Screening of acetylcholinesterase inhibitors in natural extracts by CE with electrophoretically mediated microanalysis technique
Screening of acetylcholinesterase inhibitors in natural extracts by CE with electrophoretically mediated microanalysis technique
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电泳介导微量分析技术筛选天然提取物中的乙酰胆碱酯酶抑制剂
DOI：
10.1002/elps.200600327
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发表时间：
2007-02-01
期刊：
影响因子：
2.9
通讯作者：
Kang, Jing-wu
中科院分区：
文献类型：
--
作者：
Tang, Zhong-mei;Wang, Zhao-yan;Kang, Jing-wu
本文介绍了一种从天然提取物中筛选乙酰胆碱酯酶（AChE）抑制剂的电泳介导微量分析（EMMA）方法。在该方法中，将AChE溶液以及底物和天然提取物的混合物依次注入毛细管中，并通过施加电压短时间进行电泳混合。然后，重新施加电压以将产物与未反应的底物和天然提取物分离。在UV 230 nm处测得的产物峰面积代表酶活性。 由于浸提液与底物混合，因此在测试抑制作用之前无需分离组分。如果产品的峰面积减少，则可以容易地发现天然提取物作为整体的抑制活性。这使得本发明的方法适用于筛选复杂混合物如天然提取物中的抑制剂。与目前常用的光谱法相比，本方法的主要优点是省去了光谱法所需的Ellman试剂。这不仅简化了实验过程，而且最大限度地减少了假阳性结果。此外，将分离能力与柱上酶测定相结合是一个明显的优势，可以进一步研究哪些化合物是抑制作用的原因。该方法进行了验证，使用市售的乙酰胆碱酯酶抑制剂他克林和一个小的化学库，其中包含四个乙酰胆碱酯酶抑制剂和32个天然提取物。 天然提取物中的抑制剂与本方法进行了鉴定。
An electrophoretically mediated microanalysis (EMMA) method for screening acetylcholinesterase (AChE) inhibitors in natural extracts is described. In this method, solutions of AChE and the mixture of the substrate and the natural extract were successively injected into the capillary, and mixed electrophoretically by applying a voltage for a short time. Afterwards the voltage was reapplied to separate the product from the unreacted substrate and the natural extract. The measured peak area of the product at UV 230 nm represents the enzyme activity. Since the extract is mixed with the substrate, there is no need to separate the components before testing the inhibition. The inhibitory activity of the natural extract as a whole can be easily found if the peak area of the product is reduced. This makes the present method suitable for screening inhibitors in complex mixtures, such as natural extracts. Compared to the commonly used spectrometric method for screening of AChE inhibitors, the major advantage of the present method is the elimination of Ellman reagent, which is essential for the spectrometric method. This not only simplifies the experimental procedure but also minimizes false‐positive results. Moreover, it is an obvious advantage of combining the separation power with the on‐column enzyme assay for further investigating which compound(s) is/are responsible for the inhibition. The method was validated using a commercially available AChE inhibitor tacrine and a small chemical library containing four AChE inhibitors and 32 natural extracts. Inhibitors in natural extracts were identified with the present method.