In Vivo Anti-Platelet Properties of Red Onion (Allium cepa L.

) versus Aspirin
Jhozel Kim Awao, Sigrid Shaezra Banizal, Lorie Ann Bringas, Carol Dion, Carra Louise Esteban
Trisha Ngolab, Raisa Pocais, Chriselle Poserio, Jamailah Rafael, Ryan James Suguitan, Corrieten
Yendrapati

Abstract
Introduction: CVDs are the number one cause of death globally. Most adult cardiovascular
disorders involving hypertension, cerebral hemorrhage, coronary thrombosis, arteriosclerosis and
congestive heart failure are caused by problems in the blood circulatory system as blood clotting
disorders which constitute a serious medical problem. Aspirin is the most widely used and tested
antiplatelet drug in CVD, and it is proven to be the cornerstone of antiplatelet therapy in
treatment and prevention of CVD in clinical trials in various populations. Onion (Allium cepa) is
largely universal, staple herb popular throughout history as both food and medicine and it has
been consumed for prevention of cardiovascular disorders . Onions, in general show promising
anticoagulant properties, hence it may be a candidate for an alternative ingredient in
pharmacologic agents as it is also readily available in the Philippines.
Objectives: The study aims to determine the In-vivo anti-platelet activity of red onion (Allium
cepa L.) versus Aspirin. The study specifically aims to determine the bleeding time of whole
blood of rats treated with different doses of red onion (Allium cepa L.) extract and Aspirin and to
determine the significant difference in bleeding time among the three concentrations of red onion
(Allium cepa L.) and Aspirin.
Methods: There were 30 Albino rats used in the experiment. They were divided into 5 groups,
each consisting of 6 subjects each group. A daily single dose of red onion solution, which was
made by diluting red onion extract with distilled water making a stock solution of 100mg/ml.
Page 1 of 17

The different concentrations were designated as follows: Group 1 given 250mg/kg red onion
extract, Group 2 given 500mg/kg red onion extract, Group 3 given 750mg/kg red onion
extract, Group 4- given 250mg/kg Aspirin and Group 5 given saline solution . Platelet function
was measured through bleeding time utilizing Ivys method.
Findings: Post hoc comparison using Scheffes (see appendix) test indicated that the mean score
for the control group (M=151.67, SD=7.63) was significantly different from Aspirin group
(M=734.33, SD=101.55); 750mg Allium cepa group (M=508.33, SD=104.19); 500mg Allium
cepa group (M=998.33, SD=288.26); 250mg Allium cepa group (M=429.17, SD=49.54).
Furthermore the mean score of 500mg Allium cepa group had a longer bleeding time and was
significantly different from 250mg Alium cepa extract and 750mg Allium cepa extract. The
Aspirin group however did not differ significantly from 500mg Allium cepa group. These test
results are significant at 0.05 level.
Conclusion: The findings of the current study likewise sustained the idea that red onion (Allium
cepa L.) has an in-vivo antiplatelet effects on albino rats. This demonstrated a comparable
antiplatelet effect to aspirin which shows that Allium cepa can also be used as an alternative
treatment. 50% concentration of red onion (Allium cepa L.) showed a similar antiplatelet effect
when compared to Aspirin.
KEYWORDS: Albino rats, Aspirin, Allium cepa L, Anti-Platelet

Page 2 of 17

Introduction
Cardiovascular disease (CVD) is caused by disorders of the heart and blood vessels, and
includes coronary heart disease (heart attacks), cerebrovascular disease (stroke), raised blood
pressure (hypertension), peripheral artery disease, rheumatic heart disease, congenital heart
disease and heart failure1. CVDs are the number one cause of death globally. An estimated 17.3
million people died from CVDs in 2008, representing 30% of all global deaths. Of these deaths,
an estimated 7.3 million were due to coronary heart disease and 6.2 million were due to stroke 2.
Most adult cardiovascular disorders involving hypertension, cerebral hemorrhage, coronary
thrombosis, arteriosclerosis and congestive heart failure are caused by problems in the blood
circulatory system as blood clotting disorders which constitute a serious medical problem 3.
Aspirin is the most widely used and tested antiplatelet drug in CVD, and it is proven to be
the cornerstone of antiplatelet therapy in treatment and prevention of CVD in clinical trials in
various populations 4. Aspirin is used as a primary prevention measure to aid in the prevention of
a first occurrence of CVD. It can also be used as a secondary prevention measure among
individuals who have experienced a heart attack or stroke to prevent additional cardiovascular
events 5. Aspirin induces a permanent functional defect in platelets, which can be detected
clinically as a prolonged bleeding time. This appears to be primarily, if not exclusively, due to
irreversible inactivation of a key enzyme in platelet arachidonate metabolism through acetylation
of a critical serine residue near its catalytic site. Prevention benefits of aspirin in heart disease
can be achieved with doses as low as 75150 mg daily. Aspirin at low doses decreases the
incidence of transient ischemic attacks, unstable angina, coronary artery thrombosis with
myocardial infarction, and thrombosis after coronary artery bypass grafting 6.
Herbal plants are popularly used nowadays in drug discovery due to their ancient
medicinal use. Some plant extracts even have the anticoagulant activity for treatment of CVDs 7.
Page 3 of 17

