ArticlePDF Available

IN VITRO ANTICOAGULANT POTENTIAL OF CAULERPA SP.(“LATO”), EUCHEUMA SP.(“GUSO”), ANANAS COMOSUS (“PINEAPPLE”) PEELING AND PSIDIUM GUAJAVA (“GUAVA”) LEAF EXTRACTS IN ICR MICE

Authors:
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 gusoand 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
Sig.
Eucheuma sp.
Caulerpa sp.
4.977
.594
Ananas comosus
0.000
1.000
Psidium guajava
6.765
.275
Aspirin
2.936
.942
Negative control
10.763*
.033
Caulerpa sp.
Eucheuma sp.
-4.977
.594
Ananas comosus
-4.977
.594
Psidium guajava
1.788
.991
Aspirin
-2.042
.988
Negative control
5.785
.500
Ananas comosus
Eucheuma sp.
0.000
1.000
Caulerpa sp.
4.977
.594
Psidium guajava
6.765
.275
Aspirin
2.936
.942
Negative control
10.763*
.033
Psidium guajava
Eucheuma sp.
-6.765
.275
Caulerpa sp.
-1.788
.991
Ananas comosus
-6.765
.275
Aspirin
-3.830
.843
Negative control
3.997
.819
Aspirin
Eucheuma sp.
-2.936
.942
Caulerpa sp.
2.042
.988
Ananas comosus
-2.936
.942
Psidium guajava
3.830
.843
Negative control
7.827
.244
Negative control
Eucheuma sp.
-10.763*
.033
Caulerpa sp.
-5.785
.500
Ananas comosus
-10.763*
.033
Psidium guajava
-3.997
.819
Aspirin
-7.827
.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.
REFERENCES
Alquwaizani, M., Buckley, L., Adams, C., and Fanikos,
J. (2013). Anticoagulants: a review of the
pharmacology, dosing, and
complications. Current emergency and hospital
medicine reports, 1(2), pp.83-97.
Arenajo, A.R., Ybanez, A. P., Ababan, M. M. P.,
Villajuan, C. E., Lasam, M. R. M., Young, C. P.,
and Reyes, JLA. (2017). The potential
anticoagulant property of Caulerpa lentillifera
crude extract. International Journal of Health
Sciences, 11(3).
Anderson, W., Duncan, J. G. C., and Harthill, J. E.
(1965). The anticoagulant activity of
carrageenan. Journal of Pharmacy and
Pharmacology, 17(10), 647-654.
Awtry, E., Loscalzo, J., Aspirin. (2000). Circulation;
101:1206-1218.
Bekkevold, C. M., Robertson, K. L., Reinhard, M. K.,
Battles, A. H., and Rowland, N. E. (2013).
Dehydration parameters and standards for
laboratory mice. Journal of the American
Association for Laboratory Animal Science,
52(3), 233-239.
Costa, M. S. S. P., Costa, L. S., Cordeiro, S. L.,
Almeida-Lima, J., Dantas-Santos, N.,
Magalhães, K. D., Sabry, D. A., Albuquerque, I.
R. L., Pereira, M. R., Leite, E. L., and Rocha, H.
A. O. (2012). Evaluating the possible
anticoagulant and antioxidant effects of sulfated
polysaccharides from the tropical green alga
Caulerpa cupressoides var. flabellata. Journal of
applied phycology, 24(5), pp.1159-1167.
das Neves Amorim, R. C., Gurgel Rodrigues, J. A.,
Lima Holanda, M., de Souza Mourão, P. A., and
Barros Benevides, N. M. (2011). Anticoagulant
properties of a crude sulfated polysaccharide
from the red marine alga Halymenia floresia
(Clemente) C. Agardh. Acta Scientiarum.
Biological Sciences, 33(3).
Evangelista, J. H., De Vera, M. J., Garcia, R. S., Joven,
M. G., and Nerosa, M. J. A. (2012). Preliminary
Assessment of In vitro Anticoagulant Activity
vs. Heparin 1,000 IU and Cytotoxicity of
Selected Philippine Medicinal Plants.
