基于体内过程的延龄草总皂苷抗老年痴呆研究

Senile Dementia is a neurodegenerative disease characterized by progressive memory loss and cognitive impairment, which occurs in the elderly. Currently, the world population is aging at accelerated pace. Senile Dementia is a serious danger and has becoming the fourth serious causes of death among elderly. Thus, basic research and drug development for Senile Dementia are needed..Our research indicated that Trillium tschonoskii Maxim could improve learning and memory capability, and the effects were associated with enhancement of anti-oxidase expression. Drug-containing serum after oral administration of its extract could protect cultured hippocampus neuron cell injured by β-amyloid in vitro. Analysis of steroidal saponins in Trillium tschonoskii Maxim by ultra-performance liquid chromatography coupled to mass spectrometry showed that more than 30 steroidal saponins distributed over eight steroidal aglycone skeletons existed in this herb..However, whether major steroidal saponins and/or their aglycone are (potential) bioactive components from Trillium tschonoskii Maxim, and how their pharmacokinetic characteristics and tissue distribution, whether they could pass through brain blood barrier (BBB) were not elucidated yet. All these data should be of great significance to full clarification of Trillium tschonoskii Maxim and steroidal saponins on cerebral functions and mechanism..Firstly, to study in vivo pharmacokinetics and tissue distribution of steroidal saponins and to estimate its targeting performance, LC-MS will be employed to simultaneously determine the concentration of major steroidal saponins and/or their aglycone in rat plasma and and various tissue after oral administration of total saponins. Major pharmacokinetic parameters and concentration-time curves in vivo will be calculated and compared..Secondly, to investigate on the behavior of steroidal saponins in Trillium tschonoskii Maxim in different cerebral nuclei, LC-MS method will be applied to determine major bioactive steroidal saponins and/or their aglycone in cerebral nuclei (hippocampus, cortex, striatum, thalamus and brain stem) after oral administrated total saponins. Dynamic changes will be estimated by comparing the major pharmacokinetic parameters of distribution half-life(t1/2α), eliminationhalf-life(t1/2β), area under the plasma concentration (AUC), mean retention time (MRT) and clearance(CL), respectively..Thirdly, based on above study, the influence of total saponins from Trillium tschonoskii Maxim on cerebral nuclei monoamines (5-HT, NE, E, DA), Ach which have been proved to be highly correlated to senile dementia will be investigated. Studying the kinetic behavior of drug distribution in different tissue and cerebral nuclei in vivo and influence on neurotransmitter will help discover the impact of drugs in modulating brain function and provide theoretical basis for exploitation of Trillium tschonoskii Maxim as effective and safe anti-senile dementia drug.

本研究针对当今紧迫的公共健康问题——人口迅速老龄化所带来的老年痴呆防治问题，以中药延龄草为研究对象，进行其防治老年痴呆的药效物质基础研究。前期研究通过考察延龄草的生物活性而筛选出延龄草抗多因性老年痴呆的主要有效部位为其总皂苷。本课题拟在此基础上，在“基于体内过程的中药有效成分和有效效应物质发现策略”指导下，从体内直接作用物质入手，进行延龄草总皂苷的药代动力学和组织分布研究，建立液质联用等分析方法，以入血成分(原型和/或代谢产物)为指标，对给予延龄草总皂苷后的大鼠血浆、组织样品进行定性定量分析，阐明其体内药动学行为特征和组织分布规律，研究其在与老年痴呆相关的脑核团区域性生物分布特征，并考察总皂苷对脑核团内与老年痴呆相关主要神经递质的影响，进而探讨对脑功能的影响，寻找其防治老年痴呆的效应物质（群），为开发延龄草防治老年痴呆新药提供研究基础和物质基础。

本研究以延龄草为研究对象，基于体内药代动力学和组织分布寻找和探讨中药有效成分的研究方法，结合色谱质谱分析技术，研究了皂苷polyphyllin VI的大鼠血浆药动学行为和组织分布规律；考察了D-半乳糖致AD大鼠，延龄草总皂苷（TTS）干预后，对海马、皮层、纹状体与老年痴呆相关的神经递质，谷氨酸（Glu）、 ɤ-氨基丁酸（GABA）、去甲肾上腺素（NE）、多巴胺（DA）、5-羟色胺（5-HT）的影响，探讨其对脑功能的影响，为开发延龄草防治老年痴呆新药提供研究基础和物质基础。现将本研究工作的结论要点总结如下：.1.研究了大孔吸附树脂富集TTS的工艺，建立了HPLC方法对皂苷成分（Protodioscin，Polyphyllin VII，polyphyllin VI，Polyphyllin II，Dioscin，Polyphyllin I，Trillin，Diosgenin）进行定量分析，8个皂苷总量从富集前9.82%增加到富集后22.84%，利用分光光度法，测定其总皂苷含量达到51.9%。.2.建立了UPLC/MS方法，对TTS主成分polyphyllin VI（含量16.20%）的血浆药动学进行研究。大鼠灌胃给予TTS后，Polyphyllin VI血药浓度的达峰时间在给药后8.0 h左右，自胃肠缓慢吸收，缓慢消除，且血药浓度较低，生物利用度较低。.3.UPLC/MS方法研究了大鼠灌胃给予TTS后，Polyphyllin VI的组织分布规律。其在组织药物浓度：胃＞小肠＞肝＞肾脏＞肺＞脑。在该检测条件下，主要分布在胃和小肠组织中，在脑及海马中未检出Polyphyllin VI原型成分。.4．LC-MS/MS法比较了TTS对D-半乳糖致AD大鼠海马、皮层、纹状体中神经递质Glu、GABA、NE、DA、5-HT含量的的影响。结果表明TTS干预后，将不同脑区的Glu调至正常水平，可显著提高不同脑区GABA、NE、DA、5-HT的含量，使神经递质恢复到平衡状态，从中枢神经递质的角度反映了延龄草皂苷具有增强学习记忆能力和延缓脑衰老的作用。.综上，TTS有改善大鼠学习记忆障碍和延缓脑衰老的作用，其作用可能与其恢复氨基酸类递质平衡、升高脑内单胺类神经递质有关。在脑中起作用的可能是Polyphyllin VI的原型或代谢产物，也可能是其他皂苷类成分或其他成分及其代谢产物综合作用结果。