【作者】 吴晓霞；

【导师】 于友华；

【作者基本信息】 中国中医科学院 ， 中医基础理论， 2009， 硕士

【摘要】 黄连解毒汤是中医方剂学中应用广泛的方剂之一,也是体现“异病同治”思想的有效经典方剂。黄连解毒汤是清热解毒方剂的代表方剂,主治一切实火热毒,三焦热盛之证。黄连解毒汤自古以来在临床上的应用就非常丰富,涉及多个类别的疾病,有较为成熟的临床研究背景。同时,根据黄连解毒汤的现代药理研究,证实了黄连解毒汤对糖尿病相关病理过程的干预有一定疗效。本课题旨在从黄连解毒汤治疗糖尿病的三个有效生物碱成分——药根碱、巴马汀、小檗碱为切入点,进行糖尿病大鼠体内药代动力学实验,观察这三个生物碱成分在糖尿病大鼠体内的药动学特点。同时寻找三者在正常和糖尿病大鼠体内的药动学差异,从而为黄连解毒汤“异病同治”疗效原理提供依据。本论文包括文献整理研究和实验研究两个部分。第一部分包含黄连解毒汤的历史背景、黄连解毒汤的临床应用、黄连解毒汤复方的现代药理研究、黄连解毒汤治疗糖尿病药效学研究、黄连解毒汤中各药味化学成分研究、黄连解毒汤的药代动力学研究等相关研究综述。通过文献研究可以发现黄连解毒汤临床应用广泛,同时具有抗炎、抗菌、抗内毒素、抗氧化、降脂、降血糖以及抗脑缺血、抗肿瘤等多种药理作用,是“异病同治”的代表方剂。在黄连解毒汤干预众多疾病中,其对糖尿病有明显的疗效,是目前中药治疗糖尿病的关注方剂之一,对其治疗糖尿病的机制研究有不少实验方面的探讨。其中生物碱类成分对糖尿病有明显治疗作用,主要认为小檗碱具有显著的降糖和降脂作用,药根碱和巴马汀等生物碱类成分是治疗糖尿病的有效成分。在药代动力学方面,对黄连解毒汤体内药代动力学研究主要集中在正常大鼠体内的几类药物成分的药动学分析在糖尿病大鼠体内的药动学研究只有黄芩苷和汉黄芩苷等黄酮类成分,未见生物碱类的报道。故本文提出药根碱,巴马汀和小檗碱是黄连解毒汤中具有代表性的三个有效生物碱,它们在正常大鼠体内和糖尿病大鼠体内的药动学过程如何,在生理和病理不同机体状态下是否存在药代动力学差异尚不清楚。第二部分为实验研究部分。首先,我们针对目前对于黄连解毒汤中这三个生物碱在血浆中含量的检测方法主要是HPLC方法,但与液质联用法相比存在灵敏度不够高,分析时间较长、消耗生物样品过大等不足,建立一个灵敏的检测方法进行体内药动学研究方法,这是本项研究不可或缺的前提。因此在本课题实验研究中,我们建立灵敏、特异、准确、可靠的药根碱、巴马汀、小檗碱在大鼠血清中液质联用（LC-MS/MS）分析方法。通过优化药根碱、巴马汀、小檗碱和内标延胡索乙素质谱检测条件及确定相关其他条件并开展方法学考察。在ESI（+）离子化条件下采用MRM工作模式,用m/z 338→322、351.9→308、336→319.8来分别检测药根碱、巴马汀、小檗碱,同时以m/z 356.1→192.1来检测内标延胡索乙素。药根碱在0.2 ng·mL^-1～25 ng·mL^-1、巴马汀、小檗碱在0.4 ng·mL^-1～50 ng·mL^-1呈良好线性关系。三种生物碱在高、中、低三种浓度的准确度、精密度、稳定性等均符合要求。为下一步黄连解毒汤三种生物碱在大鼠体内的药动学研究打下基础。通过大鼠腹腔注射链脲佐菌素（STZ）建立糖尿病模型大鼠后,灌胃给予正常组及糖尿病模型组大鼠黄连解毒汤,经时取血,应用上一步所建立的液质联用分析方法进行这三种生物碱含药血清的定量分析,其测定结果用DAS 2.0软件进行分析,以拟合度和AIC值作为判断标准,选择适当的房室模型,计算药动学参数。研究这三种生物碱在正常和糖尿病状态下的药动特点,并进行二者之间的药动学比较。结果表明黄连解毒汤中药根碱、巴马汀、小檗碱在正常及糖尿病大鼠体内的药时过程均符合一室开放模型,但三者分别在AUC_0-t、T_max、C_max、MRT_0-t等方面与正常组存在明显差别。从结果分析来看,药根碱在糖尿病大鼠体内药代过程与正常组大鼠相比有吸收快,血药浓度高的特点;巴马汀在糖尿病大鼠体内以消除过程缓慢为主要表现;小檗碱在糖尿病大鼠体内的吸收要好于正常组,同时会加快代谢。提示黄连解毒汤中这三个生物碱在生理和病理两个不同机体状态下药代动力学存在着一定的差异。本实验为生理和病理两个不同机体状态下黄连解毒汤的血液药动学研究提供实验依据,进而为黄连解毒汤其他成分的药动学研究和中药方剂“异病同治”物质基础研究提供借鉴。本实验所建立的液质联用（LC-MS/MS）分析方法,可以为相关中药及其方剂有效成分的检测提供高效、快速、灵敏的检测手段。更多还原