International Journal, 3(6).
Guirguis-Blake, J. M., Evans, C. V., Senger, C. A,
O'connor, E. A., and Whitlock, E. P. (2016).
Aspirin for the Primary Prevention of
Cardiovascular Events: A Systematic Evidence
Review for the US Preventive Services Task
ForceAspirin for the Primary Prevention of
Cardiovascular Events. Annals of internal
medicine, 164(12), pp.804-813.
Gurbel, P. A., Bliden, K. P., Neerchal, N. K., Chaganti,
S. K., and Tantry, U. S. (2007). Evaluation of
dose-related effects of aspirin on platelet
function. Circulation.
Hall, J. E. (2015). Guyton and Hall Textbook of
Medical Physiology E-Book. Elsevier Health
Sciences.
Hsieh, C. L., Lin, Y. C., Yen, G. C., and Chen, H. Y.
(2007). Preventive effects of guava (Psidium
guajava L.) leaves and its active compounds
against α-dicarbonyl compounds-induced blood
coagulation. Food chemistry, 103(2), 528-535.
Leite, E. L., Medeiros, M. G., Rocha, H. A., Farias, G.
G., da Silva, L. F., Chavante, S. F., de Abreu, L.
D., Dietrich, C. P., and Nader, H. B. (1998).
Structure and pharmacological activities of a
sulfated xylofucoglucuronan from the alga
Spatoglossum schröederi. Plant science, 132(2),
pp.215-228.
McLellan, D. S. and Jurd, K. M. (1992). Anticoagulants
from marine algae. Blood coagulation &
fibrinolysis, 3(1), pp.69-80.
Milić, N., Milanović, M., Kon, G. S., and Milošević, N.
(2014). Current experience and future
perspectives of bromelain application in
medicine. Medicinski časopis, 48(4), 153-158.
Oliveira, C. Z., Maiorano, V. A., Marcussi, S.,
Sant’Ana, C. D., Januário, A. H., Lourenço, M.
Journal of Agriculture and Technology Management (JATM) 21(1): 30-34 (January-June 2018)
34
p-ISSN: 2599-4875 e-ISSN: 2599-4980
©Cebu Technological University, R. Palma St. corner M.J. Cuenco Ave., Cebu City, 6000 Philippines
V., Sampaio, S. V., França, S. C., Pereira, P. S.,
and Soares, A. M. (2005). Anticoagulant and
antifibrinogenolytic properties of the aqueous
extract from Bauhinia forficata against snake
venoms. Journal of Ethnopharmacology, 98(1),
pp.213-216.
Oviedo, C. and Rodríguez, J. (2003). EDTA: the
chelating agent under environmental
scrutiny. Quimica Nova, 26(6), pp.901-905.
Painter, T. J. (1983). Algal polysaccharides. The
polysaccharides, 2, pp.195-285.
Pereira, M. G., Benevides, N. M., Melo, M. R.,
Valente, A. P., Melo, F. R., and Mourão, P. A.,
(2005). Structure and anticoagulant activity of a
sulfated galactan from the red alga, Gelidium
crinale. Is there a specific structural requirement
for the anticoagulant action?. Carbohydrate
Research, 340(12), pp.2015-2023.
Rodrigues, J. A. G., Torres, V. M., de Alencar, D. B.,
Sampaio, A. H., and Farias, W. R. L. (2009).
Extração e atividade anticoagulante dos
polissacarídeos sulfatados da alga marinha
vermelha Halymenia pseudofloresia. Revista
Ciência Agronômica. 40(2), 224-231.
Rodrigues, J. A. G., Torres, V. M., de Alencar, D. B.,
Sampaio, A. H., and Farias, W. R. L. (2010).
Heparinoides naturais isolados de rodofíceas
(Halymenia sp.) arribadas na costa cearense-doi:
10.4025/actascibiolsci. v32i3. 6282. Acta
Scientiarum. Biological Sciences 32(3), 235-242.
Rodrigues, J. A. G., Queiroz, I. N. L. D., Quinderé, A.
