![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Department of Horticulture, University of Wisconsin-Madison, Madison, WI 53706 and * Cardiology Section, H6/339 CSC, University of Wisconsin-Madison, Madison, WI 53702
2To whom correspondence should be addressed. E-mail: ilgoldma{at}facstaff.wisc.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
KEY WORDS: • onion • platelets • thrombosis • dogs
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
. Platelet aggregation plays a central role in coronary
thrombosis. Aggregation along arterial walls also contributes to the
development of atherosclerosis, a process that begins in adolescence
(2)
. A diet rich in natural platelet inhibitors may reduce
an individual’s risk of developing cardiovascular disorders involving
thrombosis. Onions (Allium cepa) and other vegetables from
the genus Allium (e.g., garlic, leek and Welsh onion)
inhibit in vitro human platelet aggregation (3)
.
Consumption of raw Welsh onion by rats has also been shown to inhibit
aggregation and other platelet functions ex vivo (4)
.
When Allium tissue is cut,
S-alk(en)yl-L-cysteine sulfoxides
(ACSO)3
are cleaved by the enzyme alliinase. Several of the products from this
and subsequent reactions, including thiosulfinates (TS) and cepaenes
(5
,6)
, inhibit in vitro platelet aggregation. The
concentration of pyruvate, a by-product of the reaction between
alliinase and ACSO, is stoichiometrically related to the quantity of
organosulfur products in a juice sample. Pyruvate concentration and in
vitro antiplatelet activity of onion juice are correlated
(7)
, suggesting that inhibition of aggregation is at least
partly sulfur dependent. Alliums and Allium organosulfur
compounds may provide further benefit to the cardiovascular system
through their abilities to inhibit lipid oxidation and lower serum
lipid and cholesterol levels (8
,9)
. Onions are also one of
the richest sources of flavonoids in the diet (10)
.
Epidemiologic studies have shown an inverse correlation between
long-term risk of cardiovascular disease and flavonoid intake
(11)
. This relationship has been attributed to the
platelet-inhibitory and antioxidant properties of flavonoids such
as quercetin (12
,13)
.
The purpose of this study was to determine whether raw onion
preparations can inhibit platelet-mediated thrombosis in vivo. A
dog model of chronic platelet-mediated thrombosis in stenosed
coronary arteries (14)
was used to this end. The effects
of intravenous (IV) onion juice and intragastric onion homogenate
administration on thrombosis and platelet aggregation were evaluated.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
The surgical procedure will be described briefly because thorough
descriptions have been published (14
,15)
. Beagles (2 y
old; Harlan Sprague Dawley, Madison, WI) of both sexes were
anesthetized, the left chest entered and the heart exposed. An
electromagnetic flow probe was placed on the circumflex coronary
artery. Distal to the probe, the artery was clamped to produce intimal
and medial damage and narrowed (60–70% diameter reduction) by placing
a plastic cylinder around the outside of the vessel. A thrombus formed
in the narrowed lumen, causing a slow (6–7 min) decline in measured
coronary blood flow. When flow reached zero, the cylinder was gently
shaken and the thrombus embolized distally, restoring coronary blood
flow. This periodic thrombus formation followed by embolization
produced cyclic flow reductions (CFR) as shown in Figure 1
. The rate of flow decline in this model is directly related to the rate
of accumulation of platelets in the narrowed lumen (16)
.
Thus, the frequency of CFR is a direct measurement of in vivo platelet
activity. This procedure conformed to guidelines from the University of
Wisconsin Research Animal Resource Center and the NIH.
|
Ten bulbs of a typical hybrid yellow storage onion cultivar "Hamlet" (Seminis Vegetable Seeds, Deforest, WI) were taken at random, chopped and juiced with a commercial juicer. The juice was incubated at 22°C for 30 min to allow ACSO lysis by alliinase and then centrifuged twice at 4000 x g for 10 min to remove pulp. The resulting supernatant (1.03 kg/L) was divided into aliquots and stored at -20°C. A new aliquot was thawed on ice immediately before use.
