Abstract:

Alzheimer's disease (AD) is the most common cause of dementia in elderly individuals. Currently, acetylcholinesterase inhibitors (AChEI) are the most effective clinical treatment for AD. AChEIs in natural products may have therapeutic potential and should be screened for use in AD treatment. The authors describe a simple and reliable method for AChEI screening by capillary electrophoresis (CE). A hexadimethrine bromide (HDB) solution was pushed into a capillary (0.015 MPa×10 s) and incubated for 5 min. The capillary was flushed with deionized water for 5 min to remove free HDB, followed by plugging with an acetylcholinesterase (AChE) solution. After a 5 min incubation, the AChE was immobilized on the positively charged coating by ion binding, and the micro-reactor was created. The substrate solution, acetylthiocholine iodide (AThC), was injected into the capillary and incubated in the micro-reactor for 1 min. The unreacted substrate and the enzymolysis product were separated by CE. Gastrodin, an important component of Gastrodia elata, can inhibit AChE activity. After a certain amount of gastrodin was spiked into the substance solution, the peak area of the product decreased. Greater peak area reduction indicated stronger inhibition of AChEI. We observed good reproducibility of the product peak, with relative standard deviation (RSD) values less than 5.3%. The micro-reactor can be reused up to 300 times, which greatly improves efficiency. When the concentration of gastrodin was 5.24 μmol/L, the inhibition rate of AChE reached 64.8%. The IC₅₀ of gastrodin was (2.26±0.14) μmol/L (R²=0.9983), which was consistent with the result of traditional UV method (2.09±0.18 μmol/L). If the function of the micro-reactor deteriorates, it can be conveniently renewed by flushing the column to remove the enzyme and repeating the AChE immobilization protocol. The proposed method is simple, efficient, and low cost, and can be used to screen AChEI from natural products, thus contributing to the improvement of AD treatment.

Key words: capillary electrophoresis (CE), acetylcholinesterase, acetylcholinesterase inhibitor, gastrodin