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

Abstract

Background Enzyme 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; α-glucosidase, α-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 α-glucosidase-, α-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. Results In the three assays analysed here, using only a buffer blank underestimated the enzyme inhibitory potential of the test sample. In the absorbance-based α-glucosidase assay, enzyme inhibition was underestimated when a sample blank was omitted for the coloured plant extracts. Similarly, in the fluorescence-based α-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. Conclusion The 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.

The role of plant‐derived natural antioxidants in reduction of oxidative stress

“…Enzyme inhibitors can form an essential class of clinical drugs for cancer, cardiovascular disease, diabetes, neurological disorders, and obesity. Therefore, natural products are extensively paid attention to due to their bioactivity and therapeutic molecules contributing to discovering the drugs from natural product extracts 127,131 . For instance, salicylates are natural ingredients in many fruits, vegetables, and spices.…”

“…Therefore, natural products are extensively paid attention to due to their bioactivity and therapeutic molecules contributing to discovering the drugs from natural product extracts. 127,131 For instance, salicylates are natural ingredients in many fruits, vegetables, and spices. Synthetic salicylates are used as the main ingredient in aspirin and other pain-alleviating medicine.…”

Influence of ultrasonic pretreatment on structural properties and biological activities of lupin protein hydrolysate

“…Inhibition of α-glucosidase activity. Determination of αglucosidase inhibitory activity of the samples was performed according to the method of Lankatillake et al (2021). In the method, 50 µL of 5 mM 4-nitrophenyl-β-D-glucopyranoside (ρNPG) solution (prepared in 0.1 M phosphate buffer, pH 6.9) was added to 100 µL of samples or standard (acarbose), followed by mixing.…”

Reusable Fluorescent Nanobiosensor Integrated in a Multiwell Plate for Screening and Quantification of Antidiabetic Drugs

“…However, these tests usually represent a high economic and time cost since they include several stages and the use of enzymes and reactants, which can sometimes be very expensive. , Specifically, the most commonly used in vitro screening approach is based on a 96-well plate assay using UV–vis spectrophotometry as the detection method. However, numerous sources of error can contribute in affecting the accuracy and reproducibility of these measurements, such as plate edge effects (temperature and evaporation deviations), turbidity, air bubbles during mixing, and so forth, as has been reported in a very recent study . An alternative to conventional methods is the use of enzyme biosensors.…”