【作者】 吕永海；

【导师】 张卫东；

【作者基本信息】 第二军医大学 ， 药物化学， 2010， 硕士

【摘要】 复方丹参片是由丹参(Radix Salviae Miltiorrhizae)、三七(Radix Notoginseng)和冰片(Borneolum Syntheticum)三味中药制成的现代中药制剂,收载于2005版中华人民共和国药典第一部,具有活血化瘀、理气止痛之功效。自20世纪70年代问世以来,复方丹参片在治疗心血管疾病方面取得了显著的成效,被广泛用来治疗心肌梗死和冠心病。临床上,关于复方丹参片改善心脏功能的机制可谓是众说纷纭,包括降低血中脂质、抑制血栓形成等。到目前为止,还没有任何一个研究能够系统地阐释复方丹参片的作用机制。另外,值得注意的是复方丹参片中化学成分众多,但并非所有的成分都是有效成分。在药效物质基础不够明确的情况下,中药复方作用机制的研究确实很难取得实质性的进展。因此,本文旨在揭示复方丹参片药效物质基础的前提下继续深入研究其治疗心肌缺血的作用机制。众所周知,中药复方是在病证结合、方证对应、理法方药一致的条件下,通过多组分作用在多靶点,融拮抗、补充、整合、调节等多种功效于一体而起到治疗作用。由此可见,中药复方的药效作用十分注重系统性和整体性。所以,对中药药效物质基础及作用机制的研究也应从整体入手。在众多的研究方法中,血清药物化学和代谢组学都十分注重整体的研究,这与中医的整体观思维不谋而合:血清药物化学方法通过分析给药后血清中的药物移行成分,研究有效成分在体内的吸收、代谢情况,用以阐明药物的整体体内过程;代谢组学方法通过分析内源性代谢物群的改变,判别机体代谢网络所处的状态,用于疾病诊断和药物作用机制的研究。在血清药物化学和代谢组学两大理论的指导下,本文采用液相色谱串联质谱等分析方法,通过整体动物实验,对复方丹参片药效物质基础及其治疗心肌缺血的作用机制进行了较系统的研究。主要研究内容如下:1)在血清药物化学理论指导下,利用快速液相色谱串联四极杆飞行时间质谱(RRLC-Q-TOF/MS),检测复方丹参片的入血成分和代谢产物,即通过比较给药前后大鼠的代谢指纹图谱,寻找并鉴定给药后血清中新出现的化学成分。2)在代谢组学理论指导下,利用超高压液相色谱串联四极杆飞行时间质谱(UPLC-Q-TOF/MS),鉴定心肌缺血的生物标志物,即通过比较造模前后大鼠的代谢指纹图谱,寻找并鉴定造模后呈现特征性改变的内源性代谢物。3)在心肌缺血模型代谢组学研究的基础上,进一步研究常见五种常见西药(异硝酸山梨酯,普奈洛尔,维拉帕米,卡托普利,曲美他嗪)与中药(复方丹参片及其有效成分)对心肌缺血大鼠的保护作用,通过中西药对比的方式,初步阐释了复方丹参片及其有效成分(丹参酮ⅡA、丹参酚酸B)治疗心肌缺血的作用机制。通过上述研究,本文在给药大鼠血清中检测到复方丹参片的14个原型成分和8个代谢产物,其中丹参醛、丹参新醌B、丹参素的糖结合物(M1)和丹参新醌B的甲基化产物(M8)等是利用LC-MS方法首次在大鼠体内检测到;在心肌缺血大鼠血浆中发现并最终鉴定了与疾病相关的22生物标志物,其中7个标志物同时出现在嘌呤代谢途径中,提示我们嘌呤代谢是心肌缺血后重点关注的对象;通过中西药对比研究,发现复方丹参片不同于任何一种西药具有其独特的治疗心肌缺血的机制,而丹参酮ⅡA和丹参酚酸B分别具有调节钙离子通道和抑制肾上腺素β受体的作用。更多还原

【Abstract】 Compound Danshen Tablets (CDTs), an herbal compound preparation consisting of Radix Salviae Miltiorrhizae, Radix Notoginseng and Borneolum Syntheticum, which was officially recorded in the 2005’s edition of Chinese pharmacopoeia, holding the functions of promoting blood circulation and removing blood stasis and regulating qi-flowing to relieve pain. It has been wildly used for the treatment of cardiovascular diseases (myocardial infarction, coronary heart disease…) via decreasing blood lipids and inhibiting thrombosis since 1970s. However, mechanisms of action of CDTs are not completely understood. In addition, there are many chemical compositions in the formula, what is important, not all the compounds are active components. During the last years, the study of Chinese medicine was limited for the lack of effective methodology. In our study, we aimed to find the active components of CDTs and explore mechanisms of action of CDTs.As we known, formulas play complex therapeutic effects through roles of multi-component on multi-target under special conditions. The efficacy of Chinese medicine attaches great importance to systematic and holistic actions. Therefore, the study of active components mechanism of Chinese medicine should start with a whole. Among the various methods, the serum pharmacochemistry and metabolomics studies are very holistic, which happens to coincide with Chinese medicine as a whole concept. Serum pharmacochemistry study aimed to explore the active components of drugs by analyzing the serum after administration to explain the whole process of drugs in vivo; metabolomics approach aimed to judge the state of body’s metabolic network by analyzing endogenous metabolites to diagnose diseases and study mechanisms of action of drugs.Based on theories of serum pharmacochemistry and metabolomics, with the help of advanced LC-MS and other modern analytical techniques, we carried out the study of pharmacodynamic material basis of CDTs and investigation of mechanisms of action for the treatment of myocardial ischemia through the animal experiment. The main content is as follows:(1) Based on serum pharmacochemistry theory, RRLC-Q-TOF/MS analysis method was established to detect components and metabolites of CDTs in rat serum after administration, which meaned to discover novel compounds in dosed rat serum by comparing the metabolic fingerprinting between the control and dosed groups.(2) Based on metabolomics theory, UPLC-Q-TOF/MS analysis method was established to explore the biomarkers of myocardial ischemic, which meaned to detect and identify significant changed metabolites by comparing the metabolic fingerprinting between the sham and myocardial ischemic model groups.(3) Based on the results of metabolomic study, we further studied the protections of five western medicines with different mechanismes of action (Verapamil, Captopril, Propranolol,Isosorbide Dinitrate and Trimetazidine) and Chinese medicines (CDTs and its active components). Finally, mechanisms of CDTs and its active components (TanshinoneIIA and Salvianolic acid B) for the treatment of myocardial ischemia were explained by comparison with western medicines.In summary, 14 components and 8 metabolites of CDTs were detected in dosed rat serum; 22 metabolites were identified as the biomarkers of myocardial ischemia; after comparative study of Chinese medicine and western medicine, CDTs has unique characteristics for the treatment of myocardial ischemia, TanshinoneIIA is a calcium antagonist and Salvianolic acid B is a beta-adrenergic blocker.更多还原