Steam-cooking rapidly destroys and reverses onion-induced antiplatelet activity.

BACKGROUND: Foods in the diet that can aid in the prevention of diseases are of major interest. Onions are key ingredients in many cuisines around the world and moreover, onion demand has trended higher over the past three decades. An important pharmacological aspect of onion is the ability to inhibit platelet aggregation. Raw onions inhibit platelet aggregation; however, when onions are boiled or heated, antiplatelet activity may be abolished. METHODS: Onion quarters were steamed for 0, 1, 3, 6, 10, and 15 min. The in vitro antiplatelet activity of a yellow hybrid storage onion was examined at these times on the blood of 12 human subjects using in vitro whole blood aggregometry. RESULTS: Contrary to findings reported for boiling, antiplatelet activity was destroyed between 3 and 6 min of steaming, and at 10 min of steaming, cooked onions stimulated platelet activity. Extracts from cooked onion had the potential to reverse the inhibitory effect on blood platelets by 25%. Responses were consistent across all donors. Total polyphenolic concentration and soluble solids were not affected by steaming time. CONCLUSIONS: The potential value of cooked onion preparations may result in destruction or reversal of antiplatelet activity, without affecting the polyphenolic concentration.

Tocotrienols-induced inhibition of platelet thrombus formation and platelet aggregation in stenosed canine coronary arteries.

BACKGROUND: Dietary supplementation with tocotrienols has been shown to decrease the risk of coronary artery disease. Tocotrienols are plant-derived forms of vitamin E, which have potent anti-inflammatory, antioxidant, anticancer, hypocholesterolemic, and neuroprotective properties. Our objective in this study was to determine the extent to which tocotrienols inhibit platelet aggregation and reduce coronary thrombosis, a major risk factor for stroke in humans. The present study was carried out to determine the comparative effects of α-tocopherol, α-tocotrienol, or tocotrienol rich fraction (TRF; a mixture of α-+γ-+δ-tocotrienols) on in vivo platelet thrombosis and ex vivo platelet aggregation (PA) after intravenous injection in anesthetized dogs, by using a mechanically stenosed circumflex coronary artery model (Folts' cyclic flow model). RESULTS: Collagen-induced platelet aggregation (PA) in platelet rich plasma (PRP) was decreased markedly after treatment with α-tocotrienol (59%; P<0.001) and TRF (92%; P<0.001). α-Tocopherol treatment was less effective, producing only a 22% (P<0.05) decrease in PA. Adenosine diphosphate-induced (ADP) PA was also decreased after treatment with α-tocotrienol (34%; P<0.05) and TRF (42%; P<0.025). These results also indicate that intravenously administered tocotrienols were significantly better than tocopherols in inhibiting cyclic flow reductions (CFRs), a measure of the acute platelet-mediated thrombus formation. Tocotrienols (TRF) given intravenously (10 mg/kg), abolished CFRs after a mean of 68 min (range 22 -130 min), and this abolition of CFRs was sustained throughout the monitoring period (50-160 min).Next, pharmacokinetic studies were carried out and tocol levels in canine plasma and platelets were measured. As expected, α-Tocopherol treatment increased levels of total tocopherols in post- vs pre-treatment specimens (57 vs 18 µg/mL in plasma, and 42 vs 10 µg/mL in platelets). However, treatment with α-tocopherol resulted in slightly decreased levels of tocotrienols in post- vs pre-treatment samples (1.4 vs 2.9 µg/mL in plasma and 2.3 vs 2.8 µg/mL in platelets). α-Tocotrienol treatment increased levels of both tocopherols and tocotrienols in post- vs pre-treatment samples (tocopherols, 45 vs 10 µg/mL in plasma and 28 vs 5 µg/mL in platelets; tocotrienols, 2.8 vs 0.9 µg/mL in plasma and 1.28 vs 1.02 µg/mL in platelets). Treatment with tocotrienols (TRF) also increased levels of tocopherols and tocotrienols in post- vs pre-treatment samples (tocopherols, 68 vs 20 µg/mL in plasma and 31.4 vs 7.9 µg/mL in platelets; tocotrienols, 8.6 vs 1.7 µg/mL in plasma and 3.8 vs 3.9 µg/mL in platelets). CONCLUSIONS: The present results indicate that intravenously administered tocotrienols inhibited acute platelet-mediated thrombus formation, and collagen and ADP-induced platelet aggregation. α-Tocotrienols treatment induced increases in α-tocopherol levels of 4-fold and 6-fold in plasma and platelets, respectively. Interestingly, tocotrienols (TRF) treatment induced a less pronounced increase in the levels of tocotrienols in plasma and platelets, suggesting that intravenously administered tocotrienols may be converted to tocopherols. Tocotrienols, given intravenously, could potentially prevent pathological platelet thrombus formation and thus provide a therapeutic benefit in conditions such as stroke and myocardial infarction.

