单胺氧化酶抑制药物体外筛选模型的建立

Construction of the screening model for monoamine oxidase inhibitors in vitro

摘要:

目的：构建一种能用于治疗神经退行性变疾病单胺氧化酶（monoamine oxidase，MAO）抑制药物的体外筛选模型。方法：采用紫外分光光度方法，以苄胺或5-羟色胺（5-hydroxytryptamine，5-HT）分别作为MAO-B或MAO-A的反应底物，测定其活性，并对MAO浓度、底物浓度、缓冲液浓度、pH值、反应时间和温度等进行优化。结果：研究确定的模型反应体系条件为：在温度37 ℃、酶预孵育时间20 min和反应时间60 min下，测定MAO-B活性最佳条件为100 mmol/L磷酸钾缓冲液（pH 7.6），MAO和底物苄胺的终浓度分别为0.15 mg/ml和2.00 mmol/L；测定MAO-A活性最佳条件为100 mmol/L磷酸钾缓冲液（pH 7.4），MAO和底物5-HT的终浓度分别为0.40 mg/ml和5.00 mmol/L。结论：建立了一种MAO抑制药物体外筛选的模型，该模型具有成本低、操作简便等特点，适合于大规模筛选MAO-B和MAO-A的抑制药物。

Abstract:

Objective：To construct a screening model for monoamine oxidase（MAO） inhibitors in the treatment of neurodegenerative diseases. Methods：Benzylamine and 5-hydroxytryptamine（5-HT） were used as MAO-B and reaction substrates and their activities were measured by UV spectrophotometry，respectively. MAO concentration，substrate concentration，phosphate buffer concentration and pH，reaction time and temperature were optimized. Results：The optimized reaction system conditions were obtained as follows：under the conditions of enzyme pre-incubation time of 20 min and reaction time of 60 min at the temperature of 37 ℃，optimum con－dition for determination of MAO-B activity was 100 mmol/L phosphate buffer（pH 7.6） and 0.15 mg/ml MAO and 2.00 mmol/L ben－zylamine，optimum condition for determination of MAO-A activity was 100 mmol/L phosphate buffer（pH 7.4） and 0.40 mg/ml MAO and 5.00 mmol/L 5-HT. Conclusions：A screening model for monoamine oxidase inhibitors in vitro is constructed successfully. Due to its low cost and simple operation，it is suitable for large-scale screening MAO-B and MAO-A inhibitors.