【作者】 续洁琨；

【导师】 姚新生； 栗原博；

【作者基本信息】 沈阳药科大学 ， 药物化学， 2006， 博士

【摘要】 茶叶是具有多种保健作用的世界性饮料之一。本研究的目的是以茶组四种植物为研究对象，在对所含的嘌呤类生物碱和茶多酚类成分比较的基础上，选择具有特征组成的可可茶(Camellia ptilophylla Chang)和苦茶(Camellia assamica var．kucha Chang et Wang)为原料，探讨富含可可碱的广东特产资源—可可茶的研究开发利用前途以及苦茶中含有的特殊嘌呤生物碱—theacrine对中枢神经系统的作用。本文采用高效液相色谱法建立了对茶叶水提取物中嘌呤生物碱及茶多酚类化合物的快速分析方法，并对可可茶、苦茶、绿茶(Camellia sinensis(L．)O．Ktze)及普洱茶(Camellia assamica(Mast．)Chang)四种茶组植物中4种嘌呤生物碱类物质和9种茶多酚类物质同时进行了定量检测。实验结果证明，与普通绿茶、普洱茶不同，可可茶中以可可碱为优势生物碱，含量高达茶叶干重的5.8％，且咖啡碱含量很低或不含咖啡碱。在确定分析方法的基础上，对173株野生可可茶植株进行了采样分析，共检测出29株完全不含咖啡碱的纯种可可茶植株，作为母本通过扦插繁殖建立纯种可可茶种苗基地，扩大天然无咖啡碱茶树的种质资源。高效液相分析结果显示苦茶中特异性嘌呤生物碱theacrine含有为1.78％，而在其他三种茶中均未检出该生物碱。通过对42g干燥苦茶样品的提取与分离，富集得到了部分theacrine(1，3，7，9-tetramethyluric acid，1)以供药理活性研究。同时，还从苦茶水提取物中分离得到caffeine(2)，theobromine(3)，(+)-catechin(4)，(-)-epigallocatechin(5)，(-)-gallocatechin(6)，(-)-epigallocatechin 3-O-gallate(7)，(-)-gallocatechin 3-O-gallate(8)，1，2，6-tri-O-galloyl-β-D-glucose(9)和gallic acid(10)。其中，化合物4～10为首次从该种中分离得到。茶叶中含有的嘌呤生物碱类物质具有显著的中枢神经药理活性，其中咖啡碱、茶碱、可可碱在临床治疗中应用广泛，但药用领域却有所不同，它们化学结构上的区别仅在于该类化合物嘌呤母核上甲基取代基的数量和位置不同。Theacrine也具有相似的嘌呤母核化学结构，但其上取代的甲基数目最多，推测其可能具有明显的药理作用，也可能与其它嘌呤类生物碱在作用上存在较大的差别。由于theacrine天然来源稀少，化学合成又比较困难，其中枢神经药理活性未见任何研究报道。苦茶的发现为研究theacrine的药理活性提供了前提条件。本文重点研究了theacrine对中枢神经系统的药理作用，包括对小鼠自主活动、强迫游泳运动、睡眠、学习记忆能力以及应激状态下实验动物脑内单胺类神经递质的影响，并将其与caffeine和theobromine进行对比，旨在探讨theacrine的中枢神经系统作用及其机制，为苦茶及其特异性生物碱theacrine的开发利用提供实验依据。研究发现，在自主活动及强迫游泳实验中，caffeine(10～30 mg／kg)显示出一定的中枢兴奋作用，而theobromine(10 mg／kg)相反具有中枢安定作用，theacrine在10 mg／kg具有微弱的中枢安定作用，而30 mg／kg下对动物行为改变作用不明显，未显示出与caffeine相似的中枢神经兴奋作用。在戊巴比妥诱导小鼠睡眠实验中，对于正常及急、慢性拘束应激小鼠，theacrine(10-30 mg／kg)均能在一定程度上延长戊巴比妥诱导的小鼠睡眠时间，而caffeine和theobromine在相同条件下显示出对睡眠时间的缩短作用。在避暗及Morris水迷宫实验中，三种受试药物均对实验动物的学习记忆能力显示出一定的增强作用，但theacrine与caffeine和theobromine相比在作用程度上没有显著差异。小鼠拘束应激实验结果显示，急性拘束应激能够普遍提高小鼠各脑区单胺类神经递质的合成与代谢。三种嘌呤生物碱类物质对正常及应激状态下小鼠脑内的单胺类递质水平有一定的升高作用，但受到剂量和脑区等多种因素显著的影响。转轮应激能够导致大鼠与临床神经厌食症和强迫症相似的行为改变以及脑内多巴胺能神经功能亢进和5-羟色胺能神经功能低下。除30 mg／kg caffeine外，三种嘌呤生物碱类化合物均对大鼠的强迫运动行为具有一定的缓解作用。三种生物碱还能够显著提高大鼠脑内5-HT含量，可能是其缓解大鼠应激状态的原因之一。但三种嘌呤生物碱对应激状态下实验动物中枢神经系统的作用以及作用的机制还有待于进一步实验证实。综合以上结果可以看出，在本实验剂量条件下theacrine对中枢神经系统没有显示出与caffeine相似的兴奋作用，但却可以显著延长戊巴比妥诱导的睡眠时间。Theacrine具有一定的增强实验动物学习记忆能力的作用。三种生物碱均能够升高正常和急性拘束应激状态下小鼠脑内的单胺类递质水平，并对转轮应激大鼠的强迫行为有一定的缓解作用，该作用可能与其提高中枢5-HT水平有关。本研究工作在化学和生物活性研究的基础上，证实了茶组植物可可茶不含caffeine的特性，并首次揭示了茶组植物苦茶中特异性嘌呤生物碱theacrine的中枢神经药理作用。本文的实验结果将为今后对茶组植物可可茶与苦茶的研究与开发提供初步的实验依据，并为系统研究和开发其他茶组植物提出了良好的思路。更多还原

