In this study an in vitro AChE inhibition

In this study, an in vitro AChE inhibition assay combined with UF-HPLC-ESI-Q-TOF/MS method was developed for rapid screening and identification of AChEI from the roots of C. chinensis Franch. Five compounds was found with AChE inhibitory activity and identified by the on-line DAD-ESI-Q-TOF/MS compared with reference standards and literatures. The inhibitory activity of these compounds was verified by in vitro AChE inhibition assay.
Experimental
Results and discussion
Conclusions In this study, an in vitro AChE inhibition assay combined with ultrafiltration HPLC-DAD-ESI-Q-TOF/MS was developed for rapid screening and identification of AChEI from extract of C. chinensis Franch. Five compounds were found to have potential AChE inhibitor activity and were confirmed by the in vitro enzyme experiments. The method was simple, rapid, sensitivity, selective and could be used for the rapid discovery of new AChEI from natural products.
Acknowledgements Financial supports from the Natural Science Foundation of Shandong Province (ZR2013HL029), Science and Technology Development Foundation of Shandong Academy of Science (2012015) and Program for Science and Technology Star for Jinan (2013030) are gratefully acknowledged.
Alzheimer’s disease (AD) is a progressive and fatal neurodegenerative disorder characterised by memory loss and behavioural disturbances., , , The worldwide prevalence of this tragic public problem has been estimated to reach 106.2million by 2050., , Various pathological hallmarks, such as accumulation of β amyloid in senile plaques, hyper-phosphorylated neurofibrillary tangles of tau protein, and loss of cholinergic activity in certain parts of brain, have been shown to be responsible for AD., , , Currently, only the non-competitive -methyl--aspartate receptor antagonist, memantine, and the cholinesterase inhibitors, tacrine, donepezil, rivastigmine, and galantamine, have been approved by the US Food and Drug Administration for the treatment of AD., , Among cholinesterase inhibitors, galantamine and donepezil selectively inhibit acetylcholinesterase (AChE), whereas rivastigmine and tacrine non-selectively inhibit both AChE and butyrylcholinesterase (BChE). The EDTA cholinergic system is vital for learning and memory consolidation and is known to be disrupted in AD., , , The synaptic cholinergic transmission is regulated by AChE and BChE enzymes that rapidly hydrolyse acetylcholine (ACh). AChE shows more hydrolytic activity than BChE does. Hence, AChE inhibitors are preferred in the treatment of AD to keep ACh levels normal. Donepezil is the most favourable AChE inhibitor since it gives a relatively positive response in AD treatment. Furthermore, compared to other AChE inhibitors, donepezil has some advantages, such as blood–brain barrier permeability, non-hepatotoxicity, the least side efficacy, and consumption once daily., Previous studies, , , , , , , , , , have shown that AChE possesses a 20Å-long, narrow gorge, which contains five separated ligand-binding sites: (1) the catalytic triad, including Ser 203, His 447, and Glu 334, which are located at the bottom of the gorge and directly participate in the catalytic cycle; (2) the oxygen anion hole containing Gly 121, Gly 122, and Ala 204 and has a role in the arrangement of hydrogen bond donors that stabilise the tetrahedral enzyme–substrate complex;, (3) the catalytic anionic site (CAS), a region where Trp86, Glu202, and Tyr337 are situated. This site is responsible for the orientation and stabilisation of the trimethylammonium group of ACh by forming cation–π interactions., , , (4) The acyl pocket covers two phenylalanine residues, 295 and 297, which bind the acetyl group of ACh. (5) The peripheral anionic site (PAS) comprises residues Tyr 72, Tyr 124, Trp 286, and Asp 74, which are located at the entrance of the narrow gorge., , Tacrine and galantamine bind to PAS, whereas some tacrine dimers, donepezil and its analogue BYYT-25, bind simultaneously to both PAS and CAS., , , , Studies suggest that AChE inhibitors should bear a core ring system that interacts with PAS, a basic centre that binds to CAS, and a linker, such as –O–, CH, CONH, and CONH(CH)n, between the core ring system and the basic centre to fulfil structural requirements., , For example, AChE inhibitors donepezil and BYYT-25 contain an indanone core ring, methylene or oxygen linkers, and benzylpiperidine or benzylpyrrolydine basic centres. The dimethoxyindanone moiety of donepezil binds to the PAS, whereas the benzylpiperidine moiety interacts with the CAS., , BYYT-25 shows similar interactions with CAS and PAS.