111111

Dental Journal
(Majalah Kedokteran Gigi)

2020 June; 53(2): 111–114

Research Report

Comparison of thrombocyte counts during the post-oral administration
of aspirin and the Holothuria scabra ethanol extract in Wistar rats (Rattus
norvegicus)

Dian Mulawarmanti and Rima Parwati Sari
Department of Oral Biology,
Faculty of Dentistry, Universitas Hang Tuah
Surabaya – Indonesia

ABSTRACT
Background: Long bleeding time is a risk factor in dental treatment, especially in patients who consume aspirin or other antithrombotic
drugs. Holothuria scabra (H. scabra) are mostly echinodermata and have been studied in Indonesia; they contain omega-3 and
glycosaminoglycans, with an influence of an antithrombotic drug. Purpose: This study aimed to investigate the thrombocyte counts
during the post-administration of aspirin and the H. scabra extract in Wistar rats (Rattus norvegicus). Methods: This study was
true experimental with a post-test control group design. The sample consisting of 30 healthy male Wistar rats (R. norvegicus) with a
bodyweight of 150–250 g was divided into three groups (n = 10). The rats in Group 1 were given sodium carboxymethyl cellulose (Na
CMC). The rats in Group 2 were given aspirin, and the rats in Group 3 were given the H. scabra ethanol extract with a 25 mg/200 g
dose as per their body weight (BW). Oral administration was given for seven days. The rats’ blood was taken on the eighth day. The
amount of thrombocyte was measured using Wright’s stain methods. The analysis of variance (ANOVA) and the Least Significant
Difference (LSD) tests were conducted for data analysis (p < 0.05). Results: The thrombocyte counts (179.00 ± 10.56) in aspirin
administration were lower than those in H. scabra (265.00 ± 18.54) and control groups (334.17 ± 13.9), with a significant difference
between the groups (p = 0.0001; p < 0.05). Conclusion: This study indicates that the oral administration of aspirin and H. scabra
decreases thrombocyte counts, whereas the administration of aspirin reduces thrombocyte counts to levels lower than those in H.
scabra in Wistar rats (R. norvegicus).

Keywords: antithrombotic; aspirin; glycosaminoglycans; Holothuria scabra

Correspondence: Rima Parwati Sari, Department of Oral Biology, Faculty of Dentistry, Universitas Hang Tuah, Jl. Arif Rahman Hakim
No. 150, Surabaya 60111, Indonesia. E-mail: rima.parwatisari@hangtuah.ac.id

INTRODUCTION

Atherosclerosis is a leading cause of vascular disease
throughout the world with manifestations of ischemic heart
disease, stroke and peripheral arterial disease. In 2010, there
were 665 people per 100,000 population with ischemic heart
disease in Central Asia per year.1 Based on the results of
the 2018 Basic Health Research report, the prevalence of
heart disease in Indonesia was 1.5%.2

Lipoproteins that form in blood vessels lead to plaque
formation in specific locations of the arteries through
inflammation, necrosis, fibrosis and intimal calcification.
The plaque causes coronary thrombosis due to acute

rupture, which can result in partial or total blockage of the
affected arteries.3 Dismissal of the thrombus will become
an embolism and obstruct the arterial system in pulmonary
embolism and brain, for example.3,4

One of the medicines used widely in the treatment
of atherosclerosis is aspirin, which functions as a long-
term antithrombotic option for oral administration for
the secondary prevention of myocardial infarction.5,6
Aspirin in small doses (100 mg per day) shows an anti-
thrombocyte activity by irreversibly inhibiting thrombocyte
by preventing thromboxane A2 (TXA2) synthesis, which
damages thrombocyte secretion and aggregation.4,7
Problems arise in the administration of aspirin in patients

