【作者】 裴利霞；

【导师】 杜冠华； 刘艾林； 方莲花； 张莉； 张天泰；

【作者基本信息】 中国协和医科大学 ， 药理学， 2008， 博士

【摘要】 中药复方是中医药理论的具体体现,但其作用机理及质量控制问题限制了中药复方的进一步使用。从中药复方药动学研究入手,研究服药后体内出现的中药成分及其代谢产物,对于认识中药复方的药效物质基础具有十分重要的作用,能够促进中药的现代化、科学化和国际化。消栓通络方由11味中药（黄芪、川芎、丹参、泽泻、山楂、三七、槐花、桂枝、郁金、木香、冰片）组成,临床上用于治疗血脂增高、脑血栓引起的精神呆滞、舌质发硬、言语迟涩、发音不清、手足发凉、活动疼痛等症,是治疗心血管疾病的传统中药复方。为开展服用消栓通络方有效成分组后主要药效成分的药动学研究,本文建立了灵敏、特异地同时定量检测方中四种化合物的分析方法。运用这种分析方法研究了服用XECG后,芦丁、金丝桃苷、槲皮素和山柰酚在大鼠体内的药动学特征。另外本文还对SAA在大鼠体内的吸收、分布、代谢及排泄,匹诺塞林在Beagle犬体内的药动学及代谢产物进行了探讨。第一部分本试验选择XECG作为研究对象,研究了大鼠口服XECG后,芦丁、金丝桃苷、槲皮素和山柰酚的药动学特征。大鼠口服剂量为300 mg/kg XECG后,5 min血中即可检测到芦丁、金丝桃苷、槲皮素、山柰酚,四种成分的达峰时间依次为0.5、0.5、4、4 h,达峰浓度分别为12.16±1.87、13.84±4.29、17.34±3.42、5.58±1.12 ng/ml。血浆药时曲线经梯形法计算,芦丁、金丝桃苷、槲皮素、山柰酚的AUC_0→24h分别为72.59±14.23、99.01±16.62、161.60±32.83、60.31±12.88 ng·h/ml。第二部分这部分实验主要是对SAA在大鼠体内的吸收、分布、代谢及排泄进行探讨,建立LC/MS/MS法测定SAA在大鼠血浆的浓度及组织分布情况。应用HPLC、LC-MSⁿ等方法从大鼠尿样及胆汁中发现了SAA的5种代谢产物。本研究还探讨了SAA对大鼠肝微粒体P450酶的影响。初步阐明了SAA在大鼠体内的吸收、分布、代谢及排泄过程,为指导新药开发和临床用药提供了一定的理论依据。1.SAA在大鼠体内的吸收动力学研究建立了测定大鼠血浆中SAA的LC/MS/MS法。线性范围为1.4-1000 ng/ml,最低定量浓度为1.4 ng/ml。绘制大鼠灌胃和静脉给予SAA后的血浆药物浓度-时间曲线,计算其主要药物动力学参数,评价大鼠给药后SAA在体内的药动学特征。2.SAA在大鼠体内的组织分布研究建立了测定大鼠体内各组织中SAA的LC/MS/MS法。实验结果发现,在大鼠体内许多组织中都能检测到SAA,表明SAA在大鼠体内广泛分布。3.SAA在大鼠体内的代谢研究应用HPLC,LC/MS等方法从大鼠尿样及胆汁中发现了SAA的5种代谢物,通过分析SAA及其代谢产物的多级质谱特征丢失碎片及质谱裂解规律,并与相关文献比较对照,推测了代谢产物的结构。SAA在大鼠体内主要经历3种代谢途径,包括甲基化、单葡萄糖醛酸结合反应,甲基化和葡萄糖醛酸结合双重反应。4.SAA在大鼠体内的排泄研究大鼠口服SAA后48 h尿排泄总量约占给药量的0.014%,粪排泄总量约占给药量的0.0022%,大鼠给药后12 h胆汁排泄总量约占给药量的0.102%。5.SAA对大鼠肝微粒体P450酶的影晌通过对CYP1A2的特异性底物非那西丁,CYP2C9的特异性底物甲苯磺丁脲,CYP2C19的特异性底物奥美拉唑,CYP2D6的特异性底物右美沙芬,CYP2E1的特异性底物氯唑沙宗,CYP3A4的特异性底物硝苯地平进行分析,评价了SAA对大鼠肝微粒体CYP1A2、CYP2C9、CYP2C19、CYP2D6、CYP2E1和CYP3A4活性的影响,结果表明:SAA对大鼠肝微粒体P450酶的各个亚型既没有明显的抑制作用,也没有明显的诱导作用。同时发现,SAA在体内对大鼠肝徽粒体P450总量及蛋白总量都没有明显的增加或减少作用。第三部分这部分研究主要是对匹诺塞林在Beagle犬体内的药代动力学及代谢产物进行探讨,建立了测定Beagle犬血浆中匹诺塞林浓度的LC/MS法,考察了匹诺塞林在Beagle犬体内的吸收动力学。Beagle犬口服100 mg/kg匹诺塞林后5 min血中即可检测到原形药,达峰时间为120 min,达峰浓度为4697.40 ng/ml。应用HPLC、LC-MSⁿ等方法从Beagle犬尿样及血样中发现了匹诺塞林的2种代谢产物并对这两种代谢产物进行鉴定。更多还原

