为药效学指标,评价二仙汤给药后不同时间点的药效学。在药效学研究的同时,基于建立的激素、脂质和鞘脂组学分析平台,结合统计学方法,开展脂质代谢组学研究;研究去卵巢大鼠血浆和肝组织紊乱的脂质代谢特征,以及二仙汤干预后不同绝经阶段去卵巢大鼠血浆和肝组织紊乱的脂质代谢特征,基于脂质合成与代谢通路,系统诠释了二仙汤调节脂代谢紊乱的作用机制,并研究不同绝经阶段病理变化和二仙汤治疗的特点。结果表明,二仙汤能有效调节双侧去卵巢引起的甾体激素和脂质的合成与代谢紊乱,其中以给药后4周发现的能够同时指征双侧去卵巢模型脂代谢紊乱和二仙汤调节作用的潜在生物标志物的数量最多,包括血浆中15种甘油酯、10种磷脂酰甘油酯和4种甾体激素,肝脏中8种甘油酯和10种磷脂酰甘油酯。除此之外,基于不同绝经阶段发现的潜在生物标志物的数量、种类、重复出现的生物标志物等研究,发现二仙汤的调节作用与时间密切相关。最后,基于反向分子对接的方法,对二仙汤进入体内的主要成分进行反向分子对接,筛选出导致代谢通路变化的潜在药效物质,构建与药效相关的内源性代谢物和外源性中药成分之间的关系(物质基础),并采用Western-blot和ELISA方法,验证了二仙汤参与调节血浆和肝脏中甘油三酯的合成通路,进一步揭示了二仙汤调节双侧去卵巢大鼠脂代谢紊乱的物质基础和作用机制,从而进一步揭示二仙汤的作用机制。
[Abstract]:Multicomponent pharmacokinetics of traditional Chinese medicine (TCM) is an important research content in clarifying the theory and mechanism of TCM. However, due to the complexity and diversity of TCM components and the complexity and uncertainty of interaction between TCM components and biological systems, the study of multicomponent pharmacokinetics of TCM has been confronted with great challenges. (2) The target compounds of pharmacokinetics research are not clear, the concentration of Chinese medicine and biological samples is low, the interference between endogenous matrix and components is large, and the lack of reference substances, which makes it difficult to establish the method of multi-component detection and quantitative determination of Chinese medicine; (3) The pharmacokinetics research of Chinese medicine is difficult. The results of pharmacokinetic studies of traditional Chinese medicine are not fully evaluated and applied because of the inconsistency of effective indexes and the unclearness of effective components.Therefore, it is necessary to use advanced analytical techniques to analyze the multi-components in traditional Chinese medicine quickly, accurately and comprehensively. The new strategy of the holistic view of TCM treatment provides technical support and scientific data for the study of multi-component pharmacokinetics of TCM in line with TCM theory. In the first part of this paper, the bottleneck problem of multi-component pharmacokinetics of traditional Chinese medicine (TCM) is the lack of in vivo components and their metabolites. A multi-component pharmacokinetic strategy was developed. Firstly, the metabolites of five main isoprenyl flavonoids from Herba Epimedii in rats were analyzed and identified by high performance liquid chromatography-high resolution mass spectrometry/mass spectrometry (HPLC-HRMSn) and mass spectrometry dendrogram similarity filtration (MTSF) techniques based on in vivo metabolism. The metabolic pathways of five main isoprenyl flavonoids were deduced. Fourteen prototype components and glucuronic acid metabolites in plasma were selected as potential pharmacokinetic indicators. High performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was used to analyze five main isoprenyl flavonoids. After intragastric administration, the main pharmacokinetic parameters of the 14 components in the plasma were characterized; since there was no reference substance for glucuronide-binding metabolites, enzymatic hydrolysis was used to convert the 14 components in the plasma samples into reference substances, and 10 potential pharmacokinetic indicators with metabolic transformation were used as the reference substances. A method of HPLC-MS/MS was established for the determination of pharmacokinetics. In the aspect of chromatographic separation, by optimizing the ratio and mixing time of the two mobile phases, the separation effect of Quaternary mobile phase was realized in binary liquid pump, and the matrix effect of cross-talk and icariin of pilocarpine A-C/icariin was eliminated. The proposed method was validated to be suitable for the analysis of biological samples and was applied to the study of 5 main isoprenyl flavonoids and Herba Epimedii wushanensis in rats after intragastric administration of 10 potential pharmacokinetic components in plasma. Kendall's tau-b hierarchical correlation analysis was used to analyze the correlation between the concentration of the target components in plasma before and after enzymatic hydrolysis and the time of administration. In addition, the level of total icariin in the enzymatic hydrolyzed plasma can reflect the exposure and dynamic changes of isoprenyl flavonoids in vivo. Therefore, icariin with high exposure and good correlation (r0.5) can be used as a pharmacokinetic index of isoprenyl flavonoids of icariin wushan. In addition, a mathematical model was established to predict the exposure of Epimedium wushanensis in rats by using the pharmacokinetic characteristics of icariin. The results showed that the predicted values were in good agreement with the experimental values. It was further confirmed that the enzymatic hydrolysis of plasma icariin could be used as a pharmacokinetic index component of isoprenyl flavonoids in Epimedium wushanensis. In the second part, the multicomponent pharmacokinetics of Xiaoxuming Decoction (AF-XXMD) was studied. 