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An in vitro AChE inhibition assay combined with UF-HPLC-ESI-Q-TOF/MS approach for screening and characterizing of AChE inhibitors from roots of Coptis chinensis Franch

https://doi.org/10.1016/j.jpba.2015.12.025Get rights and content

Highlights

  • A novel in vitro AChE inhibition assay combined with UF-HPLC-ESI-Q-TOF/MS was developed for rapid screening and identification of AChE inhibitors from roots of Coptis chinensis Franch.

  • Five AChE inhibitors were discovered and identified by UF-HPLC-DAD-ESI-Q-TOF/MS.

  • Inhibitory activities of five compounds were confirmed by in vitro AChE inhibition assays.

Abstract

In this study, an in vitro acetylcholinesterase (AChE) inhibition assay based on microplate reader combined with ultrafiltration high performance liquid chromatography-electrospray quadrupole time of flight mass (UF-HPLC-ESI-Q-TOF/MS) was developed for the rapid screening and identification of acetylcholinesterase inhibitors (AChEI) from roots of Coptis chinensis Franch. Incubation conditions such as enzyme concentration, incubation time, incubation temperature and co-solvent was optimized so as to get better screening results. Five alkaloids including columbamine, jatrorrhizine, coptisine, palmatine and berberine were found with AChE inhibition activity in the 80% ethanol extract of C. chinensis Franch. The screened compounds were identified by HPLC-DAD-ESI-Q-TOF/MS compared with the reference stands and literatures. The screened results were verified by in vitro AChE inhibition assays, palmatine showed the best AChE inhibitory activities with IC50 values of 36.6 μM among the five compounds. Results of the present study indicated that the combinative method using in vitro AChE inhibition assay and UF-HPLC-ESI-Q-TOF/MS could be widely applied for rapid screening and identification of AChEI from complex TCM extract.

Introduction

Alzheimer’s disease (AD) is an age-related neurodegenerative disease and accounting for approximately 50% of all types of dementia in elderly people [1], [2]. Acetylcholinesterase inhibitor (AChEI) have been approved for the treatment of AD in the United States and some European countries [3], [4], [5]. For the adverse side effects of marketed drugs [6], the potential acetylcholinesterase(AChE) inhibitors from natural products have attracted considerable interest for treating Alzheimer’s disease [7].

Coptidis Rhizoma (the dried roots of Coptis chinensis Franch) is known for ‘Huang Lian’ and used in the treatment of various diseases in traditional Chinese medicine(TCM), with the effects of clearing heat, drying up dampness, purging toxicosis, and detoxicification [8], [9]. Modern study indicated that Coptidis Rhizoma hold a number of alkaloids, and known to exert a variety of activities including anti-inflammatory [10], anti-diabetic [11], antioxidant effects [11], [12] and anti-AD effects [12].

The conventional strategy for the isolation and purification of bioactive compounds from natural products is time-consuming, labor-intensive, and expensive process [13], [14]. In vitro assay based on microplate reader had been used for the screening of bioactive compounds from combinatorial libraries or evaluate the activity for the extract of TCMs. However, this method couldn't give structure information of the active compounds and required multiple-step isolations for the structure elucidation [14], [15].

Ultrafiltration combined with high performance liquid chromatography–mass spectrometry(UF-HPLC-MS) was developed recently for the rapid screening and identification of bioactive compounds from complex mixtures, such as combinatorial libraries and natural product extracts [16], [17], [18], [19]. This technique has some advantages, such as fast speed, easy operation, high sensitivity and high reliability [20], [21], [22], [23] and it has been successfully applied for screening potential ligands from TCMs [15], [24], [25], [26].

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.

Section snippets

Chemicals and reagents

C. chinensis Franch were collected from medical halls in Jinan, China and were authenticated by Professor Xiao Wang, Shandong Academy of Sciences. Standards of columbamine, jatrorrhizine, coptisine, palmatine and berberine were isolated from C. chinensis Franch in our laboratories and their structures were elucidated by IR, MS, 1H and 13C NMR. The chemical structures of the 5 target compounds were shown in Fig. 1. The purity of these alkaloids were determined to be more than 98% by

optimization of incubation conditions

Favorable incubation conditions is helpful for accurate screening of UF-HPLC-DAD-MS screening procedure [28]. Incubation parameters, such as enzyme concentration, incubation time, incubation temperature and co-solvents were optimized via comparing the binding degree use tacrine as positive control.

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.

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    These authors contributed equally to this work.

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