research manuscript

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.

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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 Ivy’s method.

Findings: Post hoc comparison using Scheffe’s (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

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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 75–150 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.

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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,

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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

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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 C-

0.56 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

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water making a stock solution of 100mg/ml. Then it was stored in clean glass bottle, kept in the

refrigerator at 4°C, 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. Ivy’s

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.

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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.

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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.

Aspirin 630 915 765 735 655 706

750mg Allium cepa Extract425 400 660 540 435 585



Control 157 153 155 145 160 140

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 SEM

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

seconds) Bleeding Time ( Platelet Agent -Anti

Control 0mg Allium 250mg Allium 500mg Allium 75Aspirin

1200

1000

800

600

400

200

0

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

Sum of Squares Df Mean Square F Sig.

Between Groups 1963535.467 4 490883.867 5.000 .004

Within Groups 2454255.500 25 98170.220

Total 4417790.967 29

Post hoc comparison using Scheffe’s (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.

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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

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

products in treating CVDs.

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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

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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

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): 600–613.

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.

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APPENDIX / APPENDICES

Scheffe Post-hoc Test

Multiple Comparisons

Dependent Variable: Result

LSD

(I) Drugs (J) Drugs Mean

Differenc

e (I-J)

Std.

Error

Sig. 95% Confidence

Interval

Lower

Bound

Upper

Bound

Aspirin 75% Allium cepa

Extract

111.66667 180.89

612

.543 -

260.8959

484.2292

50% Allium cepa

Extract

-

287.66667

180.89

612

.124 -

660.2292

84.8959

25% Allium cepa

Extract

122.50000 180.89

612

.505 -

250.0625

495.0625

Control 508.3333

3*

180.89

612

.009 135.7708 880.8959

75% Allium

cepa Extract

Aspirin -

111.66667

180.89

612

.543 -

484.2292

260.8959

50% Allium cepa

Extract

-

399.3333

3*

180.89

612

.037 -

771.8959

-26.7708

25% Allium cepa 10.83333 180.89 .953 - 383.3959

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Extract 612 361.7292

Control 396.6666

7*

180.89

612

.038 24.1041 769.2292

50% Allium

cepa Extract

Aspirin 287.66667 180.89

612

.124 -84.8959 660.2292

75% Allium cepa

Extract

399.3333

3*

180.89

612

.037 26.7708 771.8959

25% Allium cepa

Extract

410.1666

7*

180.89

612

.032 37.6041 782.7292

Control 796.0000

0*

180.89

612

.000 423.4375 1168.562

5

25% Allium

cepa Extract

Aspirin -

122.50000

180.89

612

.505 -

495.0625

250.0625

75% Allium cepa

Extract

-10.83333 180.89

612

.953 -

383.3959

361.7292

50% Allium cepa

Extract

-

410.1666

7*

180.89

612

.032 -

782.7292

-37.6041

Control 385.8333

3*

180.89

612

.043 13.2708 758.3959

Control Aspirin -

508.3333

3*

180.89

612

.009 -

880.8959

-

135.7708

75% Allium cepa - 180.89 .038 - -24.1041

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Extract 396.6666

7*

612 769.2292

50% Allium cepa

Extract

-

796.0000

0*

180.89

612

.000 -

1168.562

5

-

423.4375

25% Allium cepa

Extract

-

385.8333

3*

180.89

612

.043 -

758.3959

-13.2708

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

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