3.3.3. Pancreatic Lipase Inhibition Assay

The inhibition of PL activity was determined by measuring the release of p-nitro-phenol, from p-nitrophenyl butyrate (p-NPB). In resume, 40 µL of extract solution (serial dilutions) was mixed with 20 µL of p-NPB solution (10 mM) and 40 µL of the enzyme solution (2.5 mg mL-1). Extracts and enzyme solution were prepared in phosphate buffer (0.1 M, pH 8). The enzyme substrate was dissolved in ethanol. After 20 minutes of incubation (37 °C) absorbance was read at 405 nm (Victor3 1420 multilabel plate counter, Perkin-Elmer). A stock solution of orlistat (positive control) was prepared in DMSO (10 mg mL-1 ) and further diluted with buffer. The experiment was conducted as described above. The inhibitory activity was expressed as the IC50 value (mg mL-1 dry extract).