L. G., Vairo, B. C., Mourão, P. A. D. S., and
Benevides, N. M. B. (2011). An antithrombin-
dependent sulfated polysaccharide isolated from
the green alga Caulerpa cupressoides has in vivo
anti-and prothrombotic effects. Ciência
Rural, 41(4), pp.634-639.
Rosa, M. (1972). Biological properties of carrageenan.
Journal of pharmacy and pharmacology, 24(2),
89-102.
Thompson, C. B, Diaz, D. D., Quinn, P. G., Lapins, M.,
Kurtz, S. R., and Valeri, C. R. (1983). The role
of anticoagulation in the measurement of platelet
volumes. American journal of clinical pathology
80(3):327-32.
Ullman-Culleré, M. H. and Foltz, C. J. (1999). Body
condition scoring: a rapid and accurate method
for assessing health status in mice. Comparative
Medicine, 49(3), pp.319-323.
Undas, A., Brummel-Ziedins, K. E., & Mann, K. G.
(2007). Antithrombotic properties of aspirin and
resistance to aspirin: beyond strictly antiplatelet
actions. Blood, 109(6), 2285-2292.
Weiss, H. J. (2003). Discovery of the antiplatelet effect
of aspirin: a personal reminiscence. J Thromb
Haemost. 1:1869-1875.
Witeska, M. and Wargocka, W. (2011). Disodium
EDTA used as anticoagulant causes hemolysis in
common carp blood. Turkish Journal of
Veterinary and Animal Sciences 35(2), 99-104.
Zhou, W., Abdurahman, A., Umar, A., Iskander, G.,
Abdusalam, E., Berké, B., Bégaud, B., and
Moore, N. (2014). Effects of Cydonia oblonga
Miller extracts on blood hemostasis, coagulation
and fibrinolysis in mice, and experimental
thrombosis in rats. Journal of
Ethnopharmacology, 154(1), 163-169.
ResearchGate has not been able to resolve any citations for this publication.
Article
Full-text available
Background Anticoagulants have been used in the treatment of several circulatory diseases and thrombotic disorders, and in the blood sampling for hematologic analysis. Sulfated polysaccharides (SP), which have anticoagulant properties, are found in most seaweeds, including Caulerpa spp. Objective The study generally aimed to evaluate the potential anticoagulant property of Caulerpa lentillifera. Methodology The whole plant of fresh C. lentillifera was washed thoroughly with distilled water and manually expressed to obtain the extract. C. lentillifera extract was tested in two phases. Phase one utilized nine male albino rabbits, which were randomly and equally allocated into three groups: (1) negative control (oral distilled water and subsequent in vitro mixing of extracted blood with normal saline solution), (2) positive control (oral aspirin and subsequent in vitro mixing of extracted blood with normal saline solution), and (3) experimental group (oral distilled water and in vitro mixing of extracted blood with C. lentillifera extract). Blood coagulation was evaluated by measuring the clotting time using the slide and tube methods. In phase two, peripheral blood from three apparently healthy adult dogs were used. Blood collection was performed thrice. In each collection, the sample was divided into five aliquots: (1) negative control (normal saline solution), (2) positive control (ethylenediaminetetraacetic acid [EDTA]), and (3-5) experimental treatments at 0.1, 0.15, and 0.2 ml of C. lentillifera extract. Coagulation was evaluated by measuring the clotting time using the tube method. Results Phase one results revealed significant differences on the clotting time between the negative and the positive and experimental groups (P < 0.05), and no significant differences on the clotting time between the positive and the experimental groups (P > 0.05). In phase two, all blood samples mixed with EDTA did not clot, while the negative control had an average clotting time of 2.01 min. Blood mixed with 0.2 ml of C. lentillifera extract had the longest coagulation time (15.49 min). Simple linear regression revealed a positive significant correlation (multiple R = 0.9450, R² = 0.8931, P = 0.02) implying dose-dependent anticoagulant potential. The study showed that C. lentillifera extract may have a potential anticoagulant property due to its component SP.