Dental Journal (Majalah Kedokteran Gigi) p-ISSN: 1978-3728; e-ISSN: 2442-9740. Accredited No. 32a/E/KPT/2017.
Open access under CC-BY-SA license. Available at http://e-journal.unair.ac.id/index.php/MKG
DOI: 10.20473/j.djmkg.v53.i2.p111–114

mailto:rima.parwatisari@hangtuah.ac.id
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112 Mulawarmanti et al./Dent. J. (Majalah Kedokteran Gigi) 2020 June; 53(2): 111–114

with coronary heart disease, which involves bleeding that
occurs in the gastrointestinal tract and intracranial due to
long-term use of low-dose aspirin.8 In the field of dentistry,
bleeding complications can occur after surgery/tooth
extraction, disrupting the healing process.6,9,10 Therefore,
before surgery and/or the extraction of teeth, it is vital
to carry out a laboratory examination, which includes a
thrombocyte count.11

Sea cucumber (phylum Echinodermata) has been widely
used as medicine in several countries, such as China and
Korea. In Indonesia, sea cucumbers are one of the leading
marine products that have begun to be commercially
cultivated in several regions because of their high economic
value. However, there has been limited investigation
into their benefits in the health sector.12 One type of sea
cucumber that has been widely cultivated is the sandfish
(Holothuria scabra). This biota is commonly found across
Indonesia’s coasts, with straightforward breeding. The
results of previous studies have shown that the content of
sea cucumbers include lectins, sterols, saponins (triterpen
glycosides), proteins, collagen, mucopolysaccharides,
glycosaminoglycans (GAGs), chondroitin sulfate, amino
acids, fatty acids, vitamin A, vitamin C, riboflavin,
niacin, carotenoids, minerals, polyphenols, flavonoids
and superoxide dismutase (SOD). The content of these
substances can be used as a source of protein and considered
as an anti-inflammatory, anticoagulant, anti-cholesterol and
antithrombotic agent; it can also accelerate the process of
wound healing.13

Some sea cucumber content such as GAG sulfate,
dermatan sulfate and heparin are important anticoagulants
that inhibit clot formation through interaction with
antithrombin and heparin cofactors II.14 These substances
have a mechanism that is synergistically useful in the
treatment of acute coronary syndrome (ACS).15 This is used
as the basis for selecting sandfish as an antithrombotic that
can be used as a substitute for aspirin. The purpose of this
study is to compare the administration of aspirin with the
H. scabra extract, which is given orally to the thrombocyte
count in the white rat strain of Wistar (Rattus norvegicus).

MATERIALS AND METHODS

This research is an experimental laboratory with a
randomised, completed research design. This study was
approved by the experimental animal ethics from the
Health Research Ethics Commission (KEPK) of the
Faculty of Dentistry, Universitas Hang Tuah, Surabaya
(EC/082/KEPK-FKGUHT/XII/2019). The parameter
of this study involved several thrombocytes taken from
blood preparations from mice that had been treated. Thirty
research samples were randomly divided into three groups.
The sample used had the following inclusion criteria: white
rats (R. novergicus), male Wistar strain aged 2–3 months,
bodyweight 150–200 g. The selection of experimental
animals was made based on their fur, eyes and physical

conditions, as well as their randomized group design.
Monitoring and recording were conducted to determine
whether there existed any side effects or the experimental
unit experienced any pain to be removed from the sample.
Acclimatisation was carried out before and during the study,
which involved monitoring environmental conditions, foods
and drinks.16

The H. scabra extract was obtained from a beach in East
Kalimantan (Borneo Island), Indonesia. It was transported
in freezing conditions; its internal organs were removed,
and it was washed thoroughly. Using a blender mixed
with distilled water, it was chopped into a ratio of 1:2.
The drying process was performed using the freeze–drying
method. The freeze–drying results in the form of soft,
dry preparations were mashed with mortar and pestle and
sifted using a mesh 50.17 Sodium carboxymethylcellulose
was added to ease transfer to the stomach of the rat using
the oral gavage.

