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Journal of Agriculture and Technology Management (JATM)
21(1): 30-34 (January-June 2018)
Original article
30
*corresponding author: dr.adrianpybanez@gmail.com
Received: 7 June 2018; Accepted: 24 July 2018
p-ISSN: 2599-4875 e-ISSN: 2599-4980
©Cebu Technological University, R. Palma St. corner M.J. Cuenco Ave., Cebu City, 6000 Philippines
In Vitro Anticoagulant Potential of Caulerpa sp. (“Lato”), Eucheuma sp. (“Guso”),
Ananas comosus (“Pineapple”) Peeling and Psidium guajava (“Guava”) Leaf
Extracts in ICR Mice
Althea R. Arenajo1a, Adrian P. Ybañez2a*, Jo Anne Grace F. Tan1, Charlotte E. Villajuan1, Chiqui P. Young1, Julie
Lynn A. Reyes1, Esperanza M. Del Fierro3, Alita S. Labiaga3
1College of Pharmacy, University of Southern Philippines Foundation, Lahug, Cebu City
2Gullas College of Medicine, University of the Visayas, Banilad, Mandaue City, Cebu
3Cebu Technological University- Main Campus, Cebu City
aEqual author
ABSTRACT
Anticoagulants are valuable treatments for several
circulatory diseases, thrombotic disorders and for
hematologic analysis. In this study, the in vitro
anticoagulant potential of Caulerpa sp. (“Lato”),
Eucheuma sp. (“Guso”), Ananas comosus (“Pineapple”)
peelings and Psidium guajava (“Guava”) leaf extracts
were evaluated. Samples were washed with distilled
water, air dried for 5-7 days, macerated and extracted
for 2-3 days. In vitro anticoagulant potential of the
obtained extracts was tested on 15 male ICR mice (10-
12 weeks old), and were grouped into experimental,
positive control (aspirin) and negative control (no
treatment). Coagulation time was evaluated using the
slide method. Two hundred ul of each of the extract
was placed on separate slides (in triplicate). The mice
were sedated and punctured at the facial vein. A drop of
blood was placed on each slide on top of the prepared
extract and was mixed gradually using a
microhematocrit tube. Coagulation time was recorded
when fibrin was seen to form. Results revealed that
guso and pineapple extracts had longer average
coagulation time (at least 12 minutes) than “lato” (7
minutes) and guava (5.2 minutes) extracts. One-way
analysis of variance (ANOVA) revealed significant
differences (p value = 0.022, F value = 3.314), which
were accounted by guso and pineapple extracts (p value
0.033 each) in the post-hoc analysis. The present study
showed the anticoagulant potential of the extracts from
the whole plant of guso and pineapple peelings. Further
studies must be explored in isolating specific
components of the extracts for drug development.
KEYWORDS: Ananas comosus, anticoagulant,
Caulerpa sp., Eucheuma sp., Psidium guajava
1 INTRODUCTION
Homeostasis includes maintenance of normal blood
circulation. It is characterized by the continuous flow of
blood to the different organ systems of the body. An
anticoagulant substance (heparin) present in the blood
prevents it from clotting to allow normal circulation
(Hall et al., 2015). Anticoagulants are used in the
treatment of several circulatory diseases and thrombotic
disorders, including atrial fibrillation, pulmonary
embolism, deep vein thrombosis, venous
thromboembolism, congestive heart failure, stroke,
myocardial infarction, and genetic or acquired
hypercoagulability (Alquwaizani et al., 2013). It has
also been used for treatment against snake venoms
(Oliveira et al., 2005).
Anticoagulants are used in obtaining blood samples
for routine hematological examination in human and
animal disease diagnosis. Among the commonly used
anticoagulants is the ethylenediaminetetraacetic acid
(EDTA) (Oviedo and Rodríguez, 2003). EDTA is
currently considered as an environmental pollutant
(Thompson et al., 1983). It is usually the preferred
chemical substance for blood samples for complete
blood count. EDTA has been shown to cause hemolysis
in common carp blood (Witeska and Wargocka, 2011).