Concord grape juice attenuates platelet aggregation, serum cholesterol and development of atheroma in hypercholesterolemic rabbits.

Intake of Concord grape juice (CGJ), rich in polyphenolics, inhibits platelet aggregation (PA), a risk factor for cardiovascular disease (CVD), in normocholesterolemic animals and humans. It is unclear whether CGJ can attenuate hypercholesterolemia-enhanced PA. The effects of daily CGJ consumption on hypercholesterolemia-enhanced PA and the development of atherosclerosis were investigated. Two groups of rabbits (Control and Treated; n=10 each) were fed a hypercholesterolemic diet for 48 days. Treated group then received supplemental CGJ (225mL/day) while Control group received supplemental iso-caloric sugar water for 48 days. Collagen-, collagen+epinephrine- and phorbol-12-myristate-13-acetate-induced whole blood PA responses were measured on Days 0, 48 and 96; total serum cholesterol and blood pressure were also measured. The development of aortic atheroma was quantified at the end. Both groups showed significant increases in PA and serum cholesterol at Day 48. However, at Day 96, Treated group showed significantly lower PA and development of atheroma (30.7+/-3.9% lower (p<0.001)) than Control group; Treated group also had significantly lower total serum cholesterol and blood pressure than Control group. In conclusion, daily consumption of CGJ attenuates hypercholesterolemia-enhanced PA, blood pressure, total serum cholesterol and development of atheroma in rabbits.

A continuous fluorescence assay for the determination of calcium-dependent secretory phospholipase A2 activity in serum.

BACKGROUND: Calcium-dependent secretory phospholipase A(2)-IIA (sPLA(2)-IIA) in the circulation is a marker of inflammation, associated with acute and chronic disease processes. We describe a quick, sensitive and reliable microplate continuous fluorescence assay for determining sPLA(2) activity in serum. METHODS: Liposomes composed of a fluorescent probe and varying amounts of L-alpha-phosphatidylglycerol (PG) and 1,2-dioleoyl-L-alpha-phosphatidylcholine (DOPC) were used as substrates to determine the optimal protocol for sPLA(2) activity determination without interference from serum albumin and lipoproteins. RESULTS: Hydrolysis of the labeled substrate by sPLA(2)-IIA, characterized by increase in fluorescence intensity (FI) and confirmed by end-product analysis, occurred in a time-, calcium-, and protein-dependent manner. Liposomes containing 100% PG were most suitable for measurement of sPLA(2) activity without interference from serum components; LDL produced a Ca(2+)-independent increase in FI when liposomes containing DOPC were used. The assay determined that sPLA(2) activity in serum spiked with sPLA(2)-IIA and illustrated that endogenous sPLA(2) activity was markedly higher in sera from patients with sepsis than in healthy subjects. Intra-assay and inter-assay CVs were in the ranges of 1.6-8.8% and 3.0-11.5%, respectively. CONCLUSIONS: The described method has potential for rapid and sensitive screening of sPLA(2) activity in both clinical and research settings.

Measurement of regional choroidal blood flow in rabbits and monkeys using fluorescent microspheres.

