Chapter 1

INTRODUCTION
Background of the Study
Anticoagulant, stated in Encyclopedia Britannica (2014), is an agent
that prevents blood from coagulation (clotting) by inhibiting the function of
coagulation factors present in the blood. These drugs are often used to
prevent the formation of blood clots (thrombi) in the veins. Clots which occur
in veins are composed primarily of fibrin, with small amount of platelets.
Fibrin is a protein which crosslinks and forms a meshwork to clot the blood.
Coagulation factors such as coagulation XIII help the fibrin in the coagulation
process.
According to Moll and Waldron (2013), drugs with anticoagulant
property are potentially most effective for venous clot prevention and for
cardiovascular disease involving deep vein thrombosis (DVT) in which clots
form in so-called deep veins, such as those of the legs; pulmonary embolism,
in which a clot obstructs the pulmonary artery or one of its branches;
coronary thrombosis, in which a clot obstructs a coronary artery in the heart;
and disseminated intravascular coagulation, a systemic activation of the
coagulation system that leads to the consumption of coagulation factors and
hemorrhage. Primary examples of these drugs are aspirin, coumarin, and
warfarin. Coumarin and warfarin are both vitamin K antagonists. This means
that they get their anticoagulant effect by interfering with the role vitamin K
plays in clot formation. Aspirin, on the other hand, inhibits an enzyme
(protein) known as COX1. Cyclooxygenase1 (COX1) in the platelet is involved
in the formation of chemicals that increase a platelets ability to cause
platelet aggregation. By inhibiting this enzyme, platelet aggregation is
disrupted. This results in a decreased ability to form clots.
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Moreover, there are some plants proven to exhibit anticoagulant

activity, as well as antiplatelet activity and fibrinolytic activity. Examples of
these plants are garlic, red onion, oregano, as well as plant essential oils such
as cassia oil, cinnamon bark oil and lavender oil (Moll and Waldron, 2013).
Brown seaweed (Fucus vesiculosus), for example was proven to have
antithrombin and anticoagulant activity (Silva et. Al., 2012).
Sulphated galactan is a group sulfated polysaccharide (SPS) found in
the cell wall of some members of seaweeds. These sulphated polysaccharide
are known to exhibit many biological and physiological activities
proangiogenic, antihelmitic, anticoagulation, antithrombic, antiproliferative,
anti-adhesive, hypolipidemic, antiviral, antitumor, anti-inflammatory, and
antioxidation (Shanthi et. al., 2014). As an anticoagulant and antithrombic
material, sulphated galactan displays an ability to increase the inhibition of
thrombin by antithrombin or heparin cofactor II.1,2 (Silva et. Al. 2012). In
addition, sulphated galactans are most abundant in red seaweeds (Magalhaes
et. Al., 2011)
Red seaweed (G. heteroclada) are benthic marine macroalgae
occurring naturally along the sea coast worldwide, as well as cultivated for
their applications in food, pharmaceuticals, therapeutics, and fertilizers
(Baghel et. al., 2014). They possess a blade that is leaf like, a stipe that is
stem like, and a holdfast that resembles roots like terrestrial plants.
Seaweeds contain photosynthetic pigments and use sunlight to produce food
(Almeida et. al. 2011). Red seaweed is highly valued in Asian culture due to
its availability and importance in medicine and food industry. It can prevent
diseases such as cancer, acquired immune-deficiency syndrome (AIDS),
inflammation, pain, arthritis, infection for virus, bacteria and fungus (Abasq

et. al.,2011)
In addition, Philippines is one of the intercostal country with rich source
of red seaweed. Development and advancement of knowledge with this type
of seaweed would increase the countrys economy through exporting and
importing red seaweed goods thus makes the country competitive in terms of
seaweed marketing. It can also bring business to Filipino localities engage in
production of red seaweed. Thus, innovative methods of production and
development of products originated in red seaweed is essential.
According to Glauser et. Al. (2014), sulphate galactan are often
associated with heparin which is a sulphated polysaccharide extracted in
animals and is used clinically for anticoagulation and antithrombic activities.
However, heparin has some disadvantages as it is extracted and purified
from internal organs of higher animals making its production difficult and it
also exhibits haemorrhagic like side effects. As a safe alternative source,
sulphate galactan gain much attention in the pharmaceutical industry to
develop better and safe drugs with low or less side effects. Moreover, the aim
of this study is to investigate the clot lysing activity of sulphate galactan in
red seaweed (G. heteroclada).
This study will focus on the determination of the anticoagulant activity
of red seaweed Gracilariopsis heteroclada (Zhang Et Xia) thallus extracts
using in vitro methods.

