毛细管电泳法测定天麻素对乙酰胆碱酯酶活性的抑制

Determination of gastrodin activity inhibition on acetylcholinesterase by capillary electrophoresis

阿尔茨海默症(AD)是引起中老年人痴呆最常见的疾病。目前治疗AD的药物主要为乙酰胆碱酯酶抑制剂(AChEI)。建立快速地从天然产物中筛选AChEI的方法,将对临床治疗AD产生积极的作用。该研究建立了一种简单可靠的AChEI筛选新方法。通过海美溴铵在毛细管内壁形成一段正电荷涂层,再经过离子吸附作用制备1.5 cm长的乙酰胆碱酯酶(AChE)反应器。底物碘化乙酰硫代胆碱在0.015 MPa压力下进样10 s,在微酶反应器中停留1 min后采用毛细管电泳(CE)法对底物和酶解产物进行分离。天麻素是天麻的重要药效成分之一,对AChE具有抑制作用。该研究以天麻素为例,根据加入药物前后酶解产物峰面积的差异,完成天麻素对AChE活性的抑制能力的测定。结果表明,随着天麻素浓度的增加,产物峰面积逐渐减小,对AChE的活性抑制变大。该方法所建微酶反应器产物峰面积的RSD值小于5.3%,可连续使用300次。当天麻素浓度为5.24μmol/L时,对AChE活性抑制率达到64.8%。根据加入不同浓度天麻素时的抑制率,测定出天麻素的IC50值为(2.26±0.14)μmol/L(R^2=0.9983)。与传统紫外分光光度法所得结果(2.09±0.18)μmol/L吻合较好。固定化酶微反应器的活性变差时,可以洗脱掉固定在柱上的AChE,重复酶的固定化步骤即可完成再生。该方法简单、高效,运行成本低,柱上固定的AChE酶反应器稳定性较好,可重复使用,极大地提高了工作效率,未来有望应用于各类AChEI的高通量筛选,对AD药物的研发具有积极作用。

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 5.24μmol/L,the inhibition rate of AChE reached 64.8%.The IC50 of gastrodin was(2.26±0.14)μmol/L(R^2=0.9983),which was consistent with the result of traditional UV method(2.09±0.18μmol/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.