The antithrombotic effect of aspirin (Gurbel et al.,
2007) has been known to be medically significant, as it
is a potent agent that could inhibit platelet aggregation
(Weiss, 2003). In the past 30 years, aspirin as an
anticoagulant has saved patients with cardiovascular
diseases (Awtry and Loscalzo, 2000) and has reduced
the formation of thrombus on the damaged surface of
the arterial wall (Undas et al., 2007)
Anticoagulant therapy may be expensive, and
alternative cheaper medicines from natural sources may
be explored. The search for alternative sources of
anticoagulants has risen as a result of the increasing
demand for safer anticoagulant clinical therapy (das
Neves Amorim et al., 2011). While several studies have
demonstrated the anti-thrombin and anticoagulant
properties of the marine algae Caulerpa sp. (Costa et
al., 2012; Rodrigues et al., 2009; Rodrigues et al.,
2011), only one study has specifically investigated
Caulerpa lentillifera as a potential anticoagulant
Journal of Agriculture and Technology Management (JATM) 21(1): 30-34 (January-June 2018)
31
p-ISSN: 2599-4875 e-ISSN: 2599-4980
©Cebu Technological University, R. Palma St. corner M.J. Cuenco Ave., Cebu City, 6000 Philippines
(Arenajo et al., 2017). Other natural sources, including
Eucheuma sp. (“Guso”), Ananas comosus (“Pineapple”)
peeling and Psidium guajava (“Guava”) also have
reported anticoagulant properties. However,
comparative in vitro evaluations of their potential has
not yet been performed. Hence, this study was
conducted.
2 MATERIALS AND METHODS
Research Design
The study is an experimental type. The potential
anticoagulant properties of the selected extracts were
evaluated in the ICR mice for two weeks (inclusive of
one week for animal acclimatization). Mice were
assigned into three groups: 1) negative control (no
extract), 2) positive control (aspirin) and 3)
experimental groups.
Research subjects and plant sources
Fifteen male ICR mice (10-12 weeks old;
approximately 30g) were randomly allocated into three
equal groups: experimental, positive control (aspirin)
and negative control (no treatment). The mice were
purchased from the University of San Carlos
Laboratory Animal Facility, a BAI-registered and
PALAS-accredited breeding facility. “Lato” and “guso”
were obtained from Barangay Kalawisan, Cordova,
Cebu, while pineapple peeling and guava samples were
secured from commercial sources. Species
identification was performed by a biologist from the
Biology and Environmental Studies Program of the
University of the Philippines Cebu. The study was
conducted at University of the Visayas, Gullas College
of Medicine, Cebu City.
Preparation of plant samples
Ten kilograms of fresh “guso” and “lato” and two
kilograms of fresh guava leaves and pineapple peels
were washed with distilled water to remove the foreign
materials adhering to the plant samples. A 20 cm plastic
strainer was used to hold the plant samples for one hour
to let the excess water drip. Washed samples were
transferred to a working table lined with filter paper to
remove the excess water adhering to the plant samples.
All plant materials were air dried for one to two weeks.
After air-drying, samples were minced into small sizes
(1.5 cm).
Preparation of the crude extract
The plant materials were macerated with 80% ethyl
alcohol for two days. It was then subjected to reflux
distillation for two hours to further exhaust the plant
material. After distillation, it was evaporated to syrupy
consistency to produce the extract. All extracts were
placed in a tightly closed container and kept
refrigerated at 2-80C until further use.
Animal marking and assignment to groups and
cages
Upon arrival at the animal facility, the animals
were randomly assigned to cages and marked at the
base of the tail using permanent markers for
identification. Cages were also randomly assigned to
the different treatment groups. The health of animals
was assessed by physical examination.
Animal acclimatization, monitoring, and
maintenance
The mice were acclimatized for seven days.
Commercial feeds and water were provided ad libitum.
Lighting was on a 12 hour dark-light cycle, and ambient
temperature was maintained between 24-260C.
