Abstract
Acetylcholinesterase (AChE) inhibitory activity test is one of the important areas of natural product research, which is traditionally tested by spectrophotometry. In this work, a simple high-performance liquid chromatography (HPLC) method was developed for testing AChE inhibitory activity in several natural products. Using galanthamine hydrobromide and huperzine A as positive controls, it was found that the optimal conditions for sample preparation are 7.0–8.0 for pH, 30 min for reaction time, 37 °C for reaction temperature, 1.1 × 10−3 U mL−1 for concentration of AChE, and 0.625 mM for acetylthiocholine iodide in the final reaction mixture with total volume 2 mL. The Diamonsil C18 column was used and the chromatographic conditions were mobile phase methanol–water–triethylamine (40:60:0.05, v/v/v), flow rate 1.0 mL min−1, injection volume 5 μL, run time 5 min, column temperature 37 °C, and wavelength 405 nm. The AChE inhibitory activity of anthocyanin with concentration of 10, 20, and 50 μg mL−1, blueberry extract and purple potato extract with concentration of 20, 100, and 500 μg mL−1 were further determined under this conditions. The results showed the inhibition ratios obtained from spectrophotometry were nearly adjacent and approximately lower than 10% and the values of absorbance mostly exceed 0.8, but the absorbance should ensure in the range of 0.15–0.8 to guarantee smaller relative error for spectrophotometry. While the inhibition ratios gained from HPLC method were higher than 20% and had greater difference, they had better comparability to the different concentrations of anthocyanin and its extracts. It is revealed that the HPLC method can be used for the evaluation of the AChE inhibitory activity in natural product extracts with deep color or not.
Similar content being viewed by others
References
Ahmad I, Anis I, Malik A, Nawaz SA, Choudhary MI (2003) Cholinesterase inhibitory constituents from Onosma hispida. Chem Pharm Bull 51:412–414
Campanari ML, Navarrete F, Ginsberg SD, Manzanares J, Saez-Valero J, Garcia-Ayllon MS (2016) Increased expression of readthrough acetylcholinesterase variants in the brains of Alzheimer’s disease patients. J Alzheimer’s 53:831–841. https://doi.org/10.3233/JAD-160220
Chen Y (2008) Study on the external factors affecting the rate of enzymatic reaction. Mod Agric Sci Technol 238–239
Chen Y, Carroll AD, Scampavia L, Ruzicka J (2006) Automated method, based on micro-sequential injection, for the study of enzyme kinetics and inhibition. Anal Sci 22:779–787
Cheung NS, Peng ZF, Chen MJ, Moore PK, Whiteman M (2007) Hydrogen sulfide induced neuronal death occurs via glutamate receptor and is associated with calpain activation and lysosomal rupture in mouse primary cortical neurons. Neuropharmacology 53:505–514. https://doi.org/10.1016/j.neuropharm.2007.06.014
Damar U, Gersner R, Johnstone JT, Schachter S, Rotenberg A (2016) Huperzine A as a neuroprotective and antiepileptic drug: a review of preclinical research. Exp Rev Neurother 16:671–680. https://doi.org/10.1080/14737175.2016.1175303
Du Y, Zhu B, Wu H, Chai X (2011) Characteristics of acetylcholinesterase from sea cucumber. Food Mach 27:8–12
Ellman GL, Courtney KD, Andres V Jr, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95
Enz A, Amstutz R, Boddeke H, Gmelin G, Malanowski J (1993) Brain selective inhibition of acetylcholinesterase: a novel approach to therapy for Alzheimer’s disease. Prog Brain Res 98:431–438. https://doi.org/10.1016/s0079-6123(08)62429-2
Ferreira A, Rodrigues M, Fortuna A, Falcão A, Alves G (2014) Huperzine A from Huperzia serrata: a review of its sources, chemistry, pharmacology and toxicology. Phytochem Rev 15:51–85. https://doi.org/10.1007/s11101-014-9384-y
Guardigli M, Pasini P, Mirasoli M, Leoni A, Andreani A, Roda A (2005) Chemiluminescent high-throughput microassay for evaluation of acetylcholinesterase inhibitors. Anal Chim Acta 535:139–144. https://doi.org/10.1016/j.aca.2004.12.016
Gutierres JM et al (2014) Anthocyanins restore behavioral and biochemical changes caused by streptozotocin-induced sporadic dementia of Alzheimer’s type. Life Sci 96:7–17. https://doi.org/10.1016/j.lfs.2013.11.014
Honig LS, Mayeux R (2001) Natural history of Alzheimer’s disease. Aging Clin Exp Res 13:171–182
Ichiyanagi T et al (2004) Absorption and metabolism of delphinidin 3-O-β-d-glucopyranoside in rats. Free Radic Biol Med 36:930–937. https://doi.org/10.1016/j.freeradbiomed.2004.01.005
Ingkaninan K et al (2000) High-performance liquid chromatography with on-line coupled UV, mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products. J Chromatogr A 872:31–73