Article
Full-text available
Alternative sources of anticoagulants have arisen as a result of the increasing demand for safer anticoagulant clinical therapy, and the sulfated polysaccharides of seaweeds have gained attention in biomedicine. In this study, crude sulfated polysaccharide fractions (denominated Hf1, Hf2 and Hf3) were obtained from the red marine alga Halymenia floresia and the anticoagulant properties of a soluble crude polysaccharide fraction (Hf2s) were assayed. The three differential extractions yielded 38.6%. The polysaccharides are composed mainly of galactose, with small amounts of xylose and glucose. The anticoagulant properties of Hf2s containing 53.8% sulfate and 3% protein was also compared to those of heparin (193.0 IU mg -1) by assays of activated partial thromboplastin time (APTT) and thrombin time (TT) using normal human plasma. Hf2s showed a higher anticoagulant activity (68.4 IU mg -1) than those of Hf1s and Hf3s, whose activities were 37.6 and 36.6 IU mg -1, respectively. The compound was less active than heparin, but its anticoagulant mechanism suggested that it is dependent on cofactor heparin II to inhibit thrombin activity, but not on cofactors VIII and IX. Therefore, the polysaccharide from H. floresia interfered on coagulation cascade.
Article
Full-text available
A crescente carência de heparina (HEP) motiva a busca por fontes alternativas de novos anticoagulantes naturais. Objetivou-se avaliar a atividade anticoagulante dos polissacarídeos sulfatados (PS) isolados de uma rodofícea do gênero Halymenia, nativa do litoral cearense, Brasil. Os PS totais foram obtidos por digestões consecutivas com papaína em tampão acetato de sódio 0,1 M (pH 5,0), contendo cisteína 5 mM e EDTA 5 mM, seguidas por cromatografia de troca iônica em coluna de DEAE-celulose. As frações obtidas foram concentradas por liofilização e submetidas à eletroforese em gel de agarose a 0,5%. Os ensaios anticoagulantes foram realizados pelo tempo de tromboplastina parcial ativada (TTPA), usando-se plasma de coelho e uma curva padrão de HEP (100 UI MG-1). As extrações (53,96%) mostraram diferenças marcantes durante o fracionamento e no grau de resolução dos PS. A espécie apresentou PS com atividade anticoagulante superior a HEP. O TTPA das frações modificou-se acentuadamente entre as extrações, expressando-se de maneira dose-dependente e sofrendo um acréscimo de 110,40 (1ª extração) para 143,10 UI MG-1 (3ª extração). Os resultados sugerem que a atividade anticoagulante dos PS isolados de Halymenia sp. foi promovida pela inibição da via intrínseca e/ou comum da cascata de coagulação. As modificações no TTPA possivelmente serão elucidadas pelos mecanismos de ação envolvidos na coagulação e caracterização estrutural desses compostos. Portanto, a rodofícea Halymenia sp. é uma boa fonte de heparinoides e sugerem-se estudos relacionados ao cultivo da espécie, em proteção aos bancos de algas.
Article
Full-text available
Red algae sulfated polysaccharides (SPs) have been widely described as anticoagulant and antithrombotic agents; however no description of antithrombotic activity regarding green algae SPs has been reported. Caulerpa cupressoides (Chlorophyta) has three different SPs fractions (SP1, SP2 and SP3). We investigated the effects of SP2 on thrombin activity by antithrombin and in an experimental model of venous thrombosis in rats. The inhibition of thrombin assay was evaluated using antithrombin (AT) in the presence of SP2 and the antithrombotic activity was investigated in rats with thromboplastin as the thrombogenic stimulus. The anticoagulant effects of SP2 are suggested be due to the potentiation of thrombin inhibition by antithrombin (IC50 ~ 10.0µg mL-1) and this mechanism of interaction is different when compared to other studied Caulerpa polysaccharides. SP2 exhibited antithrombotic effects at doses of 1.0 and 2.0mg kg-1 body weight, but at higher doses (>2.0mg kg-1 body weight) this polysaccharide revert the antithrombotic property. No hemorrhagic effect (2.0mg kg-1) was observed. As occurs with red algae SPs, these results indicate that green algae SPs are also capable of exhibiting different in vivo properties.