68 kinds of chemical constituents in AF-XXMD were analyzed and identified by HPLC-HRMSn and MTSF. The chemical constituents of high exposure level in plasma and brain of rats were identified. The pharmacological activities were predicted by reverse molecular docking. A high-resolution rapid liquid chromatography-mass spectrometry (RRLC-MS/MS) was developed for the determination of 21 highly exposed and predictably active compounds in rat plasma and brain tissues. Three pairs of isomers can be separated at baseline by using RRLC method for chromatographic separation; the positive and negative ion switching mode is used for mass spectrometry detection, and the mass spectrometric response of each compound is improved by adding a small amount of ammonium formate (0.08mmol l-1). The method meets the technical requirements of biological sample analysis and meets the needs of pharmacokinetic study in rat brain tissue and plasma. The pharmacokinetic characteristics and differences of plasma and target organ brain of 21 index components were studied by intragastric administration of AF-XXMD in normal rats and rats with chronic cerebral ischemia induced by bilateral common carotid artery ligation. The results showed that the pharmacokinetic characteristics of AF-XXMD in chronic cerebral ischemia model were significantly changed, including blood-brain permeability, brain dynamic changes, brain exposure and distribution in different parts of brain tissue. The therapeutic effect of AF-XXMD on chronic cerebral ischemia is related to the action of chromogenic ketone, flavonoids and alkaloids on multiple targets and multiple metabolic pathways. The technology and method of bioinformatics explore the material basis of pharmacodynamics in traditional Chinese medicine from the perspective of holistic view of Chinese medicine treatment, including the exogenous components from traditional Chinese medicine, the intrinsic components disturbed by traditional Chinese medicine in vivo, the relationship between these components and pharmacodynamics, and the research ideas and Strategies of their mechanism of action. This new strategy can quickly identify the structural information of the candidate compounds and provide effective clues for structural identification. This strategy mainly includes the following four steps: 1. An analytical method of HPLC-HRMSn was established to obtain high resolution mass fraction and multi-stage mass spectrometry data. Using MTSF technology to identify potential compounds and obtain their parent nucleus information; 2. Based on retention time and high resolution mass number, potential compounds are classified by category by discriminant analysis method; 3. Matching the parent nucleus information of potential compounds obtained by MTSF technology and discriminant analysis method, potential compounds with identical parent nucleus information are identified. The new strategy can further exclude about 41% of the irrelevant ionic information on the basis of MTSF technology, and also provide accurate structural type information of 553 candidate compounds. Finally 71 compounds were identified. Compounds. Based on the MTSF technique, we identified 13 prototype components and 150 metabolites in plasma, urine, feces and bile of rats after administration of Erxian decoction. Secondly, we established a high-throughput UHPLC-MS/MS quantitative analysis method for steroid hormones of different functional groups. In one analysis, the derivatization of carbonyl and phenolic steroid hormones was performed simultaneously. The established method requires only 500 mu L serum samples and can quantitatively analyze 20 steroid hormones within 28 minutes of a single run. The method first uses liquid-liquid extraction to extract steroid hormones from biological samples, followed by off-line extraction. Gillard P derivatization and automatic sampling procedure were used to automatically absorb two samples: first, Gillard P derivatized carbonyl steroid hormones were loaded onto the column; second, steroid hormones were used in the sample needle for on-line derivatization of sulfonyl chloride, and then the phenolic hydroxyl groups derived from sulfonyl chloride were used after the reaction. Steroid hormones were loaded onto chromatographic columns. Meanwhile, the MS switching valve cooperated closely with the sampling procedure to introduce the derived steroid hormones and remove the redundant derivative reagents. Then the two types of steroid hormone derivatives were analyzed by MS in sequence. The results showed that the linear range was 2-400. The relative standard deviation (RSD) of each compound was less than 20%, and the accuracy was between 80% and 120%. It could completely meet the requirements of steroid hormone analysis in biological samples. Thirdly, Erxian decoction was carried out bilaterally in different