After establishment of CFR, five dogs were given an IV dose of 0.09 ± 0.01 mL/kg onion juice via a catheter inserted into the femoral vein. Both before CFR and 20 min after administration of juice, 18 mL of blood was drawn through a catheter inserted into the femoral artery to a syringe containing 2 mL of 38 g/L sodium citrate. The blood was mixed with an equal volume of 9 g/L saline. Ex vivo platelet aggregation was assessed using these samples and a whole-blood electrical impedance aggregometer (Chrono-log, Havertown, PA). For each measurement, two electrodes were inserted into a tube containing the blood mixture. When a platelet agonist was added, platelets aggregated to the electrodes, causing an increase in electrical impedance. Collagen (1 and 2 mg/L), collagen (0.125 mg/L) with epinephrine (1.0 mg/L), ADP (20 µmol/L) with and without epinephrine (1.0 mg/L), and phorbol myristate acetate (PMA, 0.5 µmol/L) were used as agonists. Before and after ex vivo aggregation, results were analyzed by Student’s paired t tests. Two hours after administration of onion juice, damage to the stenosed region of the coronary artery was repeated to determine whether CFR would return.
Treatment group 2: intragastric onion homogenate.
One hour before initiating CFR, five onions of the same cultivar (Hamlet) were taken at random and quartered. One quarter of each onion was chopped and weighed. All chopped onion was placed into a food processor and homogenized together with 50 mL deionized water. The homogenate was incubated at 22°C for 30 min. The pH was then adjusted to 3.0 (the approximate acidity of the dog stomach) with HCl and the mixture was stored at 4°C until it was used <1.5 h later. CFR were initiated in six dogs. Before dissecting out the coronary artery, dogs were fitted with a gastric tube with the tip placed into the duodenum immediately distal to the gastrointestinal sphincter. Onion homogenate (2 g onion/kg) was mixed with 9 g/L saline to a total volume of 50 mL and then administered through the tube gradually over 5 min. Blood was drawn shortly before and 2.5–3 h after administration of onion to measure changes in ex vivo platelet aggregation as described for treatment group 1. Approximately 1 h after abolishment of CFR, additional damage was made to the stenosed portion of the vessel and 0.2 µg/(kg · min) epinephrine was infused for 20 min to attempt to renew CFR.
In vitro incubations.
Blood was drawn by venipuncture from six humans and six dogs through a 21-gauge butterfly needle. Nine volumes of blood were mixed with 1 volume of 38 g/L sodium citrate. The blood was then mixed with an equal volume of 9 g/L buffered saline. Onion juice (from the same preparation as that used in the IV experiments) was tested for platelet inhibition at three concentrations (1, 10 and 100 mL juice/L blood) after two different incubation times in the blood (4 and 30 min). All concentration and incubation time combinations were repeated four times in the blood of each subject. Platelet aggregation was induced by collagen (2 mg/L). Results are expressed as the percentage of platelet aggregation inhibited by onion juice. This was calculated using the ratio of the incubation results to a parallel control to which an equivalent volume of saline had been added in place of onion juice. The Mixed Procedure (SAS Institute, Cary NC) was used to estimate least-squares (LS) means and variances for all treatments. This procedure enables testing of both random and fixed variable effects in a single ANOVA. This is not possible in the general linear model ANOVA. Species and onion juice concentration were considered fixed variables, and donor nested within species was considered a random variable.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
After IV administration of onion juice, the next 1–2 thrombi that
formed in the damaged stenosed region spontaneously embolized without
shaking. Within 20 min, CFR were completely abolished in all five dogs
(Fig. 1
a). This was accompanied by significant inhibition of
ex vivo platelet aggregation induced by all of the agonists except ADP
with epinephrine (Table 1
). CFR resumed when the stenosed region was damaged further by clamping.
An additional IV administration of 0.09 mL onion juice/kg abolished the
CFR again.
|
Onion homogenate given intragastrically inhibited CFR in five dogs
within 3 h of administration (Fig. 1
b). In the sixth
dog, CFR frequency was reduced and periodic thrombi embolized
spontaneously 2 h after giving onion. CFR were not eliminated in
this dog. Ex vivo platelet aggregation by several agonists was
inhibited in the blood of the five dogs in which CFR were completely
eliminated (Table 1)
. The agonists ADP with epinephrine and ADP alone
did not induce platelet aggregation in the blood of one and two dogs,
respectively. None of the agonists elicited an aggregation response in
the blood of the dog in which CFR were not eliminated. Aggregation
induced by collagen (2 mg/L) and ADP with and without epinephrine was
not inhibited significantly. In contrast to the IV onion juice
treatment group, CFR did not return after increased vessel damage or IV
epinephrine in three of the dogs.
