Background: The search for new natural or synthetic products with antioxidant activity is commonly based on methods that involve reduction of either 2,2-diphenyl-1-picrylhydrazyl (DPPH) or 2-2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). However, the reported values of the effective concentrations are highly variable, even in controls. Herein, we optimize and validate both meth-ods of determining antiradical activity. Methods: Optimization was carried out using both a fractionated factorial design and a basic sequential simplex method, by monitoring the reduction percentage. Quercetin or Trolox were used as positive con-trol. Furthermore, for each method, linearity, precision, accuracy, robustness, plate uniformity, signal variability, and Z factor, were established. Results: The optimized conditions for the DPPH method were: DPPH 280 μM in ethanol and 15 min of reaction time in the dark. The linear range was between 7 and 140 μM with an R2 value of 0.9987. The optimized conditions for the ABTS method were: ABTS adjusted to 0.7 absorbance units, 70% concen-tration in ethanol, and a reaction time of 6 min in the dark. The linear range was found to be between 1 and 70% with an R2 = 0.9991. For both methods, the accuracy and precision were within limits and the Z factor value was higher than 0.89. The applicability of each method was assessed by analyzing eight plant extracts. Conclusion: The DPPH and ABTS reduction methods were optimized and validated on a microscale and could be expected to be implemented in any laboratory.

中文翻译：

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两种指示抗自由基活性的高通量方法的优化和验证

背景：寻找具有抗氧化活性的新天然或合成产品通常基于涉及还原 2,2-二苯基-1-三硝基苯肼 (DPPH) 或 2-2-嗪基-双(3-乙基苯并噻唑啉-6-)的方法。磺酸）（ABTS）。然而，报告的有效浓度值变化很大，即使在对照中也是如此。在这里，我们优化并验证了两种测定抗自由基活性的方法。方法：通过监测减少百分比，使用分级因子设计和基本序贯单纯形法进行优化。使用槲皮素或 Trolox 作为阳性对照。此外，还建立了每种方法的线性度、精密度、准确度、鲁棒性、板均匀性、信号变异性和 Z 因子。结果：DPPH 方法的优化条件为：DPPH 280 μM，乙醇溶液，避光反应时间 15 min。线性范围为 7 至 140 μM，R2 值为 0.9987。ABTS方法的优化条件为：ABTS调整为0.7吸光度单位，乙醇浓度为70%，避光反应时间为6 min。发现线性范围在 1 到 70% 之间，R2 = 0.9991。两种方法的准确度和精密度均在限制范围内，Z 因子值高于 0.89。通过分析八种植物提取物来评估每种方法的适用性。结论：DPPH 和 ABTS 减少方法在微尺度上进行了优化和验证，有望在任何实验室中实施。