Granados-Guzmán Graciela, Castro-Ríos Rocío, Waksman de Torres Noemí, Salazar-Aranda Ricardo
Universidad Autónoma de Nuevo León, Facultad de Medicina, Departamento de Química Analítica, Monterrey, N. L., México.
Curr Anal Chem. 2018 Oct;14(5):458-464. doi: 10.2174/1573411013666170911154755.
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.
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.
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.
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.
The method was found to be accurate, precise, selective, linear, and reliable in evaluating the antihyperglycemic activity of natural extracts in vitro.
微观体外测定法快速、简单且成本低廉，试剂用量、废弃物及实验动物使用量均有所减少。然而，其重现性较低，且与体内模型结果的相关性较差，这可能是由于各实验室方法在精度和准确性方面存在差异所致。
旨在优化并验证一种微观体外测定法，以评估α-葡萄糖苷酶活性的抑制情况，该活性可指示抗高血糖活性。
采用析因设计进行优化，同时考虑最佳抑制百分比和对照的吸光度。在掌握优化后的实验条件后，进行方法验证。
优化后的条件如下：酶浓度为0.55 U/mL；底物浓度为111.5 μM；在37°C下孵育17.5分钟。建立了阿卡波糖在100至310.2 μg/mL之间的线性范围（r2 = 0.994）。相对标准偏差（RSD）<2%，误差百分比<3%。Z因子>0.96。该方法应用于四种植物提取物，其中一种被发现具有很强的活性。
该方法在体外评估天然提取物的抗高血糖活性方面准确、精密、具选择性、呈线性且可靠。
