钩藤属 (Uncaria) 植物为茜草科 (Rubiaceae) 木质藤本，全球有 34 种，我国有11 种、1变型。《中国药典》规定中药钩藤 (Uncariae Ramulus Cum Uncis)为钩藤 U. rhynchophylla (Miq.) Miq. ex Havil.、大叶钩藤 U. macrophylla Wall.、毛钩藤 U. hirsuta Havil.、华钩藤 U. sinensis (Oliv.) Havil.或无柄果钩藤 U. sessilifructus Roxb.的干燥带钩枝茎，为临床常用的多基原中药。我国大部分地区使用的商品钩藤主要为钩藤U. rhynchophylla，其次为华钩藤U. sinensis，而某些地区亦将钩藤属其他植物作为钩藤的习用品或代用品使用，如攀茎钩藤U. scandens (Smith) Hutchins.、倒挂金钩U. lancifolia Hutchins.、平滑钩藤U. laevigata Wall. ex G. Don等。现代药理研究表明，钩藤生物碱具有良好的中枢神经系统活性，具有抗帕金森病、抗阿尔兹海默症、神经保护等作用。

中药由于其生物来源、生态环境、炮制方法、配伍应用及生产工艺等的多样性，导致其化学体系复杂，成分结构多样；此外，中药通过口服给药进入体内后会以原型与代谢产物多种形式存在，进一步导致真正药效成分难以明确。本课题组前期从钩藤（U. rhynchophylla）中分离鉴定了48个生物碱，包括22个生物碱苷元，26个生物碱葡萄糖苷，其中17个为新化合物，基本阐明了钩藤总生物碱的化学成分组成；同时建立了针对钩藤（U. rhynchophylla）中所含16个生物碱的UPLC-Qtrap-MS/MS定量分析方法，确定了16个生物碱的药代动力学特征，对比分析了16个生物碱在大鼠血浆及大脑中的含量，基本阐明了钩藤生物碱的入血和入脑成分及其药动学特点。然而大量研究表明，不同基原钩藤化学成分存在较大差别，而不同基原钩藤的体内成分在体内发生怎样的吸收代谢转化、其体内成分有何异同、哪些化学成分（群）是钩藤真正关键药效物质等问题尚不清楚，因此，对不同基原钩藤生物碱的体内成分，尤其是入血与入脑成分进行系统深入的分析，是阐释钩藤多基原入药合理性的重要有效途径。

首先，本论文对四个代表性生物碱单体，即钩藤生物碱内盐D、缝籽嗪甲醚、钩藤碱、异钩藤碱，进行了药代动力学研究与体内代谢产物分析。分别灌胃大鼠四种生物碱单体，分析药代动力学特征，发现四种生物碱吸收速率较快，体内驻留时间较短；代谢产物分析表明，钩藤生物碱内盐D共检测出23个可能的代谢产物，缝籽嗪甲醚共检测出26个可能的代谢产物，钩藤碱共检测到33个可能的代谢产物，异钩藤碱共检测到23个可能的代谢产物，且相同结构母核的生物碱具有相似的代谢途径。对生物碱单体的体内分析初步揭示了钩藤生物碱在体内吸收、代谢的规律，为不同基原钩藤总生物碱的体内研究奠定了基础。

接下来，在前期建立的针对钩藤（U. rhynchophylla）中所含的16个生物碱的UPLC-Qtrap-MS/MS方法的基础上，综合考虑不同基原钩藤中主要生物碱的种类，本论文构建了适用于多种基原钩藤中20个生物碱的UPLC-MRM-MS定量分析方法，对五种基原钩藤（钩藤U. rhynchophylla、大叶钩藤U. macrophylla、华钩藤U. sinensis、无柄果钩藤U. sessilifructus和攀茎钩藤U. scandens）总生物碱中的20个生物碱进行了定量研究，结果表明，五种基原钩藤总生物碱的化学组成差异较大，钩藤以吲哚生物碱为主，其他四种基原以氧化吲哚生物碱为主。该部分研究初步揭示了五种基原钩藤生物碱的化学成分组成，为钩藤体内成分的研究提供了化学依据。

