The Journal of Nutrition Vol. 128 No. 12 December 1998, pp. 2307-2312

Grape Juice but Not Orange or Grapefruit Juice Inhibits Platelet Activity in Dogs and Monkeys (Macaca fasciularis)^1,2

Hashim E. Osman, Nabil Maalej, Dhanansayan Shanmuganayagam, and John D. Folts³

University of Wisconsin Medical School Madison, WI, 53792

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Platelet aggregation (PA) contributes to both the development of atherosclerosis and acute platelet thrombus formation (APTF) followed by embolization producing cyclic flow reductions (CFR) in stenosed and damaged dog and human coronary arteries. In seven anesthetized dogs with coronary stenosis and medial damage, CFR occurred at 7 ± 3/30 min and were abolished 127 ± 18 min after gastric administration of 10 mL of purple grape juice/kg. Collagen-induced ex vivo whole blood PA decreased by 49 ± 9% after the abolishment of CFR with grape juice. Ten mL of orange juice/kg (n = 5) and 10 mLof grapefruit juice/kg (n = 5) had no significant effect on the frequency of the CFR or on ex vivo PA. In vitro studies have suggested that flavonoids bind to platelet cell membranes and thus may have an accumulative or tissue-loading effect over time. To test this we fed 5 mLof grape juice/kg to 5 cynomologous monkeys for 7 d. Collagen-induced ex vivo PA decreased by 41 ± 17% compared to control (pre-reatment) after 7 d of feeding. In the same 5 monkeys, neither 5 mL of orange juice/kg nor 5 mLof grapefruit juice/kg given orally for 7 d produced any significant change in PA. Grape juice contains the flavonoids quercetin, kaempferol and myricetin, which are known inhibitors of PA in vitro. Orange juice and grapefruit juice, while containing less quercetin than grape juice, primarily contain the flavonoids naringin, luteolin and apigenin glucoside. The flavonoids in grapes were shown in vitro to be good inhibitors of PA, whereas the flavonoids in oranges and grapefruit to be poor inhibitors of PA. The consumption of grape juice, containing these inhibitors of PA, may have some of the protection offered by red wine against the development of coronary artery disease (CAD) and acute occlusive thrombosis, whereas orange juice or grapefruit juice may be ineffective. Thus, grape juice may be a useful alternative dietary supplement to red wine without the concomitant alcohol intake.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Epidemiological studies show an inverse correlation between the intake of dietary flavonoids and death from coronary artery disease (Hertog et al. 1993 and 1995, Knekt et al. 1996). This may be explained in part by the antioxidant and antiplatelet properties attributed to flavonoids (Beretz et al. 1982, DeWalley et al. 1990). Platelets and oxidized low density lipoproteins (LDL) cholesterol contribute to and accelerate the rate of development of atherosclerotic coronary artery disease (Fuster et al. 1992, Steinbrecher et al. 1984 and 1987, Woolf and Davies 1992). Platelets are also the cells that initiate fatal and nonfatal myocardial infarction due to coronary thrombosis (Antiplatelet Trialists` Collaboration 1988, Chandler 1974, Davies and Thomas 1984). While antiplatelet treatment had no apparent effect on nonvascular mortality, it reduced vascular mortality by 15% and nonfatal vascular events (stroke or myocardial infarction) by 30%. Thus, the net advantage of antiplatelet treatment may hold for a wide range of types of patient with a history of occlusive vascular disease (Antiplatelet Trialists` Collaboration 1988).

It has been suggested that the daily consumption of red wine may reduce the incidence of coronary artery disease and heart attacks (Nanji 1985, Pace-Asciak et al. 1996, Renaud and Delogeril 1992). This is thought to be primarily due to the antioxidant properties, and may also be due in part to the antiplatelet properties, of the polyphenols found in red wine (Demrow et al. 1995). The antioxidant properties of the phenolic compounds in red wine reduce the propensity of LDL to undergo peroxidation and substantially prolong the lag time required for initiation of LDL oxidation (Fuhrman et al. 1995). Major portions of the phenolics in red wine are compounds called flavonoids (Havsteen 1983). These have been shown to have antioxidant and antiplatelet properties in vitro (Fuhrman et al. 1995, Tzeng et al. 1995). We have previously demonstrated that 10 mL red wine/kg (approximately three glasses), but not white wine, which contains lower amounts of flavonoids, significantly inhibits in vivo platelet activity and coronary thrombosis in the Folts cyclic flow model (Demrow et al. 1995) and in human volunteers (Folts 1998). We also demonstrated that 10 mL/kg of Welch`s^® Purple Grape Juice significantly inhibited in vivo platelet activity and experimental coronary thrombosis (Demrow et al. 1995). Grapes and grape juice contain many of the same biologically active phenolic compounds such as catechines, quercetin, kaemferol and anthocyanin that are found in red wine (Ribereau-Gayon 1972). However, citrus fruits like oranges and grapefruits and their juices contain different flavonoids, such as naringin, hesperetin and epigenin (Kefford and Chandler 1970).

