Lipase activity was measured using p-NPB as a substrate.
The method was modified from the previously described by Kim
et al. [17]. Briefly, an enzyme buffer was prepared by the
addition 30 mL of solution of porcine pancreatic lipase
(2.5 mg/mL in 10 mmol/L morpholinepropanesulphonic acid
and 1 mmol/L ethylenediamine tetraacetic acid, pH 6.8) to
850 mL of Tris buffer (100 mmol/L Tris–HCl and 5 mmol/L
CaCl2, pH 7.0). Then, either 100 mL of the plant extracts
(100 mg/mL) or Orlistat was added and incubated for 15 min
at 37 C. Ten microliter of substrate (10 mmol/L p-NPB in
dimethyl formamide) was then added and incubated for
30 min at 37 C. Lipase activity was determined by measuring
the hydrolysis of p-NPB to p-nitrophenol at 405 nm using an
ELISA reader (Biochrome, England). The inhibitory activity
(I) was calculated according to the following formula:
I% = 1 − B A − − b a × 100
where A is the activity of the enzyme without inhibitor, and a is the
negative control without inhibitor; B is the activity of the enzyme
with inhibitor, and b is the negative control with inhibitor.