4.4. ACE Inhibition Assay

The experimental model used for the assay was the inhibition of the angiotensin conversion enzyme (ACE), using a chromogenic synthetic substrate, histidine-l-hippuryl-l-leucine-chloride (HHL). The method depends on His–Leu formation by the cleavage of HHL in the presence of ACE. The formation of His–Leu was measured using the fluorescence method. The ACE inhibition potential of the different plant extracts was tested according to [34] with minor modifications. The different extracts were dissolved in methanol: water (1:1) to prepare stock solution of each extract (100 mg/mL). Serial dilutions were prepared for each test solution at concentrations 0.1, 1, 10 and 100 mg/mL.

The test solutions were prepared by adding 40 µL enzyme solution (2 mU ACE prepared in 0.1 M Na borate buffer) to 20 µL of each tested dilution of each sample and then incubated at 37 °C for 10 min. then, 40 µL HHL substrate (0.8 mM/L) were added to the solution. The test solutions were all incubated at 37 °C for 1 h and 60 µL 0.5 M sodium hydroxide were then added to stop the reaction. Blank solutions were prepared for each sample by adding buffer solution instead of the enzyme solution. The control solution was prepared by using each specified solvent instead of the sample. Triplicates were run for each sample. Experiments were set in 96 well microplates. The formation of His–Leu was measured using the florescence method, using the FLUOstar OPTIMA plate reader (BMG Labtech Inc., Offenburg, Germany) at excitation and emission wavelengths of 360 nm and 500 nm, respectively. The percentages of inhibition (% I ± SD) were calculated using the following formula:

The IC50 ± SD (n = 3) values were calculated in mg/mL by linear interpolation.