Liu Wei, Yang Yadi, Cheng Xuemei, Gong Can, Li Shuping, He Dandan, Yang Li, Wang Zhengtao, Wang Changhong
Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine and The MOE Key Laboratory for Standardization of Chinese Medicines and The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines and Shanghai R&D Center for Standardization of Chinese Medicines, Shanghai 201203, China.
Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine and The MOE Key Laboratory for Standardization of Chinese Medicines and The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese Medicines and Shanghai R&D Center for Standardization of Chinese Medicines, Shanghai 201203, China.
J Pharm Biomed Anal. 2014 Jun;94:215-20. doi: 10.1016/j.jpba.2014.02.004. Epub 2014 Feb 18.
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are legitimate therapeutic targets for Alzheimer's disease. The classical approach for screening potential AChE/BChE inhibitors was developed by Ellman. However, the background color of compounds or plant extracts remained uncertain and frequently interfered with the detection of the secondary reaction, thereby easily yielding false positive or false negative results. Rapid, selective, and sensitive ultra-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry method was developed and used for the detection of AChE and BChE inhibition by directly determining the common product, choline (Ch). Proper separation was achieved for choline and chlormequat (internal standard) within 1.2min via isocratic elution (0.1% fromic acid:methanol=98:2) on an HSS T3 column following a simple precipitation of proteins for sample treatment. The relative standard deviations of the intra- and inter-day precisions were below 7.34 and 9.09%, respectively, whereas the mean accuracy for the quality control samples was 100.31±10.93%. The method exhibited the advantages of small total reaction volume (100μL), short analysis time (1.2min), high sensitivity (LOQ of 0.036μM for Ch), and low cost (little consumption enzymes of 0.0035 and 0.008unitmL(-1) for AChE and BChE, and substrates of 5.505 and 7.152μM for ACh and BCh in individual inhibition, respectively), and without matrix effect (90.00-105.03%). The developed method was successfully applied for detecting the AChE and BChE inhibitive activities for model drugs, including galanthamine, tacrine, neostigmine methylsulfate, eserine, as well as β-carboline and quinazoline alkaloids from Peganum harmala.
乙酰胆碱酯酶（AChE）和丁酰胆碱酯酶（BChE）是治疗阿尔茨海默病的合理靶点。Ellman开发了筛选潜在AChE/BChE抑制剂的经典方法。然而，化合物或植物提取物的背景颜色仍然不确定，经常干扰二级反应的检测，从而容易产生假阳性或假阴性结果。开发了快速、选择性和灵敏的超高效液相色谱结合电喷雾电离串联质谱法，通过直接测定共同产物胆碱（Ch）来检测AChE和BChE抑制作用。在对样品进行简单的蛋白质沉淀处理后，采用等度洗脱（0.1%甲酸：甲醇 = 98:2），在HSS T3柱上1.2分钟内实现了胆碱和矮壮素（内标）的良好分离。日内和日间精密度的相对标准偏差分别低于7.34%和9.09%，而质量控制样品的平均准确度为100.31±10.93%。该方法具有总反应体积小（100μL）、分析时间短（1.2分钟）、灵敏度高（Ch的定量下限为0.036μM）和成本低（AChE和BChE的酶消耗量分别为0.0035和0.008单位mL(-1)，个体抑制中ACh和BCh的底物浓度分别为5.505和7.152μM）以及无基质效应（90.00 - 105.03%）等优点。所开发的方法成功应用于检测模型药物（包括加兰他敏、他克林、甲硫酸新斯的明、毒扁豆碱）以及骆驼蓬中的β-咔啉和喹唑啉生物碱的AChE和BChE抑制活性。
