【作者】 郭宇洁；

【导师】 刘建勋；

【作者基本信息】 中国中医科学院 ， 中西医结合基础， 2009， 博士

【摘要】 中药方剂是中医辨证论治、理法方药体系的重要组成部分,组方配伍是中药方剂研究的核心。随着医学模式和疾病谱的不断变化,需要更多新的中药方剂以应对越来越多的疾病挑战,中药方剂如何进行组方成为目前面临的关键问题,对中药方剂组方设计方法进行研究具有重要意义。本文提出新中药方剂的组方设计方法并对其进行验证。在中医药理论指导下,选择中药经典名方,吸纳新的有效中药,化裁而出中药方剂基本方;将正交实验设计应用于中药方剂组方设计中的两个关键环节,即药味配伍和剂量配比。以典型的药理模型和特异性药效指标为判断标准,通过数学分析,在众多的比例中筛选出饮片层面的最佳药味配伍和剂量配比;运用化学分离和制剂分析技术,获得工艺稳定、质量可控的化学最佳制剂,再进行系统药效实验,验证其药效有效性,体现中药复方化学的整体研究理念;在此基础上进行系统药效学及药代动力学研究,探讨中药方剂作用、作用途径和中医药理论的相关性,推动方剂学科的发展。以治疗偏头痛的麻芎舒痛方为研究对象,对中药方剂组方设计方法进行验证,初步证明了中药方剂组方设计方法的可行性和科学性,为中药复方的配伍理论及应用提供有力支撑。1基于药效学基础的麻芎舒痛方组方依据和剂量筛选本文首先对中药复方的组方依据和剂量筛选进行药效学研究。以临床疗效为基础,以《中华人民共和国药典》为依据,拟定中药复方基本药味和用药范围。以镇痛效应为观察指标,通过正交试验,观察不同配比和不同剂量的中药复方药物对3种偏头痛动物模型的药效影响,筛选出中药复方药味配伍及最佳剂量配比。1.1治疗偏头痛中药复方药物不同剂量配伍对硝酸甘油型偏头痛大鼠行为学的影响观察不同药味配伍和不同剂量配比的中药复方对硝酸甘油型偏头痛大鼠挠头次数的影响。结果显示麻芎舒痛方（样本十）对偏头痛大鼠的挠头次数有最佳抑制作用。与模型对照组对比,统计学具有显著性差异（P<0.05）。1.2治疗偏头痛中药复方药物不同剂量配伍对热板法小鼠镇痛作用的研究观察在热板法小鼠模型实验中,不同药味配伍和不同剂量配比的中药复方的镇痛作用。结果显示（样本二、样本八和样本十）对小鼠热板法致痛时间有明显的延长效应。与模型对照组对比,统计学具有显著性差异（P<0.05）。1.3治疗偏头痛中药复方药物不同剂量配伍对2%甲醛所致双相疼痛镇痛效应影响观察不同药味配伍和不同剂量配比的中药复方的对2%甲醛所致双相疼痛镇痛效应的影响。结果显示样本六、样本七和样本十对2%甲醛致痛所产生的Ⅱ相疼痛时间有明显延长效应。与模型对照组对比,统计学具有显著性差异（P<0.05）。综上所述,以3种动物模型的综合药理效应为实验依据,进行统计学和方差分析,筛选出包含治疗偏头痛的著名药对和经典药队的中药复方一麻芎舒痛方,为治疗偏头痛的中药复方提供了组方依据和优化剂量。2不同治则配伍的中药方剂药效研究采用硝酸甘油诱发的实验性偏头痛大鼠模型,以与偏头痛相关的5-HT作为药效学重要指标之一,观察不同治则组成的中药复方对大鼠脑组织和血清中的多种指标的影响,进一步确证麻芎舒痛方的药理作用。对正常对照组、模型组、阳性药对照组和中药复方治则配伍组在偏头痛大鼠血清中5-HT、CGRP和ET-1的含量进行统计分析,结果表明:与模型组相比,天麻、川芎配伍组可以显著降低偏头痛大鼠血清中5-HT含量（P<0.05）。麻芎舒通方水提物可以显著降低偏头痛大鼠血清中5-HT和ET-1含量（P<0.05）。麻芎舒通方醇提物可以显著降低偏头痛大鼠血清中CGRP、ET-1和5-HT含量（P<0.05）。对正常对照组、模型组、阳性药对照组和中药复方治则配伍组在偏头痛大鼠脑组织中5-HT含量进行统计分析,结果表明:与模型对照组相比,天麻川芎配伍组、麻芎舒通方水提物和麻芎舒通方醇提物均可以显著降低偏头痛大鼠脑组织中在5-HT含量（P<0.05）。对不同治则配伍的中药方剂进行药效研究,结果显示:以平肝降火、活血行气、散通络止痛为治则的麻芎舒通方药效优于天麻、川芎配伍组。不同的提取工艺具有不同的药理效应,麻芎舒通方乙醇提取优于水煎提取。3中药复方（麻芎舒痛方）的药学研究如何正确地评价中药制备工艺的合理与优劣,是目前困扰中药研究的问题之一。中药复方药学研究是中药复方研究的基础,尽可能地将发挥药效和发挥疗效的物质基础提取出来,才能更好地运用于临床,服务于患者。最佳制备工艺研究是中药复方药学研究的关键环节。3.1川芎不同提取部位治疗偏头痛的药效学研究本文在比较关键的药物或不好确定药物提取路线时,以药效学试验为依据,对川芎多种提取物进行比较,将中药化学-药理效应相结合,并采用HPLC-ECD技术测定偏头痛大鼠血液和脑组织中的5-HT含量,最终确定川芎乙醇提取;通过对其余四味药物化学成分和药理成果的分析,确定四味药物醇提。3.2中药复方提取路线和提取方式的确定以君臣药物的有效成分为指标,对复方的混合提取方式和单独提取方式进行考察,天麻素混和提取/单独提取的比值是102.42%,葛根素混和提取/单独提取的比值是108.54%,说明混合提取更有利于天麻和葛根中有效成分的提取。阿魏酸混和提取/单独提取的比值是99.46%,说明对于阿魏酸来说,混合提取和单独提取基本相当。从大生产和成本考虑,川芎亦选择混合提取方式。3.3麻芎舒痛方的制备工艺研究以L₉（3⁴）正交试验进行最佳工艺条件的筛选和验证,确定了复方的最佳制备工艺,即五味药物以8倍量60%的乙醇提取3次,每次2小时。此工艺在保证君药有效成分提取的同时,兼顾了两味臣药有效成分的提取。五味药物同时提取,具有成本降低、工艺简单有效之优点,有利于大生产。4中药复方（麻芎舒痛方）的制剂质量控制4.1仪器与试药高效液相色谱仪:Waters600泵,Waters 996检测器,Waters717自动进样器,Millennium³² Login工作站;薄层扫描仪,型号:TRANSILLUMINATOR 2020D;空气压缩泵,TOSHIBA Corporation生产;阿魏酸、天麻素、葛根素、川芎对照药材和白芷对照药材为对照品或对照药材。4.2 TLC色谱鉴别照中国药典薄层色谱法（附录ⅥB）试验,对川芎、天麻和葛根进行TLC定性鉴别。川芎的薄层色谱鉴别采用川芎对照药材,以正己烷.醋酸乙酯（9∶1）为展开剂,供试品色谱中,在与对照药材色谱相应的位置上,显相同颜色的荧光斑点;葛根的薄层色谱鉴别采用葛根素对照品,以氯仿-甲醇-水（7∶2.5∶0.25）为展开剂,供试品色谱中,在与对照品色谱相应的位置上,显相同颜色的荧光斑点;白芷的薄层鉴别采用白芷对照药材,以石油醚（60-90℃）-乙醚（1∶1）为展开剂,。供试品色谱中,在与对照品色谱相应的位置上,显相同颜色的荧光斑点。阴性对照均无干扰。4.3含量测定4.3.1样品中阿魏酸的含量测定色谱柱:SymmetryC18（3.9×150 mm,5μm）;流动相A:乙腈-0.05%醋酸水溶液,流动相B:甲醇。0～45 min,A由100%至60%,B由0%至40%,45～50 min,A由60%至0%,B由40%至100%;检测波长为323 nm;流速:1.0ml·min^-1;柱温:30℃;阿魏酸在0.02715～0.4887μg范围内,峰面积和进样量呈良好的线性关系（γ=0.9998,n=7）;阿魏酸样品回收率为98.22%,RSD为1.30%（n=6）;本品每1g含川芎以阿魏酸(C₁₀H₁₀O₄)计,不得少于0.18 mg。4.3.2样品中天麻素的含量测定色谱柱:Symmetry C18（3.9×150 mm,5μm）;流动相:乙腈-0.05%冰醋酸水溶液（2:98）;检测波长为220nm;流速为0.6 ml·min^-1;柱温:室温;天麻素在0.1032～1.0320μg范围内,峰面积和进样量呈良好的线性关系（γ=1.0000,n=7）;天麻素样品回收率为97.81%,RSD为1.49%（n=6）;本品每1g含天麻以天麻素(C₁₃H₁₈O₇)计,不得少于1.92 mg。4.3.3样品中葛根素的含量测定色谱柱:SymmetryC18（3.9×150 mm,5μm）;流动相A:乙腈-0.05%醋酸水溶液,流动相B:甲醇。0～45min,A由100%～60%,B由0%～40%,45～69min,A由60%～0%,B由40%～100%;检测波长为250nm;流速:1.0 ml·min^-1;柱温:30℃;葛根素在0.164～2.05μg范围内,峰面积和进样量呈良好的线性关系（γ=0.9998,n=9）;葛根素样品回收率为97.87%,RSD为1.57%（n=6）;本品每1g含葛根以葛根素(C₂₁H₂₀O₉)计,不得少于13.18 mg。5中药复方中多成分在体肠吸收研究本文初步考察了中药复方中川芎、天麻、葛根的有效成分阿魏酸、天麻素和葛根素在体肠吸收行为。复方配伍和单味药材的肠吸收行为进行比较,阿魏酸的单位面积肠吸收量增加,天麻素和葛根素的单位面积肠吸收量减少,体现出中药复方配伍的协同或拮抗作用;当药物浓度增大时,阿魏酸的单位面积肠吸收量基本相当,天麻素和葛根素的单位面积肠吸收量随浓度增大而增加,药物的吸收属于被动吸收范畴;基于天麻素在复方配伍时天麻素的肠吸收量有较大降低,故将不同药物与天麻进行配伍研究,旨在寻求抑制天麻素吸收的药材来源。天麻分别与川芎、白芷配伍时,天麻素的肠吸收率均达到90%左右。而天麻与葛根配伍时,天麻素的吸收率为46%,天麻素的肠吸收有较大程度的降低,说明复方配伍时抑制天麻素肠吸收的药物是葛根。6 LC-MS-MS法对中药复方中多种有效成分的定性研究本文应用LC-MS-MS法对中药复方（麻芎舒痛方）中多种有效成分进行定性研究。建立了多种成分于同一液相色谱图中的梯度洗脱系统,以LC-MS-MS负离子扫描方式对复方样品Krebes-Ringer’s液中的多种成分进行定性研究,共明确了川芎和葛根中的8种成分,分别为川芎中的阿魏酸、葛根中的4′,7-二葡萄糖苷大豆苷元、3′-羟基葛根素、葛根素、3′-甲氧基葛根素、葛根素木糖苷、大豆苷、大豆黄素。在定性研究中推测出结构的一种成分是滨蒿内酯（Scoparone）,推测它有可能是麻芎舒痛方在共同的提取过程中产生的一种新成分。查阅文献滨蒿内酯的植物来源多为菊科植物滨蒿Artemisia scoparia Waldst.et Kit.花、种子,茵陈蒿A.capillaris Thunb.茎、叶,芸香科植物印度枸（木桔）Aegle marmelos Corr.根和伞形科植物木柴胡Bupleurumfruticescens L.地上部,本复方中没有这些植物,滨蒿内酯（Scoparone）的来源需要进一步确认。LC-MS-MS法对中药复方的定性研究为其物质基础研究和更深入的体内代谢研究打下基础。7研究的创新性◆提出“将多种药理模型与特异性指标相结合,以药味配伍和剂量配比为筛选依据”的中药方剂组方设计研究方法,并对麻芎舒痛方的药味配伍和剂量配比进行筛选,验证了组方设计的可行性和合理性;◆选择与疾病相关的特异性药效指标成分,以最佳制剂进行药效试验验证和药代动力学探索性研究,探讨中药方剂作用和中药复方配伍的相关性;◆建立了多种成分于同一液相色谱图中的梯度洗脱系统,探讨麻芎舒痛方中多成分的在体肠吸收行为,对麻芎舒痛方药物相互配伍规律进行探索性研究,为进一步的药代研究打下基础。更多还原

【Abstract】 Design Studies of the Fractional Optimization of TCM Formula（MXST）A Traditional Chinese Medical formula（TCM formula） is one of the most important parts of the Chinese medical science.Studies on composing prescriptions and drug compatibility are the core of TCM formulas.With the changes of medical mode,more and more new TCM formulas are required.It is the key question to design the prescriptions.This paper puts forward and verifies a new design method of new TCM formulas.Orthogonal experiment design is