OBJECTIVE: To develop a quantitative measure of regional variation in choroidal blood flow (ChBF). METHODS: Five million 15-microm fluorescent microspheres were injected into the left ventricles of 4 rabbits and 3 monkeys. The fixed globes were bleached, flat mounted, and photomicrographed. After image analysis to locate each microsphere, regional densities and blood flow were determined. RESULTS: Regional variation in ChBF was clearly evident. In the rabbit, a high density of spheres was seen in the visual streak. This was surrounded by a middle peripheral area of low sphere density and a far peripheral region of moderately high density. In the monkeys, sphere density was markedly greater in the macula compared with the periphery. Contour plots produced lines of constant flow that were oval and extended farther nasally than temporally in the monkeys. The ratio of central to peripheral ChBF was much greater in the monkeys than in the rabbits. CONCLUSION: Quantitative assessment of regional ChBF can be performed using a modification of the fluorescent microsphere impaction method. CLINICAL RELEVANCE: This method of determining regional ChBF will be useful for studying the vascular effects of pharmacologic agents and for characterizing animal models of human disease involving the outer retina.

Modulation of endotoxin-induced cardiopulmonary dysfunction by S-nitroso-albumin.

Nitric oxide (NO) is an endogenous vasodilator and modulator of inflammation. During endotoxemia, the beneficial effects of NO are overwhelmed by the inflammatory cascade, resulting in a functional depletion of NO. S-nitroso-albumin (S-NO-alb) exists as a novel and highly stable NO thiol complex that slowly releases NO into the vascular micro-environment. Using a porcine model, we examined the ability of intravenous S-NO-alb to modulate cardiopulmonary dysfunction characteristic of endotoxemia. Pigs were anesthetized, instrumented for standard cardiopulmonary function measurements, and randomly assigned to receive: (i) albumin + saline; (ii) albumin + LPS; or (iii) S-NO-alb + LPS. Cardiopulmonary parameters were evaluated every 30 min and ex vivo phorbol myristate acetate (PMA)-stimulated superoxide release was serially determined as a marker of in vivo neutrophil priming. Lung myeloperoxidase (MPO) activity was measured as a marker of neutrophil migration into the lung. LPS-induced cardiopulmonary dysfunction was characterized by a sustained elevation in mean pulmonary arterial pressure, pulmonary vascular resistance, and peak intratracheal pressure, as well as a reduction in cardiac index, stroke volume index and PaO(2) over 6 h. Pretreatment with S-NO-alb attenuated LPS-induced cardiopulmonary dysfunction without adversely affecting systemic hemodynamics. Moreover, S-NO-alb blunted the LPS-induced hypoxemic response and reduced neutrophil activation. S-NO-alb did not, however, attenuate LPS-induced increases in lung MPO. Our results suggest that S-NO-alb can selectively modulate endotoxin-induced pulmonary dysfunction, attenuate neutrophil priming and block the early mortality (40%) in this model.

Effects of ethanol and other constituents of alcoholic beverages on coronary heart disease: a review.

Moderate consumption of alcoholic beverages has been shown to reduce the incidence of coronary artery disease (CAD) compared to abstinence and heavy drinking. Epidemiologic studies suggest that any form of alcoholic beverage offers this protection. However, several studies have shown that the polyphenolic compounds found in many alcoholic beverages are more effective than pure alcohol for improving specific biologic factors known to contribute to atherosclerotic development. The alcoholic beverage polyphenols improve endothelial function, protect LDL from oxidation, inhibit platelet activity, inhibit vascular smooth muscle cell (VSMC) proliferation and migration, and reduce the activation of monocytes in the post-prandial period. Not only does pure alcohol not provide these benefits, but it has also been shown to act as a pro-oxidant, impair endothelial function, and possibly be proinflammatory. In studies comparing red wine, white wine, beer, and liquor, the red wine and dark-colored beer seem to provide more protection than liquor, even though the alcohol contents (per serving) are similar. The results suggest that the type of alcoholic beverage consumed does make a difference because pure alcohol cannot fully account for the beneficial effects.

Potential health benefits from the flavonoids in grape products on vascular disease.