Article
Full-text available
Seaweeds are a source of several biopolymers widely used in cosmetics, food, and pharmaceuticals. Among them are sulfated polysaccharides, which have several biological/pharmacological activities, such as antioxidant and anticoagulant activities. In the present study, four sulfated polysaccharides, denominated CCB-F0.3, CCB-F0.5, CCB-F1.0, and CCB-F2.0, were obtained from the chlorophyte Caulerpa cupressoides var. flabellata through proteolytic digestion, followed by acetone fractionation and molecular sieving in Sephadex G-100. Chemical analyses showed that CCB-F0.5 had the highest sulfate/sugar ratio (0.73), whereas CCB-F1.0 exhibited the lowest ratio (0.23). Polysaccharides from C. cupressoides displayed a heterogeneous constitution of monosaccharides, with galactose as the main sugar unit (except for CCB-F2.0). The presence of sulfated polysaccharides was confirmed by electrophoretic and infrared analyses. Sulfated polysaccharides showed no activity in superoxide and hydroxyl radical scavenging; however, they did demonstrate total antioxidant capacity and ferrous chelating activity. Caulerpa polysaccharides also exhibited anticoagulant activity in the intrinsic (activated partial thromboplastin time (aPTT) test) and extrinsic pathway (prothrombin time (PT) test). In the aPTT test, all polysaccharides displayed considerable dose-dependent activity. A significant result was the aPTT activity of the polysaccharides CCB-F0.3 and CCB-F0.5, which was similar to that of Clexane®, a commercial low molecular weight heparin. In addition, CCB-F0.3 and CCB-F0.5 showed PT activity. Sulfated polysaccharides from C. cupressoides are therefore promising antioxidant agents in preventing the formation of reactive oxygen species and for their possible use in anticoagulant therapy.
Article
Background: Cardiovascular disease (CVD) is the leading cause of death in the United States. Purpose: To update a systematic review about the benefits of aspirin for the primary prevention of cardiovascular events in adults aged 40 years or older and to evaluate effect modification in subpopulations. Data sources: MEDLINE, PubMed, Cochrane Central Register of Controlled Trials (January 2008 to January 2015), and Cochrane Database of Systematic Reviews. Study selection: Two investigators independently reviewed 3396 abstracts and 65 articles according to prespecified criteria. All included trials evaluated aspirin for the primary prevention of cardiovascular events. Data extraction: Two investigators assessed study quality; data were abstracted by 1 reviewer and checked by a second. Data synthesis: Two good-quality and 9 fair-quality randomized, controlled trials were identified. In analyses of all doses, aspirin reduced the risk for nonfatal myocardial infarction (MI) (relative risk [RR], 0.78 [95% CI, 0.71 to 0.87]) but not nonfatal stroke; aspirin showed little or no benefit for all-cause or cardiovascular mortality. Benefits began within the first 5 years. Older adults achieved greater relative MI reduction, but no other effect modifications were found in analyzed subpopulations. In trials with aspirin doses of 100 mg or less per day, the reduction in nonfatal MI benefit persisted (absolute risk reduction, 0.15 to 1.43 events per 1000 person-years) and a 14% reduction in nonfatal stroke benefit was noted, but no benefit was found for all-cause mortality (RR, 0.95 [CI, 0.89 to 1.01]) or cardiovascular mortality (RR, 0.97 [CI, 0.85 to 1.10]). Limitation: Evidence for aspirin in primary prevention is heterogeneous and limited by rare events and few credible subgroup analyses. Conclusion: The beneficial effect of aspirin for the primary prevention of CVD is modest and occurs at doses of 100 mg or less per day. Older adults seem to achieve a greater relative MI benefit. Primary funding source: Agency for Healthcare Research and Quality.
