A new TLC bioautographic assay for qualitative and quantitative estimation of lipase inhibitors.

Abstract

Lipase inhibitory assays based on TLC bioautography have made recent progress; however, an assay with greater substrate specificity and quantitative capabilities would advance the efficacy of this particular bioassay. To address these limitations, a new TLC bioautographic assay for detecting lipase inhibitors was developed and validated in this study. The new TLC bioautographic assay was based on reaction of lipase with β-naphthyl myristate and the subsequent formation of the purple dye between β-naphthol and Fast Blue B salt (FBB). The relative lipase inhibitory capacity (RLIC) was determined by a TLC densitometry with fluorescence detection, expressed as orlistat equivalents in millimoles on a per sample weight basis. Six pure compounds and three natural extracts were evaluated for their potential lipase inhibitory activities by this TLC bioautographic assay. The β-naphthyl myristate as the substrate improved the detection sensitivity and specificity significantly. The limit of detection (LOD) of this assay was 0.01 ng for orlistat, the current treatment for obesity. This assay has acceptable accuracy (92.07-105.39%), intra-day and inter-day precisions [relative standard deviation (RSD), 2.64-4.40%], as well as intra-plate and inter-plate precisions (RSD, 1.8-4.9%). The developed method is rapid, simple, stable, and specific for screening and estimation of the potential lipase inhibitors.