In the dog, monkey, a nd human we have shown that 5 ml/kg of red wine or 5-10 ml/kg of purple grape juice but not orange or grapefruit juice inhibits platelet activity, and protects against epinephrine activation of platelets. Red wine and purple grape juice enhances platelet and endothelial production of nitric oxide (Fitzpatrick et al., 1993, Parker et al., 2000). This is thought to be one of the mechanisms whereby purple grape juice significantly improved endothelial function in 15 patients with coronary artery disease. The consumption of purple grape juice by the patients also offered increased protection against LDL cholesterol oxidation, even though all the patients were also taking another antioxidant vitamin E, 400 IU/day. The number of people and animals in these studies was small; however, each one acted as their own control as measurements were made in each before, and then after consumption of red wine or purple grape juice. Thus these studies are thought to be significant. We feel that the results of these studies are encouraging and justify further research on larger numbers of subjects. This suggests that the flavonoids in purple grape juice and red wine may inhibit the initiation of atherosclerosis by one or more of the mechanisms described above. It will take years to fully characterize the potential benefits of daily consumption of red wine or purple grape juice for maintaining a healthy heart. Based on the existing evidence of antiplatelet and antioxidant benefits and improved endothelial function from red wine and purple grape juice, it seems reasonable to suggest that moderate amounts of red wine or purple grape juice be included among the 5-7 daily servings of fruits and vegetables per day as recommended by the American Heart Association to help reduce the risk of developing cardiovascular disease.

Differential effects of grape ( Vitis vinifera ) skin polyphenolics on human platelet aggregation and low-density lipoprotein oxidation.

Antioxidant and antiplatelet properties of grape products are thought to be responsible for observed antiatherosclerotic effects. Diverse classes of phenolics are derived from the seed and skin (GSK) of grapes. The relative contributions of the classes of phenolics to observed properties of grape products are unknown. In this paper, GSK fractions were used to examine effects on platelet aggregation, low-density lipoprotein (LDL) oxidation in vitro, and relative binding of phenolics to LDL. GSK was separated into six fractions (fractions 1-6), and primary phenolics were characterized using high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Fractions 4, 5, and 6, enriched in polygalloyl polyflavan-3-ols (PGPFs) with 3-6, 4-8, and 6-15 degrees of polymerization, respectively, inhibited platelet aggregation. Fractions 1-3, containing various amounts of oligosaccharides, hydroxycinnamic acids, anthocyanins, flavanols, and low molecular weight PGPFs, significantly increased platelet aggregation. Fractions 4-6 were most effective in binding LDL and inhibiting LDL oxidation. Fractions 5 and 6 exhibited the greatest inhibition of platelet aggregation and LDL oxidation, suggesting that polymeric PGPFs are responsible for the beneficial effects of grape products. Conversely, phenolics in fractions 1-3 may reduce the net biological potency of the grape products and have undesirable effects on cardiovascular disease risk factors.

Antithrombotic properties of the thromboxane A2/prostaglandin H2 receptor antagonist S18886 on prevention of platelet-dependent cyclic flow reductions in dogs.

A potent thromboxane A2/PGH2 (TP)-receptor antagonist, S18886, was evaluated for its antithrombotic property in a dog model of acute periodic platelet-mediated thrombosis in stenosed coronary arteries with endothelial damage. After thrombosis had been obtained in 11 dogs, S18886 (300 microg/kg bolus) was administered IV. Heart rate, systemic blood pressure, and coronary blood flow were continuously recorded. Ex vivo whole blood platelet aggregation (PA), blood pH, hematocrit, platelet count, PO2, PCO2, and bleeding times were measured before and 30 minutes after administration of S18886. S18886 completely inhibited thrombosis in all dogs in approximately 5-10 minutes. No change in heart rate, blood pressure, pH, PO2, PCO2, platelet count, or bleeding time and a slight but significant elevation in hematocrit occurred. Infusion of epinephrine IV after complete inhibition of thrombosis by S18886 partially restored thrombosis in 3 of the 11 dogs. PA induced by collagen (4 microg/mL), collagen (0.25 microg/mL) plus epinephrine (1 microg/mL), collagen (1 microg/mL) plus epinephrine (1 microg/mL), ADP (40 microM) plus epinephrine (1 microg/mL), and phorbol 12-myristate 13-acetate (0.5 nM) were attenuated by 90 +/- 8% (P < 0.005), 98 +/- 2% (P < 0.05), 78 +/- 6% (P < 0.005), 70 +/- 10% (P < 0.005), and 28 +/- 8% (P < 0.05), respectively. In conclusion, S18886 is a potent platelet inhibitor that attenuates in vivo platelet-dependent thrombosis in the experimental dog model and reduces ex vivo platelet aggregation.