【Abstract】 Tea is the most popular beverage known to have beneficial effects on health in the world. On basis of comparison of chemical constituents of purine alkaloids and catechins in four species of Camellia Sect. Thea, the research discussed the future of development and utilization to C. ptilophylla, a special tea plant abundant in theobromine, and investigated the effects of theacrine, a peculiar purine alkaloid in C. assamica var. kucha, on central nervous system.In this paper, a RP-HPLC method was established to analyze the purine alkaloids and catechins in water extract of tea leaves. Four purine alkaloids and nine catechins in C. assamica var. kucha, C. ptilophylla, C. sinensis and C. assamica were identified and quantified at the same time. The results indicated theobromine, different from C. sinensis and C. assamica, is the major alkaloid (5.8 %) present in dry leaves of C. ptilphylla, which contains no or a small amount of caffeine. Furthermore, the HPLC method was used to detect caffeine content of 173 wild grown trees of C. ptilphylla, 29 caffeine-free tea trees obtained which could be cutting propagated and resolve the problem of commercial production of caffeine free tea.A large content substance was isolated from C. assamica var. kucha. Its structure was elucidated as theacrine (1, 3, 7, 9-tetramethyluric acid, 1) through spectroscopic analysis. Meanwhile, other 9 compounds were obtained from the extract of kucha leaves, which were caffeine (2), theobromine (3), (+)-catechin (4), (-)-epigallocatechin (5), (-)-gallocatechin (6), (-)-epigallocatechin 3-O-gallate (7), (-)-gallocatechin 3-O-gallate (8), 1, 2, 6-tri-O-galloyl-β-D-glucose (9) and gallic acid (10). Among these, compounds 4～10 were isolated from the plant for the first time.Purine alkaloids present in tea have significant effects on CNS. Caffeine, theophylline and theobromine are all widely used in clinical therapy. Even though these kinds of compounds have similar chemical structures, their effects on CNS may be much different. Compared with caffeine, the effects of theacrine on CNS have never been studied since its scarcity in nature and difficulty of artificial synthesis. Therefore, we investigated some behavioral and CNS effects of theacrine including ambulatory activity, forced swimming test, pentobarbital-induced sleep time, step-through test and Morris water maze test. The effects of theacrine on monoamine transmitters in brain of animals were also studied by animal models of restraint stress and activity stress. By comparison with caffeine and theobromine, the effects of theacrine on CNS could be illustrated.In the ambulatory activity and forced swimming tests, caffeine (10-30 mg/kg) could produce a remarkable increase in activity, but theobromine (10 mg/kg) responded with a reversed effect compared with caffeine. Theacrine at the dose of 10 mg/kg also showed some weakly sedative effect, whereas did not process significant effect at 30 mg/kg. In test of pentobarbital induced sleep time, theacrine at 10 or 30 mg/kg could reliably prolong the sleep time of mice in either normal or acute and chronic stress condition, whereas caffeine and theobromine increased the arousal level of mice at the same time. The step-through task and Morris water maze test results revealed that theacrine, caffeine and theobromine all could improve the learning and momory abilities.The results of restraint stress suggested that the levels of monoamine transmitters in brain of mice generally increased after restraint stress. Three purine alkaloids could increase NE, 5-HT and 5-HIAA levels in brain cortex and NE in hypothalamus in both normal and stress mice, but the action of theacrine and theobromine were much weaker than caffeine at the same dose. The activity stress paradigm could make rats have abnormal behaviors such as anorexia nervosa and compulsive behavior similar to those observed in humans. Except for 30 mg/kg caffeine, all three purine alkaloids could ameliorate the abnormal behaviors of activity stress rats. Neurochemical results showed that administration of three purine alkaloids could increase 5-HT levels in brain of activity stress rats which may account for their protective effect on behavior of stress rats. The difference of effects of three alkaloids on central nonoamine transmitters and the mechanisms involved still required further study.The chemical and bioactive studies in this academic dissertation confirmed caffeine-free characters of C. ptilophylla, and firstly revealed effects of theacrine obtained from C. assamica var. kucha on central nervous system. Our findings will provide faithful theoretical and experimental evidences for research and development of C. ptilophylla and C. assamica var. kucha in future, and also inaugurate a new way to explore systemically the other plants’ resources of Camellia Sect. Thea.更多还原