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The in vitro anticoagulant

effect of Allium cepa L.


aqueous extract

Jessilyn S. Daliri
Pamela Camille A. Dagman
Hannah Faye D. Libatique
Ronalyn Pastor
July Anne Simon
BS PHARMACY 4A

I. Significance/Relevance of the Study


This study will be a significant endeavor in investigating and exploring
the use of Allium cepa L. to patients with problems in the blood circulatory
system. This study will also be a significant attempt on combating major

morbidity and mortality rate caused by blood clotting disorders by obtaining


information on the usefulness of Allium cepa extract as a supportive
therapy. Thus, a positive result of this study will serve as an identification of
a novel, effective anticoagulant agent that improves the management of
blood clotting disorders in a wide range of patients. Furthermore, this
research work would give an idea to the people about the significant
medicinal use of Onion and would serves as an avenue for further discovery
in research and clinical purposes.

II. Rationale and Background


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. Blood clotting
disorders constitute a serious medical problem, and antithrombotic,
anticoagulative and thrombolytic reagents from various sources have been
investigated as solutions.
Anticoagulants are substances or agents which prevent or retard
clotting of blood, by removing calcium. It could be used in vivo (inside the
body) or in vitro (outside the body).

Allium species, especially garlic and onion, contain biologically active


substances affecting blood clotting. The organosulfur compounds present in
Allium species have been reported to be strong anticoagulants (Goldman
et.al., 1996). This magic drug is well known for its: fibrinolytic effects
(Chutani and Bordia, 1981), hemodynamic and hemostatic effects (Brosche
et al., 1990), platelet effects (Apitz-Castro et al., 1992), immunologic effects
(Chisty et al., 1996), lipid-lowering effects (Ide and Lau, 1997), antiartherosclerotic effects (Koscielny et al., 1999), anti-oxidative effects (Borek,
2001), anti-cancer effects (Milner, 2001), vascular effects (Ashraf et al.,
2004), anti-microbial effects (Benkeblia, 2004), haematological effects
(Fazlolahzadeh et al., 2011) and hepatoprotective effects (Ugwu and Omale,
2011).

Haemostasis is the process that retains the blood within the vascular
system during periods of injury. The coagulation mechanism may be thought
of as a complex series of cascading reactions involving development of
enzymes from their precursor (zymogens, procoagulants proenzymes). Most
of the substances necessary for coagulation are present in an inert form and
must be converted to an activated state. As one enzyme is formed it then
becomes available to convert the next zymogen to its activated enzyme
(serine protease). This process continues until a fibrin meshwork clot has
formed. In addition to the zymogens, protein cofactors and membrane

phospholipids surfaces, calcium ions play an active role in the final


development of the fibrin clot.

In the hope of finding a treatment/therapeutic remedies for blood


clotting disorders, which is cost-efficient, effective, and safe conceived the
idea of doing an interventional study to determine the effects of Allium cepa
extract to the normal blood clotting time of human blood.

III. OBJECTIVES
The main objective of this study is to analyse and evaluate the
anticoagulant properties of Allium cepa. Specifically, this study aims to
accomplish the following:
1. To determine whether or not the plant extract of Allium cepa induce an
effect on the normal blood clotting time on human.
2. To formulate a product that contains Allium cepa as an active ingredients
3. To make use of Allium cepa as an alternative /supplementary
anticoagulant agent to improve and/or prevent cardiovascular diseases.
4. To recommend Allium cepa as an anticoagulant to medical practitioners
and researchers for the improvement of this study, so that it may
contribute to pharmaceutical applications and various health services.

IV. Materials and Methods


a. Procedures

Plant Extract
Freshly procured red onion (Allium cepa) bulbs were cut into small
slices, crushed, and dried in an oven at 70oC. Fifty (50) grams of the dried
samples were pulverized, and suspended in 100 ml of boiling water (100 oC)
for 30 min. The extract was centrifuged at 12,000 x g for 30 min, and the
supernatant was used for this experiment. (Kim et. al., 2014)
0.5 mL (eq. to 0.25 g) was introduced to the first test tube, 1 mL (eq.
to 0.5 g) into the second, and 2 mL (eq. to 1 g) into the third test tube.
Sample Collection and Analysis
The participants had been chosen according to the following criteria:
having

normal

PT,

not

suffering

from

any

cardiovascular

diseases

(hypertension, congestive heart failure or coagulation disorders such as;


Hemophilia A or B) or diabetes, not recently using NSAIDs and not obese,
alcoholics or smokers and also free from dyslipidemic disorders. (Taj Eldin et.
al., 2013)

Verbal consent was obtained from ten (10) human volunteers. Blood
samples were extracted with the help of medical technologists. Blood
samples of shortlisted donors
were subjected to basic hematology screening tests to exclude those with
abnormal results.

2 mL of the blood samples obtained from healthy individuals will be


introduced into three test tubes containing different volumes (0.5 mL, 1 mL
and 2 mL) of Allium cepa extract. 2 mL of blood samples will also be
introduced into three test tubes of EDTA/Trisodium citrate and water, the
EDTA being in same concentrations as the Allium cepa.
Packed cell volume (PCV), Platelet count, total white blood cell count,
activated partial thromboplastin time (APTT), prothrombin time (PT) and
haemoglobin

concentration

were

analyzed

using

standard

methods.

(Cheesbrough, 2000)
b. List of materials needed
Mortar and pestle
Whatman No. 1 filter
paper
Funnel
Erlenmeyer flask
Beaker (50 mL, 100
mL, 250 mL)
Dropper
c. List of reagents (include quantity)
Red onion (Allium cepa L.) bulbs
Distilled water
EDTA/Trisodium citrate
d. List of machines/ equipment needed
Analytical balance
Centrifuge
Sucking pump
Oven

Test tube (Small and


big)
Test tube rack
Stirring rod
Goggles
Gloves
Face masks

V. Government or non-government people to work


with
e. Include fields of specialization
National Museum (Botany Division)
4J Clinical Diagnostic Laboratory
f. identify relevant experience of the
helping you carrying out the project

person/organization

in

The Botany division of National Museum offers identification of botanical


specimens and charges according to degree of taxonomic rank educidated.
(Dr.

Domingo

A.

Madulid,

Division

Chief,

527-1218

botany@nationalmuseum.gov.ph)
The blood samples obtained from the 10 participants will be screened
with the help of 4J Clinical Diagnostic Laboratory.

VI. Total Estimated Budget


The total estimated budget for this study is about Php 10,00015,000,
which depends on the positive or negative results of the experimentation.
Onion bulbs to be propagated Php 120 (1 kg)
Authentication of plant samples Php 4000 (w/ fare)
Screening of blood samples Php 2000

VII. Duration of the Project


The expected duration of this project is 3-6 months, which depends on the
positive or negative results of the experimentation.
This study involves:

1. Seek participants for the extraction of blood samples to be used in the


study

Screening of the blood samples to determine if the participants

have reached the criteria set (1-2 weeks)


2. The preparation of the plant sample to be authenticated by National
Museum (1 week)
3. Experimentation
a. Preparation of the sample
b. Blood extraction
c. Analysis