Beddings were changed every three days. Body score,
appearance and behavior (Ullman-Culleré and Foltz,
1999; Bekkevold et al., 2013) were monitored.
Preparation and administration of aspirin and
distilled water
The amount to be administered was computed
based on the bodyweight (at 10 ml/kg). Aspirin and
distilled water were administered orally at 24 and 12
hours prior to blood collection using a gauge 16 ball
point gavage needle. Gavage needles were disinfected
and flushed with distilled water for three times in
between administrations. For the positive control
(aspirin), one-fourth of a 300 mg-aspirin tablet was
crushed and mixed in 75 ml distilled water and was
orally administered at a dose of 5 mg/kg (Zhou et al.,
2014).
Sedation, blood samples and evaluation of
coagulation
The mice were sedated using a combination of
tiletamine/zolazepam (Zoletil) administered
intraperitoneally. After sedation, the facial vein was
punctured using a hematocrit tube. One drop of blood
was placed on each slide containing 0.2 ml of the
experimental extracts (accomplished in triplicates) and
was mixed gradually using a hematocrit tube.
Coagulation time was recorded once fibrin was seen.
Longest coagulation time was pegged at 12 minutes as
it was observed that the slides would dry up after such
time.
Data processing and analysis
Observations were manually recorded in a tally
sheet. Gathered data were encoded in Microsoft Excel
and imported into statistical software. Data was
analyzed using one way analysis of variance (ANOVA)
with post-hoc analysis by Tukeys method. The
significance level was set at 5%.
Ethical consideration
The procedures performed in this study were
guided by the principles of animal welfare, Animal
Welfare Act of the Philippines (RA 8485) and AO 45 of
the Bureau of the Animal Industry. The study was also
approved by the Institutional Animal Care and Use
Committee.
Journal of Agriculture and Technology Management (JATM) 21(1): 30-34 (January-June 2018)
32
p-ISSN: 2599-4875 e-ISSN: 2599-4980
©Cebu Technological University, R. Palma St. corner M.J. Cuenco Ave., Cebu City, 6000 Philippines
Committee of Pet Science Laboratory Animal
Facility and Veterinary Clinic, Cebu City, Philippines.
3 RESULTS AND DISCUSSION
The whole plant of guso and pineapple peeling
extracts were found to have the longest average clotting
time (at least 12 minutes), followed by the “lato” (seven
minutes). Guava leaf extract was also found to prolong
clotting time (5.2 minutes) compared to the negative
control (0.9 minute), but almost the same result with
that of the positive control (aspirin) (Table 1). Guso
has been shown to have an anticoagulant effect because
of its carrageenan component (Anderson et al., 1965;
Rosa, 1972; McLellan and Jurd, 1992).
Pineapple contains a complex enzyme, bromelain,
which may have an anticoagulant property. Studies
have been conducted using stems (Milić et al., 2014)
and fresh fruit juice extract (Evangelista et al., 2012),
but it appears that the present study is the first to
explore pineapple peels as a potential source of
anticoagulant. On the other hand, Hsieh et al. (2007)
also found an anticoagulant property of guava.
Statistical analysis revealed significant differences
(p value 0.022, F value 3.314), which were accounted
guso and pineapple extracts (p value = 0.033 each) in
the post-hoc analysis (Table 2). The results indicate that
guso and pineapple had longer clotting time, suggesting
better anticoagulant properties than the other
treatments.
Aspirin has properties that can reduce the ability of
the blood to clot, and thus it is often used in the
treatment of conditions associated with blood clots,
including heart attacks (Guirguis-Blake et al., 2016).
The present study showed that the studied extracts
could have anticoagulant properties that can be equal to
or much better than aspirin.
