基于GABAaR靶点的药用植物蓬莱葛中枢神经抑制药效物质基础及作用机理研究

Anxiety and insomnia have become an epidemic in modern society, and the mechanism is associated with the disorder of central nervous system (CNS) excitement and inhibition. At present, agonists of GABAaR (γ-aminobutyric acid A receptor) are widely used in clinical treatment because of its activities and clear target, thus GABAaR has become a key target of central nervous inhibition for drug design. Gardneramine was separated from root of Gardneria multiflora Makino, and it has a central nervous inhibitory effect. Although Gardneria multiflora Makino has a long history , its chemical constituents and mechanisms are not informative. According to the unique structure and activities of gardneramine, the study will present therapeutic basis including active experiments, determine the active compositions, and reveal their active patterns based on target GABAaR, which all with the help of Chinese traditional medicine chemistry, pharmaceutical analysis, pharmacology, biology and other modern technologies. Therefore, the prupose of this research lies in discovering the constituents of effective substances, active characteristics and mechanism. Also this research will provide necessary data for the innovation of basic research, and will provide new skeleton for drug design.

焦虑症、失眠症已成为现代社会流行病，其发生机制与中枢神经兴奋、抑制平衡失调密切相关。目前GABAaR（γ-aminobutyric acid A receptor）激动剂因其活性明显、靶点明确而在临床得到广泛应用，进而GABAaR已成为新型中枢神经抑制药物设计的关键靶点。Gardneramine来源于药用植物蓬莱葛，体内表现出良好的中枢神经抑制活性。蓬莱葛虽有民间用药历史，但其化学物质组成、作用机制均不祥。为深入系统揭示该药用植物的药效物质基础与作用机理，课题采用中药化学、药物分析、药理学、生物学等现代技术方法，阐述蓬莱葛作用中枢神经的药效活性，确定其活性物质组成，探讨其基于GABAaR靶点的作用模式，从而揭示蓬莱葛的活性物质基础及作用机理。课题目的及意义在于全面揭示蓬莱葛的药效物质组成、活性特点及机理，为其创新基础研究提供必要的数据支持，为新药设计提供新骨架、新结构。

本项目为研究蓬莱葛中单萜吲哚生物碱的中枢神经抑制活性物质及机制研究，本课题对蓬莱葛的化学成分进行了系统研究，运用硅胶柱色谱法、凝胶柱色谱法、ODS柱色谱法、HPLC制备等方法从中分离得到45个化合物，应用MS、UV、IR、1D和2D-NMR等方法，结合其理化性质，已鉴定出41个化合物。本研究构建了体内神经抑制药效筛选药理模型，通过动物行为学实验考察有效组分及物质对于模型大鼠焦虑的改善情况，评价和验证了乙酸乙酯萃取部位和有效组分Ⅰ、Ⅱ、Ⅲ的中枢神经抑制活性。乙酸乙酯萃取部位和有效组分Ⅰ、Ⅱ、Ⅲ能显著增加EPM开放臂的活动距离和进入开放臂的次数；显著增加OFT中央区域活动距离和活动总距离；显著增加穿箱次数TT值。实验结果提示有效部位和有效组分能够一定程度上改善慢性应激诱导的大鼠焦虑行为并增加焦虑大鼠的自主活动能力。ELISA检测和免疫组化结果表明，上述有效部位和有效组分均能显著增加大鼠脑内GABA含量。因此，我们推断蓬莱葛中的乙酸乙酯萃取部位和有效组分Ⅰ、Ⅱ、Ⅲ的抗焦虑药效机制可能与增加大鼠脑内GABA含量有关。此外，对蓬莱葛中的主要活性成分蓬莱葛胺进行药动学与组织分布研究，建立了测定蓬莱葛胺的专属、简单、灵敏的UHPLC-MS/MS大鼠体内分析方法，总结了给药后蓬莱葛胺在大鼠体内的药动学行为，提供了蓬莱葛胺的主要药动学参数及在各组织的分布情况。本研究完成了预期目标，为蓬莱葛中枢神经抑制药效物质基础及作用机制研究提供了理论依据。