2.5. In Vitro Assay for ACE-Inhibitory Activity

ACE-inhibitory activity was evaluated through RP-HPLC and assayed using the modified spectrophotometric method described by Cushman and Cheung [35] and Wu and Ding [36]. In brief, 15 mM HHL was dissolved in 100 mM Na-borate buffer (pH 8.3) supplemented with 300 mM NaCl. Rabbit lung ACE was dissolved in the same buffer at a concentration of 53.2 mU/mL. A mixture containing 75 μL of ACE solution and 75 μL of the sample with a 5000-Da MWCO membrane (Millipore, Burlington, MA, USA) was incubated at 37 °C for 10 min, to which was added 75 μL of HHL solution, and the mixture was incubated for a further 30 min. The reaction was halted by addition of 250 μL of 1 N hydrochloric acid, and 10 μL of this solution was injected directly into a Luna C₁₈ analytical column (4.6 × 250 mm², particle size: 5 μm; Phenomenex, Torrance, CA, USA) to separate the substrate HHL and product hippuric acid (HA) liberated through hydrolysis of HHL. The column was eluted with a mobile phase of 0.1% trifluoroacetic acid in methanol and water (50/50, v/v) at a constant flow rate of 0.8 mL/min using a pump (model L-7100, Hitachi, Tokyo, Japan) and monitored at 228 nm using an ultraviolet (UV) spectrophotometer (UV-VIS detector 118, Gilson Medical Electronics, Villiers-le-Bel, France). Finally, inhibition activity was calculated using the following formula.

where Ec is the absorbance with addition of the buffer instead of the test sample (control), Es is the absorbance when the sample was added to the reaction mixture (sample), and Eb is the absorbance when the stop solution was added before the reaction occurred (blank). The IC₅₀ value was defined as the concentration of peptide in milligrams per millilitre required to reduce 50% of the absorbance peak height of the HA (50% inhibition of ACE), which was determined through regression analysis of ACE inhibition (%) versus the log 10 (peptide concentration, mg/mL) curve and constructed using at least six separate analyses to calculate the concentration. Captopril (positive control) exhibited the highest significant ACE-inhibitory activity (IC₅₀ value = 0.0069 μM). All data presented in this paper are the average of three repeats or mean ± standard deviation (SD).