In vitro pancreatic lipase inhibition assay

The pancreatic lipase activity was measured titrimetrically with a pH-Stat (Metrohm, Switzerland) at pH 8.5 and 37 °C using olive oil emulsion as substrate and in the presence of 4 mM NaDC and Turkey pancreatic colipase previously purified according to Rathelot and colleagues [29]. One lipase unit corresponds to 1 μmol of fatty acid released per minute.

In this assay, RaEO was dissolved in DMSO and was used to evaluate its inhibitory effect on lipase. To evaluate the pancreatic lipase inhibitory activity, TPL was preincubated at room temperature for 1 h with various RaEO concentrations. The reaction medium contained 20 μL of RaEO and 60 μL of enzyme. After preincubation, 40 μL from the reaction mixture were used to evaluate the residual pancreatic lipase activity, as previously indicated. Pure control having 100% enzyme activity was conducted by replacing the essential oil with DMSO. Blank for pure control having 0% enzyme activity was conducted with DMSO and by replacing the enzyme with buffer. On the other hand, THL was used as a positive control for this test. The lipase inhibition (% inhibition) was calculated in comparison with the initial activity, measured in the absence of inhibitors. The IC₅₀ values were calculated from plots of log concentration of essential oil versus percentage inhibition curves using Sigma Plot 12.1 (IL, USA).