Moderate consumption of red wine is associated with a decrease in the incidence of cardiovascular events1 and the cardioprotective effects have been partially attributed to polyphenolic compounds present in purple grapes. The bioactivity of polyphenols includes antioxidant and free radical-scavenging properties that lead to a decrease in LDL oxidation,2 an increase in HDL level,3 prevention of platelet aggregation,4 and leukocyte adhesion.5 Grapes contain a variety of antioxidants, including resveratrol, quercetin, catechin, epicatechin, and proanthocyanidins,6 with resveratrol present mainly in grape skin and proanthocyanidins present in seeds. Interestingly, purple grape juice has been shown to be effective in improving lipid and antioxidant profiles at a much lower dose of polyphenols as compared with wines.7 The growing interest in the presumed benefits of wine in protecting against coronary heart disease, the inherent limitations in promoting alcohol consumption, and the high sugar content of purple grape juice, has lead to further exploration of alternative purple grape-derived products. Purple grape extracts are commercially available and one component of the over 5 billion dollars that Europeans spend annually on herbal supplements. Like many supplements, little is known about their specific bioactivity.
The protective effects of flavonoids in biologic systems are ascribed to their capacity to chelate metal catalysts,8 activate antioxidant enzymes,9 reduce α-tocopherol radicals,10 and inhibit oxidases.11 Specific flavonoids interact with arachidonic acid metabolism and inhibit platelet thromboxane A2 production.12 Red wine polyphenols have been shown to increase endothelial nitric oxide (NO) synthase (eNOS) expression and subsequent endothelial NO release.13 The effects of beverages derived from purple grapes may be independent of alcohol content as extracts of grape seeds and skins exhibit endothelium-NO-dependent relaxing activity in vitro.14 Importantly, it has been reported that the majority of the polyphenolic content in grape beverages is derived from the grape seed and skin.15,16 A previous study found that components of grape seed and skin, when present in combination as in red wine or juice, exhibit greater antiplatelet effect17; however, the mechanism for this and its other vasoactive effects are unknown. Therefore, this study examined the effect of extracts from distinct components of grape plants on related thrombotic and inflammatory properties in platelets.
MATERIALS AND METHODS
Phorbol myristate acetate (PMA), prostaglandin E, and lucigenin (bis-N-methylacridinium nitrate) were purchased from Sigma Company (St. Louis, MO). Fibrinogen was purchased from Enzyme Research Laboratories (South Bend, IN). GPRP (GPRP Fibrin Antipolymerant) was purchased from PolyPeptide Laboratories (Torrance, CA). Thrombin receptor activating peptide (TRAP) was purchased from Phoenix Pharmaceuticals, Inc. (Belmont, CA). Dihydrorhodamine 123 (DHR) was purchased from Cayman Chemical Company (Ann Arbor, MI). Grape seed and skin extracts were provided by Melaleuca Inc. (Idaho Falls, ID). The content and quantification of total phenolics in grape seed and skin extracts were previously characterized.15-17
Preparation of Grape Seed and Grape Skin Extracts
We used concentrations of extracts as described in the text. Extracts were prepared as previously described.17 The extracts were dissolved in solution [3 dimethyl sulfoxide (DMSO): Preservative-free saline (PF-saline)].
Preparation of Washed Platelets
Blood samples, using acid/citrate/dextrose (ACD) as anticoagulant (1:9 vol/vol), were obtained from healthy volunteers who had provided informed consent (BUSM IRB) and were not on medications or nutritional supplements for 2 weeks prior to blood draw. Platelet-rich plasma (PRP) was obtained after centrifugation at 150 × g for 20 minutes at 24°C. PRP was incubated with grape seed (GSD) (50 mg/L, 100 mg/L) and skin (GSK) (250 mg/L, 500 mg/L) extracts for 30 minutes at 24°C. After the addition of the equal volume of platelet washing buffer (with PGE2 in a 1:10,000 ratio) the PRP was centrifuged (350 × g, 15 minutes, 24°C) and the platelet pellet was resuspended in Tyrode's solution.18 Platelet counts were determined using a Coulter counter, model ZM (Coulter Electronics, Miami, FL). For NO measurements and aggregation the platelet counts were adjusted to final concentration of 2.5 × 105 platelets/μL, for superoxide measurements 3.5 to 4 × 105 platelets/μL. All the platelet incubations in this study were carried out at room temperature and ambient pO2.