Grape seed and skin extracts inhibit platelet function and release of reactive oxygen intermediates.

Red wine and purple grape juice contain polymeric flavonoids with antioxidant properties believed to be protective against cardiovascular events but the alcohol and sugar content of these beverages has curtailed their medicinal use. Acute cardiac events are also associated with enhanced inflammation and thrombosis. In this study, the extracts from grape skins or seeds were examined for their anti-inflammatory properties and effect on platelet release of reactive oxygen intermediates. Incubation of platelets with seed or skin extract led to a decrease in platelet aggregation from 68.8+/-19.8% to 45+/-3.6% for seeds and to 27+/-7.2% for skin, respectively (P<0.05). Platelet incubation with grape skin or seed extracts led to a marked decrease in superoxide release from 73+/-6.2 to 2+/-3.4 for grape seeds and to 0.33+/-0.57 for grape skin (chemilum. units; P<0.05) as well as a significant increase in radical-scavenging activity, decrease in reactive oxygen species release by confocal microscopy, and enhanced platelet NO was measured using an NO-sensitive microelectrode. These effects were dose dependent for both grape extracts. Coincubation with seeds and skins led to additive inhibition of platelet aggregation, enhanced NO release, and prevented superoxide production. Incubation with seed or skin extracts led to an immediate attenuation of release of the inflammatory mediator, soluble CD40 ligand. Thus, the extracts from purple grape skins and seeds inhibit platelet function and platelet-dependent inflammatory responses at pharmacologically relevant concentrations. These findings suggest potentially beneficial platelet-dependent antithrombotic and anti-inflammatory properties of purple grape-derived flavonoids.

Grape seed and grape skin extracts elicit a greater antiplatelet effect when used in combination than when used individually in dogs and humans.

Grape products, rich in polyphenolics, inhibit platelet aggregation (PA), a risk factor for coronary artery disease. We postulated that combining extracts of grape seed (GSD) and grape skin (GSK), primary sources of grape polyphenolics, individually shown to inhibit PA, might enhance their individual antiplatelet effects. This hypothesis was examined in vitro (human platelets) and ex vivo (dog platelets) by studying the effects of the extracts on collagen-induced whole blood PA. In vitro, threshold concentration of only GSD, individually incubated with blood, significantly inhibited PA; PA was inhibited by 12.7 +/- 3.5% (P < or = 0.01). No significant changes in Pa were observed with threshold concentrations of GSK, used individually. In two dose combinations, GSD and GSK inhibited PA 40.5 +/- 10.1% (P < or = 0.005) and 96.5 +/- 3.1% (P < or = 0.001). In the ex vivo study, seven dogs were fed threshold doses of GSD or GSK individually, in combination or in combination with a proprietary enzyme blend (EB; thought to enhance bioavailability) for 8 d. PA was measured before and after each treatment. PA measurements were also repeated 24 h after the final dose of GSD + GSK + EB. Feeding the extracts individually did not affect PA, whereas feeding them in combination inhibited PA by 31.9 +/- 7.1% (P < or = 0.05). Feeding EB in addition to GSD + GSK inhibited PA by 56.2 +/- 8.1% (P < or = 0.005); 24 h later, PA was still inhibited by 31.5 +/- 10.5% (P < or = 0.05), suggesting a residual antiplatelet effect from the administration of the final dose. The results suggest that the components of GSD and GSK, when present in combination as in red wine, grape juice or in a commercial preparation containing both extracts, exhibit a greater antiplatelet effect than when present individually.