A new thin-layer chromatography–direct bioautography assay for the qualitative and quantitative determination of peroxidase inhibitors in plant extracts

Abstract

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.