Mechanism and specificity of angiotensin converting enzyme inhibition activity by syzygium polyanthum wight (WALP.) leaves

Abstract

Syzygium polyanthum is an ethnomedicinal plant used for the treatment of hypertension. This study investigates the anti-hypertensive property of this plant using angiotensin-converting enzyme (ACE) inhibition assay. The ability to inhibit this enzyme in vitro indicates the ability to decrease blood pressure. This study aims to determine the ACE inhibitory activity of S. polyanthum leaves, its inhibition specificity, mechanism and the possible bioactive compound. S. polyanthum leaves were dried, ground, and then macerated in a bath-sonicator with four different solvents; water, methanol, ethyl acetate, and hexane producing four types of extracts, the aqueous (ASP), methanolic (MSP), ethyl acetate (EASP) and hexane (HSP) extracts. Each extract (100 µg/ml) was initially screened using ACE inhibition assay according to Cushman and Cheung method and then compared with the standard drug, captopril (2.06 ng/ml). The most active extract was further tested at a range of concentrations (1 to 1000 µg/ml) to determine its potency and then tested for the mechanism and inhibition specificity using zinc chloride, bovine serum albumin (BSA), α-chymotrypsin, and trypsin. The phytochemical composition in the most active extract was then analysed using Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-QTOF/MS). In silico docking analysis was then performed between the major identified compound in the most active extract with the ACE. ASP at 100 μg/ml exhibited the highest inhibition activity (69.43 ± 0.60%) compared to MSP (41.63 ± 0.15%), EASP (9.62 ± 1.60%) and also HSP (45.40 ± 0.15%). From the dose-response curve for ACE inhibition activity of ASP, the inhibitory concentration of ASP that causes 50% of ACE inhibition activity (IC50) was 41 μg/ml. ACE inhibition activity by ASP was significantly reduced by the presence of BSA, indicative of interaction of ASP with albumin. ACE inhibition activity by ASP was not significantly affected with the presence of zinc chloride, indicating that its inhibitory activity on ACE was non-dependent of zinc at the ACE active site. There was no significant inhibition by ASP on other enzymes such as α-chymotrypsin and trypsin. There were 26 compounds identified in ASP with the following major compounds; 1-galloyl-glucose (21%), feroxin A (12%), nilocitin (10 %), 2,6-di-O-galloyl-β-D-glucose (9%), 1-O-galloylpedun-culagin (7%), 5-desgalloylstachyurin (6%), and yakuchinone A (6%). Molecular docking analysis showed that 1-galloyl-glucose has lower binding energy (-7.7 kcal/mol) with the ACE, as compared to the standard drug, captopril (-5.6 kcal/mol); indicative of good interaction between 1-galloyl-glucose and the ACE. In conclusion, this study showed that the enzyme inhibition activity by S. polyanthum leaves extract was specific towards ACE. This inhibition possibly occurs via protein precipitation which was non-dependent to the chelation with zinc at the ACE active site with 1-galloyl-glucose, suggested as the potential bioactive compound.