ACE Inhibition Assay

ACE inhibitory activity was evaluated using the ACE activity assay test (Sigma-Aldrich CS0002). This assay is based on the hydrolysis of angiotensin I by the ACE to yield active angiotensin II. This test uses a synthetic fluorogenic peptide as the substrate, and the measured fluorescence is directly proportional to the ACE activity present. Briefly, all reagents were diluted in the assay buffer, according to the kit instructions. A standard curve was prepared in 100 μL of assay buffer per well ranging from 0 to 8 nmol for calculating the enzymatic activity. A total of 10 μL of 1 (final concentration of 1 μM), an inhibitor of ACE activity (lisinopril, Cayman Chemical) (a final concentration of 12 nM), a positive control of the ACE (provided by the kit), and a negative control (assay buffer) were pipetted into a 96-well black opaque plate. Then, 40 μL of the ACE, provided in the kit, were added to the sample and control wells and incubated at 37 °C for 5 min. The reaction was initiated by adding 50 μL of the fluorogenic substrate to experimental, control, and blank sample wells. The reaction was carried out at 37 °C, and fluorescence was read every minute for 5 min using a microplate reader (BioTek Cytation 5 Cell Imaging Multimode Reader, Agilent). The absorbance wavelengths of excitation and emission were set at 320 and 405 nm, respectively. All analyses were performed in duplicate. To calculate the enzymatic activity, a linear regression was determined.