Porcine pancreatic lipase type 2

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 activity of α-amylase was measured using a modified method [33 (link)]. Briefly, the tested sample and α-amylase solution (0.2 U/mL) were incubated at 37 °C for 30 min. Next, 2% soluble starch solution was added to the mixture, and the incubation was continued for another 20 min at 37 °C. HCl (1 M) was added to terminate the enzymatic reaction, followed by iodine reagent (5 mg/mL). Acarbose (200 μg/mL) was used as a positive control. The absorbance was measured at 620 nm, and the percent of inhibition was calculated.
Pancreatic lipase: The pancreatic lipase activity was performed using PNPP as substrate [34 (link)]. PNPP was used as a substrate in a solution containing: 40 mg PNPP in isopropanol added to 50 mM Tris–HCl buffer (pH 8.0), 40 mg gum Arabic, 80 mg sodium deoxycholate, and Triton X-100. Orlistat (50 μg/mL) was used as a positive control. Briefly, 20 μL of the tested sample was put into 96-well plates, and lipase enzyme solution (10 mg/mL; porcine pancreatic lipase type II, Sigma-Aldrich) was freshly prepared in 50 mM Tris-HCl buffer (pH 8.0), stirred until fully dissolved and was then added 80 μL to all tests. After 37 °C for 15 min, the substrate solution was added at 37 °C for 25 min. Absorbance was recorded at 405 nm.