【Abstract】 Huanglianjiedu Decoction is one of the widely applied Chinese medical formulas. It is also the effective formula which can reflect the thought of "the same syndrome for Different Disease".Huanglianjiedu Decoction is a typical one in clearing away the heat-evil and expelling superficial evils.From time immemorial,Huanglianjiedu Decoction has been widely applied in clinical to treat several diseases. So it has a more mature clinical research background. According to the modern pharmacological research ,Huanglianjiedu Decoction can interferes the pathological process of diabetes mellitus in a certain degree effectively.So in this thesis, we pay attention to the three effective alkaloids in Huanglianjiedu Decoction---jatrorrhizine、palmatine、berberine, study the pharmacokinetics of the three alkaloids in rats serum after an intragastrical（i.g.） administration of Huanglianjiedu Decoction to rat ,observe the pharmacokinetic character of the three alkaloids in diabetic rats serum,find out the pharmacokinetics difference of the three alkaloids in two models.The work of this thesis were separated to two parts:document sorting and experimental study.The first part contains the historical background of Huanglianjiedu Decoction,the clinical application of Huanglianjiedu Decoction,the modern pharmacological research of Huanglianjiedu compound,the mechanism study on the treatment of diabetes mellitus by Huanglianjiedu Decoction,the chemical composition of Huanglianjiedu Decoction and the pharmacokinetic study of Huanglianjiedu Decoction .We found Huanglianjiedu Decoction was widely used in clinical which played many roles on disease treatment, such asanti-inflammatory,antimicrobial,antiendotoxin,anti-oxidant,lipid-lowering,hypoglycemi c action,anti-cerebral ischemia, antitumor etc.Among them Huanglianjiedu Decoction had significant curative effects on diabetes mellitus.There were many experimental researches of the mechanism study on the treatment of diabetes mellitus by Huanglianjiedu Decoction.The alkaloid of Huanglianjiedu Decoction acted effectively on diabetes mellitus.It was considered that berberine has significant lipid-lowering and hypoglycemic function.In addition, jatrorrhizine and palmatine were also effective components.In pharmacokinetics,the research of Huanglianjiedu Decoction focused on pharmacokinetic analyse of some kind of medicine components in the normal rats in vivo.In diabetic rats,there were only the pharmacokinetics researchs of baicalin and wogonoside, the research of alkaloids has not been reported. So we suggested that Jatrorrhizine、palmatine、berberine are three effectively alkaloids in Huanglianjiedu Decoction,the pharmacokinetics process of them in normal rats and diabetic rats are still unclear. And we still don’t know whether their pharmacokinetics are different in the different body state between physiology and pathology.The second part is experimental study.Earlier publications had described methods on analysis of jatrorrhizine、palmatine and berberine in plasma were HPLC mostly.Compared with LC-MS/MS, HPLC was less sensitive and more time and biomedical samples consuming etc. But a sensitive detection method was a prerequisite to the pharmacokinetic evaluation. So, we plan to establish a sensitive and reliable high-performance liquid chromatography-tandem mass sepctrometry （LC-MS/MS） method on simultaneous determination of jatrorrhizine, palmatine and berberine in rat serum. The optimal ionization and fragmentation conditions,as well as HPLC ones,to detect jatrorrhozine,palmatine and berberine were developed and validated. The detection was performed by MRM mode via electrospray ionization （ESI） source operating in the positive ionization mode.The precursor-to-product ion transitions were at m/z 338→322 for jatrorrhozine , m/z 351.9→308 for palmatine, m/z 336→319.8 for berberine and m/z 356.1→192.1 for tetrahydropalmatine（IS） respectively. The method was linear over the concentration range of 0.2-25 ng·mL^-1 for jatrorrhozine,0.4-50 ng·mL^-1 for palmatine and berberine. The method was validated according to the requirements.It laies the foundation for pharmacokinetic study of jatrorrhizine、palmatine and berberine after oral administration of Huanglianjiedu Decoction.The diabetic model of Sprague-Dawleg rats were established by injecting streptozotocin（STZ） to intraperitoneal. After the Huanglianjiedu Decoction was administered to rats of normal group and the diabetic group through ig route, LC-MS/MS method has been applied to the pharmacokinetic study of the three alkaloids in rat serum. DAS procedure was used to process concentration-time data. To research the pharmacokinetic characters of the three alkaloids in physiology state and pathology state,the results indicate that the pharmacokinetic process of the three alkaloids after oral administration of Huanglianjiedu Decoction were both fitted to be a one-compartment model. But they were different in AUC_0-t, T_max, C_max , MRT_0-t between nomal rats and diabetic rats. The results showed that jatrorrhizine in the diabetic group was absorbed more quickly and got higher serum concentration than rats of normal group; palmatine is more slowly during the process of elimination in the diabetic group; berberine was absorbed better and metabolize more quickly than normal group. It proved that there was different between physiology state and pathology state in pharmacokinetic of the three alkaloids.The experiment was the basis study for pharmacokinetic of Huanglianjiedu Decoction in physiology state and pathology state. Furthermore, it also provided reference for pharmacokinetic study of other compounds of Huanglianjiedu Decoction and material base research of "the same syndrome for Different Disease". In the present study,a highly seletive LC/MS/MS method was developed. It could be modified to make it applicable for determination of other alkaloids of TCM in serum.更多还原