Research Paradigm
Independent Variable

Dependent Variable

Red Seaweed Thallus

Extract

Anticoagulant
Activity

50 uL
100 uL
150 uL
Control

Prothrombin
Time (s)

General and Specific Objectives

The general objective of the study is to determine the anticoagulant
activity of red seaweed (G. heteroclada) thallus extracts on human plasma
using in vitro methods.
Specifically, the study aims to determine:
1. The prothrombin time of human plasma without addition of red seaweed
thallus extracts,
2. The prothrombin time of human plasma when added with 50 uL red
seaweed thallus extract,
3. The prothrombin time of human plasma when added with 100 uL red
seaweed thallus extract,
4. The prothrombin time of human plasma when added with 150 uL red
seaweed thallus extract,
5. If there is a significant difference in the prothrombin time of human plasma
when added with different concentrations of red seaweed thallus extracts: (a)
50 uL, (b) 100 uL and (c) 150 uL.
Hypothesis
There is no significant difference in the prothrombin time of human
plasma when added with different concentrations of red seaweed thallus
extracts: (a) 50 uL, (b) 100 uL and (c) 150 uL.
Significance of the Study
The purpose of the study is to promote the diversity of algae according
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to their functional properties with regards to their high contribution to the

society. The consumption of seaweeds is highly valued in Asian culture due to
its medicinal properties. Seaweed, as a natural food, has vastly contributed to
the community with its availability and potential use in medicine and various
industries. Furthermore, the significance of this study is for people to be fully
aware of the importance of seaweed and its underlying elements, and how it
would affect our lives.
The study is contributed to:
Medical Laboratory Science Students. The study about the
versatility of seaweeds due to its valuable components leads us to this study.
The potential anticoagulant activity of G. heteroclada proves another
essential activity from another diverse specie of red algae. Research students
in pursuit of this study could develop innovative methods that will improve
the anticoagulant mechanism of this red algae specie. The study we have
conducted could be a basis for their research activity about the anticoagulant
activity present in a Gracilaria spp. and create advanced studies.
Researchers. The potential anticoagulant activity found in the
Gracilaria spp. can encourage other researchers to create other studies in
proving the biological activity found in other unknown species of seaweeds
especially to some red algae for the improvement of knowledge of the society
and advancement of our country. In the future, we are able to compete with
other developing countries and develop new therapeutic product that are
natural and chemical-free. We can rely on the sources we produce in this
country and use it to innovate our industry, particularly in medicinal and food
industry. With the profound knowledge of the researchers, we can attain a
better country and a better life. And with the support of our government, we
can achieve these goals.
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Community. Seaweed industry has already started in other regions of

the country. Considering that we have large seaweed producing provinces in
the Philippines. Most of the seaweeds gathered by natural means are dried
and exported to other countries, where they process it for industrial and
consumer product. The industry can help the localities to engage in business
with a collaboration of the government, encourage, support and give them
the information they needed on how to manage the production of seaweeds.
In seaweed farming, the proper water management and fertilization would
produce a good-quality seaweeds, giving a good market value-added product.
Seaweed farmers could boost their income by trading their products with
other communities. The income gathered in this industry could benefit the
family in supporting their needs.
Economy. Findings, discoveries, and innovations gathered throughout
this study can serve as our edge in competing with other developing
countries. Moreover, the collaborative effort we collaborative effort we can
acquire with the other countries can help us develop innovative resources to
acquire better production and high quality of seaweeds.
Country. Philippines, comprised of group of islands which are
surrounded by bodies of water and yet we lack the ability to compete with
other developing countries in case of seaweed industry. The diversity of
seaweeds corresponding to their valuable components found in the
Philippines are needed to develop the production of a high-quality products to
be able to sustain the demand of the market. If we continue a trading system
between other countries, we can develop a good relationship with each other,
thus giving us the trust to market our goods to them. We could develop
innovative strategies to produce a new product and manufacture our own