最后，对五种基原钩藤总生物碱的体内成分进行研究。分别灌胃大鼠五种基原钩藤总生物碱，对五种基原钩藤总生物碱在大鼠血浆、尿液、粪便、大脑和脑脊液中的原型和代谢产物的定性分析表明，在所有生物样本中共检测到465个体内成分（钩藤：202个，大叶钩藤：208个，华钩藤：140个，无柄果钩藤：114个，攀茎钩藤：60个），五种基原钩藤体内共有成分仅有8个，多数体内成分仅在某一种基原钩藤中被检测到，表明五种基原总生物碱的体内成分具有较大差异性。同时采用建立的UPLC-MRM-MS定量分析方法，对大鼠血浆与大脑中的20个生物碱原型成分进行定量测定，对比分析五种基原钩藤总生物碱在大脑与血浆中含量的异同，结果表明，20个生物碱均可被吸收入血并透过血脑屏障，但不同结构类型、不同立体异构和取代基的生物碱入血和入脑的难易有所不同。五种基原钩藤总生物碱的体内成分表明钩藤U. rhynchophylla总生物碱的体内成分与其他四种基原的相似度最低，其特有成分以吲哚生物碱为主，其他四种基原特有成分以氧化吲哚生物碱为主。上述研究初步揭示了不同基原钩藤总生物碱体内成分的异同，为不同基原钩藤体内药效物质的深入研究提供了重要参考。

The genus Uncaria (family Rubiaceae) has 34 species in the whole world, and 11 species and 1 variety are found in China. According to the Chinese Pharmacopoeia, Uncariae Ramulus Cum Uncis (“Gou-Teng” in Chinese), a famous Traditional Chinese Medicine (TCM) with multiple origins, is derived from the dried hook-bearing stems and branches of Uncaria rhynchophylla (Miq.) Miq. ex Havil., Uncaria macrophylla Wall., Uncaria hirsute Havil, Uncaria sinensis (Oliv.) Havil., and Uncaria sessilifructus Roxb.. The commercial Uncariae Ramulus Cum Uncis used in most areas of China is mainly derived from U. rhynchophylla, followed by U. sinensis, while other species of the genus Uncaria, such as Uncaria scandens (Smith) Hutchins, Uncaria lancifolia Hutchins, Uncaria laevigata Wall. ex G. Don, and so on, are also used as the habitual products or substitutes for Uncariae Ramulus Cum Uncis in some areas. The modern pharmacological studies have shown that the alkaloids of Uncariae Ramulus Cum Uncis have good biological activities, such as anti-Parkinson's disease, anti-Alzheimer's disease and neuroprotection, and so on.

Due to the diversity of biological sources, ecological environments, compatibility and production processes, the chemical compositions of TCMs are much complicated. Moreover, the various forms of metabolites after orally administration results in the more difficulty in clarification of the effective components of TCMs. In our previous study, 48 alkaloids were isolated and identified from U. rhynchophylla, of which 26 were alkaloidal glucosides and 17 were new compounds, which generally clarified the chemical constituents of U. rhynchophylla alkaloids. In addition, a UPLC-Qtrap-MS/MS method was also established for the quantitative analysis of 16 alkaloids of U. rhynchophylla, and the contents of 16 alkaloids in plasma and brain, as well as the pharmacokinetic profiles of the 16 alkaloids, were determined. A large number of studies have shown that the chemical compositions of Uncariae Ramulus Cum Uncis from different origins vary greatly, but the in vivo chemical components of Uncariae Ramulus Cum Uncis from different origins are still unclear. Therefore, a systematic and in-depth analysis of the in vivo components of the alkaloids of Uncariae Ramulus Cum Uncis from different origins should be carried out.

Based on the UPLC-Qtrap-MS/MS method for 16 alkaloids of U. rhynchophylla, a UPLC-MRM-MS method for the quantitative analysis of 20 alkaloids was established, and the contents of 20 alkaloids in the total alkaloids of Uncariae Ramulus Cum Uncis from five origins, including U. rhynchophylla, U. macrophylla, U. sinensis, U. sessilifructus and U. scandens, were determined and provided a chemical basis for further study of their in vivo components.

Then, the pharmacokinetic study and in vivo metabolite analysis of four representative alkaloids, including rhynchophyllionium D, geissoschizine methyl ether, rhynchophylline and isorhynchophylline, were investigated. It was found that the four alkaloids were absorbed, reached to the maximum blood concentration and excreted rapidly. A total of 23 possible metabolites of rhynchophyllionium D, 26 possible metabolites of geissoschizine methyl ether, 33 possible metabolites of rhynchophylline and 23 possible metabolites of isorhynchophylline were detected, and the alkaloids with same nucleus structures showed similar metabolic pathways. The in vivo analysis of the four alkaloids revealed the metabolic pathways of representative alkaloids, and laid a foundation for the study of in vivo components of total alkaloids of Uncariae Ramulus Cum Uncis.

