4.7. Determination of Basil Extracts’ Inhibition Potential against Enzymatic Activities Using Spectrophotometric Assays

The in vitro collagenase inhibition assay was performed following the manufacturer’s instructions. Each O. basilicum extract was tested at 3.33 mg/mL. The 1,10-phenanthroline was used as a positive inhibitor. The rate of enzymatic hydrolysis of the (N-(3-[2-Furyl]acryloyl]-Leu-Gly-Pro-Ala) (FALGPA) substrate was monitored by its absorbance change at 345 nm (Infinite 200 PRO spectrophotofluorimeter, Tecan, Austria). All the reactions were performed in triplicate.

The anti-acetylcholinesterase (anti-AChE) activity of basil extracts was measured using a microplate assay based on the Ellman method, as described previously [67]. Basil extracts (20 μL) at different concentrations (3.3 mg/mL, 1.65 mg/mL, 8.825 mg/mL) were added to the reaction mixtures. Hydrolysis of acetylthiocholine was monitored for 20 min by measuring the absorbance at 412 nm in the Tecan Infinite M200 Pro microplate reader. Tacrine, 100 nM (Sigma Aldrich, ≥99%), was used as a positive control for inhibitory activity. All reactions were performed in triplicate.

The inhibitory potential of basil extracts on pancreatic lipase activity was determined by a colorimetric assay as previously described [68], but with a minor modification to adapt it to 96 well-plates. Porcine pancreatic lipase type II (Sigma Aldrich, L3126-25G) was suspended in Tris-HCl buffer (2.5 mmol, pH 7,4 with 2,5 mmol NaCl) at a concentration of 200 units/mL. p-Nitrophenyl palmitate (PNPP), a PL substrate, was dissolved in Tris-Na deoxycholate buffer (50 mM Tris-HCl pH 8, 5 mM Na deoxycholate) to a final concentration of 320 µM. Each tested extract was preincubated with PL for at least 10 min at 37 °C before adding the substrate mixture to begin the reaction, which was also maintained at 37 °C. By comparing the lipase activity of PL with and without the extract, the percentage of PL’s residual activity was calculated for each extract. Orlistat, a well-known PL inhibitor, served as the positive control. The solvent’s final concentration was fixed and did not increase beyond 5% [69].

The antidiabetic properties of basil extracts were determined by the Caraway–Somogyi method [70,71] with minor modifications. Acarbose, also known as an α-amylase inhibitor, was used as a standard. In a 96-well microplate, 25 μL of basil extract or acarbose of a certain concentration and 50 μL of 0.5 mg/mL α-amylase solution prepared in 20 mM phosphate buffer (pH 6.9 with 0.006 M NaCl) were mixed and preincubated for 10 min at 37 °C. Afterwards, 50 μL of starch solution (0.05%) was added to initiate the reaction and the mixture was incubated for another 10 min at 37 °C. After the incubation, 25 μL of 1 M HCl was added to stop the reaction, followed by 100 μL of iodine–potassium iodide solution. A blank (sample without α-amylase), a positive control (only solvent with α-amylase), and a negative control (only solvent without α-amylase) were also prepared, and the reactions were performed using the protocol described above.

Absorbances of the samples (As), blanks (Ab), and positive (AC+) and negative (AC−) controls were read at 630 nm.