2.7. Pancreatic Lipase Inhibition Assay

Pancreatic lipase activity was measured using the method previously reported by Kim et al. [22]. An enzyme buffer was prepared by the addition of 6 μL of porcine pancreatic lipase solution (Sigma-Aldrich, St. Louis, MO, USA) in a buffer (10 mM morpholinepropanesulfonic acid and 1 mM EDTA, pH 6.8) to Tris buffer (169 μL, 100 mM Tris-HCl, and 5 mM CaCl₂, pH 7.0). Aliquots (20 μL) of each sample and orlistat at 100 μg/mL were then mixed with 175 μL enzyme buffer and incubated for 15 min at 37 °C with 5 μL of the substrate solution (10 mM p-NPB (p-nitrophenyl butyrate) in dimethylformamide). The enzymatic reaction mixtures were incubated at 37 °C for 35 min. Pancreatic lipase activity was determined by measuring the hydrolysis of p-NPB to p-nitrophenol, with absorption being recorded at 405 nm using a microplate reader (Model Infinite M200 PRO; Tecan, Austria). Inhibition of lipase activity was expressed in terms of the percentage reduction in absorbance when porcine pancreatic lipase was incubated with the test compounds, and calculated using the following Equation (4)

where A is the lipase activity without inhibitor, a is the negative control without inhibitor, B is the lipase activity with inhibitor, and b is the negative control with inhibitor. The results are expressed as an average (n = 4).