Briefly, appropriate dilutions (0–200 lL) of the extracts and
500 lL of 0.02 M sodium phosphate buffer (pH 6.9 with
0.006 M NaCl) containing porcine pancreatic a-amylase (EC
3.2.1.1) (0.5 mg/mL) were incubated at 25 C for 10 min. Then,
500 lL of 1%starch solution in 0.02 Msodium phosphate buffer
(pH 6.9 with 0.006 M NaCl) was added to the reacting mixture.
Thereafter, the reaction mixture was incubated at 25 C for
10 min and 1.0 mL of dinitrosalicylic acid (DNSA) was added.
Then the reactionwas stopped by incubating in a boiling water
bath for 5min and later cooled to room temperature. The reaction
mixture was then diluted by adding 10 mL of distilled
water, and absorbance was measured at 540 nm in a spectrophotometer
(Worthington, 1993). The reference sample included
all other reagents and the enzyme with the exception
of the test sample. The a-amylase inhibitory activity was expressed
as percentage inhibition.
Inhibition ð%Þ ¼ ½ðAbsref AbssampleÞ=Absref 100
where Absref = absorbance of the reference; Abssample = absorbance
of the test samples.