3.9.2. In Vitro α-Amylase Assay

The inhibition of α-amylase was measured using our previously published protocol using 96 well microplates [18]. Pure compounds (10 μL, 0.43 mM) and 140 μL of 1◦ saline were added to each well. Subsequently, 45 μL of 1% starch and α-amylase (10 mg/mL) was added. The reaction mixture was then incubated for 40 min at 25 ◦C followed by the addition of 50 μL of 3, 5-dinitrosalicylic acid (1%) in 20% Rochelle's salt. The reaction mixture was incubated for 1 h at 50 ◦C, and the absorbance of each well plate was recorded at 540 nm. The absorbance of each reaction mixture was plotted in the analytical curve previously obtained for dextrose (25, 50, 75, 100, 125, 150, 200 μg/mL). The results were expressed as a percentage of inhibition.

### 3.9.3. In Vitro α-Glucosidase Assay

The α-glucosidase inhibitory activity was evaluated according to a previously published protocol [19]. The assay mixture consisted of 70 μL of 100 mM phosphate buffer (pH 6.8), 10 μL (0.67 mM) of compound dissolved in DMSO, and 20 μL of 1 U/mL αglucosidase solution were added to 96 well plates in triplicates. The plate was incubated at 37 ◦C for 15 min, followed by the addition of 20 μL of the p-nitrophenyl α-Dglucopyranoside substrate. The reaction mixture was incubated at 40 ◦C for 30 min, and then 50 μL of 0.1 M Na2CO3 solution was added. The absorbance was recorded at 405 nm using a microplate reader, and results were expressed in percentage of inhibition. All experiments were conducted in triplicate.

### *3.10. In Silico Docking Study*

Purified compounds were docked with porcine pancreatic α-amylase (PDB ID: 1OSE) and α-glucosidase crystalline structure (PDB ID:3A4A) of isomaltase from *Saccharomyces cerevisiae*. The complex acarbose with porcine pancreatic α-amylase was removed using Biovia Discovery studio 4.5 software (Dassault Systems BIOVIA, Discovery Studio Modeling Environment, Release 4.5, San Diego, CA, USA, 2015). The methodology for the active site prediction, protein structure, and ligand preparations for molecular docking was provided in detail in our previous study [18]. Compounds were docked using the Lamarckian genetic algorithm in the AutoDock 4.2 Program (ADT, version: 1.5.6). The 2D visualization of ligand-protein interactions was analyzed using DS 4.5 (Dassault Systems), while PyMOL molecular graphics system (PyMOL Molecular Graphics System, San Carlos, CA, USA) was used to better visualize the 3D interactions between ligands and receptors.
