Background: Microscale in vitro assays are fast, simple, and inexpensive, with reduced reagent quantities, waste, and experimental animal use. However, they have low reproducibility and low correlation with the results of in vivo models, possibly due to differences in precision and accuracy in methodologies between laboratories. Objective: The objective was the optimization and validation of an in vitro assay, carried out on microscale, to assess the inhibition of α-glucosidase activity, which is indicative of antihyperglycemic activity. Methods: The optimization was carried out using a fractional factorial design taking into account the best inhibition percentage and the absorbance of the controls. With the optimized experimental conditions in hand, we carried out method validation. Results: The optimized conditions were as follows: enzyme concentration, 0.55 U/mL; substrate concentration, 111.5 μM; and 17.5 min incubation at 37°C. A linear range between 100 and 310.2 μg/mL of acarbose (r2 0.994) was established. The RSD was <2% and the % error was <3%. The Z factor was >0.96. This method was applied to four plant extracts, one of which was found to be very active. Conclusion: The method was found to be accurate, precise, selective, linear, and reliable in evaluating the antihyperglycemic activity of natural extracts in vitro.

中文翻译：

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评估α-葡萄糖苷酶抑制活性的微量体外方法的优化和验证

背景：微量体外检测快速、简单且价格低廉，减少了试剂数量、浪费和实验动物的使用。然而，它们的重现性低，与体内模型结果的相关性低，可能是由于实验室之间方法学的精度和准确性存在差异。目的：目的是优化和验证在微尺度上进行的体外测定，以评估 α-葡萄糖苷酶活性的抑制，这表明抗高血糖活性。方法：使用部分因子设计进行优化，考虑到最佳抑制百分比和对照的吸光度。有了优化的实验条件，我们进行了方法验证。结果：优化条件如下：酶浓度，0.55 U/mL；底物浓度，111.5 μM；在 37°C 下孵育 17.5 分钟。建立了 100 和 310.2 μg/mL 阿卡波糖 (r2 0.994) 之间的线性范围。RSD <2%，% 误差 <3%。Z 因子 > 0.96。该方法应用于四种植物提取物，其中一种被发现非常活跃。结论：该方法在体外评价天然提取物的降血糖活性方面准确、准确、选择性、线性、可靠。