Chapter
This chapter reviews algal polysaccharides. Fossil evidence indicates that many of the algae have changed very little in the hundreds of millions of years and it may be somewhat reckless to assume that the evolution of algal glycan structures has proceeded exactly in parallel with morphology. As new fossil evidence comes continually to light, ideas about the phylogenesis of algae are constantly being revised and there are inevitably areas of disagreement among authorities. There is unanimous agreement that the blue-green algae (Cyanophyta) are by far the oldest and there is reasonable evidence that the red algae (Rhodophyta) appeared next and the brown algae (Phaeophyta) last. In view of the very primitive morphology of algae, one might expect that the structures of the glycans would also be simple. In fact, the reverse seems to be true. Many algal glycans surpass even the gum exudates of terrestrial plants in apparent complexity. Some disorder might perhaps be expected in the glycans of organisms that are low on the evolutionary scale, because it implies that the biosynthetic enzymes are low in specificity rather than large in number. The apparent structural complexity of some algal glycans is increased by a special feature of the reproductive cycle of many algae, whereby the organism passes successively through gametophytic (haploid, male and female) and sporophytic (diploid) forms. There is growing evidence that many algal glycans exist in the native state, at least partly, as proteoglycans and that accepted procedures for isolation usually entail some degradation of these more complex macromolecules, with the liberation of the glycan moiety.
Article
Bromelain is a complex mixture of different thiol endopeptidases which is derived from the pineapple stems (Ananas comosus, fam. Bromeliaceae). Bromelain is used in food industry for meat tenderizing, beer purification etc. A great number of studies conducted on cell cultures and experimental animals indicate that bromelain possesses antiedematous, analgesic, anti-coagulant, anti-inflammatory, immunomodulatory and hepatoprotective effect. However, there are only a few clinical studies which, under the clinical conditions, undoubtedly prove the therapeutic effects of bromelain in obesity treatment, sinusitis, tooth extraction pain, osteoarthritis, pityriasis rosea, burns and cardiovascular diseases such as angina pectoris, transient ischemic attack (TIA) or thrombophlebitis. The studies of chemoprotective activity have just been started, while the studies on humans have not been conducted yet. All studies confirm that bromelain is well tolerated as well as that it causes few side effects. Therefore, it is necessary to conduct new studies that would confirm the role of bromelain in the therapy of certain pathological changes. An overview of the clinical studies in which some bromelain pharmacological effects have been confirmed is presented in this paper as well as selected in vitro and in vivo studies which are the base for further clinical research.
Article
Cydonia Oblonga Miller (COM) is traditionally used in Uyghur medicine for the prevention of cardiovascular disease. The present study is designed to explore the effects of COM extracts on models and markers of thrombosis and related biomarkers. 20, 40, 80mg/kg/day COM aqueous extracts and 5mg/kg/day aspirin, orally for 14 days were compared to untreated controls in mice on bleeding and clotting times, using the tail cutting and glass slide methods and for death rates in collagen-epinephrine pulmonary thrombosis, thrombolysis in vitro and euglobulin lysis time (ELT). In rats, common carotid artery FeCl3-induced thrombus and inferior vena cava thrombosis occlusion time, plasma concentrations of thromboxane B2 (TXB2) and 6-keto-prostaglandine F1α (6-keto-PGF1α) were measured. Compared to controls, COM extracts dose-dependently prolonged bleeding by 2.17, 2.78 and 3.63 times, vs. aspirin 2.58, and the clotting time by 1.44, 2.47 and 2.48 times, vs. aspirin 1.91. COM reduced pulmonary embolus mortality by 27, 40 and 53%, vs. 47% for aspirin. COM dose-dependently increased thrombolysis by 45, 55 and 63%, vs. 56% for aspirin, and shortened ELT to 71, 61 and 43%, vs. 43% for aspirin. In rats, venous occlusion time was prolonged. Arterial and venous thrombus weights were dose-dependently reduced in COM groups. TXB2 decreased and 6-keto-PGF1α increased with COM and aspirin, with an association between 6-keto-PGF1α/TXB2 and arterial or venous thrombus weight for all products, and for occlusion time with COM but not for aspirin. We confirm the experimental effects of COM on hemostasis and thrombosis. Further exploration of putative clinical effects appear justified.