2.3. α-Glucosidase Enzymatic Assay

The α-glucosidase enzyme inhibition activities were assessed employing a modified method derived from a prior study [19]. In the α-glucosidase assay, acarbose used as the standard drug, and the reaction mixture was carefully prepared as follows: 50 µL of 0.1 M phosphate buffer (pH 7.0), 25 µL of 0.5 mM 4-nitrophenyl α-D-glucopyranoside (dissolved in 0.1 M phosphate buffer, pH 7.0), 10 µL of each test sample (at concentrations of 62.5, 125, 250, 500, and 1000 µg/mL), and 25 µL of α-glucosidase solution. To prepare the α-glucosidase solution, a stock solution of 1 mg/mL was diluted in 0.01 M phosphate buffer (pH 7.0) to yield a final concentration of 0.04 Units/mL, just before the assay. Following meticulous assembly, the reaction mixture underwent a 37°C incubation for 30 minutes. Termination of the reaction was accomplished by introducing 100 µL of 0.2 M sodium carbonate solution. The enzymatic hydrolysis of the substrate was gauged by monitoring the liberation of p-nitrophenol within the reaction mixture, assessed at 410 nm utilizing a microplate reader. The IC₅₀ calculation was based on linear regression analysis by plotting the value of sample concentrations (X) and the percentage of inhibition (Y). The combination index was calculated by using CompuSyn Software [20], employing the following equations to categorize the extract interactions: synergistic (CI ≤ 1), additive (CI = 1), and antagonistic (CI > 1).

At this point, (D₁) and (D₂) represent the combination doses of Syzygium polyanthum extract and Muntingia calabura extract, respectively, that give an IC₅₀ effect. Moreover, (Dx)₁ and (Dx)₂ reflect the single doses of Syzygium polyanthum extract and Muntingia calabura extract that elicit the same effect [21].