Pancreatic Lipase Inhibition Assay

The pancreatic lipase inhibitory activity was measured using 4-methylumbelliferyl oleate (4-MU oleate) (Sigma, St. Louis, MO, USA), as a substrate, as previously described by Zhang et al. [48 (link)]. A volume of 5 μL of sample solution, or Orlistat (Sigma, St. Louis, MO, USA) used as positive control, was mixed with 50 μL of 0.1 mM 4-MU oleate, 20 μL of 0.1 M citrate-Na₂-HPO₄ buffer (pH 7.4) and 25 µL of porcine pancreatic lipase. The mixture was then incubated at 37 °C for 10 min, and the fluorescence intensity of 4-MU, released by the lipase activity, was measured at excitation and emission wavelengths of 320 nm and 450 nm, respectively, using a fluoroskan micro-plate reader (Fluoroskan Ascent FL, Thermo Electron Corporation, Vantaa, Finland). The inhibition of pancreatic lipase activity was determined in percentage at different samples’ concentrations.