Research on R. novergicus began with acclimatisation
for seven days in a laboratory environment. Before being
treated, the Wistar rats fasted for about 18 hours, albeit
they were given drinking water. Sick rats were excluded
from the study. The white rats were divided into three
groups. Group 1 consisted of the control group that was
given Na CMC. Group 2 included the treatment group
and was given an aspirin dose of 1.8 mg/200 g as per BW,
which was based on the conversion of an aspirin dose to
rats (200 g) as an antithrombotic of 100 mg/day. Group
3 consisted of the treatment group and was given the H.
scabra extract at a dose of 25 mg/200 g as per BW, which
was based on previous research on the benefits of H. scabra
on thrombocyte counts.18 The treatment was carried out for
seven days by giving a single dose. After blood sampling,
the animal was terminated with cervical dislocation.

Thrombocyte count examination was performed on the
eighth day, using the Wright wipe method. This method
was carried out by adding 0.1 g of Wright powder dissolved
with 60 ml methanol to 0.1 g of Wright reagent (Merck®
Paint No. 1.01383.0500, Darmstadt, Germany). Afterwards,
it was stored in glass bottles and kept in a closed cupboard
to avoid sunlight. The solution was used after 10 days of
storage. In addition to Wright staining, the use of a buffer
solution was needed to fix the Wright smear with a pH
of 6.4. The Wright smear procedure was performed by
dripping the Wright solution onto the preparation until all
smears were inundated. Furthermore, the buffer solution
was dripped until all swabs were flooded and left for 5–12
min. The smear was rinsed with water, and the back of the
dirty smear was cleaned of the remaining dye. Preparations
of peripheral blood smear were left to dry in the air.18
Observations were made by two people with five visual
fields using a light microscope (Olympus® CX21, Japan)
with 400× magnification. Using the Statistical Package for
Social Sciences version 23 (IBM® 2015, New York, United
States), the one-way analysis of variance (ANOVA) was
performed (p < 0.05), followed by the post-hoc Least
Significant Difference (LSD) test (p < 0.05).

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113Mulawarmanti et al./Dent. J. (Majalah Kedokteran Gigi) 2020 June; 53(2): 111–114

RESULTS

The histological section of the thrombocyte in each group
can be seen in Figure 1. The mean and standard deviation
of the thrombocyte counts in each group describe the
differences between the groups (Figure 2). The application
of sandfish extract (H. scabra) at a dose of 25 mg/200
g as per BW reduced the thrombocyte counts (265.00 ±
18.54) compared with the control group (334.17 ± 13.93).
However, it did not reduce the thrombocyte counts similar
to the aspirin group (179.00 ± 10.56). The ANOVA test
identified a significant difference in the mean of the
thrombocyte counts. The LSD test showed that the results
were significantly different (p < 0.05) between Groups K
and A (p = 0.001; p < 0.05), Group K with HS Group 25
(p = 0.001; p < 0.05), and Group A with HS Group 25 (p
= 0.001; p < 0.05). These findings illustrate a significant
difference between the administration of aspirin and H.
scabra.

DISCUSSION

This study found that compared with the administration of
aspirin, which can prevent bleeding after antithrombotic/
anticoagulant medication, some thrombocytes did not

decrease significantly. Normally, thrombocytes are
associated with the initiation of the coagulation process,
whereby their reaction to damage to blood vessels becomes
the main target in haemostasis. Thrombocyte hyperactive
reaction triggers side effects in coronary artery disease,
which results in thrombosis.4 The antithrombocyte effect
of aspirin involves inhibiting the synthesis of thromboxane
A2 (TXA2) from arachidonic acid in thrombocytes because
of the irreversible acetylation process and inhibition of
cyclooxygenase – an essential enzyme in the synthesis
of prostaglandins and thromboxane A2.19 The presence
of these obstacles causes a decrease in the number of
thrombocytes present in the blood. This is indicated by
the significant difference between the control group and
the aspirin-administered group, where the thrombocytes
in the latter group have thrombocyte counts that are much
lower than those in the controls.

