Establishment of an HPLC method for testing acetylcholinesterase inhibitory activity and compared with traditional spectrophotometry
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.
KeywordsAcetylcholinesterase Alzheimer’s disease High-performance liquid chromatography Anthocyanin
The authors express gratitude to Excellent Young Science and Technology Talent Plan (No. 5625) and Guizhou Province High Level Creative Talents Cultivation (No. 4033) for providing the financial support to conduct this project.
Compliance with ethical standards
Conflict of interest
There are no conflicts of interest to declare.
- Chen Y (2008) Study on the external factors affecting the rate of enzymatic reaction. Mod Agric Sci Technol 238–239Google Scholar
- 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 22:779–787Google Scholar
- 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 CrossRefGoogle Scholar
- Du Y, Zhu B, Wu H, Chai X (2011) Characteristics of acetylcholinesterase from sea cucumber. Food Mach 27:8–12Google Scholar
- 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 CrossRefGoogle Scholar
- Irth H, Oosterkamp AJ, Tjaden UR, Greef JVD (1995) Strategies for on-line coupling of immunoassays to high-performance liquid chromatography. Trends Anal Chem 14:355–361Google Scholar
- 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 24:238–241Google Scholar
- Miguel MG (2011) Anthocyanins: antioxidant and or anti-inflammatory activities. J Appl Pharm Sci 1:7–15Google Scholar
- United Nations, Department of Economic and Social Affairs, Population Division (2007) World population prospects: the 2006 revision. Demographic Profiles, vol 10, pp 147–156Google Scholar
- Pan W (2006) Biochemistry. People’s Medical Publishing House, BeijingGoogle Scholar
- 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 8:400–404Google Scholar
- 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 Google Scholar
- Si S, Zhang Y (2007) Drug screening: method and practice. Chemical Industry Press, BeijingGoogle Scholar
- 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 28:156–160Google Scholar
- Zhou Y (2014) Analyzing enzymatic reaction methods and affecting factors of enzymatic reaction rate. Chin J Health Manag 71–72Google Scholar
- Zou L, Quan M, Cheng Y, Li L (2005) Research progress of acetylcholinesterase inhibitors. Food Sci 26:105–108Google Scholar