High-performance liquid chromatography-mass spectrometry-based acetylcholinesterase assay for the screening of inhibitors in natural extracts

被引:78
作者
de Jong, CF [1 ]
Derks, RJE [1 ]
Bruyneel, B [1 ]
Niessen, W [1 ]
Irth, H [1 ]
机构
[1] Vrije Univ Amsterdam, Sect Analyt Chem & Appl Spectroscopy, Dept Chem & Pharmaceut Sci, Fac Sci, NL-1081 HV Amsterdam, Netherlands
关键词
label free screening; MS; acetylcholinesterase; natural products; Narcissus; continuous-flow assay;
D O I
10.1016/j.chroma.2006.01.059
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The present paper describes a High-performance liquid chromatography-mass spectrometry (LC-MS) methodology for the screening of acetylcholinesterase (AChE) inhibitors in natural extracts. AChE activity of sample components is monitored by a post-column biochemical assay that is based on the separate, sequential mixing of AChE and acetylcholine, respectively, with the HPLC eluate. AChE inhibitors are detected by measuring a decrease of product formation using electrospray MS. Ammonium bicarbonate was used as buffer in order to achieve optimum compatibility between biochemical assay and MS detection conditions. The assay is robust and stable for over 13 h and compares favourably with other AChE assays in terms of stability and sensitivity. IC50 values of 9-aminoacridine, galanthamine, gallamine, (-)-huperzine A and thioflavin T were determined to be 0.12, 0.38, 6.4, 0.46 and 3.2 mu M, respectively. The assay was used to effectively identify an AChE inhibitor present in a crude extract of Narcissus c.v. "Bridal Crown". (c) 2006 Elsevier B.V. All rights reserved.
引用
收藏
页码:303 / 310
页数:8
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