menopausal stages based on lipomics. To study the regulating effect of ovarian rats, sham operation group, bilateral ovariectomy model group, Erxian Decoction group and nilestriol group were set up. Body weight, blood lipid index (cholesterol, high density lipoprotein, low density lipoprotein and triglyceride) and liver oil red O staining were used as pharmacodynamic indexes to evaluate the effect of Erxian decoction. Pharmacodynamics at different time points after ovariectomy. At the same time, based on the established analysis platform of hormones, lipids and sphingolipids, combined with statistical methods, lipid metabolism of ovariectomized rats was studied; the characteristics of lipid metabolism in plasma and liver tissue disorders and plasma of ovariectomized rats at different stages after intervention of Erxian Decoction were studied. Based on the pathway of lipid synthesis and metabolism, the mechanism of Erxian Decoction in regulating lipid metabolism disorder was systematically interpreted, and the pathological changes in different menopausal stages and the characteristics of Erxian Decoction in treatment were studied. The results showed that Erxian Decoction could effectively regulate the synthesis and substitution of steroid hormones and lipids induced by bilateral ovariectomy. Among the metabolic disorders, the largest number of potential biomarkers, including 15 glycerides in plasma, 10 phosphatidylglycerides and 4 steroid hormones, 8 glycerides in the liver and 10 phosphatidylglycerides, were found four weeks after administration, indicating both lipid metabolism disorders and the regulatory effects of Erxian Decoction in bilateral ovariectomized rats. According to the research on the quantity, species and repeated biomarkers of potential biomarkers found in different menopausal stages, it is found that the regulating effect of Erxian Decoction is closely related to time. Finally, based on the method of reverse molecular docking, the main components of Erxian Decoction were docked by reverse molecular docking, and the metabolic pathways were screened out. The relationship between endogenous metabolites and exogenous components of traditional Chinese medicine was constructed. Western-blot and ELISA methods were used to verify the role of Erxian Decoction in regulating the synthesis pathway of triglycerides in plasma and liver, further revealing the regulation of Erxian Decoction on lipid metabolism disorders in bilateral ovariectomized rats. The material foundation and mechanism of action are further revealed in the mechanism of Erxian Decoction.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285
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本文编号：2208835
[Abstract]:Multicomponent pharmacokinetics of traditional Chinese medicine (TCM) is an important research content in clarifying the theory and mechanism of TCM. However, due to the complexity and diversity of TCM components and the complexity and uncertainty of interaction between TCM components and biological systems, the study of multicomponent pharmacokinetics of TCM has been confronted with great challenges. (2) The target compounds of pharmacokinetics research are not clear, the concentration of Chinese medicine and biological samples is low, the interference between endogenous matrix and components is large, and the lack of reference substances, which makes it difficult to establish the method of multi-component detection and quantitative determination of Chinese medicine; (3) The pharmacokinetics research of Chinese medicine is difficult. The results of pharmacokinetic studies of traditional Chinese medicine are not fully evaluated and applied because of the inconsistency of effective indexes and the unclearness of effective components.Therefore, it is necessary to use advanced analytical techniques to analyze the multi-components in traditional Chinese medicine quickly, accurately and comprehensively. The new strategy of the holistic view of TCM treatment provides technical support and scientific data for the study of multi-component pharmacokinetics of TCM in line with TCM theory. In the first part of this paper, the bottleneck problem of multi-component pharmacokinetics of traditional Chinese medicine (TCM) is the lack of in vivo components and their metabolites. A multi-component pharmacokinetic strategy was developed. Firstly, the metabolites of five main isoprenyl flavonoids from Herba Epimedii in rats were analyzed and identified by high performance liquid chromatography-high resolution mass spectrometry/mass spectrometry (HPLC-HRMSn) and mass spectrometry dendrogram similarity filtration (MTSF) techniques based on in vivo metabolism. The metabolic pathways of five main isoprenyl flavonoids were deduced. Fourteen prototype components and glucuronic acid metabolites in plasma were selected as potential pharmacokinetic indicators. High performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was used to analyze five main isoprenyl flavonoids. After intragastric administration, the main pharmacokinetic parameters of the 14 components in the plasma were characterized; since there was no reference substance for glucuronide-binding metabolites, enzymatic hydrolysis was used to convert the 14 components in the plasma samples into reference substances, and 10 potential pharmacokinetic indicators with metabolic transformation were used as the reference substances. A method of HPLC-MS/MS was established for the determination of pharmacokinetics. In the aspect of chromatographic separation, by optimizing the ratio and mixing time of the two mobile phases, the separation effect of Quaternary mobile phase was realized in binary liquid pump, and the matrix effect of cross-talk and icariin of pilocarpine A-C/icariin was eliminated. The proposed method was validated to be suitable for the analysis of biological samples and was applied to the study of 5 main isoprenyl flavonoids and Herba Epimedii wushanensis in rats after intragastric administration of 10 potential pharmacokinetic components in plasma. Kendall's tau-b hierarchical correlation analysis was used to analyze the correlation between the concentration of the target components in plasma before and after enzymatic hydrolysis and the time of administration. In addition, the level of total icariin in the enzymatic hydrolyzed plasma can reflect the exposure and dynamic changes of isoprenyl flavonoids in vivo. Therefore, icariin with high exposure and good correlation (r0.5) can be used as a pharmacokinetic index of isoprenyl flavonoids of icariin wushan. In addition, a mathematical model was established to predict the exposure of Epimedium wushanensis in rats by using the pharmacokinetic characteristics of icariin. The results showed that the predicted values were in good agreement with the experimental values. It was further confirmed that the enzymatic hydrolysis of plasma icariin could be used as a pharmacokinetic index component of isoprenyl flavonoids in Epimedium wushanensis. In the second part, the multicomponent pharmacokinetics of Xiaoxuming Decoction (AF-XXMD) was studied. 68 kinds of chemical constituents in AF-XXMD were analyzed and identified by HPLC-HRMSn and MTSF. The chemical constituents of high exposure level in plasma and brain of rats were identified. The pharmacological activities were predicted by reverse molecular docking. A high-resolution rapid liquid chromatography-mass spectrometry (RRLC-MS/MS) was developed for the determination of 21 highly exposed and predictably active compounds in rat plasma and brain tissues. Three pairs of isomers can be separated at baseline by using RRLC method for chromatographic separation; the positive and negative ion switching mode is used for mass spectrometry detection, and the mass spectrometric response of each compound is improved by adding a small amount of ammonium formate (0.08mmol l-1). The method meets the technical requirements of biological sample analysis and meets the needs of pharmacokinetic study in rat brain tissue and plasma. The pharmacokinetic characteristics and differences of plasma and target organ brain of 21 index components were studied by intragastric administration of AF-XXMD in normal rats and rats with chronic cerebral ischemia induced by bilateral common carotid artery ligation. The results showed that the pharmacokinetic characteristics of AF-XXMD in chronic cerebral ischemia model were significantly changed, including blood-brain permeability, brain dynamic changes, brain exposure and distribution in different parts of brain tissue. The therapeutic effect of AF-XXMD on chronic cerebral ischemia is related to the action of chromogenic ketone, flavonoids and alkaloids on multiple targets and multiple metabolic pathways. The technology and method of bioinformatics explore the material basis of pharmacodynamics in traditional Chinese medicine from the perspective of holistic view of Chinese medicine treatment, including the exogenous components from traditional Chinese medicine, the intrinsic components disturbed by traditional Chinese medicine in vivo, the relationship between these components and pharmacodynamics, and the research ideas and Strategies of their mechanism of action. This new strategy can quickly identify the structural information of the candidate compounds and provide effective clues for structural identification. This strategy mainly includes the following four steps: 1. An analytical method of HPLC-HRMSn was established to obtain high resolution mass fraction and multi-stage mass spectrometry data. Using MTSF technology to identify potential compounds and obtain their parent nucleus information; 2. Based on retention time and high resolution mass number, potential compounds are classified by category by discriminant analysis method; 3. Matching the parent nucleus information of potential compounds obtained by MTSF technology and discriminant analysis method, potential compounds with identical parent nucleus information are identified. The new strategy can further exclude about 41% of the irrelevant ionic information on the basis of MTSF technology, and also provide accurate structural type information of 553 candidate compounds. Finally 71 compounds were identified. Compounds. Based on the MTSF technique, we identified 13 prototype components and 150 metabolites in plasma, urine, feces and bile of rats after administration of Erxian decoction. Secondly, we established a high-throughput UHPLC-MS/MS quantitative analysis method for steroid hormones of different functional groups. In one analysis, the derivatization of carbonyl and phenolic steroid hormones was performed simultaneously. The established method requires only 500 mu L serum samples and can quantitatively analyze 20 steroid hormones within 28 minutes of a single run. The method first uses liquid-liquid extraction to extract steroid hormones from biological samples, followed by off-line extraction. Gillard P derivatization and automatic sampling procedure were used to automatically absorb two samples: first, Gillard P derivatized carbonyl steroid hormones were loaded onto the column; second, steroid hormones were used in the sample needle for on-line derivatization of sulfonyl chloride, and then the phenolic hydroxyl groups derived from sulfonyl chloride were used after the reaction. Steroid hormones were loaded onto chromatographic columns. Meanwhile, the MS switching valve cooperated closely with the sampling procedure to introduce the derived steroid hormones and remove the redundant derivative reagents. Then the two types of steroid hormone derivatives were analyzed by MS in sequence. The results showed that the linear range was 2-400. The relative standard deviation (RSD) of each compound was less than 20%, and the accuracy was between 80% and 120%. It could completely meet the requirements of steroid hormone analysis in biological samples. Thirdly, Erxian decoction was carried out bilaterally in different menopausal stages based on lipomics. To study the regulating effect of ovarian rats, sham operation group, bilateral ovariectomy model group, Erxian Decoction group and nilestriol group were set up. Body weight, blood lipid index (cholesterol, high density lipoprotein, low density lipoprotein and triglyceride) and liver oil red O staining were used as pharmacodynamic indexes to evaluate the effect of Erxian decoction. Pharmacodynamics at different time points after ovariectomy. At the same time, based on the established analysis platform of hormones, lipids and sphingolipids, combined with statistical methods, lipid metabolism of ovariectomized rats was studied; the characteristics of lipid metabolism in plasma and liver tissue disorders and plasma of ovariectomized rats at different stages after intervention of Erxian Decoction were studied. Based on the pathway of lipid synthesis and metabolism, the mechanism of Erxian Decoction in regulating lipid metabolism disorder was systematically interpreted, and the pathological changes in different menopausal stages and the characteristics of Erxian Decoction in treatment were studied. The results showed that Erxian Decoction could effectively regulate the synthesis and substitution of steroid hormones and lipids induced by bilateral ovariectomy. Among the metabolic disorders, the largest number of potential biomarkers, including 15 glycerides in plasma, 10 phosphatidylglycerides and 4 steroid hormones, 8 glycerides in the liver and 10 phosphatidylglycerides, were found four weeks after administration, indicating both lipid metabolism disorders and the regulatory effects of Erxian Decoction in bilateral ovariectomized rats. According to the research on the quantity, species and repeated biomarkers of potential biomarkers found in different menopausal stages, it is found that the regulating effect of Erxian Decoction is closely related to time. Finally, based on the method of reverse molecular docking, the main components of Erxian Decoction were docked by reverse molecular docking, and the metabolic pathways were screened out. The relationship between endogenous metabolites and exogenous components of traditional Chinese medicine was constructed. Western-blot and ELISA methods were used to verify the role of Erxian Decoction in regulating the synthesis pathway of triglycerides in plasma and liver, further revealing the regulation of Erxian Decoction on lipid metabolism disorders in bilateral ovariectomized rats. The material foundation and mechanism of action are further revealed in the mechanism of Erxian Decoction.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285
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本文编号：2208835
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