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

) versus Aspirin
Background and rationale
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.
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
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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,
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.
Goals and 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
i.
The bleeding time of whole blood of rats treated with:
a. 50% concentration of red onion (Allium cepa L.) extract
b. 75% concentration of red onion (Allium cepa L.) extract
c. Aspirin
ii.
The significant difference in bleeding time between the two concentrations of red
onion (Allium cepa L.) and aspirin
Research Design
An experimental design will be used. The main outcome of this study will be to compare
the in-vivo anti-platelet properties of red onion (Allium cepa L.) to the anti-platelet properties of
aspirin. The antiplatelet properties will be assessed using bleeding time as a parameter. The
animals (rats) will be randomly divided into 4 groups with each group having a close range of
weight and age group. Group A: Will receive 50% concentration of red onion extract. Group B:
Will recieve 75% concentration of red onion extract, Group C: Will receive Aspirin and Group
D: will receive Normal saline (control)

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Rats

Group A - 50%
red onion extract

w/ or w/o outcome

Group B - 75%
red onion extract

w/ or w/o outcome

Group C Aspirin

w/ or w/o outcome

Group D - Saline
(control)

w/ or w/o outcome

Fig 1 Experimental Study

Methodology
Onion Extract Preparation

The plant components will be dried in an oven at 40 C for 48 hours and ground to fine
powder. 10 grams of the dried powder will be mixed with 50 ml. methanol and will be extracted
overnight at 4C. The rude red onion extracts will be centrifuged at 1500 x g for 10 minutes at
4C. the supernatant will be then filtered using a Whatman No. 1 filter to remove all residual
plant debris. The filtrates solvent ( methanol) will be removed in a rotavapor evaporator at 40C,
and the residues will be kept at 4C. The subsequent residue will be mixed with 10 ml.
Deionized distilled water, will be frozen at -80C and freeze dried. The freeze-dried extracts will
be stored in the dark at 4C.12
Aspirin stock solution
100mg of aspirin will be weighed using an electronic weighing balance and will be
transferred into a beaker containing 2ml of normal saline with spatula. Concentration stock
solution of 50mg/ml is now prepared and will be stored in a glass bottle with cover.13
Procedure
16 animals (rats) will be divided into groups A, B, C and D with 4 rats in each group
having a close range of weight. The extract, aspirin solution and normal saline will be
administered to the subjects respectively using a cannula fitted into the nozzle or a 1ml syringe
thereby enhancing administration. Appropriate volume of solutions will be delivered directly into
the esophagus of the rats with the aid of the cannula. Group A: Red onion extract at 50% daily x
14 days, Group B: Red onion extract at 75% daily x 14 days, Group C: Aspirin solution
250mg/kg x14 days, Group D: Normal saline 0.5mg daily x 14 days 13
Testing
Bleeding time will be used as parameter of platelet function as to primary hemostasis.
Ivys method will be implemented: the rat will be placed on a restrainer. Tail passed through one
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end of the opening. Tail will be disinfected, wiped, dried and pricked with sterile lancet about
4mm deep. Stop clock will be started and oozing blood will be wiped with filter paper at 15
second intervals and repeated every 15 seconds on fresh spots of filtered paper until bleeding
stops. 13
Safety of research subjects/participants
With the use of commercially available aspirin, the rats will be closely monitored for any
untoward side effects of the drugs.

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Data management and statistical analysis

Table 1.a will present the data of bleeding time of rats with the use of various concentrations of

onions in 50% and 75% concentration. The table below shows how the data will be presented.
Experimental
conditions
Red onion extract
at
50%
conc.
(Group A rats)
Red onion extract
at
75%
conc.
(Group B rats)

Rat 1

Bleeding time (in seconds)

Rat 2
Rat 3

Mean
Rat 4

Table 1.b will present the data of bleeding time of rats with the use aspirin 100mg in 2 ml normal

saline. The table below shows how the data will be presented.
Experimental
conditions
Aspirin 50mg/ml
(Group C rats)

Rat 1

Bleeding time (in seconds)

Rat 2
Rat 3

Mean
Rat 4

Table 2 will illustrate the significant differences in bleeding time among various concentrations

of red onion and aspirin. The table below shows how the data will be presented.
Experimental
condition
Normal Saline 0.5
mg (Group D rats)
Red onion extract
at
50% conc.
(Group A rats)
Red onion extract
at
75%
conc.
(Group B rats)
Aspirin 50mg/ml
(Group C rats)

