2.12. Protease Inhibition Assay

This procedure was performed as per the protocol of Dharmalingam and group, with a slight modification. For this, 2 mL of reaction mixture consisting of trypsin (0.06 mL), Tris HCl buffer (1 mL; 20 mM; pH 7.4) and fractionated extracts of the plant (1 mL) was incubated for 10 min at 37 °C. Afterward, casein (1 mL of 0.65% w/v) was added, and the mixture was further re-incubated for 20 min. Subsequently Perchloric acid (2 mL; 2 M) was added to that mixture, a cloudy suspension was obtained and centrifuged at 7830 rpm for 20 min. The absorbance of the supernatant liquid was measured at 280 nm. Tris-HCl buffer solution was used as a control. The formula that was used for the calculation of % inhibition is mentioned below [31].

In this Ac denotes the absorbance of control sample whereas at shows the absorbance related with test sample.