4.6. Enzyme Assay

The ACE inhibiting capability was evaluated according to the method described by Wang et al. ([14], which employs the hippuryl-histidilleucina tripeptide (Hip-His-Leu or HHL) as the substrate and evaluates the formation of hippuric acid (HA) by liquid chromatography (HPLC-DAD). Briefly, the commercial enzyme ACE and the substrate HHL (Sigma-Aldrich^®) were dissolved in borate buffer (NaOH and boric acid) 100 mmol·L⁻¹, pH 8.3, supplemented with 300 mmol·L⁻¹ NaCl; their concentrations were 0.2 U/mL and 5 mmol·L⁻¹, respectively. Meanwhile, HA and captopril (standard ACE inhibitor) were dissolved in distilled water. The enzyme was pre-incubated in the presence or absence of inhibitor (isolated microginins or captopril) for 30 min at 37 °C, and the reaction was initiated by addition of substrate. After 30 min, the reaction was stopped by adding 250 μL of hydrochloric acid, and the sample was directly injected into the chromatographic system. The concentrations of the inhibitors chosen for the experiment were: 0.1 μM, 0.2 μM, 0.4 μM and 0.8 μM for captopril (Sigma^®; IC₅₀ = 0.021 ± 0.013 μM) and 5 μg·mL⁻¹, 10 μg·mL⁻¹, 50 μg·mL⁻¹, 100 μg·mL⁻¹, 500 μg·mL⁻¹ and 800 μg·mL⁻¹ for microginins. The separation was performed in a reversed phase column (Luna C18(2); 150 nm × 4.6 mm, 5 μm; Phenomenex^®) using (A) water with 0.05% trifluoroacetic acid (TFA) and 0.05% triethylamine, and (B) acetonitrile as mobile phase; the ratio of solvent A:B was 7:3. The isocratic elution was used and the formation of HA was monitored at 226 nm. The reaction system was: 10 μL HHL (5 mmol·L⁻¹), 10 μL ACE (0.2 U/mL), 40 μL inhibitor and 40 μL borate buffer 100 mM (pH = 8.3). The buffer was incubated in the thermomixer for 5 min at 37 °C, the inhibitor (isolated microginins or captopril) and the enzyme were added. After 5 min, the substrate was added to start the reaction.

The inhibitory rate was calculated, according to Wang et al. [14], by I% = (A − B)/A × 100%; where A is the peak area of HA without adding ACE inhibitors; and B is the peak area of HA with the addition of ACE inhibitors [14]. All data presented are the average of three repeats; the data are presented as means ± SEM.