View larger version (24K): [in this window] [in a new window]   Fig 1. Schematic diagram of an impedance aggregometer. Two electrodes immersed in whole blood measure the impedance. Upon addition of the platelet agonist, platelets aggregate onto the electrodes and cause an increase in impedance, which is recorded on a strip chart recorder. The change in impedance is proportional to the ex vivo platelet activity.

View larger version (79K): [in this window] [in a new window]   Fig 2. The top two tracings show phasic arterial blood pressure and circumflex blood flow at slow recording paper speed. Damage to the vessel wall and stenosis around the coronary artery caused platelets to aggregate in the narrowed lumen producing cyclic flow reductions (CFR). In the center, epinephrine (Epi) 16.0 mg/kg was infused for 20 min. The increase in frequency of the CFR is a direct indication of an increase in in vivo platelet activity caused by the epinephrine. Two h after 10 mL purple grape juice/kg was given by stomach tube, the CFR were abolished and did not return when Epi was infused (bottom two tracings).

View larger version (73K): [in this window] [in a new window]   Fig 3. The top two tracings show phasic arterial blood pressure and circumflex blood flow at a slow recording paper speed. Damage to the vessel wall and stenosis around the coronary artery caused platelets to aggregate in the narrowed lumen producing cyclic flow reductions (CFR). Two h after 10 mLorange juice/kg was given by stomach tube, the frequency of the CFR were not significantly decreased (bottom two tracings) compared to pretreatment.

View larger version (64K): [in this window] [in a new window]   Fig 4. The top two tracings show phasic arterial blood pressure and circumflex blood flow at a slow recording paper speed (6 mm/min). Damage to the vessel wall and stenosis around the coronary artery caused platelets to aggregate in the narrowed lumen producing cyclic flow reductions (CFR). Two h after 10 mLgrapefruit juice/kg was given by stomach tube, the CFR frequency were not significantly decreased (bottom two tracings) compared to pretreatment.

View larger version (84K): [in this window] [in a new window]   Fig 5. The change in impedance caused by collagen-induced platelet before and after feeding the monkey 10 mL of grape juice/kg for 7 d.

In this study, we first elected to compare, in our established dog model of experimental coronary artery stenosis and thrombosis, the in vivo platelet inhibitory properties of the polyphenolic compounds in purple grape juice, orange juice and grapefruit juice. Secondly, we chose to compare the ex vivo antiplatelet effects of grape juice, orange juice and grapefruit juice in cynomologous monkeys (Macaca fasciularis) fed each of the three beverages for 7 d. As in vitro studies show that flavonoids bind to cell membranes (Gryglewski et al. 1987), we postulated that a subthreshold dose of grape juice fed for 7 d may have an accumulative or a tissue loading effect. Five mL of grape juice/kg is not effective in inhibiting platelets when given as a single dose; however, feeding this lower dose for seven consecutive days may cause an accumulation of the flavonoids such that it may effectively inhibit platelets activity.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