Measurement of Platelet Nitric Oxide Production and Aggregation
Aggregation was studied in washed platelets as previously described18 using a platelet aggregometer (Model PAP-4, Bio/DATA Corporation, Horsham, PA) and simultaneous measurement of NO release using an NO-selective micro-electrode. Platelet NO production was quantified as the integrated signal detected by the micro-electrode following platelet activation with 20 μM TRAP.
Measurement of Platelet Superoxide Release
Aggregation-dependent superoxide production from stimulated platelets was determined as previously described.19 Superoxide release of washed platelets was measured with lucigenin (250 μM) in a lumiaggregometer (whole blood lumiaggregometer, Model 500-Ca, Chrono-Log Corp). In some experiments, the superoxide scavenger Tempol was incubated with grape skin or seed extracts prior to superoxide measurement. PMA was used as the platelet agonist as it leads to enhanced superoxide generation.19
Measurement of Soluble CD40 Ligand
Platelet aggregation-induced release of soluble CD40 ligand (sCD40L) was measured after TRAP stimulation after preincubation with grape seed and skin extracts for 20 minutes at room temperature. After 3 minutes of aggregation, the samples were centrifuged (10900 × g) for 2 minutes at 4°C. The supernatant was collected and stored at -20°C until measurements were performed. Soluble CD40L was measured using an enzyme-linked immunoassay (Chemicon, Temecula, CA).
DPPH Free Radical-Scavenging Activity
The free radical-scavenging activities of grape seed and skin extracts were examined based on their ability to bleach the stable radical 2,2-diprenyl-1-picrylhydrazyl (DPPH).20 This assay estimates the potential anti-radical activity of antioxidants. Absorbance at 515 nm was determined after 30 minutes at room temperature and the scavenging activity was calculated as a percentage of the radical reduction. All experiments were performed in triplicate. The radical-scavenging activity was calculated using the equation:
where A0: A515 of DPPH without sample; A: A515 of sample and DPPH; Ab: A515 of sample without DPPH.
Platelet Measurement of Reactive Oxygen Species Release by Confocal Microscopy
Fluorescence confocal images were obtained using a two-photon laser scanning microscope. For the measurement of platelet fluorescence induction, 200 × 103/μL cells in HEPES buffer were loaded on a slide with 20 μM dihydrorhodamine (DHR-123) for 10 minutes in presence and absence of seed and skin extracts. Baseline (T = 0 minutes) readings were collected. Platelets were stimulated with 25 μM TRAP and images were captured at 10 minutes. Images were analyzed and quantified using NIH image software and fluorescence ratios at baseline versus 10 minutes were compared.
All data are presented as mean ± SEM. One-way parametric ANOVA was performed with SigmaStat version 2.03. A statistically significant difference was assumed with a value of P < 0.05.
The Effect of Grape Seed and Skin Extracts on Platelet Aggregation
Previously, it has been shown in vivo and in vitro that juice from purple grapes inhibits platelet aggregation.21 To determine the components responsible for this property, the effect of grape skins and seeds on platelet aggregation was measured. As shown in Figure 1, the incubation of platelets with grape seed or skin extracts in the presence of 20 μM TRAP led to a dose-dependent inhibition of extent of aggregation. Collagen, thrombin, and ADP-induced platelet aggregation after incubation PRP with grape seed and skin extracts had a similar effect (data not shown).
The Effect of Grape Seed and Skin Extracts on Platelet-Derived Nitric Oxide Release
Consumption of purple grape juice enhances platelet NO release21 and red wine increases eNOS expression.13 To determine whether these properties exist with the isolated extracts, stimulation-dependent release of NO was determined. As seen in Figure 2, in the presence of 20 μM TRAP there is a dose-dependent enhancement of release of platelet-derived NO for both grape skin and seed extracts. As with aggregation, there was a greater increase in platelet NO production after extract coincubation. Maximal effect of NO release was from 76 ± 19 (a.u.) with vehicle control to 385 ± 57 (a.u.; n = 5; *P < 0.05) for the combination of 100 mg/L of grape seed and 500 mg/L of grape skin. The same effect was found with other well-known platelet activators including ADP and collagen but total amount platelet NO release was half as compared with TRAP suggesting that thrombin receptor stimulation causes enhanced platelet NO release.
The Effect of Grape Seed and Skin Extracts on Platelet Release of Superoxide
Superoxide is produced by aggregating platelets19,22 and exogenous superoxide augments platelet activation.23 To determine whether grape skin and seed extracts decrease platelet superoxide release, platelets were stimulated with PMA (0.15 μM) in the presence of lucigenin (250 μM). As shown in Figure 3, grape skin and seed extracts dramatically inhibited platelet superoxide release. In the presence of grape skin extract alone or the combination of grape seed and skin extracts, superoxide release was almost completely eliminated.
