基于活性筛选和谱效表征的益智仁抗阿尔茨海默病药效物质基础研究

In this study, based on traditional Chinese medicine theory, screening of active components as the strating point, exploration of the changes of endogenous biomarkers after drug intervention as the goal, we applied mutiple modern scientific technology and methods such as chemical fingerprints, serum pharmacochemistry, pharmacokinetics and pharmacodynamics, determination of endogenous biomarkers, pharmaceutical statistics, to explore the " therapeutic material basis -mechanism" of the traditional Chinese medicine Alpinia oxyphylla. Senile dementia pharmacological model induced by Aβ was established to screen the active site through the whole animal behavior study; and then we investigated the in vitro and in vivo chemical fingerprints to determine the active components using serum pharmacochemistry and liquid chromatography-mass spectrometry method. After the determination of active components, we studied the pharmacokinetics-pharmacodynamic characteristics of thses active components to reveal the relationship between the dynamic chromatograms of serum serum pharmaceutical chemistry and pharmacodynamic results, and obtain the spectrum-effect characterization. Additionally, we researched the regularity of changes of endogenous biomarker (cholinesterase activity) before and after drug administration, to reveal the therapeutic material basis and targets of Alpinia oxyphylla in treatment of Alzheimer's disease, realizing the integration of Chinese medicine material basis and mechanism research. The aim of the study was to establish the evaluating method for “Alzheimer's disease - cholinesterase activity - traditional Chinese medicine curative effect”, according to it, we could obtain a new method to study the active components of Chinese medicine and new mode to solve the key scientific problems in modernization of traditional Chinese medicine.

本研究以中医药理论为指导，活性成分筛选为切入点，药物干预后内源性生物标记物变化规律探索为目标，综合应用中药指纹图谱、血清药物化学、药动学-药效学、内源性生物标记物测定、药学统计学等现代科学技术和方法，以益智仁为研究对象，对传统中药“药效物质基础-作用机制”展开深入探索。建立Aβ诱导的老年痴呆药理模型，通过整体动物行为学考察，筛选活性部位；建立活性部位体内外化学成分指纹图谱，通过血清药物化学和液-质联用技术，确定活性成分；考察活性成分的药动学-药效学特征，将血清药物化学动态图谱与药效结果相关联，建立谱效关系；在此基础上，研究给药前后内源性生物标记物胆碱酶活性变化规律，揭示益智仁治疗阿尔茨海默病的药效物质基础和作用靶点，实现中药药效物质基础与作用机制研究的一体化。本研究力求建立阿尔茨海默病-胆碱酶活性-中药疗效相关性评价方法，开启研究中药药效物质基础新方法和解决中药现代化关键科学问题新模式。

本项目在前期工作基础上，建立了脑室注射Aβ1-42致小鼠痴呆模型，通过整体动物行为学和生化指标的测定考察药物对于模型小鼠学习记忆能力的恢复情况，评价和验证了益智三氯甲烷和正丁醇萃取部位抗阿尔茨海默病的活性。益智三氯甲烷和正丁醇部位萃取物能显著提高Y迷宫实验中小鼠自发交替反应率；显著提高主动逃避实验中小鼠的反应正确率及缩短4日总时间；显著缩短空间探索实验中小鼠逃避潜伏期、延长定位航行实验中小鼠在目标象限的游泳时间及增加跨越平台的次数。ELISA检测结果表明，上述萃取物能显著增加小鼠GSH-Px水平，降低大脑MDA的水平，降低海马中AChE水平以及Aβ1-42的含量，且能改善小鼠脑部皮质和海马的神经元细胞形态。综上所述，益智三氯甲烷和正丁醇萃取部位能显著改善Aβ1-42诱导的学习记忆障碍，结合生化指标和病理切片研究结果，其作用机制与改善中枢胆碱能神经系统功能、自由基清除、小胶质细胞激活以及清除淀粉样蛋白沉积有关。通过对益智三氯甲烷和正丁醇部位萃取物中化学成分的质谱鉴定，指认了10个化合物，主要为萜类和黄酮类化合物。对黄酮类成分采用15 种不同极性的大孔吸附树脂进行富集，进一步评价其体外抗氧化活性。结果表明，HP20型 大孔吸附树脂能有效分离纯化益智中的总黄酮，益智总黄酮表现出良好的体外抗氧化活性。对诺卡酮、白杨素、和杨芽黄素进行药动学研究，对比给药后三种成分在正常大鼠与Aβ1-42致痴呆模型大鼠体内药动学行为差异，提供活性成分的主要药动学参数。. 本研究完成了预期目标，为益智改善Aβ1-42诱导的学习记忆障碍、抗阿尔茨海默病活性的药效物质基础的研究提供了理论依据。