Kinetic Characterization of Inhibition of Angiotensin Converting Enzyme by Proanthocyanidins Extracted from Vitis Vinífera L. cv. País

摘要

The kinetics inhibition of angiotensin converting enzyme (ACE) by the proanthocyanidins (PAs) extracted from seed and skin grapes of Vitis vinifera L País, an ancestral Chilean grape was evaluated, as a possible natural source to produce a natural hypertensive pharmaceutical drug. The skin and seed grapes were separately extracted with an acetone/water (2:1 v,v) solution and then purified by size exclusion chromatography using a Toyopearl 40 HW-F resin packed column. The extracts were characterized in their levels of PAs by acid catalysis depolymerization with phloroglucinol followed by the detection of the extension and terminal subunits by HPLC. The mean degree of polymerization (mDP), reaction yield (%) and average molecular weight (aMW) of the extracts were determined. The seed and skin extracts (0.001; 0.005; 0.01; 0.1 and 1 mg/mL) inhibition on ACE was evaluated by hyppuric acid (HA) determination from the hydrolysis of hippuryl-histidyl-leucine (HHL) by ACE with quantitative HPLC separation. The kinetic parameters were calculated by adjusting the data to the Lineweaver-Burk, Eadie-Hofstee and Lagmuir equations. Structural compositions differed significantly between both extracts; indeed, those from skin do not exhibit epicatechin (EC) and epicatechin-gallate (ECG); whereas epigalocatechin (EGC) was not detected in seed extracts. Also, skin extracts have higher mDP (34.1) than seed extracts (7.6). The effective concentration to produce 50% of inhibition (EC_(50)) was determined. The inhibition on ACE was higher with skin (EC_(50) = 0.075uM) than with seed extracts (EC_(50) = 0.124uM). The inhibition by PAs from skin and seed showed a mixed inhibition mechanism. At all inhibitor concentrations the apparent saturation constant (K_(mapp)) was increasing and the apparent maximum rate (V_(mapp)) was decreasing. This behaviour could be explained by the chain size and conformation of the PAs through ACE.