In vitro incubations.
The results of the mixed ANOVA of the incubation data demonstrated that
dog platelets in whole blood responded to much smaller concentrations
of onion juice than did human platelets (Table 2
). All concentrations of onion juice were capable of inhibiting
collagen-induced platelet aggregation in dog blood. Only the
highest concentration of juice (100 mL/L, final concentration)
significantly inhibited aggregation in human blood. The level of
platelet inhibition was significantly different in human and dog blood
at the lowest concentration of juice (1 mL/L) and for the highest
concentration of juice after the 4-min incubation. Inhibition of
aggregation was consistently higher for 30-min incubations than for the
corresponding 4-min incubations with the same concentration of onion
juice. However, for both species and all onion juice concentrations,
there were no significant differences in inhibition after incubations
for 4 and 30 min with the exception of 100 mL juice/L human blood
(P < 0.01).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
.
Thiosulfinates (TS) have been implicated as a primary source of the
antiplatelet property of alliums. In one study, nearly all of the in
vitro antiplatelet activity of raw garlic was attributable to these
compounds (18)
. Variation in the quantities of the
alk(en)yl substituents of ACSO gives rise to the differences in the
smell and taste of raw Allium species and cultivars. TS
display differential antiplatelet activity in vitro, which is dependent
upon the alk(en)yl substituents (5)
. The
IC50 value (concentration at which 50% of
platelet aggregation is inhibited) of synthetic propyl propaneTS
(PPTS), a compound found in raw onion and garlic, is 0.27 mmol/L in
human blood. Intravenous administration of >0.3 mmol/L PPTS had no
effect on CFR or ex vivo platelet aggregation when administered to two
dogs. Within 5 min of administration, a strong smell of raw garlic came
from the respirator ventilating the dogs, suggesting that either the
compound or its metabolite was being respired from the dogs’ lungs.
When PPTS (0.3 mmol/L) was incubated with dog blood in vitro, it
inhibited collagen-induced platelet aggregation effectively
(unpublished data).
The CFR model is an on-line in vivo bioassay for platelet
aggregation. Although TS have thus far not inhibited
platelet-mediated CFR as they do in vitro platelet aggregation,
whole onion juice containing considerably less TS is an effective
inhibitor. Onions contain many other organosulfur compounds
(19)
and flavonoids, particularly quercetin
(20)
. These may contribute synergistically to the
inhibition of CFR and ex vivo aggregation. Cooked onions containing a
known level of quercetin fed to humans over a period of 7 d did
not significantly alter ex vivo platelet aggregation (21)
.
Cooking the onion may have prevented ACSO lysis by alliinase,
preventing the synthesis of organosulfur platelet inhibitory compounds.
Alternatively, it may have caused volatilization and transformation of
the TS and other organosulfur compounds present in raw tissue, possibly
lowering the potential for inhibition of aggregation and synergism with
flavonoids.
It was apparent in the group 1 dogs that a much smaller amount of IV
onion juice was needed to inhibit CFR than was predicted by results
from in vitro incubations with human blood. CFR experiments testing
fruit juices in dogs have effectively predicted the outcome of ex vivo
platelet aggregation after consumption of juices by monkeys
(22)
and humans (23)
. This was not the case
with onion juice; thus, an experiment was performed to compare the
effectiveness of onion juice at inhibiting human and dog platelet
aggregation. The results indicated that raw onion juice can inhibit
platelet aggregation in vitro in dog blood at much lower concentrations
than in human blood (Table 2)
. Chronic ingestion of onions can cause
hemolytic anemia in dogs (24)
. The heightened sensitivity
of dog platelets to onion may be related to a secondary effect of hemolytic anemia. For future in vivo and ex vivo studies of
onion-induced inhibition of platelet aggregation, it may be more
appropriate to use an animal species with an in vitro response more
similar to that of humans. There is an apparent lack of
dose-response relationship for onion juice and aggregation of dog
platelets. For both incubation times, a dosage of 1 mL/L onion juice
resulted in greater inhibition of aggregation than 10 mL/L. However,
the differences in LS means of the two dosages for each incubation time
were not significant (P > 0.1).