PART A: In vivo studies of experimental coronary thrombosis in dogs

Surgical preparation.  The surgical preparation has been described in detail in previous publications (Demrow et al. 1995, Folts 1991a and 1991b). Seventeen adult mongrel dogs (supplied by the animal care facility of the University of Wisconsin-Madison) of either sex were anesthetized with 5 mg/kg of sodium pentabarbital given through the brachiocephalic artery, and the chest opened at the fifth intercostal space. The left circumflex coronary artery was dissected out and an electromagnetic flow probe placed around the artery to measure coronary blood flow. Distal to the flow probe, the artery was clamped three times with a special surgical clamp to produce intimal and medial damage. A plastic cylinder of appropriate inside diameter was placed around the artery to produce a 70 ± 5% reduction in arterial diameter. Platelets aggregate periodically in the area of the stenosed and damaged arterial lumen gradually forming an occlusive platelet thrombus that causes coronary blood flow to decline to zero. A pressure gradient then builds up across the stenosed lumen, dislodging the platelet thrombus and causing it to fragment and embolize distal to the stenosed site. If the thrombus does not embolize spontaneously, the plastic cylinder is shaken gently to dislodge the thrombus. This causes a sudden restoration of coronary blood flow. This acute platelet mediated thrombus formation followed by embolization causes cyclic flow reductions (CFR). The frequency of the CFR is a direct indication of in vivo platelet activity (Maalej et al. 1998). The periodic decline in coronary blood flow produces regional ischemia, with S-T segment changes in the ECG. We elected to study the intragastric administration of three commercially available juices on thrombus formation: 100% Welch`s<sup>®</sup> purple grape juice (Welch`s, Concord, MA), 100% Minute Maid^®orange juice (Minute Maid, Chicago, IL), and 100% Ocean Spray^® grapefruit juice (Ocean Spray, Plymouth, MA). The dogs and the juices to be studied were selected randomly. The investigation conforms with the guidelines for the care and use of laboratory animals by the US National Institute of health (NIH publication 85-23) and the University of Wisconsin Research Animal Resource Center.

Group 1: Dogs receiving purple grape juice.  Seven randomly selected dogs were prepared as described above. CFR due to acute platelet mediated thrombus formation followed by embolization occurred at a rate of 7 ± 3/30 min during a control 30 min observation period. Before treatment, a control blood sample was drawn for ex vivo platelet aggregation studies (9 mL of blood was added to 1 mL of 3.8% sodium citrate). The blood was studied in a Chrono-Log^® whole blood platelet aggregometer (Chrono-Log Inc.) using collagen as the platelet agonist. Each dog was then administered 10 mL 100% Welch`s^® grape juice/kg of by a stomach tube over a 10-min period. The CFR were continuously monitored for 2 1/2 h or until the CFR were abolished. Two hours after the grape juice was administered, a second blood sample was obtained and studied in the aggregometer. If the CFR were abolished by the purple grape juice, 0.8 mole epinephrine · kg^-1 · min^-1 was infused IV for 20 min. This test was performed to see if elevated plasma epinephrine could increase platelet activity and renew the CFR (Folts and Rowe 1988).

Group 2: Dogs receiving orange juice.  Five randomly selected dogs were prepared and studied as described above. CFR due to acute platelet mediated thrombus formation occurred during control observation period. A control blood sample was drawn, for ex vivo whole blood aggregation, before orange juice was administered. Each dog was then administered 10 mL Minute Maid^® 100% orange juice/kg by stomach tube over a 10-min period. The CFR were continuously observed for 2-4 h and then a second blood sample was obtained for ex vivo whole blood platelet aggregation.

Group 3: Dogs receiving grapefruit juice.  Five more dogs were prepared as described above. CFR due to acute platelet mediated thrombus formation, followed by embolization occurred during the 30 min control observation period. A control blood sample was drawn, for ex vivo whole blood aggregation, before grapefruit juice was administered. Each dog was then administered 10 mLOcean Spray^® 100% grapefruit juice/kg by a stomach tube over a 10- min period. The CFR were monitored for 2-4 h and then a second blood sample was obtained for ex vivo whole blood aggregation.

PART B: Feeding studies in monkeys

Ex Vivo Platelet Aggregation Studies.  Nine mL of whole blood was drawn into a syringe containing 1 mL of 3.9% sodium citrate as an anticoagulant. The blood was then added to an equal volume of preservative-free saline. A 1 mL aliquot was added to a cuvette with a siliconized stir bar and warmed to 37°C. Then incremental portions of 2 mMADP or 1 mg collagen/mLwas added to aliquots of blood and placed in the aggregometer (Fig. 1). The change in impedance produced was a measure of platelet aggregation and was observed for 8 min.

Phenolic content.  The total polyphenolic content of the three juices were assayed by the Folin-Ciocalteu assay. Folin-Ciocalteu reagent was prepared by diluting a stock solution (Fisher Scientific Inc.) with distilled water (1:10 by volume). A sample of juice or gallic acid standards (50 mL) was added to 5 mL of reagent in a test tube followed by 4 mL of 75 g Na₂CO₃ /L. The tube was stirred and kept at ambient temperature for 2 h. Absorbance at 675 nm was recorded for the juices and gallic acid standards (Folin and Ciocalteu 1927).

