Study On Acetylcholinesterase Inhibitor Screening

In recent years, liquid chromatography-mass spectrometry technology (LC-MS) has achieved greatly development among modern analytic techniques. The application of LC-MS in the field of natural pharmaceutical chemistry has greatly promoted the development of the related subjects, such as pharmaceutical analysis, drug discovery and modernization of traditional Chinese medicine. In this study, establishment of a new method for in vitro screening of acetylcholinesterase inhibitors based on immobilized enzyme and a new screening method for acetylcholinesterase inhibitors based on LC-MS were developed. This method can provide methodology reference for distinguish and rapid discovery of acetylcholinesterase inhibitors for marine medicinal organism. The main studies and results are summarized as follows.1. In this study, an economical method for rapid in vitro screening of acetylcholinesterase inhibitors (AChEIs) is developed, which is using Ellman’s reaction-based assay with reusable immobilized AChE. AChE-immobilized aminated silica gel microspheres are applied to develop new screening method. Firstly, the experimental conditions are optimized. And then, the inhibitory activity on immobilized AChE of tacrine and eserine (positive controls) is tested to validate the effectiveness of the new method. Multiple technical parameters of this new method are also evaluated. Finally, the inhibitory activity on AChE of monomeric compounds and natural products is evaluated by the proposed new method. The results show that the obtained results also demonstrate that this new method is sensitive and specific for rapid screening of AChEIs. Good method validation and multiple parameters evaluation results are obtained. The proposed method has many advantages:good applicability, continuous reuse of the immobilized AChE, with reliable results, etc. All the above demonstrate that this method is a powerful tool for identification of AChE inhibitors from natural products or chemical libraries produced by combinatorial chemistry.2. A method based on high performance liquid chromatography-mass spectrometrywas(LC-ESI-Trap/MS) established for the screening and identification of acetylcholinesterase inhibitors(tacrine and eserine). Firstly, with the mass spectrometry analysis method and optimized incubation conditions, a method based on LC-ESI-Trap/MS was developed for the screening and identification of acetylcholinesterase inhibitors. Secondly, the developed method was successfully applied for screening of acetylcholinesterase inhibitor in the ethyl acetate extract of Sea urchins and ulva. Under the optimized conditions, acetylcholinesterase inhibitors compounds in medical organism were successfully separated. This method was more sensitive and selective compared to using Ellman’s reaction-based assay, and it was more suitable for the determination of low concentration of acetylcholinesterase inhibitors in medical organism. Therefore, LC-ESI-Trap/MS was an effective method for rapid screening and identification of acetylcholinesterase inhibitor, which could provide methodology support for rapid finding acetylcholinesterase inhibitor from medical organism.