基于色谱联用技术的开心散抗老年痴呆活性成分的筛选及作用机制研究

Alzheimer's disease (AD) is one of the five major deseases in the 21st century. Searching for an effective drug for AD has become the research focus of the medical community in the world. Drug screening is the starting point and the decisive key step in the process of drug innovation. Kai-xin-san, as a classical prescription of TCM, has definite pharmacological effects on AD, but the specific active ingredient and action mechanism is not clear. Based on the experience of the research and development of AD drug, kai-xin-san was taken as an example in this project.The theory and technology of various disicipline such as analytical sceince, TCM,cell biology and moleccular biology, were combined. The latest achievements of hyphenated liquid chromatography, target cell extracion-HPLC-MS and balance dialysis-HPLC-MS drug screening methods were integrated to construct a set of multi-targeted screening toolbox suitable for TCM; At first cell-based drug screening was carried out, secondly multi-targeted molecular-based screening was done,the two methods complement and confirm each other, then the active ingredients in kai-xin-san were found with an unbiased, sensitive and speed screening. The primary correlation of bioactive compounds-pathways-targets were discussed according to the experimental data from chemistry, molecular biology and cell pharmacology,and the elementary mechanism of kaixinsan may be spelled out for AD. The implementation of the project can provide a research paradigm for the study of material base and mechanism of TCM.

老年痴呆（AD）为21世纪的五大重点疾病之一，寻找治疗AD的有效药物已成为世界医学界的研究热点。药物筛选是创新药物研究过程的起点和具有决定意义的关键步骤。中药经方开心散有明确的抗AD药理作用，但其具体活性成分与作用机制尚不明确。本项目基于抗AD药物的研发经验，以开心散为例，融合现代分析科学、中药学、细胞生物学和分子生物学等学科的理论和技术，整合色谱联用技术（HPLC-MS）、靶细胞提取-HPLC-MS和平衡透析-HPLC-MS在药物筛选中的最新成果，构建一套适用于中药的多指标筛选体系；先从细胞水平进行整体初筛，再从分子水平进行多指标靶向筛选，二者互为补充、互为印证，实现开心散中抗AD活性成分的无偏、灵敏、快速发现；综合化学、分子生物学和细胞药理学等实验数据，探讨活性成分-途径-靶点之间的相关性，从整体上阐明其作用机制。项目的实施，能为中药活性成分筛选及其作用机制的研究提供一个较好的范例。

由于中药作用的整体性、化学成分的多样性和作用机制的复杂性，中药药效物质及其作用机制的研究仍然是制约中药现代化进程的瓶颈之一。本项目以抗AD的中药经方开心散为例，整合细胞水平筛选、分子水平筛选、色谱联用等技术的最新成果，构建了一套适用于中药等复杂体系中活性成分的多指标筛选体系，针对治疗AD的“多靶点”，快速发现其 “多成分”，探讨作用的“多途径”，从整体上分析了开心散抗AD的药效物质及其作用机理。.主要内容与结果：.采用UPLC-QTOF/MS技术快速鉴定了开心散中主要化合物：人参，54个；远志，39个；茯苓，66个；石菖蒲，87个。为后续的药物筛选工作提供分析依据。.对人参、远志、石菖蒲、茯苓的定量分析指标和质量控制方法进行了探讨，为开心散及各单味药的质量控制提供了实验依据。.细胞水平筛选：开心散及各单味药对SH-SY5Y细胞有显著增殖和保护作用；筛选到4个人参皂苷、10个远志化合物、13个茯苓组分和16个石菖蒲活性成分。它们可能通过细胞膜或细胞内部的相关受体，酶，离子通道等起到促进细胞活力和保护作用。.分子水平筛选：精选三个靶点（AchE、MAO-B和NMDA受体），采用亲和超滤质谱（UF-MS）技术进行靶向筛选。从开心散及其单味药中筛选得到了数十种抗AD的活性成分，包括：人参皂苷；远志皂苷、远志寡糖酯、远志酮；石菖蒲挥发油、生物碱；茯苓三萜酸。活性成分与各靶点之间是一种多成分多途径多靶点的多维网络结构。.综上，我们发现了开心散抗AD作用的活性成分群，它们通过抗氧化损伤、胆碱能体系和阻断神经元毒性损伤等途径作用于AchE、MAO-B、NMDA受体等靶点而发挥抗AD的药理活性。开心散抗AD作用体现了中药典型的多成分、多靶点、多途径的整体作用特点。项目的实施能为中药活性组分的发现和作用机理的研究提供一种较好的思路和方法，为开心散的现代应用提供科学依据，为抗AD新药研发提供前期基础。