FREE-RADICALS SCAVENGING ACTION AND ANTI-ENZYME ACTIVITIES OF PROCYANIDINES FROM VITIS-VINIFERA - A MECHANISM FOR THEIR CAPILLARY PROTECTIVE ACTION

The scavenging by procyanidines (polyphenol oligomers from Vitis vinif era seeds, CAS 85594-37-2) of reactive oxygen species (ROS) involved i n the onset (HO degrees) and the maintenance of microvascular injury ( lipid radicals R degrees, RO degrees, ROO degrees) has been studied in phosphatidylcholine liposomes (PCL), using two different models of fr ee radical generation: a) iron-promoted and b) ultrasound- induced lip id peroxidation. In a) lipid peroxidation was assessed by determinatio n of thiobarbituric acid-reactive substances (TBARS); in b) by determi nation of conjugated dienes, formation of breakdown carbonyl products (as 2,4-dinitrophenylhydrazones) and loss of native phosphatidylcholin e.In the iron-promoted (Fenton-driven) model, procyanidines had a rema rkable, close-dependent antilipoperoxidant activity (IC50 = 2.5 mu mol /l), more than one order of magnitude greater than that of the monomer ic unit catechin (IC50 = 50 mu mol/l), activity which is due, at least in part, to their metal-chelating properties. In the more specific mo del b), which discriminates between the initiator (hydroxyl radical fr om water sonolysis) and the propagator species of lipid peroxidation ( the peroxyl radical, from autooxidation of C-centered radicals), procy anidines are highly effective in preventing conjugated diene formation in both the induction (IC50 = 0.1 mu mol/l) and propagation (IC50 = 0 .05 mu mol/l) phases (the scavenging effect of a-tocopherol was weaker , with IC50 of 1.5 and 1.25 mu mol/l). In addition, procyanidines at 0 .5 mu mol/l markedly delayed tire onset of the breakdown phase (48 h), totally inhibiting during this time the formation of degradation prod ucts (the lag-time induced by alpha-tocopherol was only of 24 h at 10 mu mol/l concentration). The HO degrees entrapping capacity of these c ompounds was further confirmed by UV studies and by electron spin reso nance (ESR) spectro-scopy, using DMPO as spin trapper: procyanidines m arkedly reduce, in a dose-dependent fashion, the signal intensity of t he DMPO-OH radical spin adduct (100 % inhibition at 40 mu mol/l). The results of the second part of this study show that procyanidines, in a ddition to free radical scavening action, strongly and non-competitive ly, inhibit xanthine oxidase activity, the enzyme which triggers the o xy radical cascade (IC50 = 2.4 umol/l). In addition procyanidines non- competitively inhibit the activities of the proteolytic enzymes collag enase (IC50 = 38 mu mol/l) and elastase (IC50 = 4.24 mu mol/l) and of the glycosidases hyaluronidase and beta-glucuronidase (IC50 = 80 mu mo l/l and 1.1 mu mol/l), involved in the tunzover of the main structural components of the extravascular matrix collagen, elastin and hyaluron ic acid. All these findings provide a strong molecular basis for the c apillary protective action of procyanidines, which is probably a pluri centric mechanism, based on radical quenching and antioxidant effects and on the inhibition of some key enzymes of the microvascular endotht elium and extravascular matrix.