【Abstract】 Herbology is traditionally one of the more important modalities utilized in traditional Chinese Medicine,however,their unclear pharmacological mechanisms and the difficulties in quality control restrict their wider clinical use.Clarifying the effective compounds in Chinese multiherb remedy,which can be achieved by pharmacokinetic and metabolism study,has an important role in realizing the modernization,scientific research,and internationalization of traditional Chinese Medicine.XiaoShuanTongLuo,composed of eleven herbs（Milkvetch Root,Szechwan Lovage Rhizome,Danshen Root,Hawthorn Fruit,Sanchi,Turmeric Root Tuber,Common Aucklandia Root,Pagodatree Flower,Oriental Waterplantain Rhizome,Cassia Twig, Borneol） is a famous Chinese traditional prescription with better curative effect on cardiovascular diseases,acroparalysis and other symptoms arising from apoplexy.To understand the oral absorption and the metabolism of the active constituents of XiaoShuanTongLuo Effective Components Group,a specific and sensitive analytical method was established,to simultaneously determine rutin,hyperoside,quercetin and kaempferol,which were applied to analyze the pharmacokinetic study of XiaoShuanTongLuo Effective Components Group.In addition,absorption,tissue distribution,metabolism and excretion of SAA were studied.Whereas,pharmacokinetics and metabolism of pinocembrin were also studied in this dissertation.PartⅠXiaoShuanTongLuo Effective Components Group was studied in this dissertation.A sensitive method was developed,which was further applied to assess the pharmacokinetics of rutin,hyperoside,quercetin,kaempferol in rats after oral administration of XiaoShuanTongLuo Effective Components Group.After single oral doses of 300 mg/kg to rats,rutin,hyperoside,quercetin and kaempferol all appeared in rat plasma at 5min after administration.The peak plasma concentration occurred at 0.5,0.5,4,4 h with the C_max12.16±1.87,13.84±4.29,17.34±3.42,5.58±1.12 ng/ml,respectively.The AUC_0→24h were 72.59±14.23,99.01±16.62,161.60±32.83,60.31±12.88 ng·h/ml. PartⅡPharmacokinetics and metabolism of SAA were studied,including absorption,tissue distribution,metabolism and excretion in rats.A sensitive analytical method was established for SAA in rat plasma,which was further applied to assess the pharmacokinetics and tissue distribution of SAA in rats receiving a single oral dose of SAA.Using the multi-stage MS（MSⁿ） method to analyze SAA and its metabolite of urine and bile in rats,the characteristic fragment ions were obtained.LC/MSⁿ method was eatablished to identify five metabolites.The effect of SAA on P450 enzymes in rat liver microsomes was evaluated for the first time.These studies provided scientific data for the new drug development and clinic use.1.Absorption pharmacokinetics in ratsTo investigate the absorption pharmacokinetics of SAA in rats,a sensitive analytical method was firstly established and validated for SAA in rat plasma,which was further applied to assess the pharmacokinetics of SAA in rats receiving a single oral dose of SAA.Linear calibration curves were obtained over the range of 1.4-1000 ng/ml for SAA in rat plasma.The lower limit of quantification was 1.4 ng/ml.Plasma concentration data were analyzed after i.v.or i.g.adminstration of SAA.2.Tissue distribution in ratsA LC/MS/MS method was developed to determine SAA in rat tissues.The results showed that SAA was distributed widely after oral administration to rats and could be found in many tissues.3.Metabolism of SAA in ratsMetabolites in rat urine and bile were investigated.A total of five metabolites were found in urine and bile by HPLC with ion trap mass spectrometric detection.Using the multi-stage MS（MSⁿ） method analysis of SAA and its metabolite,the characteristic fragment ions were obtained.A LC/MSⁿ method was eatablished to identify five metabolites.There were three main possible metabolic pathways of SAA in rat.In the first pathway SAA was absorbed into the blood and conjugated with methyl group.In the second pathway SAA was absorbed into the blood and conjugated with glucuronic acid group.In the third pathway SAA was absorbed into the blood and conjugated with both glucuronic acid group and methyl group.4.Excretion of SAA in ratsSAA was excreted approximately 0.102%into the bile in rats after 12 h post-dose.In feces,0.0022%was recovered after 48 h post-dose.In urine,0.014%was recovered after 48 h post-dose in rats. 5.The effect of SAA on P450 enzymes in rat liver microsomesUsing the CYP-specific substrate,the effect of SAA on CYP450s（CYP1A2, CYP2C9,CYP2C19,CYP2D6,CYP2E1 and CYP3A4） was evaluated both in vivo and in vitro studies.The data showed SAA neither induced nor inhibited the activities of CYP450s in rat liver microsomes.It was also found that SAA had no significant effect on rat liver microsomal protein and CYP450 content.PartⅢPharmacokinetics and metabolism of pinocembrin were studied in this part.A sensitive analytical method was established for pinocembrin in Beagle dog plasma, which was further applied to assess the pharmacokinetics of pinocembrin in Beagle dog receiving a single oral dose of pinocembrin.After single oral doses of 100 mg/kg to Beagle dog,pinocembrin appeared in Beagle dog plasma at 5min after administration. The peak plasma concentration occurred at 120 min with the C_max 4697.40 ng/ml.Using the multi-stage MS（MSⁿ） method to analyze the metabolite of urine and plasma in Beagle dogs,the characteristic fragment ions were obtained.LC/MSⁿ method was established to identify two metabolites.更多还原