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Anti-thrombotic effect of proanthocyanidin, a purified ingredient of grape seed

https://doi.org/10.1016/j.thromres.2004.07.015Get rights and content

Abstract

Introduction: Moderate and regular consumption of wine reduces the risk of acute coronary thrombotic events. The mechanism of the anti-thrombotic effect of wine is not clear. Extract or purified ingredients of grapes have not yet been studied for anti-thrombotic effect. Materials and methods: Anti-thrombotic effect of proanthocyanidin, a highly purified ingredient of grape seed, was assessed by a shear-induced thrombosis test in vitro and by a laser-induced thrombosis test in the mouse carotid artery, in vivo. Results and conclusions: Intravenously (20 mg/kg body weight, BW) or orally (2×200 mg/kg BW) administered proanthocyanidin significantly inhibited the laser-irradiation induced thrombus formation in the carotid artery (both P=0.01). Subsequent to oral administration of proanthocyanidin, in vitro platelet reactivity to shear stress has been inhibited. The latter suggests that the in vivo anti-thrombotic effect of proanthocyanidin may be due to a direct inhibitory effect on platelets.

Introduction

Prevention of life-style related atherothrombotic diseases such as myocardial infarction and stroke is important and urgent social task in the developed countries. Epidemiological studies provided irrefutable evidence for the causative role of inappropriate diet both in the development and clinical outcome of thrombotic diseases. Regular consumption of diet or nutritional supplements of experimentally proven anti-thrombotic effect may offer effective and economical way of preventing thrombotic events.

Mortality from cardiovascular diseases is significantly lower in French than in people from other countries having similar high fat diet [1]. It has been suggested that the French's habitual and large amount of red wine consumption is responsible for the lower cardiovascular mortality [2]. This so-called “French Paradox” has accelerated laboratory studies for finding anti-thrombotic fruits and vegetables and for identifying the effective ingredients. Grapes and grape products have been studied for platelet inhibitory and anti-thrombotic effects [3], [4], [5], [6], [7]. Other studies showed that 3-week intake of proanthocyanidin, a purified substance of grape seeds, protected from myocardial infarction subsequent to reperfusion [8], [9], [10]. The aim of the present study was therefore to assess the anti-thrombotic activity of purified proanthocyanidin by experimental models, which reflect arterial thrombogenesis.

The common technique of assessing anti-thrombotic effect of foods and vegetables is to use animal models. By the Folts' model of coronary stenosis, Demrow et al. [3] and Osman et al. [4] demonstrated that red wine (1987 Chateauneuf-du-Pape) and Welch's natural purple grape juice inhibited thrombogenesis in vivo. Freedman et al. [6] and Shanmuganayagam et al. [7] made unsuccessful attempts to identify the active material(s).

In arterial thrombotic diseases such as cardiovascular disease and stroke, platelets play a pivotal role. The conventional in vitro platelet function test is the platelet aggregometry, which measures activation and aggregation response of platelets to different stimuli such as collagen, adenosine diphosphate or epinephrine. However, platelet aggregation induced by high shear forces is more relevant to pathology than the agonists-induced platelet aggregation test [11], [12]. Further, because of the effect of all anticoagulants on platelets and coagulation, a test, which is performed from native, non-anticoagulated blood has much more relevance to in vivo than those conventional tests, which use anticoagulated blood [13], [14], [15], [16], [17], [18], [19], [20]. In this study, a shear-induced in vitro platelet function test from native blood sample was used for screening followed by the helium–neon laser-induced in vivo thrombogenesis test performed in the carotid artery of atherosclerosis-prone mice.

Section snippets

Chemicals

Purified proanthocyanidin preparation (Gravinol-S, GSE) was donated by Kikkoman (Japan); GSE was composed of 89.3% proanthocyanidins, which contained 6.6% dimer, 5.0% trimer, 2.9% tetramer and 74.8% oligomers and polymers bigger than pentamer, 6.6% monomeric flavanols (2.5% (+)-catechin, 2.2% (−)-epicatechin, 1.4% (−)-epigallocatechin and 0.5% (−)-epigallocatechin gallate), 2.24% water, 1.06% protein and 0.8% ash. Sodium pentobarbital was purchased from Abbott. Lab. (USA) and Evans blue dye

Effect of intra-arterial proanthocyanidin

Results are shown in Fig. 3. A 2 mg/kg BW had no significant anti-thrombotic effect (30 0541±55 922 vs. 26 7081±20 602; proanthocyanidin vs. controls), but 20 mg/kg BW significantly inhibited thrombus formation (30 0541±55 922 vs. 13 6051±16 998; proanthocyanidin vs. controls).

Effect of oral proanthocyanidin

Results are shown in Fig 4. A single administration of 200 mg/kg BW did not inhibit thrombus formation (262 229±44 447 vs. 22 3014±32 149; proanthocyanidin vs. controls) but repeated (×2) 200 mg/kg BW dose (400 mg/kg BW)

Discussion

Grapes, their ingredients and grape products were shown to inhibit platelet aggregation in vitro and thrombus formation in vivo [3], [4], [5], [6], [7]. Earlier studies in rodents have shown that intake of proanthocyanidin, a purified ingredient of grape seeds protects from myocardial infarction after reperfusion injury [8], [9], [10]. However, the chemical characteristic and the mechanism of action of the effective ingredient(s) of grapes have not yet been clarified. There are numerous

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