【作者】 随志刚；

【导师】 刘忠英；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 乌头类中药川乌、草乌、附子均为中国药典收录的传统的中药,它们的主要活性成分和毒性成分均为乌头碱、中乌头碱和次乌头碱等C19双酯二萜型乌头类双酯型生物碱。因此,研究三种乌头类双酯型生物碱在体内代谢和组织分布,对于阐明乌头类中药药理学毒理学作用机理,指导临床合理用药具有重要意义。本论文研究了乌头碱、中乌头碱和次乌头碱质谱裂解规律,建立了基于LC/MSn的结构鉴定方法,从三种乌头类双酯型生物碱质谱裂解规律中找出了与结构特异性相关的“诊断离子”,对主要活性基团的电离活性进行排序,并将其用于代谢产物的结构鉴定。在上述研究的基础上,对这三种乌头类双酯型生物碱的体内代谢产物进行了结构鉴定,从家兔消化道内容物中发现了97种代谢产物,其中乌头碱有15类33个代谢产物;中乌头碱的32种代谢产物均可从乌头碱的代谢产物找到分子量相差14的对应化合物,次乌头碱的32种代谢产物也都能从乌头碱的代谢产物找到分子量相差30的对应化合物,说明这三种生物碱具有基本相同的代谢规律。其中65种代谢产物尚未见报道。采用液相色谱和三重四极杆型质谱联用仪,优化色谱和质谱条件,选用MRM模式,建立了测定动物组织中乌头碱、中乌头碱和次乌头碱的液相色谱?串联质谱(LC/MS/MS)法。并考察了组织中这三种乌头类双酯型生物碱的分布情况,结果表明,三种生物碱均主要集中在胆和肝脏内,在脾、肾、心、肺、脑中也有分布,三种生物碱在脏器中的浓度由大到小依次为胆、肝、脾、肾、心、肺、脑。更多还原

【Abstract】 Radix Aconiti, Radix Aconiti Kusnezoffii and Radix Aconiti Lateralis Preparata are traditional Chinese herbs which have extensive activities in anti-inflammatory, abirritation and anti-rheumatism. At the same time, they are well known for their acute and high toxicity, for example, in the causation of severe arrhythmias leading to death. Aconitine, Mesaconitine and Hypaconitine are pharmacologically improved to be the main active ingredients and toxic ingredients as well in the herbs. So, it is very necessary to research the metabolism and distribution of Aconitine, Mesaconitine and Hypaconitine. The aim of this work is to investigate the Metabolism and tissue distribution of them in vivo using the advanced LC/MSn and LC/MS/MS methods.1 The fragmentation pathway of Aconitine, Mesaconitine and HypaconitineThis work explained formation of main diagnostic fragment ions by using LCQ mass spectrometer and shown that [M+H-60]+ was specifically relevant to C8-acetoxyl group, [M+H-18]+ to C3-hydroxyl group and [M+H-60-32-28]+ to C15-hydroxyl group. At the same time, we also discuss the fragmentation sequence of main groups and deduced that C8-acetoxyl group is the first active group in fragmentation, C1-methoxy group the second and C6- methoxy group the third。2 Identification of the metabolites of Aconitine, Mesaconitine and Hypaconitine in rabbits The characteristic fragmentation of Aconitine, Mesaconitine or Hypaconitine wereobtained by analyzing their behavior in multi-stage MS（Msn）. The fragmentation pathways can provide theoretical support to help us to identify the infinite structure of metabolites. Metabolites in the gastrointestinal tract, feces, urine and bile of rabbits administrated a single oral dose of 2mg·Kg-1 Aconitine, Mesaconitine or Hypaconitine were investigated respectively. A total of 97 metabolites were found except the unchanged drug. A number of 33 metabolites of aconitine were analyzed in 15 kinds as fellows: （1）Metabolites which MW less 14 than aconitine （aMD14-1～aMD14-6）, we deduced that aMD14-1, aMD14-3 or aMD14-6 were demethylate of methoxy group in C1, C6 or C16; aMD14-2 and aMD14-4, one was demethylate of methoxy group in C18 and the other can not be identified; aMD14-5 was 8-formyl-14-benzoyl-aconine. （2）Metabolites which MW more 16 than aconitine (aM_P16-1～aMP16-6), we deduced that aM_P16-1 or aM_P16-3 were 3′-hydroxyl aconitine or 4′-hydroxyl aconitine; aMP16-6 was 2′-hydroxyl aconitine; aMP16-4和aMP16-5 can not be identified; aM_P16-2 may be a produce of hydroxylation occurred in methoxy group. （3）Metabolite which MW less 42 than aconitine （aMD42）, we deduced it as 14-benzoyl-aconine. （4）Metabolite which MW less 104 than aconitine （aMD104）, we deduced it as 8-acetyl -aconine. （5）Metabolites which MW less 30 than aconitine （aMD30-1～aMD30-5）, aMD30-3 was deduced as indaconitien, aMD30-5 was deduced as deoxyaconitine and others can not be identified. （6）Metabolite which MW more 28 than aconitine （aMP28）, we deduced it as 8- butyryl -aconine. （7）Metabolites which MW more 30 than aconitine （aMP30-1, aMP30-2）. Their structures can not be deduced. （8）Metabolites which MW more 182 than aconitine （aMP182-1 , aMP182-2）. They were deduced as 8-pentadecanoyl-14-benzoyl-aconine or 8- （13-methyl-tetradecanoyl）-14-benzoyl- aconine. （9）Metabolites which MW more 196 than aconitine （aMP196-1, aMP196-2）. They were deduced as 8-palmityl -14-benzoyl-aconine or 8–（14-methyl-pentadecanoyl）-14-benzoyl- aconine. （10）Metabolite which MW more 224 than aconitine （aMP224）. It was deduced as 8-stearoyl-14-benzoyl-aconine. （11）Metabolites which MW more 316 than aconitine （aMP316-1 and aMP316-2）. Their structures can not be deduced. （12）Metabolites which MW more 168 than aconitine （aMP168-1, aMP168-2）. They were deduced as 8-myristoyl-14-benzoyl- aconine or 8-（12-methyl-tridecanoyl）-14-benzoyl-aconine. （13）Metabolite which MW more 218 than aconitine （aMP218）. It was deduced as 8-linolenoyl-14-benzoyl-aconine. （14）Metabolite which MW more 220 than aconitine （aMP220）. It was deduced as 8–linoleoyl- 14-benzoyl-aconine. （15） metabolites which MW more 222 than aconitine （aMP222-1, aMP222-2）. It was deduced as 8–oleoyl-14-benzoyl-aconine or 8-（11-methyl- jecericanoyl）- 14-benzoyl-aconine. Mesaconitine and Hypaconitine have the similar metabolism pathways with the Aconitine, and their metabolites were identified by analyzing the chromatography and mass spectra and comparing with reference substance.3 Tissue distribution of Aconitine, Mesaconitine and Hypaconitine in rabbitsA sensitive LC/MS/MS method was development with MRM mode to simultaneously determine Aconitine, Mesaconitine and Hypaconitine in biological samples of rabbits. The tissue distribution of Aconitine, Mesaconitine and Hypaconitine was investigated at 4 h Post-dose in rabbits. The result shows that the order of the concentration of Aconitine, Mesaconitine and Hypaconitine for high level to light level in the seven main tissues all are: gallbladder, liver, spleen, kidney, heart, lung, brain.更多还原