Rat 1

Bleeding time (in seconds)

Rat 2
Rat 3

Mean
Rat 4

For tables 1a and 1b, Mean and Standard Deviation will be used to determine the
bleeding time of rats with the use of red onion and aspirin.
For table 2, to determine significant differences in bleeding time among concentrations of
red onion and aspirin, One-way method of Analysis of variance (ANOVA) will be used. All data
will be expressed as means standard error of means (SEM) and will be analyzed by ANOVA.
Comparisons with the control group will be made using One-way ANOVA. Differences will be
considered significant if P < 0.05. If there will be significant differences, clusters will show
where the differences will lie.
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Ethical considerations
This research protocol will be submitted with the letter of permission to conduct the
experimentation to the Philippine Animal Welfare Society prior to the conduct of the study. The
research protocol will also be reviewed by the SLU Research Ethics Committee.
Problems anticipated
According to some studies, in- vivo testing has an indirect approach in measuring drug
absorption. The first pass metabolism is a basis for in-vivo testing to function as an indirect
approach to measure drug absorption, and at times a poor approach. Post absorption process can
alter plasma profiles. However, in-vivo is still considered as the gold standard for testing 12.
Aside from that, we do not foresee any major difficulties in implementing this project.
Dissemination of results and publication policy
This research protocol will be subjected for dissemination and publication to the SLU
Research Journal.
Disclosure of potential conflicts of interest
The investigators declare that there is no potential source of conflict of interest.
Financing of project and other support
This study will be financed by the investigators.
Collaboration with other scientist
The investigators will collaborate with NSRU and BSU-Veterinary School.
Project team and management plan
The project team shown in table 3 consists of the principal investigator and co-principal
investigators. The team is composed of second year students of SLU School of Medicine. All
team members will contribute in writing the report, as well as contribute to the needs of the team
as well as provide manpower.
Table 3 - Research team
Researcher
1 Bringas, Lorie Ann

Institutional Affiliation
SLU Principal Investigator

2 Awao, Jhozel Kim

SLUCo-Principal
Investigator
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Roles
She will lead the team meeting and the
writing and submission of reports.
Besides being the team leader, She will
be responsible in editing the research
She is responsible for the safety of
research subjects and participants.
Responsible in proof reading the research

3 Banizal, Sigrid Shaezra

SLUCo-Principal
Investigator

4 Dion, Carol

SLUCo-Principal
Investigator

5 Esteban, Carra Louise

SLUCo-Principal
Investigator

She is in charge of identifying ethical

considerations the team may encounter.

6 Ngolab, Trisha

SLUCo-Principal
Investigator

She is tasked with finding out references

that will be essential to the research.

7 Pocais, Raisa

SLUCo-Principal
Investigator
SLUCo-Principal
Investigator
SLUCo-Principal
Investigator

She is in charge in formulating the

methodology
She is in charge in formulating the
methodology
She will help work with the other
investigators for managing and analyzing
data.

SLUCo-Principal
Investigator
SLUCo-Principal
Investigator

He is responsible for the dissemination of

results and publication policy.
She will help work with the other
investigators for managing and analyzing
data.

8 Poserio, Chriselle
9 Rafael, Jamailah

10 Suguitan, Ryan James

11 Yendrapati, H. Corrieten

She is in charge of finding out potential

conflicts of interests, as well as the
disclosure of such conflicts
She will help work with other
investigators for managing and analyzing
data.

Table 4 - Management Plan

The table 4 below shows a list of project activities with the dates they will be performed.
Management of finances

December 13 to 16, 2014

Pretest

January 19 to 24, 2015

Actual Experiment

January 26 to February 6, 2015

Interpretation and Analysis of Results

February 7 to February 21, 2015

Final Report

February 22 to March 15, 2015

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November 2014).
2. WHO Media centre . Cardiovascular diseases.
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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
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Anticoagulant Activity of Red Onion (Allium cepa L.). Sudan Journal of Medical
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ASIKA E.C,IDONIJE B.O, OKHAI .O.,IRIBHOGBE I.O. Preliminary investigation of

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