The mechanism of onion inhibition of platelet-mediated thrombosis
in dogs remains unclear. TS and other organosulfur compounds found in
raw onion react quickly with free sulfhydryl groups (19)
.
They may react with cysteine residues on platelet receptors or other
transmembrane, intracellular or serum proteins involved in clotting.
PMA-induced ex vivo platelet aggregation was inhibited in both
treatment groups. This suggests that an active component of raw onion
may inhibit protein kinase C–dependent aggregation ex vivo. Daily
consumption of garlic has been shown to reduce human serum levels of
thromboxane A2, a key compound in the clotting
cascade, by 80% (25)
. Aspirin functions similarly by
inhibiting cyclooxygenase production of thromboxane
A2 (26)
. This inhibition can be
overcome by IV epinephrine in dogs (27)
. CFR did not
return after IV epinephrine in three of the dogs given onion
intragastrically. Future studies comparing the effects of organosulfur
and flavonoid fractions of onion will help to identify the active
components.
|
FOOTNOTES |
|---|
3 Abbreviations used: ACSO,
S-alk(en)yl-L-cysteine sulfoxides; CFR,
cyclic flow reductions; IV, intravenous; LS means, least-squares
means; PMA, phorbol myristate acetate; PPTS, propyl propaneTS; TS,
thiosulfinates.
Manuscript received April 24, 2001. Revision accepted July 3, 2001.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Fuster V. F., Badimon L., Badimon J. J. & Chesebro J. H. (1992) Mechanisms of disease: the pathogenesis of coronary artery disease and the acute coronary syndromes. N. Engl. J. Med. 326:242-250.[Medline]
2.
McGill H. C., McMahan A., Zieske A. W., Tracy R. E., Malcom G. T., Herderick E. E. & Strong J. P. (2000) Association of coronary heart disease risk factors with microscopic qualities of coronary atherosclerosis in youth. Circulation 102:374-379.
3. Goldman I. L., Schwartz B. S. & Kopelberg M. (1995) Variability in blood platelet inhibitory activity of Allium (Alliaceae) species accessions. Am. J. Botany 82:827-832.
4.
Chen J. H., Chen H., Tsai S. J. & Jen C. J. (2000) Chronic consumption of raw but not boiled Welsh onion juice inhibits rat platelet function. J. Nutr. 130:34-37.
5. Briggs W. H., Xiao H., Parkin K. L., Shen C. & Goldman I. L. (2000) Differential inhibition of human platelet aggregation by selected Allium thiosulfinates. J. Agric. Food Chem. 48:5731-5735.[Medline]
6. Morimitsu Y., Morioka Y. & Kawakishi S. (1992) Inhibitors of platelet aggregation generated from mixtures of Allium species and/or S-alk(en)yl-L-cysteine sulfoxides. J. Agric. Food Chem. 40:368-372.
7. Goldman I. L., Kopelberg M., Debaene J.E.P. & Schwartz B. S. (1996) Antiplatelet activity of onion (Allium cepa) is sulfur dependent. Thromb. Haemostasis 76:450-452.[Medline]
8. Yin M. & Cheng W. (1998) Antioxidant activity of several Allium members. J. Agric. Food Chem. 46:4097-4101.
9. Augusti K. T. (1990) Therapeutic and medicinal values of onions and garlic. Rabinovich H. Brewster J. eds. Alliums and Allied Crops 3:93-108 CRC Press Boca Raton, FL. .