Qualitative analysis of the major types of flavonoids in the juices was performed by high performance liquid chromatography (HPLC) using a diode array detector. Reverse phase of HPLC (C-18 column, 5 mm particle size, 4.6 x 2.5 mm) was performed using a linear gradient from water/acetic acid (975:25 by vol., solvent A) to 100% methanol (solvent B) over 40 min at a flow rate of 1 mL/min. The UV spectra of major peaks were used to classify the flavonoids as flavonols, anthocyanidins, flavonones, flavones and proanthocyanidines.

Statistical methods.  All data values are reported as means ± SD. The statistical significance of the difference between measurements (pre- vs. posttreatment) was obtained from the two-tailed paired Student's t-test.

	RESULTS

Abstract Introduction Methods Results Discussion References

PART A: In vivo studies of experimental coronary thrombosis in Dogs

Group 1: Dogs receiving purple grape juice.  The CFR averaged 7 ± 3/30 min during a control 30 min observation period. The CFR were abolished in all seven dogs 127 ± 18 min after receiving purple grape juice by stomach tube. Figure 2 shows a representative tracing. Collagen-induced (1 mg/mL) ex vivo platelet aggregation response was decreased by 48 ± 9% after grape juice. Thus purple grape juice produced a significant decrease in platelet activity (P < 0.04). The infusion of epinephrine did not produce a renewal of CFR in any dog.

Group 2: Dogs receiving orange juice.  The CFR averaged 6 ± 3/30 min before and 5 ± 4/30 min 2 h after the orange juice was given by stomach tube. A representative tracing is shown in Figure 3. There was no significant change in ex vivo and in vivo platelet aggregation due to orange juice administration. Because the CFR were not abolished, the IV infusion of epinephrine was not done.

Group 3: Dogs receiving grapefruit juice.  The CFR averaged 8 ± 4/30 min at control and decreased to an average of 5 ± 3/30 min 2 h after the grapefruit juice by stomach tube, showing no significant change. A representative tracing is shown in Figure 4. There was also no significant change in ex vivo platelet aggregation. Hence, grapefruit juice did not produce any detectable inhibition of platelet activity. Because the CFR were not abolished, the IV infusion of epinephrine was not done.

There was no significant change in heart rate or blood pressure during the 2 h of observation following administration of any of the three juices.

PART B: Feeding studies in monkeys

Purple grape juice.  After the 7 d of feeding grape juice, the collagen-induced (1 mg/mL) platelet aggregation decreased by 41 ± 17% (P = 0.003) compared to control. A representative tracing is shown in Figure 5. The ADP-induced (2 µmol/L) aggregation also decreased by 31 ± 10% (P = 0.03) (Table 1). Therefore, feeding monkeys for 7 d half the dose of purple grape juice that inhibited acute platelet mediated thrombosis in dogs significantly inhibited ex vivo platelet activity.

Grapefruit juice.  After 7 d of feeding grapefruit juice, there was no significant change in the collagen-induced (1 mg/mL) platelet aggregation response (19 ± 10%, P = 0.44). There was also no significant change in the ADP-induced (2 µmol/L) platelet aggregation response (11 ± 7%, P = 0.13) (Table 1).

Orange juice.  After 7 d of feeding orange juice, there was no significant change in the collagen-induced (1 mg/mL) platelet aggregation response (9 ± 5%, P = 0.34). There was also no significant change in the ADP-induced (2 µmol/L) platelet aggregation response (4 ± 11%, P = 0.06) (Table 1).

Phenolic concentration.  Quantitative results from the Folin-Ciocalteu assay showed that grape juice had a greater total polyphenolic concentration than orange juice or grapefruit juice. The gallic acid equivalents in g/L juice were 2.26, 0.75 and 0.86, respectively. Qualitative analysis indicated that there were large differences in the classes of flavonoids present in the juices. Grape juice contained flavonols and anthocyanidins and there was no evidence that these flavonoids were present in the orange juice or grapefruit juice. The major peaks in orange juice and grapefruit juice were flavanones and flavones.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The polyphenolic compounds, called bioflavonoids or phytochemicals, are widely distributed in the plant kingdom, but are not found in animal food sources (Cook and Samman 1996). Plant preparations containing flavonoids have been used for centuries as herbal remedies for a variety of diseases and found to have an impact on allergy, arthritis and cancer (Middleton and Kandaswami 1993). The pioneering work of Ruszynak and Szent-Gyorgi (1936) that demonstrated the capacity of extracts of Capsicum annum and citrus lemon fruits to cure subcutaneous capillary bleeding, resulted in the elucidation of the active principle(s) in these plant extracts and led to the identification of a remarkable number of pharmacological effects of these flavonoids. Many of them have antioxidant, anti-inflammatory and platelet inhibitory effects (Cook and Samman 1996, Frankel et al. 1993, Gryglewski et al. 1987, Kinsella et al. 1993). However, many of such studies have only shown the medicinal properties of flavonoids in vitro. Thus, it is important to study the benefits of flavonoids in vivoto establish their clinical relevance.