Philippines, a tropical country, have a variety of herbal plants 8. Onion (Allium cepa) is largely
universal, staple herb popular throughout history as both food and medicine and it has been
consumed for prevention of cardiovascular disorders 9. Onions, in general show promising
anticoagulant properties, hence it may be a candidate for an alternative ingredient in
pharmacologic agents as it is also readily available in the Philippines. Red onion (Allium cepa L.)
has been used since ancient times for the treatment of many diseases

10

.Red onion also showed

significant antithrombotic activity 11

Red onions are native to Asia and the Middle East and have been cultivated for over five
thousand years. The onion, known scientifically as Allium cepa, is, on the surface, a humble
brown, white or red, paper-thin skinned bulb. The word onion comes from the Latin word unio,
which means "single," or "one"reflecting of the onion plant producing a single bulb, unlike its
cousin, the garlic, that produces many small bulbs. The name also describes the onion bulb when
cut down the middle; it is a union (also from unio) of many separate, concentrically arranged
layers.
In observational studies, involving research using human subjects has prompted that most
of the cardiovascular benefits have been demonstrated in the form of a planned overall diet. In all
of these observational diet-based studies, participants with the greatest intake of vegetables
(including onions) gain the most health benefits. Multiple studies show onion to be a food that
provides protection for the heart and blood vessels when consumed in a diet that is rich
especially in flavonoid-containing vegetables and fruits. The benefits of onion in this overall
dietary context, extends to prevention of heart attack.
Most studies on the anti platelet properties of red onion are in-vitro. In vivo studies are
present, however their focus was on the strength of its anti platelet activity. In addition to this,

Page 4 of 17

the potency and efficacy of red onion extract in terms of its anti-platelet properties has not yet
been compared to anti platelet medications like Aspirin.
The study aims to determine the In-vivo anti-platelet activity of red onion (Allium cepa
L.) versus Aspirin. The study specifically aims to determine the bleeding time of whole blood of
rats treated with different doses of red onion (Allium cepa L.) extract and Aspirin and to
determine the significant difference in bleeding time among the three concentrations of red onion
(Allium cepa L.) and Aspirin.

Methods
Ethical statement
All animal experiments conformed to the Animal welfare act of the Philippines RA 8485.
Study design
Five groups of six albino rats each were studied: A- 250 mg/ml Aspirin, B- 250/ml dose
of Allium cepa extract, C- 500 mg/ml dose of Allium cepa extract, D- 750/ml dose of Allium
cepa extract, E- control. The rats were randomly selected into treatment groups.
Onion Extract Preparation
400g of ground sample was macerated and soaked in 2000ml of methanol for 48hrs. The
mixture was separated by sieving with a clean glass bottle. The extract was concentrated using a
rotary evaporated. The sample was stored in a clean glass bottle. The sample was then diluted
using distilled water. 12
Aspirin stock solution
500mg of aspirin was weighed using an electronic weighing balance and was transferred
into a beaker containing 5ml of normal saline with spatula. Concentration stock solution of
100mg/ml was prepared and stored in a glass bottle with cover.13
Page 5 of 17