Irth H, Oosterkamp AJ, Tjaden UR, Greef JVD (1995) Strategies for on-line coupling of immunoassays to high-performance liquid chromatography. Trends Anal Chem 14:355–361
Kong J, Chia L, Goh N, Chia T, Brouillard R (2003) Analysis and biological activities of anthocyanins. Phytochemistry 64:923–933. https://doi.org/10.1016/s0031-9422(03)00438-2
Krikorian R, Shidler MD, Nash TA, Kalt W, Vinqvist-Tymchuk MR, Shukitt-Hale B, Joseph JA (2010) Blueberry supplementation improves memory in older adults. J Agric Food Chem 58:3996–4000. https://doi.org/10.1021/jf9029332
Liu H, Wang M, Wu S, Zheng Y, Zhang F (2012) The studies of filter to endophytic fungi from Huperzia serrata by the acetylcholinesterase activity screening model. Strait Pharm J 24:238–241
Marco L, Carreiras M (2006) Galanthamine, a natural product for the treatment of Alzheimer’s disease. Recent Pat CNS Drug Discov 1:105–111. https://doi.org/10.2174/157488906775245246
Masson P, Schopfer LM, Bartels CF, Froment MT, Ribes F, Nachon F, Lockridge O (2002) Substrate activation in acetylcholinesterase induced by low pH or mutation in the pi-cation subsite. Biochim Biophys Acta 1594:313–324
Miguel MG (2011) Anthocyanins: antioxidant and or anti-inflammatory activities. J Appl Pharm Sci 1:7–15
United Nations, Department of Economic and Social Affairs, Population Division (2007) World population prospects: the 2006 revision. Demographic Profiles, vol 10, pp 147–156
Pan W (2006) Biochemistry. People’s Medical Publishing House, Beijing
Perry EK (1986) The cholinergic hypothesis—ten years on. Br Med Bull 42:63–69
Pervin M, Hasnat MA, Lee YM, Kim DH, Jo JE, Lim BO (2014) Antioxidant activity and acetylcholinesterase inhibition of grape skin anthocyanin (GSA). Molecules 19:9403–9418. https://doi.org/10.3390/molecules19079403
Qiu C, Liu X, Lin X, Ren H, Wu M (2008) Study on optimization of conditions for analyzing activity of acetylcholinesterase derived from crucian. Food Sci 8:400–404
Rainer M (1997) Galanthamine in Alzheimer’s disease. CNS Drugs 7:89–97
Rhee IK, Appels N, Luijendijk T, Irth H, Verpoorte R (2003) Determining acetylcholinesterase inhibitory activity in plant extracts using a fluorimetric flow assay. Phytochem Anal 14:145–149. https://doi.org/10.1002/pca.695
Romero A, Cacabelos R, Oset-Gasque MJ, Samadi A, Marco-Contelles J (2013) Novel tacrine-related drugs as potential candidates for the treatment of Alzheimer’s disease. Bioorg Med Chem Lett 23:1916–1922. https://doi.org/10.1016/j.bmcl.2013.02.017
Shang Z, Lu Z, Shang X (2016) Study on extraction technique and stability of the red pigment in jujube peel by microwave method. J Guizhou Normal Univ (Natural Edition) 34:84–88. https://doi.org/10.16614/j.cnki.issn1004-5570.2016.02.018
Sharma RJ, Gupta RC, Singh S, Bansal AK, Singh IP (2016) Stability of anthocyanins- and anthocyanidins-enriched extracts, and formulations of fruit pulp of Eugenia jambolana (‘jamun’). Food Chem 190:808–817. https://doi.org/10.1016/j.foodchem.2015.06.029
Shipp J, Abdel-Aal ESM (2010) Food applications and physiological effects of anthocyanins as functional food ingredients. Open Food Sci J 4:7–22
Si S, Zhang Y (2007) Drug screening: method and practice. Chemical Industry Press, Beijing
Tampi RR, Tampi DJ, Ghori AK (2016) Acetylcholinesterase inhibitors for delirium in older adults. Am J Alzheimer’s Dis Other Dement 31:305–310. https://doi.org/10.1177/1533317515619034
Wei H, Shen J, Wu W, Zhao J, Zhan Z (2009) Purification, biochemical properties and insecticides susceptibility of acetylcholinerase from housefly (Musca domestica L.). J Agro Environ Sci 28:156–160
Wen Y, Chen H, Zhou X, Deng Q, Zhao C, Gong X (2016) A polyamide resin based method for adsorption of anthocyanins from blackberries. New J Chem 40:3773–3780. https://doi.org/10.1039/c6nj00054a
Zhou Y (2014) Analyzing enzymatic reaction methods and affecting factors of enzymatic reaction rate. Chin J Health Manag 71–72
Zou L, Quan M, Cheng Y, Li L (2005) Research progress of acetylcholinesterase inhibitors. Food Sci 26:105–108
Acknowledgements
The authors express gratitude to Excellent Young Science and Technology Talent Plan (No. [2017]5625) and Guizhou Province High Level Creative Talents Cultivation (No. [2015]4033) for providing the financial support to conduct this project.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
There are no conflicts of interest to declare.
Rights and permissions
About this article
Cite this article
Wang, XY., Liang, Q., Chen, HG. et al. Establishment of an HPLC method for testing acetylcholinesterase inhibitory activity and compared with traditional spectrophotometry. Chem. Pap. 72, 2255–2264 (2018). https://doi.org/10.1007/s11696-018-0459-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11696-018-0459-x