Superoxide can limit the bioavailability of NO. To determine the relative contribution and interaction of release of NO and superoxide in the setting of grape extracts, platelets were incubated with the membrane permeable superoxide scavenger Tempol in the presence or absence of varying extract concentrations. Lower extract concentrations were used in these experiments to detect a difference as extract inhibition of superoxide release was found in previous experiments to be highly efficient. Superoxide dismutase metabolizes superoxide, but has poor membrane permeability. Tempol, however, is a stable, cell-permeable superoxide dismutase mimetic that functions as a superoxide trap. As seen in Figure 4, incubation with Tempol decreased superoxide release in either the presence or absence of the extracts. Tempol scavenges superoxide, which can quench endogenous NO. Therefore, Tempol can potentiate NO-mediated responses in the presence of enhanced superoxide levels by increasing NO availability. Surprisingly, in our experiments Tempol had no effect on NO release with or without the extracts (Table 1) suggesting that the augmentation of NO by grape seed and skin extracts is not caused by its attenuation of superoxide. These data suggest that the effect of Tempol is NO independent.
Grape Seed and Skin Extracts and Platelet Release of Soluble CD40 Ligand
The release of reactive oxygen intermediates can alter inflammatory processes mediated by platelets. Therefore, the effect of grape skin and seed extracts on aggregation-induced release of the inflammatory protein sCD40L was determined. For TRAP-induced platelet aggregation and as seen in Figure 5, grape seed and skin extracts inhibit the release of platelet sCD40L. We have determined that 2 hours after thrombin stimulation, platelets release increased sCD40L. Although longer time for release would likely show greater changes due to the extracts, we report the immediate effects to parallel the measurements of reactive oxygen and nitrogen species.
The Effect of Grape Skin and Seed Extracts on Radical-Scavenging Activity
Because superoxide release may be mediated by direct antioxidant effects, the direct free radical-scavenging activity of grape skin and seed extracts was determined by radical reduction using DPPH. As seen in Figure 6, there is a dose-dependent increase in radical-scavenging activity for both grape skins and seeds.
The Effect of Grape Skin and Seed Extracts on Platelet Release of Reactive Oxygen Intermediates
Fluorescent probe dihydrorhodamine (DHR-123) can be used to quantify platelet release of reactive oxygen intermediates. Following treatment with grape seed and skin extracts, activation-induced platelet clumping and induction of fluorescence by confocal microscopic imaging is significantly attenuated (Fig. 7).
Accumulating evidence supports the protective role of grape-derived polyphenols in cardiovascular diseases. Two chemical classes of flavonoids, the flavan-3-ols (catechins and proanthocyanidins) and the anthocyanins, are the natural antioxidants present at the highest concentration in purple grape juice and red wine. According to some studies, the content of anthocyanins and proanthocyanidins are much greater in red as compared with white wines.24 In the berry, the anthocyanins are localized in the skins, similarly to other phenolics of grape such as the resveratrols and the flavonols, whereas the flavan-3-ols are contained both in the skins and seeds.25 Polyphenolic compounds, such as resveratrol, are naturally present at high concentration in grape skin, seeds, and red wine. All phenolic compounds are highly unstable and theirs level in wine and juice are affected by numerous processing conditions (crushing, pressing, sulfite addition, skin contact, oak aging).26 Studies have shown27 that most flavonoids are effective antioxidants in a wide range of chemical oxidation systems being capable of scavenging peroxyl radicals, hydroxyl radicals, and peroxynitrite. More recently the ability of procyanidins from grape seeds to prevent peroxynitrite attack on endothelial cells and enhanced endothelium-dependent relaxation in human artery has been demonstrated28 and the threshold for relaxation by proanthocyanidins oligomers was between 0.5 and 4 μg/mL.29 Several studies have shown that proanthocyanidins are potent scavengers of peroxyl and hydroxyl radicals that are generated in the reperfused myocardium after ischemia.30,31 This suggests that the cardioprotective effects could be attributed, at least in part, to the ability of proanthocyanidins to scavenge hydroxyl and peroxyl radicals.