product.
Scope and Limitations
The study was conducted in a research laboratory of Loreto D. Tupaz
builiding in Central Philippine University on May 21, 2015. Red seaweed was
obtained from UP Brackishwater Aquaculture Center (UP BAC), Leganes, Iloilo,
and extracted using mortar and pestle. Blood was extracted from three (3)
healthy volunteer donors via venipuncture, and centrifuged to obtain platelet
poor plasma (ppp). The three individuals were randomly picked from the
group, and screened for normal prothrombin time. Prothrombin time was
determined using the method described by McPherson and Pincus (2011).
Moreover, the variable of interest is only limited to the anticoagulant
activity of red seaweed G. heteroclada (Zhang et Xia) thallus extracts on
human plasma using in vitro methods. Anticoagulant activity is represented
by prothrombin time.
Definition of Terms
Anticoagulant. Anticoagulant is any substance that prevents blood
clotting. (Essential 1800 Medical Words Dictionary, 2014).
In the study, the anticoagulant activity of red seaweed thallus extract
was determined. Anticoagulant activity was the dependent variable of the
study and was measured using prothrombin time.
Centrifugation. Centrifugation is the process of separating fractions
of systems in a centrifuge. (The Dictionary of Cell and Molecular Biology,
2012)
In the study, centrifugation was carried out to separate the blood cells
from plasma in order to obtain pure platelet plasma (ppp) for prothrombin
time test.
Extract. Extract is a concentrated preparation of a drug obtained by
removing the active constituents with suitable solvents,evaporating all or
nearly all of the solvent,adjusting the residual mass or power to the
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prescribed standard. (Stedman's Medical Dictionary for the Health

Professioins and Nursing, 2012)
In the study, the thallus extract of G. heteroclada was obtained by
mortar and pestle, and the extracts were distributed to the plasma samples
in different concentrations.
In vitro. In vitro means literally in glass, or in the test tube, taking
place outside the body of the organism, as in vitro tests or in studies
involving isolated perfused organs, isolated cells, or subcellular preparations.
(Dictionary of Toxicology, 2014)
In the study, the anticoagulant activity of red seaweed thallus extract
was determined using in vitro methods. A temperature of 37 degrees Celsius
was maintained in a water bath.
Plasma. Plasma is the pale yellow fluid part of blood in which the
cellular components are suspended: red blood cells or (erythrocytes), white
blood cells (leukocytes) and blood platelets (thrombocytes). (Mosbys
Dictionary of Medicine and Health Professions, 2010)
In the study, human plasma was obtained from three (3) healthy
volunteer donors via venipuncture using 10 cc syringes. Plasma samples
were further centrifuged to obtain platelet poor plasma (ppp).
Prothrombin time. Prothrombin time or PT is the time required for
clotting after thromboplastin and calcium are added in optimal amounts to
blood of normal fibrinogen content; if prothrombin is diminished, the clotting
time increases: used to evaluate the extrinsic clotting system. (Stedmans
Medical Dictionary for the Health Professions and Nursing, 2012)
In the study, prothrombin time was used to measure the anticoagulant
activity of red seaweed thallus extracts, and was based on the method
described by McPherson and Pincus (2011). Thromboplastin reagent was
added to facilitate clotting, and stopwatch was used to record the time it took
for a clot to form.
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Thallus. Thallus is a simple plant or fungus body that is devoid of

roots,stems,and leaves.(Stedman's Medical Dictionary for the Health
Professioins and Nursing, 2012)
In the study, the thallus extract of G. heteroclada was obtained by
mortar and pestle, and stored in an air tight container prior to testing.
Thromboplastin. Thromboplastin is a substance released from
damaged tissue to start the extrinsic coagulation pathway, intrinsic
thromboplastin produced by the interaction of several factors during
coagulation. (Mosbys Dictionary of Medicine and Health Professions, 2010)
In the study, thromboplastin was the reagent used in the prothrombin
time test described by McPherson and Pincus (2011). When added to the
plasma sample, thromboplastin served to facilitate the clotting of blood.
Venipuncture. Venipuncture means the puncture of a vein, usually to
withdraw blood or inject a colution. (Stedmans Medical Dictionary for the
Health Professions and Nursing, 2012)
In the study, venipuncture was performed to obtain human plasma
from three (3) healthy volunteer donors using 5 cc syringes. Blood samples
were put in citrate tubes then centrifuged to obtain platelet poor plasma
(ppp).