Experimental procedure:
The extract, aspirin solution and normal saline were administered to the subjects
respectively using a cannula fitted into the nozzle or a 1ml syringe thereby enhancing
administration. Appropriate volume of solutions: A- 0.34 ml aspirin B- 0.32 ml extract per rat C0.56 ml extract per rat D- 1 ml extract per rat and E- for control were delivered directly into the
esophagus of the rats with the aid of the cannula. The administration of extracts was performed
for 14 days at 10 am daily. The animals were tested in a home cage.
Administration of substances directly into the mouth or by orogastric or nasogastric
gavage is common in laboratory animal medicine and research. The oral route is economical,
convenient and relatively safe. Gavage (esophageal or gastric) is often used in research settings,
instead of mixing in water or food to ensure precise and accurate dosing of animals. 14
Experimental animals
Female and male rats of 2-3 weeks age, mean weight of 112.73 grams ranging from 90.5
g-178.8 g.. The rats were indicated to be free of known viral, bacterial and parasitic pathogens.
The albino rats were purchased in Benguet State University College of Veterinary Medicine
The animals were housed in clean metal cages and maintained in a well ventilated animal
house with constant 12-hr light-dark schedule, both of which were provided by the researchers in
cooperation with veterinary students from Benguet state University. The animals were fed with
standard rat pellet diet and were allowed with free access to clean drinking water. The subjects
were given five days to habituate in the study site.
There were 30 Albino rats used in the experiment. They were divided into 5 groups, each
consisting of 6 subjects each group. Groupings of the rats were randomly done.
All the rats were fed equally and continually throughout the period of the experiment. A daily
single dose of red onion solution, which was made by diluting red onion extract with distilled
Page 6 of 17

water making a stock solution of 100mg/ml. Then it was stored in clean glass bottle, kept in the
refrigerator at 4C, which were given in different doses per orem to the subjects using a gavage
tube every 8AM for two weeks. The different concentrations were designated as follows: Group
1 given 250mg/kg red onion extract, Group 2 given 500mg/kg red onion extract, Group 3
given 750mg/kg red onion extract, Group 4- given 250mg/kg Aspirin and Group 5 given saline
solution13 .
Testing
Bleeding time was used as parameter of platelet function as to primary hemostasis. Ivys
method was implemented: the rat was placed on a restrainer. Tail passed through one end of the
opening. Tail was disinfected, wiped, dried and pricked with sterile lancet about 4mm deep. Stop
clock was started and oozing blood was wiped with filter paper at 15 second interval and
repeated every 15 seconds on fresh spots of filtered paper until bleeding stops. 13
Statistical methods
Mean and Standard Deviation was used to determine the bleeding time of rats with the
use of red onion and aspirin. One way between-groups analysis of variance (ANOVA) was
conducted to determine the anti-platelet properties of the different red onion (allium cepa L.)
concentrations of 250mg, 500mg and 750mg in vivo. For the comparisons with the control
group, One-way ANOVA was used. Values were expressed as means SEM. P < 0.05 was
considered as the limit of significance (Garcia-Manzano et al., 2001). Analyses were performed
using IBM SPSS Statistics 21.
The result was confirmed by post hoc comparison using the Scheffe test.

Page 7 of 17

Results
This section discusses the data collected and the statistical results which the findings
undergone.
The animals health status was monitored throughout the experiments. The rats were in
good physical condition throughout the experiment as there were no pertinent manifestations that
indicated the absence of deformities and the rats also coped well in the current environmental
setup. 30 rats were used in this study in which they were divided into 5 groups which contain
different concentrations of Allium cepa extract 250mg, 500mg, 750mg, Aspirin and the control
group respectively.
All the rats were in good physical health. However some of the rats suffered minor
wounds that was noticed during the transfer of cage from one to another.
The group then carefully and meticulously transferred the cage during the successive
experimental trials. They were observed and monitored for any further bleeding but in the end no
rat suffered potential life threatening conditions.
There were 6 trials done for each group. The bleeding time was recorded in which
different concentrations of red onion extract, aspirin and the control was used.

Page 8 of 17

Table 1. Bleeding time (sec) of the different concentrations of Allium cepa extract (250mg,
500mg, 750mg) versus the control groups (Aspirin and PNSS) in rats.

Bleeding Time
seconds)
(

Anti
-Platelet Agent

Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6

Aspirin

630

915

765

735

655

70

750mg

Allium cepa 425

400

660

540

435

6
58

Extract
500mg

Allium cepa 630

990

890

1500

900

5
10

Extract
250mg

Allium cepa 405

495

400

450

360

80
46

Extract
Control

157

153

155

145

160

5
14

0
Above table shows the data collected on bleeding times for the three concentration
groups of red onion extract, Aspirin and a control group. There were 6 trials done for each group.
Figure 1 shows the average bleeding times for the different groups.