The cyclic flow model, which has been described as a good on-line bioassay for in vivo platelet activity (Bush and Shebuski 1990), has been used to study and predict which platelet inhibitors would be successful in clinical trials (Folts 1995). The CFR observed in the dog model were also observed in the peripheral arteries of patients with intermittent claudication (Folts et al. 1982) and in the diseased coronary arteries of patients with unstable angina (Eichhorn et al. 1991). The CFR observed in the dog are thought to correlate well with acute ischemic syndromes in humans (Hisao et al. 1993). Thus the fact that purple grape juice inhibits in vivo platelet activity and abolishes the CFR and periodic acute regional ischemia has potential relevance to patients with atherosclerosis and intermittent claudication or unstable angina.

In our study, a single dose of purple grape juice significantly inhibited ex vivo and in vivo platelet activity and abolished CFR in the stenosed coronary arteries of seven dogs. Feeding half that dose of purple grape juice for 7 d significantly attenuated ex vivo platelet aggregation in the monkeys. On the other hand, neither grapefruit nor orange juice had any significant effect on platelet activity or the coronary artery CFR in the dogs or on the ex vivo platelet aggregation in the monkeys.

In the present study, we compared the inhibitory effect of purple grape juice to orange and grapefruit juices in dogs and monkeys. In the dogs, the grape juice significantly decreased in vivo and ex vivo platelet activity. It would appear that feeding 1/2 the minimal acute effective dose of grape juice to monkeys for 7 d significantly inhibits platelet activity. It may be that the active ingredients in grape juice build up over a period of time in the platelet by a tissue loading or accumulative effect. The equivalent doses of grapefruit juice and orange juice did not inhibit ex vivo platelet activity after 7 d. We hypothesize that the effect of purple grape juice on platelets may be due to its specific content of polyphenolic compounds, which differs from those in citrus fruit.

Each of these three fruits contains a variety of flavonoids. Entirely different groups of flavonoids are found in citrus fruits like oranges and grapefruit compared to grapes. Naringen, naringenin and hesperidin, which are flavones, are commonly found in oranges and grapefruit (Kefford and Chandler 1970). Flavonoids most commonly found in grapes are catechin, epicatechin, dihydro quercetin, kaempherol and cyanidins (Mazza and Miniati 1993). Unlike the citrus fruits, grapes and red wine predominantly contain flavonols (Ribereau-Gayon 1972).

It appears that something, possibly the flavonols, in grape juice inhibits in vivo platelet activity better than the flavones in orange juice or grapefruit juice. This seems consistent with studies on structure-activity relationships that suggest that the flavonoids in grapes may be better platelet inhibitors than those found in orange or grapefruit (Brandi 1992). Flavonols such as quercetin and rutin are present in grape juice in larger amounts than grapefruit and orange juices (Ribereau-Gayon 1972, Mazza and Miniati 1993). Flavonols have also been shown to disaggregate platelet thrombi that were adhering to the blood supefused endothelial surface in vitro (Gryglewski et al.). Moreover these flavonols have a strong ability to bind to proteins, especially to those in platelet membrane (Mazza and Miniati 1992, Singleton 1981). Flavonoids in red wine and grape juice are also good antioxidants and thus protect the antioxidant capacity of serum (Singleton 1981, Whitehead et al. 1995) in some cases better than vitamin E on a molar basis (Frankel et al. 1993).

In summary, purple grape juice, but not equal amounts of orange juice or grapefruit juice, significantly inhibits both ex vivo (whole blood platelet aggregometer) and in vivo platelet activity and experimental coronary thrombosis. Thus, the daily consumption of grape juice may reduce the rate of development of atherosclerotic narrowing of the coronary arteries as well as the incidence of potentially fatal coronary thrombosis. Purple grape juice may be as effective as red wine in protecting patients with CAD from coronary thrombosis and heart attacks. Purple grape juice is likely to be better than wine for those who cannot or should not drink alcoholic beverages.

	FOOTNOTES

Manuscript received 15 April 1998. Initial reviews completed 13 May 1998. Revision accepted 4 August 1998.

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