Sea cucumbers contain GAGs and omega-3s, which
play a vital role in the thrombogenic process.13 One of
the GAGs that plays a role in antithrombotics is dermatan
sulfate. Through the formation of complex covalent
bonds with heparin-II (HCII) cofactors, dermatan
sulfate selectively inhibits thrombin action, thereby
preventing vascular thrombosis.20 Heparin/heparan sulfate
contained in GAGs consists of 20–100 units of N-acetate
D-glucosamine α disaccharide associated with glucuronic
acid. The molecular mechanism of heparin/heparan sulfate
as an anticoagulant can bind and increase the inhibitory
activity of plasma protein antithrombin against several
serine proteases from the coagulation system, the most
important of which are factor IIa (thrombin), Xa and IXa.21
Although the anticoagulant mechanism is more dominant,
heparin/heparan sulfate also has antithrombotic properties.
Antithrombotic mechanisms occur with the release of
Tissue Factor Pathway Inhibitors (TFPI) associated with
the molecular weight and sulfate content of heparin.22
The GAGs found in the echinoderm cannot help in
concluding that antithrombotic properties are strong. Some
studies summarised by Pavão23 show that heparin-like
polymers in ascidians and molluscs are different from low
anticoagulant ability, as well as significant antithrombotic
and anti-inflammatory activities; however, they do not
cause bleeding.

Figure 1. Histological section of thrombocytes (green arrows) during post-administration of aspirin (A), the Holothuria scabra
extract (HS) and control in Wistar rats. Staining of Wright’s methods. Observation using a light microscope at 400x×
magnification.

Figure 2. Mean and standard deviation of the thrombocyte
counts in each group. *Significance difference: p =
0.05.

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114 Mulawarmanti et al./Dent. J. (Majalah Kedokteran Gigi) 2020 June; 53(2): 111–114

Clinical results in humans show that omega-3 from
marine biota can function as antithrombotic. Omega-3 fatty
acids in marine life containing eicosapentaenoic acid (EPA)
and docosahexaenoic acid (DHA) consumption 2–4 g/day
and cause optimal anti-atherosclerotic, anti-inflammatory
and antithrombotic effects.24 DHA has a quicker start in
inhibiting thrombocyte aggregation induced by adenine
diphosphate (ADP). However, both EPA and DHA are
incorporated into thrombocyte phospholipids by inhibiting
the formation of arachidonic acid (AA), which can help in
reducing thrombocyte aggregation by lowering thrombocyte
procoagulant metabolites arising from AA synthesis. EPA
also competes with AA in the cyclooxygenase pathway,
which directly and indirectly reduces the formation of
TXA2 pro-aggregatory AA metabolites.24 This was
proved in studies with thrombocyte counts in H. scabra
administration compared with the control group.

A previous study mentioned that the presence of a
barrier in TXA2 and thrombocyte aggregation triggers
bleeding.19 In their multinational study in the United States,
Akintoye et al.25 conducted a placebo-controlled trial
involving 1,516 patients, who were given perioperative
fish oil (EPA-DHA), 8–10 g/day for 2–5 days before
surgery and then 2 g/day postoperatively. Compared with
the placebo, the administration of fish oil did not show
any bleeding.25 Akintoye et al.’s study is in line with
this study’s results whereby the administration of the H.
scabra ethanol extract containing EPA–DHA was not
proved to increase perioperative bleeding; conversely,
it even reduced the amount of blood transfusion. High
omega-3-PUFA administration is associated with a lower
risk of bleeding. Conclusions from this study indicate that
the oral administration of aspirin and H. scabra decreases
thrombocyte counts, whereas the administration of aspirin
reduces thrombocyte counts to lower levels than those in
H. scabra in Wistar rats (R. Novergicus).

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