Table 1. Average clotting time (minutes) of male ICR blood mixed with Caulerpa sp., Eucheuma sp., Ananas comosus peeling
and Psidium guajava extracts
Group
Mean
SD
Eucheuma sp.(“Guso”)
12.0
0.0
Caulerpa sp. (“Lato”)
7.0
5.7
Ananas comosus (Pineapple) peeling
12.0
0.0
Psidium guajava (Guava) leaf
5.2
9.4
Positive Control (Aspirin)
6.2
10.0
Negative Control (Normal saline solution)
0.9
0.3
Table 2. Post-hoc analysis of the coagulation time between Eucheuma sp., Caulerpa sp., Ananas comosus, Psidium guajava and
control groups
Group
Mean Difference
Std. Error
Sig.
Eucheuma sp.
Caulerpa sp.
4.977
3.070
.594
Ananas comosus
0.000
3.070
1.000
Psidium guajava
6.765
3.070
.275
Aspirin
2.936
3.256
.942
Negative control
10.763*
3.256
.033
Caulerpa sp.
Eucheuma sp.
-4.977
3.070
.594
Ananas comosus
-4.977
3.070
.594
Psidium guajava
1.788
3.070
.991
Aspirin
-2.042
3.256
.988
Negative control
5.785
3.256
.500
Ananas comosus
Eucheuma sp.
0.000
3.070
1.000
Caulerpa sp.
4.977
3.070
.594
Psidium guajava
6.765
3.070
.275
Aspirin
2.936
3.256
.942
Negative control
10.763*
3.256
.033
Psidium guajava
Eucheuma sp.
-6.765
3.070
.275
Caulerpa sp.
-1.788
3.070
.991
Ananas comosus
-6.765
3.070
.275
Aspirin
-3.830
3.256
.843
Negative control
3.997
3.256
.819
Aspirin
Eucheuma sp.
-2.936
3.256
.942
Caulerpa sp.
2.042
3.256
.988
Ananas comosus
-2.936
3.256
.942
Psidium guajava
3.830
3.256
.843
Negative control
7.827
3.432
.244
Negative control
Eucheuma sp.
-10.763*
3.256
.033
Caulerpa sp.
-5.785
3.256
.500
Ananas comosus
-10.763*
3.256
.033
Psidium guajava
-3.997
3.256
.819
Aspirin
-7.827
3.432
.244
Journal of Agriculture and Technology Management (JATM) 21(1): 30-34 (January-June 2018)
33
p-ISSN: 2599-4875 e-ISSN: 2599-4980
©Cebu Technological University, R. Palma St. corner M.J. Cuenco Ave., Cebu City, 6000 Philippines
In a study by Rodrigues et al. (2011), the
anticoagulant property of a related species Caulerpa
cupressoides var. lycopodium was found to be dose-
dependent. The aforementioned study also extracted
sulfated polysaccharides (SP) from the selected
seaweed.
SPs are structural components of the cell wall of
marine algae, in which they are found in high
concentrations (Painter et al., 1983; Pereira et al., 2005;
Rodrigues et al., 2009; Rodrigues et al., 2010). The use
of these molecules as alternative sources of
anticoagulants is justified by the fact that algae are
phylogenetically distant from mammals, significantly
reducing contamination by viral particles (Leite et al.,
1998).
Another study evaluated the in vitro anticoagulant
activity of SP fractions from red alga Halymenia
pseudofloresia using citrated rabbit plasma and
observed marked changes in activated partial
thromboplastin time (APTT) (Rodrigues et al., 2009).
The fractions obtained in the first (464.20, 211.60,
103.50 and 101.70 IU mg-1) were more active
compared to those from the third extraction (137.10,
96.50 and 89.20 IU mg-1). Its actions were considered
superior to the existing heparin standard (100 IU mg-1)
and to SPs from the same-genus species Halymenia sp.
(Rodrigues et al., 2010).
4 CONCLUSION
The ethanolic extracts of guso and pineapple
peelings apparently have in vitro anticoagulant
potential. The potential of these extracts as an in vitro
anticoagulant for diagnostic purposes and as a potential
therapy for thrombotic disorders can be explored in
vivo. Further studies in isolating specific components
of the extracts for drug development are also
recommended.
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