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J Chromatogr A. 2018 Aug 17;1563:135-143. doi: 10.1016/j.chroma.2018.05.069. Epub 2018 May 30.

Online screening of acetylcholinesterase inhibitors in natural products using monolith-based immobilized capillary enzyme reactors combined with liquid chromatography-mass spectrometry.

Author information

1
Institute of Pharmaceutical Analysis, College of Pharmacy, Jinan University, Guangzhou, 510632, China.
2
Institute of Pharmaceutical Analysis, College of Pharmacy, Jinan University, Guangzhou, 510632, China; Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine & New Drug Research, Jinan University, Guangzhou, 510632, China.
3
Division of Bioanalytical Chemistry, AIMMS Research Group Biomolecular Analysis, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.
4
Institute of Pharmaceutical Analysis, College of Pharmacy, Jinan University, Guangzhou, 510632, China; Division of Bioanalytical Chemistry, AIMMS Research Group Biomolecular Analysis, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.
5
Institute of Pharmaceutical Analysis, College of Pharmacy, Jinan University, Guangzhou, 510632, China; Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine & New Drug Research, Jinan University, Guangzhou, 510632, China. Electronic address: qiqinxtu@163.com.
6
Institute of Pharmaceutical Analysis, College of Pharmacy, Jinan University, Guangzhou, 510632, China; Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine & New Drug Research, Jinan University, Guangzhou, 510632, China. Electronic address: jzjjackson@hotmail.com.

Abstract

In order to develop a direct and reliable method for discovering lead compounds from traditional Chinese medicines (TCMs), a comparative online ligand fishing platform was developed using immobilized capillary enzyme reactors (ICERs) in combination with liquid chromatography-mass spectrometry (LC-MS). Methacrylate-based monolithic capillaries (400 μm I.D. × 10 cm) containing epoxy reactive groups were used as support to immobilize the target enzyme acetylcholinesterase (AChE). The activity and kinetic parameters of the AChE-ICER were investigated using micro-LC-UV. Subsequently, ligand fishing and identification from mixtures was carried out using the complete AChE-ICER-LC-MS platform. For efficient distinction of true actives from false positives, highly automated comparative analyses were run alternatingly using AChE-ICERs and negative control-ICERs, both online installed in the system. After washing unbound compounds to the waste, bound ligands were eluted from the AChE-ICER to a trapping loop using a denaturing solution. The trapped ligands were further separated and identified using LC-MS. Non-specific binding to the monolith support or non-functional sites of the immobilized enzyme was investigated by exposing analytes to the negative control-ICER. The specificity of the proposed approach was verified by analyzing a known AChE inhibitor in the presence of an inactive compound. The platform was applied to screen for AChE inhibitors in extracts of Corydalis yanhusuo. Eight compounds (columbamine, jatrorrhizine, coptisine, palmatine, berberine, dehydrocorydaline, tetrahydropalmatine and corydaline) with AChE binding affinity were detected and identified, and their AChE inhibitory activities were further verified by an in vitro enzymatic inhibition assay. Experimental results show that the proposed comparative online ligand fishing platform is suitable for rapid screening and mass-selective detection of AChE inhibitors in complex mixtures.

KEYWORDS:

Acetylcholinesterase; Comparative online ligand fishing; Immobilized capillary enzyme reactor; Monolith; Traditional Chinese medicines

PMID:
29866504
DOI:
10.1016/j.chroma.2018.05.069
[Indexed for MEDLINE]

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