used during the formulation design of a new TCM formula which is composed of a classical prescription and effective TCM.Two key links in a new TCM formula are the best dose design and drug compatibility,which is obtained by using classical animal model and special pharmacology index; The best chemical preparation is obtained by using the chemical and analysis techniques.System studies on the pharmacology embody the whole view of chemistry of compound prescriptions.System researches on the pharmacology and Pharmacokinetics and the correlation with TCM theory promote the subject development of the TCM prescription.By taking MXSTF as a research object,exploratory researches on TCM formula prove the feasibility and scientific.1 The base of prescription formulation and dose screening on the basis of pharmacodynamics of MXSTFFormula screening by the research of pharmacodynamics was studied firstly.The basic recipe is drafted based on the clinical efficacy and PRC codex.Analgesic effect was the observation index by the orthogonal design.Efficacy impact was studied by TCM formulas with different proportions and doses on three animal models of migraine,then the best drug compatibility and dose ratio were screened.1.1 Influences on the model rats of migraine induced by nitroglycerin by TCM formulas with different proportions and dosesThe influences on the model rats of migraine induced by nitroglycerin by TCM formulas with different proportions and doses were observed.Results:The tenth sample（MXSTF） has the best analgesic effect on the model rats of migraine induced by nitroglycerin.Compared to the model control group,it is showed statistical difference（P<0.05）.1.2 Studies on the pain time of model mouse induced by hot plate by TCM formulas with different proportions and dosesThe studies on the pain time of model mouse induced by hot plate by TCM formulas with different proportions and doses were observed.Results:Pain Time was obviously postponed on the model mouse induced by hot plate because of the second sample、the eighth sample and the tenth sample.Compared to the model control group,it is showed statistical difference（P<0.05）.1.3 Influences on the delay time of model mouse of induced by 2%formaldehyde by TCM formulas with different proportions and dosesThe influences on the delay time of model mouse of induced by 2%formaldehyde by TCM formulas with different proportions and doses were observed.Results:Delay time was obviously longer then other samples on the model mouse ofⅡ-phase-pain induced by 2%formaldehyde because of the sixth sample、 the seventh sample and the tenth sample.It is showed statistical difference when compared to the model control group（P<0.05）.In a word,the base of prescription formulation and dose screening of the TCM formula curing migraine were set up according to these pharmacological effects,which include the famous drug pair and classical drug team.2 Studies on the pharmacology of TCM formulas with different therapeutic principle compatibilityThe influence on the contents of index components was observed by TCM formulas with different therapeutic principle compatibility in rat’s brain tissue and serum.The model rats of migraine induced by nitroglycerin and special pharmacology index were used during the experiment in order to verify the pharmacological action of MXSTF.The contents of 5-HT、CGRP and ET-1 in rat’s serum were statistically analyzed among normal control group、positive drug control group and TCM formulas groups with different therapeutic principle compatibility.Compared with model group,there were significant differences in 5-HT level from the group of Rhizoma Gastrodiae and Rhizoma Chuanxiong（P<0.05）,in 5-HT and ET-1 levels from MXSTF water extract（P<0.05）,and in 5-HT、ET-1和CGRP levels from MXSTF alcohol extraction（P<0.05）.The levels of 5-HT in rat’s brain tissue were statistically analyzed among normal control group、positive drug control group and TCM formulas groups with different therapeutic principle compatibility. The results show that there were obvious differences among Rhizoma Gastrodiae and Rhizoma Chuanxiong group、MXSTF water extract and MXSTF alcohol extraction when compared with model group（P<0.05 ）.The pharmacological actions show that MXSTF is superior to Rhizoma Gastrodiae and Rhizoma Chuanxiong group and MXSTF alcohol extraction is better than MXSTF water extract.3 Studies on the pharmacy of MXSTFThe pharmacy studies is the base of the study of TCM formula.In order to improve clinical effect,the main material basis of drug must be extracted as much as possible.Technology is a key link to the study on the pharmacy of TCM formula.It is one of the issues that how to correctly evaluate the preparation process of Chinese medicine.3.1 Effects on the pharmacodynamic of different ingredients extracted from ChuanxiongPharmacodynamic experiments were as indexes when the route of drug extraction is difficult to determine.Many ingredients were extracted from Chuanxiong.All of them were compared with the pharmacodynamics of migraine.The mode with the combination of Chinese Medicine Chemistry and Pharmacology of TCM is used to determine the extraction route of Chuanxiong.The content of 5-HT was determined by electrochemical method during migraine.Then chuanxiong