Next, the in vivo components after orally administration of the total alkaloids of Uncariae Ramulus Cum Uncis from five origins were investigated and analyzed comparatively. The prototypes and metabolites of the total alkaloids of Uncariae Ramulus Cum Uncis from five origins in rat plasma, urine, feces, brain and cerebrospinal fluid were characterized, and a total of 465 in vivo constituents were detected in all samples, including 202 from U. rhynchophylla, 208 from U. macrophylla, 140 from U. sinensis, 114 from U. sessilifructu and 60 from U. scandens. Only eight constituents were detected in all five origins of Uncariae Ramulus Cum Uncis, while most constituents were only detected in one origin, suggesting the in vivo components of the total alkaloids of Uncariae Ramulus Cum Uncis from the five origins were different greatly. Furthermore, a quantitative analysis of the 20 alkaloids in the plasm and brain after orally administration of the total alkaloids of Uncariae Ramulus Cum Uncis from five origins were investigated, and the results showed that all the 20 alkaloids can be absorbed into blood and passed through the blood-brain barrier, but the alkaloids with different structural nucleus, stereoisomers and substituent groups might be different in the ability of absorption and passing through the blood-brain barrier. Generally, the in vivo components of the total alkaloids of U. rhynchophylla that were characterized by indole alkaloids, was the least similar to the other four origins that were characterized by oxindole alkaloids. The above results revealed the similarities and differences of in vivo components of Uncariae Ramulus Cum Uncis from different species, and laid a foundation for the further study on the in vivo effective components of Uncariae Ramulus Cum Uncis.

第一章 文献综述 1
1.1 钩藤生物碱类化学成分分析及其质谱裂解规律研究概况 2
1.1.1 不同基原钩藤生物碱类成分对比分析 2
1.1.2 钩藤生物碱的质谱裂解规律 4
1.2 钩藤生物碱的体内过程研究概况 6
1.2.1 钩藤生物碱体内代谢产物研究概况 6
1.2.2 钩藤生物碱的血浆药代动力学研究概况 9
1.2.3 小结 11
第二章 四种典型钩藤生物碱单体的体内成分分析 17
2.1 实验仪器与材料 17
2.1.1 实验仪器 17
2.1.2 药品与试剂 18
2.1.3 实验动物 18
2.2 四种典型钩藤生物碱单体的血浆药代动力学分析 19
2.2.1 实验方法 19
2.2.2 结果与讨论 21
2.2.3 小结 30
2.3 四种典型钩藤生物碱单体的体内代谢产物定性分析 31
2.3.1 实验方法 31
2.3.2 结果与讨论 33
2.3.3 小结 104
第三章 五种基原钩藤总生物碱的制备及MRM定量分析 105
3.1 不同基原钩藤总生物碱的制备 105
3.1.1 实验仪器与材料 105
3.1.2 实验方法 106
3.1.3 结果与讨论 108
3.2 不同基原钩藤总生物碱中20个化合物的含量测定 112
3.2.1 实验仪器与材料 113
3.2.2 实验方法 114
3.2.3 结果与讨论 117
3.3 小结 121
第四章 五种基原钩藤总生物碱的体内成分分析 123
4.1 实验仪器与材料 123
4.1.1 实验仪器 123
4.1.2 药品与试剂 124
4.1.3 实验动物 124
4.2 五种基原钩藤总生物碱体内原型及代谢产物的定性分析 124
4.2.1 实验方法 124
4.2.2 结果与讨论 125
4.3 五种基原钩藤总生物碱单次给药后体内成分的定量分析 130
4.3.1 实验方法 130
4.3.2 结果与讨论 131
4.4 小结 138
第五章 结论与展望 141
参考文献 143
附录A 20个生物碱在血浆、大脑中含量的汇总 149
附录B UPLC-QTOF-MS检测到的五种基原钩藤总生物碱体内成分 151
致谢 185
北京大学学位论文原创性声明和使用授权说明 187
个人简历、在学期间发表的学术论文与研究成果 189

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