Thin-layer chromatography (TLC) hyphenated to bioassays is a modern tool used for discovery of drugs with diverse biological activities from plant extracts which justifies the continuous need for developing such methods. A new TLC–bioautography assay for detecting peroxidase enzyme inhibitors was developed and validated in this study. Peroxidases are related to pathogenesis of many diseases such as neurodegenerative disorders and rheumatoid arthritis and are considered as inflammatory markers. The developed TLC–bioautography method was based on reaction of peroxidase with hydrogen peroxide and the subsequent formation of blue color by the reaction between the released oxygen and benzidine. Compounds with peroxidase inhibition activity are observed as yellowish spots against a blue background. The developed method was validated according to the ICH guidelines. Quantitative estimation of peroxidase inhibitors was achieved by applying image analyses techniques, showing good linearity (R² = 0.998), accuracy (% recovery 96.6–99.7%), intra-day precisions (RSD% = 0.95–2.35) and inter-day precision (RSD% = 1.80–3.20) as well as low LOD (0.28 μg/spot) and LOQ (0.85 μg/spot). The developed method was applied to different Juniperus species fractions as a demonstrative illustration. Overall, the method proved to be efficient, simple and rapid for identification of peroxidase inhibitors in complex matrices and can be applied in high-throughput screening of plant extracts.

中文翻译：

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一种新的薄层色谱直接生物自显影法，用于定性和定量测定植物提取物中的过氧化物酶抑制剂

联用于生物测定的薄层色谱（TLC）是一种现代工具，可用于从植物提取物中发现具有多种生物活性的药物，这证明了不断开发这种方法的必要性。在这项研究中开发并验证了一种新的TLC-生物自显影技术检测过氧化物酶抑制剂的方法。过氧化物酶与许多疾病的发病机理有关，例如神经退行性疾病和类风湿关节炎，被认为是炎症标志物。发达的TLC-生物自显影方法是基于过氧化物酶与过氧化氢的反应，随后通过释放的氧与联苯胺之间的反应而形成蓝色。观察到具有过氧化物酶抑制活性的化合物在蓝色背景下呈淡黄色斑点。根据ICH指南验证了开发的方法。通过应用图像分析技术对过氧化物酶抑制剂进行定量估计，显示出良好的线性（R²  = 0.998），准确度（回收率96.6–99.7％），日内精度（RSD％= 0.95–2.35）和日间精度（RSD％= 1.80–3.20）以及低LOD（0.28μg/点） ）和LOQ（0.85μg/点）。所开发的方法被应用于不同的杜松属植物种级分作为例证。总的来说，该方法被证明是高效，简单，快速的用于鉴定复杂基质中过氧化物酶抑制剂的方法，可用于植物提取物的高通量筛选。