ASSESSMENT OF α-AMYLASE INHIBITION ACTIVITY BY AN OPTIMIZED AND VALIDATED IN VITRO MICROSCALE METHOD

Metabolic disorders, including hyperglycemia, characterize type-2 diabetes. One of the treatment methods used for postprandial hyperglycemia includes using potential therapeutic agents to inhibit α-amylase activity. This study utilized fractional design and the simplex method to optimize in vitro microscale assay inhibition conditions using Miller’s reaction. In addition, the effect of substrate concentration on enzyme activity was analyzed. Enzyme concentration of 0.15 U mL-1 and pre- and post-incubation times of 7.2 and 5.5 min, respectively, in water bath (15.6 min) equipment, were set up for optimized condition for the enzyme activity. Analytical validation was performed based on different international guidelines. Km was found to be 0.38 mg mL-1. Linearity was obtained at the acarbose concentration of 1.5 µg mL-1 and 5 µg mL-1. The IC50 for the positive control was found to be 0.6 µg mL-1. The relative standard deviation and Z value were found to be <4% and >0.93, respectively. Additionally, the optimized assay was applied to extracts from five different plants. Two plant extracts (Zanthoxylum fagara and Chrysactinia mexicana) inhibited α-amylase activity. The optimized and validated method was accurate, precise, and linear.

2型糖尿病以包括高血糖在内的代谢紊乱为典型特征。针对餐后高血糖的治疗策略之一，是通过潜在治疗剂抑制α-淀粉酶（α-amylase）的活性。本研究采用部分设计（fractional design）与单纯形法（simplex method），依托米勒反应（Miller’s reaction）优化体外微量抑制测定的反应条件。此外，本研究还分析了底物浓度对酶活性的影响。最终确立的酶活性测定优化条件为：酶浓度0.15 U·mL⁻¹，预孵育与后孵育时间分别为7.2 min与5.5 min，采用总孵育时长15.6 min的水浴装置完成实验。本研究依据多项国际指南开展分析验证，测得米氏常数（Km）为0.38 mg·mL⁻¹。该方法在阿卡波糖浓度1.5 µg·mL⁻¹至5 µg·mL⁻¹范围内呈现良好线性关系。阳性对照的半抑制浓度（IC₅₀）为0.6 µg·mL⁻¹，相对标准偏差与Z值分别小于4%与大于0.93。此外，将该优化验证后的测定方法应用于5种不同植物的提取物，发现Zanthoxylum fagara与Chrysactinia mexicana两种植物提取物可抑制α-淀粉酶活性。经优化与验证的该测定方法具备良好的准确性、精密度与线性特征。