Figure 1. Bleeding time of the different concentrations of Allium cepa extract (250mg, 500mg,
750mg) versus the control groups (Aspirin and PNSS). The values represent the mean

Page 9 of 17

SEM

1200
1000
800
600
400
200
0

Aspirin

75
0mg Allium50
0mg Allium25
0mg Allium Control

Statistical Analysis
A one way between-groups analysis of variance (ANOVA) was conducted to determine
the anti-platelet properties of the different red onion (allium cepa L.) concentrations of 250mg,
500mg and 750mg in vivo shown in Figure 1 . Result (Table 2) shows statistically significant
difference between the 5 groups [F (4, 25) 28.725, p= 0.004] at 0.05 level. This result was
confirmed by post hoc comparison using the Scheffe test.
Table 2. Results of one way Analysis of Variance
ANOVA
Between Groups
Within Groups
Total

Sum of Squares
1963535.467
2454255.500
4417790.967

Df
4
25
29

Mean Square
490883.867
98170.220

F
5.000

Sig.
.004

Post hoc comparison using Scheffes (see appendix) test indicated that the mean score for
the control group (M=151.67, SD=7.63) was significantly different from Aspirin group
(M=734.33, SD=101.55); 750mg Allium cepa group (M=508.33, SD=104.19); 500mg Allium
cepa group (M=998.33, SD=288.26); 250mg Allium cepa group (M=429.17, SD=49.54).
Furthermore the mean score of 500mg Allium cepa group had a longer bleeding time and was
significantly different from 250mg Alium cepa extract and 750mg Allium cepa extract. The
Page 10 of 17

Aspirin group however did not differ significantly from 500mg Allium cepa group. These test
results are significant at 0.05 level.

Page 11 of 17

Discussion
The shortest bleeding time was obtained with the use of 250mg Allium cepa (410
seconds), this is followed by 750mg Allium cepa (500 seconds), then Aspirin (775 seconds) and
the longest is 500mg Allium cepa (1000 seconds). The three concentrations of red onion (Al.
cepa L.) have anti-platelet activities. The 25% and 75% extract concentration of Allium cepa L.
have lesser anti-platelet activity when compared to the platelet medication Aspirin. As for the
50% extract concentration of red onion no statistical significance was observed in its anti-platelet
activity when compared to Aspirin. 50% concentration of red onion (Allium cepa L.) can be used
as an alternative source of antiplatelet agent.
The result is supported by various in- vivo studies already conducted that focused and
showed the effects of Allium cepa as an antiplatelet. 15,16Members of Allium family, especially
garlic and onion, have been used as traditional medicines to treat a variety of diseases including
cardiovascular problems. The beneficial properties of Allium cepa have been attributed to their
phytoconstituents that has the ability to inhibit platelet aggregation and thromboxane formation
17.

The findings of the current study likewise sustained the idea that red onion (Allium cepa
L.) has an in-vivo antiplatelet effects on albino rats. This demonstrated a comparable antiplatelet
effect to aspirin which shows that Allium cepa can also be used as an alternative treatment. 50%
concentration of red onion (Allium cepa L.) showed a similar antiplatelet effect when compared
to Aspirin.
The study, however, had certain limitations that of which involved indirect measurement
of the anti-platelet properties of Red Onion (Allium cepa L.) which could limit its
generalizability. Extensive studies are necessary to further solidify the outcome extracted from
the evidence on the effects of Allium cepa as an alternative to aspirin in preventing thrombotic
Page 12 of 17

events, nevertheless the study presented a significance to the development of therapeutic

products in treating CVDs.

Page 13 of 17

References
1. Cardiovascular diseases. http://www.who.int/topics/cardiovascular_diseases/en/ (accessed 27
November 2014).
2. WHO
Media

centre.

Cardiovascular

diseases.

http://www.who.int/mediacentre/factsheets/fs317/en/ (accessed 27 November 2014).

3. Taj Eldin IM, Majed M. Abdalmutalab, Hajir M. Izzalddeen. Evidence for an in vitro
Anticoagulant Activity of Red Onion (Allium cepa L.). Sudan Journal of Medical
Sciences.2011;6(2):85-88
4. Yuxiang Dai and Junbo Ge. Clinical Use of Aspirin in Treatment and Prevention of
Cardiovascular Disease. .2012;():7
5. Arch G. Mainous, Rebecca J. Tanner, Ronald I. Shorr and Marian C. Limacher. Use of Aspirin
for Primary and Secondary Cardiovascular Disease Prevention in the United States. Journal of
the American Heart Association.2014;():10
6. Katzung Bertram, Masters Susan, Trevor Anthony. Basic and Clinical Pharmacology . 12th ed.
United States of America. McGraw-Hill Companies, Inc.; 2012
7. Narjis Hadi Mansoor Al-Saadi. In VItro Study of the anticoagulant activity of some plant
extracts. Indian Journal of Applied Research.2013;3(7):120-122
8. Taj Eldin IM, Majed M. Abdalmutalab, Hajir M. Izzalddeen. Evidence for an in vitro
Anticoagulant Activity of Red Onion (Allium cepa L.). Sudan Journal of Medical
Sciences.2011;6(2):85-88
9. J.H. Evangelista, et al.. Preliminary Assessment of In vitro Anticoagulant Activity vs. Heparin
1,000I.U. and Cytotoxicity of Selected Philippine Medicinal Plants. Inte rnational Journal of
C hemical and Environm ental Engineering.2012;3(6):371-376
10. C. Davison, R. A. Levendal and C. L. Frost. Cardiovascular benefits of an organic extract of
Tulbaghia violacea: Its anticoagulant and anti-platelet properties. Journal of Medicinal Plants
Research.2012;6(33): 4815-4824
11. Kanae Hyodo, Izumi Horii , Masaru Nishino , John C Giddings , Junichiro Yamamoto . The
antithrombotic effects of onion filtrates in rats and mice . .2011;3(6):319-325
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12. C. Davison, R. A. Levendal and C. L. Frost. Cardiovascular benefits of an organic extract of

