2.8. Renin Inhibition Assay

The renin-inhibitory activity of samples was determined using a previously described method [26]. Briefly, 20 µL of the substrate, 160 µL of assay buffer, and 10 µL of distilled water were mixed and added to the background well. Then, 20 µL of the substrate, 150 µL of assay buffer, and 10 µL of water were mixed into the control (uninhibited) wells, whereas the sample (inhibited) wells contained same reagents except that the water was replaced with 10 µL of samples (0.5 mg/mL assay concentration). This was followed by the addition of 10 µL of renin solution (dissolved in the assay buffer) to the control and sample wells to initiate enzyme reaction; the microplate was shaken for 10 s to ensure adequate mixing of the reagents and then incubated at 37 °C for 10 min in the dark. Enzyme catalytic activity was measured as the fluorescence intensity (FI) measured at excitation and emission wavelengths of 340 and 490 nm, respectively. Enzyme inhibition was calculated as follows after subtracting the FI of the background well from the control and sample wells: