2.5. Angiotensin I-converting-enzyme (ACE) inhibitory activity

There are several methods to determine the ACE inhibitory activity which can be used to determine the ACE activity. However, the method utilized here is a spectrophotometric method based on the commonly used method introduced by Cushman and Cheung (1971).¹⁶ This method is simple, sensitive, and rapid, requiring no solvent extraction and can, therefore, be used for high-throughput screening of ACE inhibitors. ACE inhibitory activity was assayed by measuring the release of HA (Hippuric acid) from the substrate HHL.

Various types of substrates and methods used to analyze ACE inhibition activity are following: Cushman and Cheung Method using a substrate hippuryl-histidyl-leucine (HHL), Holmquist method using a substrate furanacryloyl-tripeptide, Elbl and Wagner method using a substrate benzoil-[l-14C] glicyl-l-histidine-l-leucine, Carmel and Yaron method using a substrate o-aminobenzoylglycyl-p-nitrophenylalanilproline, and Lam method using 3-hydroxybutyrylglycyl-glycyl-glycine as substrate. Methods to measure the results of enzymatic reactions are spectrophotometric, fluorometric, high-performance liquid chromatography, electrophoresis, and radiochemistry. A Radiometric assay using the labeled angiotensin I substrate, wherein the release of radioactive histidine-leucine which serves as an enzymatic activity index.¹⁷ Carmel and Yaron (1977–1978) developed a measurement method of the ACE inhibitory activity using an o-aminobenzoylglycine-p-nitrophenylalanylproline as a substrate and then hydrolyzed into o-aminobenzoylglicyl.¹⁸ Holmquist et al. in 1979 developed a method using FAPGG as a substrate. This method is based on the absorption spectrum blue shift that occurred on the substrate hydrolysis produces dipeptide and furanacryloyl-blocked amino acid.¹⁹ Many of the methods described above require expensive instrumentation and a large volume of organic solvents,²⁰ so utilized a simple, sensitive, and rapid, requiring no solvent extraction method as described by Jimsheena and Gowda, 2009.²¹

The assay mixture contained 125 μL of a 0.05 M sodium borate buffer (pH 8.2), containing 0.3 M NaCl, 50 μL of 5 mM HHL and 25 μL of ACE (2.5 Milli units (mU)), which was pre-incubated with different sample concentrations of the plant inhibitor. The reaction was stopped after incubation at 37 °C for 30 min by the addition of 0.2 mL of 1 M HCl. Pyridine (0.4 mL) was added followed by 0.2 mL of BSC (the order of addition of reagents is critical) The solution was slowly mixed using a vortex mixer and cooled on ice. The yellow colour developed was measured at 410 nm using UV–visible spectrophotometer (UV-1700 Shimadzu, Japan). One unit of ACE activity is defined as the amount of enzyme, which releases 1  μmol of HA per min at 37 °C (Degree Celsius) and pH 8.2.²¹

The degree of ACE inhibition (%) was calculated with the following equation

Where.

A1 is absorbance of the ACE solution without an inhibitor (Plant extract and fractions)

A2 is absorbance of the tested sample of extract and fractions

A3 is absorbance of HHL solution (a buffer was added instead of the ACE solution and sample)

The IC₅₀ value is defined as the concentration of inhibitor required to decrease the HA peak area by 50% (indicating 50% inhibition of ACE), and was calculated using a non-linear regression from a plot of activity versus inhibitory concentration of at least five separate concentrations. To obtain the standard deviation during the assay, each assay was performed in triplicate.