Tulbaghia violacea: Its anticoagulant and anti-platelet properties. Journal of Medicinal Plants
Research.2012;6(33): 4815-4824
13. ASIKA E.C,IDONIJE B.O, OKHAI .O.,IRIBHOGBE I.O. Preliminary investigation of
antithrombotic activities of methanolic seed extracts of Garcinia Combogia in rats . Annals of
Biological Research.2011;2(3):333-346
14. Turner P, Brabb T, Pekow C and Vasbinder MA.

Administration of Substances to

Laboratory Animals: Routes of Administration and Factors to Consider. J Am Assoc Lab

Anim Sci. 2011 Sep; 50(5): 600613.
15. Jia-Huey Chen, Hsiun-ing Chen, Shun-Jen Tsai et al. (2000). Chronic Consumption of Raw
but not Boiled Welsh Onion Juice Inhibits Rat Platelet Function. The American Society for
Nutritional Sciences. Journal of Nutrition, 1(30): 34-37.
16. Park, H.J, Cho, H.R, Jin, J.C. Onion (Allium cepa L) peel extract has anti-platelet effects in
rat platelets. SpringerOpen Journal. 2013;4(17): 14-20.
17. Hyodo, K, Horii, I, Nishino, M, Giddings, J, Yamamoto, J. The antithrombotic effects of
onion filtrates in rats and mice. ScirpOrg Journal. 2011;3(6): 319-325.

APPENDIX / APPENDICES
Scheffe Post-hoc Test
Multiple Comparisons
Dependent Variable: Result
LSD
(I) Drugs
(J) Drugs

Mean

Std.

Difference

Error

Sig.

95%

Confidence

Interval
Lower

Upper

Bound
-

Bound
484.2292

(I-J)
Aspirin

75% Allium cepa

111.66667

Page 15 of 17

180.89

.543

Extract
50% Allium cepa

612
180.89

Extract
25% Allium cepa

287.66667
122.50000

Extract
Control

508.33333
*

75%

Allium

.124

260.8959
-

84.8959

612
180.89

.505

660.2292
-

495.0625

612
180.89

.009

250.0625
135.7708

880.8959

.543

260.8959

.037

484.2292
-

-26.7708

Aspirin

612
180.89

50% Allium cepa

111.66667
-

612
180.89

Extract

399.33333

612

cepa Extract

771.8959

25% Allium cepa

Extract
Control

10.83333

180.89

396.66667
*

50%

Allium

383.3959

612
180.89

.038

361.7292
24.1041

769.2292

.124

-84.8959

660.2292

Aspirin

287.66667

75% Allium cepa

399.33333

612
180.89

.037

26.7708

771.8959

Extract
25% Allium cepa

612
180.89

.032

37.6041

782.7292

Extract
Control

612
180.89

.000

423.4375

1168.562

.505

5
250.0625
361.7292
-37.6041

410.16667
796.00000
*

Allium

612
180.89

cepa Extract

25%

.953

Aspirin

612
180.89

75% Allium cepa

122.50000
-10.83333

612
180.89

.953

495.0625
-

Extract
50% Allium cepa

612
180.89

.032

383.3959
-

Extract

410.16667

612

cepa Extract

Page 16 of 17

782.7292

Control
Control

Aspirin

385.83333

180.89

.043

13.2708

758.3959

612
180.89

.009

508.33333

612

880.8959

135.7708

-24.1041

75% Allium cepa

180.89

Extract

396.66667

612

.038

769.2292

50% Allium cepa

180.89

Extract

796.00000

612

.000

25% Allium cepa

180.89

Extract

385.83333

612

*. The mean difference is significant at the 0.05 level.

Page 17 of 17

.043

1168.562

423.4375

5
-

-13.2708

758.3959