is extracted by ethanol;Other drugs were also extracted by ethanol.3.2 Determination of the extraction route and the extraction wayUsing effective components of monarch drug and ministerial drugs as a parameter,the effects of mixed extracting and separate extracting were observed.The ratio of mixed extracting/separate extracting for Gastrodin is 102.42%,the ratio of mixed extracting/separate extracting for Puerarin is 108.54%.The dates showed that mixed extracting is more beneficial to the extraction of Tianma and Gegen.The ratio of mixed extracting/separate extracting for Ferulic acid is 99.46%.The mixed extracting is equal to separate extracting for Ferulic acid,so the mixed extracting was chosen for Chuanxiong. 3.3 Studies on the technology of MXSTFThe optimum mixture extraction process by L₉（3⁴） is as follows:80%alcohol extracting third,adding 8times amount of alcohol into Chinese medical materials,once 2 hours.The method has features of low cost,simple process and effectiveness,which will be advantageous to extend and use the formula.4 Quality control of TCM formula（MXSTF）4.1 Instrument and testing drugHPLC:Waters 600 pump,Waters 996 detertor,Waters 717 auto sampler,Millennium³² Login;TLC scanner,TRANSILLUMINATOR 2020D;air compressor pump,TOSHIBA Corporation.Reference substance:Ferulic acid,Gastrodin,Puerarin;Reference drugs:Rhizoma Chuanxiong,Radix Angelicae Dahuricae.4.2 TLC chromatographic identificationn-Hexane-ethyl acetate（9:1） was used for Rhizoma Chuanxiong with crude standard as reference; Trichloromethane- methanol-water（7:2.5:0.25） was used for Radix puerariae lobatae;Petroleum ether-Ether（1:1） was used for Radix Angelicae Dahuricae with crude standard as reference.4.3 Determination4.3.1 Determination of Ferulic acid in sampleColumn:SymmetryC₁₈（3.9×150 mm,5μm）;The mobile phase A was acetonitrile-0.05%acetic water, the mobile phase B was methanol.0-45 min,A from 100%to 60%,B from 0%to 40%;45 - 50 min,A from 60%to 0%,B from 40%to 100%;The detection wavelength was 323 nm;Column temperature was 30℃;The flow rate was 1.0 ml·min^-1;A good linearity of Ferulic acid presented over the range of 0.02715-0.4887μg（γ=0.9998,n=7）;The average recovery rate for the content determination was up to 98.22%,RSD=1.30%（n=5）;Determination of Ferulic acid in the preparation was not less than 0.18 mg per one gram.4.3.2 Determination of Gastrodin in sampleColumn:SymmetryC₁₈（3.9×150 mm,5μm）;The mobile phase was acetonitrile-0.05%acetic water （2:98）;The detection wavelength was 220 nm;The flow rate was 0.6 ml·min^-1;Column temperature was room temperature;A good linearity of Gastrodin presented over the range of 0.1032-1.0320μg（γ=1.0000, n=7）;The average recovery rate for the content determination was up to 97.81%,RSD=1.49%（n=6）; Determination of Gastrodin in the preparation was not less than 1.92 mg per one gram.4.3.3 Determination of Puerarin in sampleColumn:SymmetryC₁₈（3.9×150mm,5μm）;The mobile phase A was acetonitrile-0.05%acetic water, the mobile phase B was methanol.0-45 min,A from 100%to 60%,B from 0%to 40%;45 - 69 min,A from 60%to 0%,B from 40%to 100%;The detection wavelength was 250 nm;Column temperature was 30℃;The flow rate was 1.0 ml·min^-1;A good linearity of Puerarin presented over the range of 0.164-2.05μg（γ=0.9998,n=9）;The average recovery rate for the content determination was up to 97.87%, RSD=1.57%（n=6）;Determination of Puerarin in the preparation was not less than 13.18 mg per one gram.5 Studies on Absorption Kinetics of Multi-composition by Intestinal Backflow in vivoIntestinal absorption behaviors of Ferulic acid、Gastrodin and Puerarin were observed in this thesis. Compared to single herb,the uptakes amount per unit area of Ferulic acid increase in the TCM formula, while the uptake amounts of Gastrodin and Puerarin per unit area decrease.Synergistic or antagonistic effect was shown between compatibility in TCM formula.With drug concentration increasing,the uptake amounts of Gastrodin and Puerarin were increased.The absorption way fits the passive transport mechanism;Based on the great decrease of Gastrodin in TCM formula,the studies of compatibility of Tianma with different drugs were carried out.The absorption rate of Chuanxiong and Tianma、Baizhi and Tianma were all for 90%,while the absorption rate of Gengen and Tianma was 46%.Therefore Gengen is the herb source which restrains the absorption of Gastrodin in intestinal.6 Qualitative Study on Multi-composition in TCM Formula by LC-MS-MSThis paper established gradient elution system which can include multi-composition in the same chromatogram.Eight kinds of elements of Chuanxiong and Gengen dissolved in Krebes -Ringer’s solution were determined with LC-MS-MS negative ion scan mode,which are Ferulic acid、daidzein 4’-7-diglucoside、3’-OH Puerarin、Puerarin、3’-OCH₃ Puerarin、Puerarin xyloside、Daidzin and Daidzein respectively.It needs to further research on two unknown materials and Scoparone.The source of Scoparone needs to be further determined because the TCM formula do not include the plants which Scoparone coming from,namely Artemisia scoparia Waldst.et Kit.、A.capillaris Thunb.、Aegle marmelos Corr.and Bupleurum fruticescens L.Qualitative study by LC-MS-MS is as a basis for further studies on the material base and metabolism in vivo.更多还原