Establishment of an HPLC method for testing acetylcholinesterase inhibitory activity and compared with traditional spectrophotometry
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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.
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