Cytation 3 hybrid microplate reader

An ELISA reader was used to assess the inhibition of α-glucosidase by 1,3,5-trisubstituted-2-thioxoimidazolidin-4-ones derivatives at various doses (1000, 100, 10, and 1 µg/mL) according to Peytam et al. [10 (link)]. The α-glucosidase enzyme (EC3.2.1.20, Saccharomyces cerevisiae (20 U/mg), Acarbose, and substrate (p-nitrophenyl-β-D-glucopyranoside) were provided by Sigma-Aldrich. Other chemicals were of the highest purity grade and purchased from Merck India. Enzyme was prepared in a potassium phosphate buffer (pH 6.8, 50 mM). Compounds 4a–b, 5a–b, 7a–b, and Acarbose were dissolved in DMSO (10% final conc.). The different concentrations of the synthesized derivatives/Acarbose (20 µL), enzyme solution (20 µL), and potassium phosphate buffer (135 µL), were added in the 96-well plate and incubated at 37 °C for 10 min. Then, the substrate (25 µL, 4 mM) was added to the mentioned mixture and allowed to incubate at 37 °C for 20 min. Finally, the change in absorbance at 405 nm was monitored using a Cytation 3 hybrid microplate reader (BioTek, Winooski, VT, USA). The control and standard agents were DMSO and Acarbose, respectively. GraphPad prism 6.0 was used to calculate the IC₅₀ values of the investigated compounds from the nonlinear regression curve (Systat Software, Inc. Tools for Science) (https://www.graphpad.com/scientific-software/prism/ (accessed on 1 September 2022)).

Based on the reported method of Lossow et al., rat small intestine α-glucosidase (EC 3.2.1.20) was prepared. The in-vitro activity was determined by the measurement of 4-nitrophenol which was released from para-nitrophenyl α-D glucopyranoside [44 (link), 45 (link)]. The preparation of 200 µL was performed as follows: the enzyme solution (190 µL, 0.15 units/ml), different concentrations of target compounds (1, 10, 20, 50, 100, 500 and 1000 µM (5 µL), potassium phosphate buffer. Final compounds were dissolved in DMSO (not exceed than 5% of final volume) and pre-incubated at 37 °C, p-nitrophenyl glucopyranoside and then substrate (5 µL, 3 mM), was added to the enzyme solution and incubated for further one hour at 37 °C. Finally, by using Cytation 3 hybrid microplate reader (BioTek, USA) any change in the absorbance was measured at 405 nm. By using GraphPadprism 6.0 (SanDiego, California, USA) (https://www.graphpad.com/scientificsoftware/prism/) was used to obtain IC₅₀ values of tested compounds.