The regulation of platelet activation by pharmacological agents that modulate a variety of platelet functions has proven a successful approach for the prevention of thrombosis. In the face of a multi-billion dollar per year business in dietary supplements, there is a growing interest in the study of the actual biologic effects of dietary consumption of natural substances in the prevention of atherothrombotic disease. Nitric oxide is a known mediator of cardiovascular processes including its effects on regulation of platelet aggregation and reactive oxygen intermediates.32-34 In this study, the effect of extracts from grape seeds and skins on platelet function, NO, and superoxide release were determined. Our results show both grape skin and seed extracts lead to a dose-dependent inhibition of aggregation and enhanced release of platelet NO generation. These findings are consistent with a recent canine study where, after skin and seed extract oral supplementation, ex vivo platelet aggregation was inhibited.17,35 It was observed in both in vitro incubation studies with human platelets (whole blood) and ex vivo feeding studies in the dog and humans.35 A limitation of our study is that the experiments were performed in vitro; however, in our studies inhibition of platelet aggregation is consistent with the findings of a recent report demonstrating comparable inhibition of platelet aggregation after extract supplementation with 30 mg/kg/d for 8 weeks.36 In our study, the most marked effect of these extracts was their potent attenuation of platelet superoxide release. Platelets, like endothelial cells, contain not only the L-arginine-NO synthase pathway but membrane-bound NAD(P)H oxidases37,38 although the specific source of platelet-derived superoxide is not completely defined. As shown in Figure 3, grape skins and seeds produce a dose-dependent inhibition of stimulation-induced platelet superoxide release.
To determine if the increase in NO release was due to the extracts inhibition of superoxide, Tempol, a membrane permeable intracellular reactive oxygen species scavenger was used. Tempol interacts with oxygen-derived free radicals including superoxide anions, hydroxyl radicals, and peroxynitrite39,40 and its incubation with grape extracts showed that Tempol (Fig. 4) significantly reduced superoxide release in a dose-dependent manner. Superoxide anions react with NO and can inhibit responses mediated by platelet NO release. As Tempol chelates superoxide anions, it can potentiate NO-mediated responses. Because Tempol had no effect on platelet-derived NO release, this suggests that the superoxide attenuating effects of grape extracts were independent of their effects on NO. Other factors may be altering these results, however, as the rate constant for the NO-superoxide reaction is high and the concomitant formation of other reactive species must be considered. There are several mechanisms that control NO bioavailability including the NO/cGMP signaling pathway; NO-dependent phosphorylation of platelet vasodilator-stimulated phosphoprotein; generation of prostaglandins; and level of platelet NO synthase expression and substrate availability. Nevertheless, the effect of superoxide on platelet NO in the setting of these extracts warrants further study. To further define the antioxidant (reducing) capacities of grape seed and skin extracts, the free radical-scavenging properties of the grape compounds were examined by determining their capacity to scavenge the free radical DPPH. As shown in Figure 6, GSD and GSK inhibited DPPH radical in a concentration-dependent manner suggesting a potent free radical-scavenging effect. The oxidant-sensitive fluorophore, DHR123, was used to detect the effect of grape seed and skin extracts on the platelet reactive oxygen species generation. As shown in Figure 7, presence of seed, skin, or combination markedly attenuated platelet activation-induced fluorescence. These results suggest that some of the marked effects on superoxide release are due to antioxidant or radical scavenging properties of the extracts.
Approximately 95% of sCD40L in blood originates from platelets and is involved in thrombosis and vascular inflammation.41 Recent studies have generated clinical interest in the use of sCD40L levels as a marker of thrombotic risk.42 Other grape product-derived substances including resveratrol have been reported to suppress oxidative damage and inflammation.43 We recently demonstrated that consumption of purple grape juice for 2 weeks suppresses CD40L in patients with stable coronary disease.44 We therefore wanted to examine the anti-inflammatory potential of grape seed and skin on the release of sCD40L from platelets. As seen in Figure 5, grape seed and skin extracts inhibit the release of platelet sCD40L.
In conclusion, these studies have shown that extracts from purple grape components have significant antioxidant and anti-thrombotic properties that lead to inhibition of platelet activation, increased platelet NO release and radical scavenging activity, as well as decreased superoxide/reactive oxygen intermediate production and release of platelet sCD40L. Although additional studies are required to fully evaluate the precise mechanism and components of grape skin and seeds that are responsible for the marked effects, these finding have implications for understanding the protective effect of red wine and purple grape products on cardiovascular diseases. Seed and skin extracts may be a viable choice as a flavonoid source as they lack the detrimental effects associated with sugar and alcohol intake.
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Keywords:© 2005 Lippincott Williams & Wilkins, Inc.
aggregation; flavonoids; nitric oxide; platelets; superoxide