10. Hertog M.G.L., Feskens P.C.H., Hollman M. B., Katan M. B. & Kromhout D. (1993) Intake of potentially anticarcinogenic flavonoids and their determinants in adults in the Netherlands. Nutr. Cancer 20:21-29.[Medline]
11. Hertog M.G.L., Kromhout D., Aravanis C., Blackburn H., Buzina R., Fidanza F. & Giampaoli S. (1995) Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study. Arch. Intern. Med. 155:381-386.[Abstract]
12. Beretz A., Cazenave J. P. & Anton R. (1982) Inhibition of aggregation and secretion of human platelets by quercetin and other flavonoids: structure-activity relationships. Agents Actions 12:382-387.[Medline]
13. DeWhalley C. V., Rankin S. M., Hoult J.R.S., Jessup W. & Leake D. S. (1990) Flavonoids inhibit the oxidative modification of low density lipoproteins by macrophages. Biomed. Pharmacother. 39:1743-1750.
14. Folts J. (1991) An in vivo model of experimental arterial stenosis, intimal damage, and periodic thrombosis. Circulation 83(suppl. IV):3-14.
15.
Demrow H. S., Slane P. R. & Folts J. D. (1995) Administration of wine and grape juice inhibits in vivo platelet activity and thrombosis in stenosed canine coronary arteries. Circulation 91:1182-1188.
16. Maalej N., Holden J. & Folts J. D. (1998) The effect of shear stress on acute platelet thrombus formation in stenosed canine carotid arteries: an in vivo quantitative study. J. Thromb. Thrombolysis 5:231-238.[Medline]
17.
Folts J. D., Schafer A. I., Loscalzo J., Willerson J. T. & Muller J. E. (1999) A perspective on the potential problems with aspirin as an antithrombotic agent: a comparison of studies in an animal model with clinical trials. J. Am. Coll. Card. 33:295-303.
18. Lawson L. D., Ransom D. K. & Hughes B. G. (1992) Inhibition of whole blood platelet-aggregation by compounds in garlic clove extracts and commercial garlic products. Thromb. Res. 65:141-156.[Medline]
19. Block E. (1992) The organosulfur chemistry of the genus Allium—implications for the organic chemistry of sulfur. Agnew. Chem. Int. Ed. Engl. 31:1135-1178.
20. Fenwick G. R. & Hanley A. B. (1990) Chemical composition. Rabinovich H. Brewster J. eds. Alliums and Allied Crops 3:18-31 CRC Press Boca Raton, FL. .
21. Janssen K., Mensink R. P., Cox F.J.J., Harryvan J. L., Hovenier R. & Hollman P.C.H. (1998) Effects of the flavonoids quercetin and apigenin on hemostasis in healthy volunteers: results from an in vitro and a dietary supplement study. Am. J. Clin. Nutr. 67:255-262.[Abstract]
22.
Osman H. E., Maalej N., Shanmuganayagam D. & Folts J. D. (1998) Grape juice but not orange or grapefruit juice inhibits platelet activity in dogs and monkeys (Macaca fasciularis). J. Nutr. 128:2307-2312.
23.
Keevil J. G., Osman H. E., Reed J. D. & Folts J. D. (2000) Grape juice, but not orange juice or grapefruit juice, inhibits human platelet aggregation. J. Nutr. 130:53-56.
24. Munday R. & Manns E. (1994) Comparative toxicity of prop(en)yl disulfides derived from Alliaceae: possible involvement of 1-propenyl disulfides in onion-induced hemolytic anemia. J. Agric. Food Chem. 42:959-962.
25. Ali M. & Thomas M. (1995) Consumption of a garlic clove a day could be beneficial in preventing thrombosis. Prostoglandins Leukot. Essent. Fatty Acids 53:211-212.
26.
Roth G. J., Stanford N. & Majerus P. W. (1975) Acetylation of prostaglandin synthase by aspirin. Proc. Natl. Acad. Sci. U.S.A. 72:3073-3076.
27. Folts J. D. & Rowe G. G. (1988) Epinephrine reverses aspirin inhibition of in vivo platelet thrombus formation in stenosed dog coronary arteries. Thromb. Res. 50:507-516.[Medline]
This article has been cited by other articles:
|
|
 |
|
  P.F. Cavagnaro, M.M. Sance, and C.R. Galmarini Effect of Heating on Onion (Allium cepa L.) Antiplatelet Activity and Pungency Sensory Perception Food Science and Technology International, December 1, 2007; 13(6): 447 - 453. [Abstract] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
