3.5. Assay of ACE Activity and Inhibitory Activity

ACE activity was evaluated by measuring the amount of hippuric acid (HA) [6]. The assay mixture (0.3 mL) consisted of 130 µL BBS buffer pH 8.3 (0.1 mM, containing 0.3 M NaCl), 40 µL HHL (5 mM), and different amounts of the free and immobilized ACE. Incubation was carried out at 37 °C for 10 min and was terminated by addition of HCl (0.1 mL, 1 M). The HA content of the mixture was determined by RP-HPLC (Agilent 1260) at 228 nm with a DAD detector. One unit of ACE enzyme activity (U) was defined as the amount of enzyme catalyzing HHL to generate 1 µM of HA per minute under the experimental conditions.

The ACE inhibitory activity were measured as described above, with the sample replacing the BBS buffer. The ACE inhibition percentage was calculated using Equation (3) [10]:

where A₁ and A₂ are the peak areas of HA in the control group and with inhibitor, respectively.