Porcine pl

Chrysin, porcine PL and p-nitrophenyl palmitate (p-NPP) were procured from Sigma Aldrich®, India. The rat normal chow was procured from Krishna Valley AgroTech, Maharashtra, India and fructose was obtained from Tate & Lyle, United Kingdom. Orlistat was provided as a gift sample by Macleods Pharmaceuticals Ltd., Mumbai, India. Intralipid® 20% was procured from a local pharmacy. The kits for the estimation of triglycerides (TG) and cholesterol were procured from Erba Mannheim®, Germany. All other chemicals used in this study were of analytical grade.

The ability of the extracts to inhibit porcine PL was determined using the method earlier reported by Eom et al. [28 (link)] with a slight modification. In this assay, PL hydrolyzes p-nitrophenyl butyrate to form p-nitrophenol which is measured spectrophotometrically. The enzyme solution was prepared by adding 30 µL (10 units) of porcine PL (Sigma, USA) in 10 mM morpholine propane sulfonic acid and 1 mM EDTA (pH 6.8) to 850 µL of Tris buffer (100 mM Tris-HC1 and 5 mM CaCl₂, pH 7.0). Thereafter, 100 µL of appropriate concentrations (20, 40, 60, 80 µg/mL) of the extracts or orlistat (positive control) were mixed with 880 µL of the enzyme solution and incubated for 10 min at 37°C. After incubation, 20 µL of the substrate solution (10 mM of p-nitrophenyl butyrate in dimethylformamide) was added to initiate the hydrolytic reaction, which was allowed to proceed for 20 min at 37°C. A reference test (without the extract) was included in the assay. The absorbance of the p-nitrophenol produced from the hydrolysis of p-nitrophenylbutyrate was measured at 405 nm. The inhibition of PL activity by the extracts was expressed as % inhibition thus:
% Inhibition = [(A405_reference–A405_sample)÷A405_reference]×100.
Where A405_reference is the absorbance of the reference without the extract, and A405_sample is the absorbance of test containing the extract.