高等学校化学学报 ›› 2013, Vol. 34 ›› Issue (5): 1121-1126.doi: 10.7503/cjcu20120772

• 分析化学 • 上一篇    下一篇

基于固定化酶的乙酰胆碱酯酶抑制剂体外筛选模型的建立

陈晨1,2, 史倩1, 陈军辉1, 张茹潭1, 李鑫1, 郑立1, 王小如1,3   

  1. 1. 国家海洋局第一海洋研究所现代分析技术及中药标准化重点实验室, 青岛 266061;
    2. 上海海洋大学水产与生命学院, 上海 201306;
    3. 厦门大学化学化工学院, 厦门 361005
  • 收稿日期:2012-08-21 出版日期:2013-05-10 发布日期:2013-05-10
  • 通讯作者: 陈军辉, 男, 博士, 副研究员, 主要从事天然产物化学方面的研究. E-mail: jhchen@fio.org.cn E-mail:jhchen@fio.org.cn
  • 基金资助:

    国家自然科学基金(批准号: 20905017)和国家"八六三"计划项目(批准号: 2007AA092001-10)资助.

Establishment of a New Model for in Vitro Screening of Acetylcholinesterase Inhibitors Based on Immobilized Enzyme

CHEN Chen1,2, SHI Qian1, CHEN Jun-Hui1, ZHANG Ru-Tan1, LI Xin1, ZHENG Li1, WANG Xiao-Ru1,3   

  1. 1. Key Lab of Analytical Technology Development and Standardization of Chinese Medicines, The First Institute of Oceanography, State Oceanic Administration, Qingdao 266061, China;
    2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China;
    3. College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China
  • Received:2012-08-21 Online:2013-05-10 Published:2013-05-10

摘要:

以可重复使用的固定化酶代替游离态酶, 建立一种基于比色分析的乙酰胆碱酯酶(AChE)抑制剂体外筛选新模型. 采用以氨基化硅胶为载体固定的AChE优化了实验条件, 用AChE抑制剂阳性对照物他克林和毒扁豆碱对该模型进行验证, 还对模型技术参数进行评价, 并将新模型用于单体化合物及天然产物粗提物AChE抑制活性评价. 结果表明, 最佳实验条件为: 固定化酶用量55 μL, 底物浓度5 mmol/L, 甲醇、 乙醇及体积分数不高于6%的二甲基亚砜水溶液均可作为样品溶剂; 模型验证及模型技术参数评价结果良好, 该模型对AChE抑制剂筛选有较好的特异性和灵敏度, 可用于筛选AChE抑制剂. 该模型具有适用性强、 固定化酶可重复使用及结果可靠等优点, 是单体化合物及天然产物粗提物AChE抑制剂活性评价的有效方法.

关键词: 固定化酶, 乙酰胆碱酯酶抑制剂, 靶酶抑制剂筛选

Abstract:

An economical model for rapid in vitro screening of acetylcholinesterase(AChE) inhibitors was developed, which used Ellman's reaction-based assay with reusable immobilized AChE. AChE-immobilized aminated silica gel microspheres were applied to develop new screening method. Firstly, the experimental conditions were optimized. And then, the inhibitory activity on immobilized AChE of tacrine and eserine(positive controls) was tested to validate the effectiveness of the new model. Multiple technical parameters of this new model were also evaluated. Finally, the inhibitory activity on AChE of monomeric compounds and natural products was evaluated by the proposed new model. The results show that the optimal conditions included the followings: 55 μL of immobilized AChE as the appropriate dose, 5 mmol/L acetylthiocholine iodide as substrate, methanol and ethanol as well as dimethyl sulfoxide which was under 6% as sample solvent. The obtained results also demonstrated that this new model was sensitive and specific for rapid screening of AChE inhibitors. Good model validation and multiple parameters evaluation results were obtained. The proposed model has many advantages: good applicability, continuous reuse of the immobilized AChE, with reliable results, etc. This model is a powerful tool for identification of AChE inhibitors from natural products or chemical libraries produced by combinatorial chemistry.

Key words: Immobilized enzyme, Acetylcholinesterase inhibitor, Target enzyme inhibitor screening

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