Screening natural product extracts for potential enzyme inhibitors: protocols, and the standardisation of the usage of blanks in α-amylase, α-glucosidase and lipase assays

BackgroundEnzyme assays have widespread applications in drug discovery from plants to natural products. The appropriate use of blanks in enzyme assays is important for assay baseline-correction, and the correction of false signals associated with background matrix interferences. However, the blank-correction procedures reported in published literature are highly inconsistent. We investigated the influence of using different types of blanks on the final calculated activity/inhibition results for three enzymes of significance in diabetes and obesity; alpha -glucosidase, alpha -amylase, and lipase. This is the first study to examine how different blank-correcting methods affect enzyme assay results. Although assays targeting the above enzymes are common in the literature, there is a scarcity of detailed published protocols. Therefore, we have provided comprehensive, step-by-step protocols for alpha -glucosidase-, alpha -amylase- and lipase-inhibition assays that can be performed in 96-well format in a simple, fast, and resource-efficient manner with clear instructions for blank-correction and calculation of results.ResultsIn the three assays analysed here, using only a buffer blank underestimated the enzyme inhibitory potential of the test sample. In the absorbance-based alpha -glucosidase assay, enzyme inhibition was underestimated when a sample blank was omitted for the coloured plant extracts. Similarly, in the fluorescence-based alpha -amylase and lipase assays, enzyme inhibition was underestimated when a substrate blank was omitted. For all three assays, method six [Raw Data - (Substrate+Sample Blank)] enabled the correction of interferences due to the buffer, sample, and substrate without double-blanking, and eliminated the need to add substrate to each sample blank.ConclusionThe choice of blanks and blank-correction methods contribute to the variability of assay results and the likelihood of underestimating the enzyme inhibitory potential of a test sample. This highlights the importance of standardising the use of blanks and the reporting of blank-correction procedures in published studies in order to ensure the accuracy and reproducibility of results, and avoid overlooked opportunities in drug discovery research due to inadvertent underestimation of enzyme inhibitory potential of test samples resulting from unsuitable blank-correction. Based on our assessments, we recommend method six [RD - (Su+SaB)] as a suitable method for blank-correction of raw data in enzyme assays.

Automated summary

The choice of blanks and blank-correction methods in enzyme assays significantly impacts the accuracy and reproducibility of results, with some common methods underestimating enzyme inhibitory potential. Standardizing the use of blanks and reporting of blank-correction procedures in published studies is crucial to avoid inadvertent underestimation of enzyme inhibitory potential and ensure research accuracy and reproducibility. Based on the study findings, method six [RD - (Su+SaB)] is recommended as a suitable blank-correction method for enzyme assays.