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IN VITRO STUDY OF ANTI-THROMBOTIC ACTIVITY OF LATO (Caulerpa
lentillifera)
A Research Presented to the Faculty of the
College of Pharmacy and Medical Technology
In Partial Fulfillment of the Requirements for the Degree of
Bachelor in Medical Laboratory Science
CASAMAYOR, Melvin Lloyd
CERVANTES, Maria Gaye Margarette
CHAM, Freysie Kate
CHANG, Alyssa Jame
CHUA, Mary Elizabeth
COFREROS, Edrylle
CORONEL, Lovely Therese
FABIAA, Angelica Florence
July 2015
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APPROVAL SHEET
This research entitled,
IN VITRO STUDY OF ANTI-THROMBOTIC ACTIVITY OF LATO (Caulerpa
lentillifera)
Prepared and submitted by CASAMAYOR, Melvin Lloyd, CERVANTES,
Maria Gaye Margarette, CHAM, Freysie Kate, CHANG, Alyssa Jame,
CHUA, Mary Elizabeth, COFREROS, Edrylle, CORONEL, Lovely Therese,
and FABIAA, Angelica Florence has been approved as partial
fulfillment of the requirements for the Degree Bachelor in Medical
Laboratory Science.
CHRISTINE ALOG-VILLANUEVA, RMT, MSMT Research Adviser
PANEL OF EXAMINERS
Approved by the College of Pharmacy and Medical Technology
Committee on Oral Examination on July 4, 2015.
FERNANDO CHRISTIAN JOLITO III, RMT
Panelist
JOSE G. PEREZ, JR., RMT, MSMT MA. DEANNA B. JOLITO, RMT, MSMT
Panelist Panelist
Acknowledged:
ZESIL GAY E. GELLE, RMT, MSMT Dean
College of Pharmacy and Medical Technology
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IN VITRO STUDY OF ANTI-THROMBOTIC ACTIVITY OF LATO (Caulerpa
lentillifera)
Casamayor, Melvin Lloyd; Cervantes, Maria Gaye Margarette; Cham,
Freysie Kate;
Chang, Alyssa Jame; Chua, Mary Elizabeth; Cofreros, Edrylle;
Coronel, Lovely Therese; Fabiaa, Angelica Florence
ABSTRACT
Background: If a clot, or thrombus, obstruct the blood flow,
thrombosis will take place which can be serious and even cause
death. Most cardiovascular diseases such as hypertension, cerebral
hemorrhage, coronary thrombosis, arteriosclerosis and congestive
heart failure are caused by blood clotting disorders. Caulerpa
species contain sulfated polysaccharides (SPs) which is reported to
have the potential anti-thrombotic activity.
Objectives: This study aimed to determine the Activated Partial
Thromboplastin Time (APTT) and Prothrombin Time (PT) before and
after administration of Caulerpa lentillifera extract (50%, 75%,
100% concentrations), positive control, and negative control.
Methodology: Respondents used in the study were screened
according to normal Body Mass Index, cholesterol levels and blood
sugar levels. Blood samples were obtained and prepared for testing.
Activated Partial Thromboplastin Time (APTT) and Prothrombin Time
(PT) were determined at baseline and after administration of
Caulerpa lentillifera extracts (100%, 75% and 50%), positive
control and negative control into the human plasma to determine the
APTT and PT using a thromboanalyzer.
Results and Discussion: PT and APTT were prolonged after
treatment of different concentrations of Caulerpa lentillifera
extracts (50%, 75%, and 100%). In APTT test, there is a significant
difference between positive control and 50% and 100% Lato extract;
however 75% Lato extract is comparable with the positive control.
While in PT test, all concentration differ significantly with the
positive control (Aspirin).
Conclusion and Recommendations: Caulerpa lentillifera extract is
considered a potent antithrombotic agent based on prolonged PT and
APTT. A similar study may be conducted using in vivo assay instead
of in vitro. Additionally, more trials and replicates can be used
in the study.
Keywords: Caulerpa lentillifera, antithrombotic, prothrombin
time, activated partial thromboplastin time
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ACKNOWLEDGEMENT
The researchers would like to express utmost and heartfelt
gratitude to the
following persons:
Mrs. Ma. Deanna B. Jolito, Research I Adviser, for imparting her
knowledge and
guiding us throughout the preparation of our study.
Mrs. Christine A. Villanueva, Research II Adviser, for
insightful comments and
advices during the preparation and the conduct of the
experiment.
Mr. Jose Perez, Jr., for teaching the researchers the proper way
to do and write
the research paper, and sharing his wit and experiences.
Mr. Bernard Simundo, for supervising and accommodating the
researchers in the
Research Laboratory.
Mrs. Cherry Rose Haro, for being patient and considerate in
providing the
researchers the materials needed for the conduct of the study
and for validating the
gathered data.
Mrs. Grace Hope Gallego, for allotting her spare time as the
researchers
phlebotomist and helping during the conduct of the study.
Mrs. Jenalyn Faith Caras, who possess her expertise in the field
of Hematology,
for the untiring support and assistance during the conduct of
the study.
Mr. Joselito Bolivar for sharing his mathematical skills in
formulating and solving
the necessary solutions of the researchers study.
Ms. Elsa Juanillo, for sharing the researcher her expertise in
Hematology, for
enlightening the minds of the researcher on the principles and
concepts of the study.
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Chemistry Department, for providing the researchers with
chemicals and
apparatus needed.
Medical Technology Laboratory Stockroom personnel, for providing
the
researchers with the materials needed during the conduct of the
study and for allowing
the researchers to perform the experiment in the Medical
technology Laboratory.
For the family of the researchers, for the untiring support
morally, spiritually,
emotionally, most importantly, financially.
This paper will never be possible without the presence of mind,
intelligence, and
flexibility given by Almighty God.
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TABLE OF CONTENTS
Title Page
CHAPTER
1. INTRODUCTION
Background and Rationale of the Study 1
Objectives of the Study 2
Hypothesis of the Study 3
Theoretical Framework 3
Conceptual Framework 4
Significance of the Study 4
Scope and Limitations 5
Definition of Terms 5
2. REVIEW OF RELATED LITERATURE
Introduction 7
Caulerpa species 8
Antithrombosis 9
Prothrombin Time Test 11
Activated Partial Thromboplastin Time 12
Summary of Review of Related Literature 13
3. METHODOLOGY
Research Design and Purpose of the Study 15
Sampling and Sample Size Determination 15
Plant Identification 16
Collection and Preparation of Plant Extract 17
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Preparation of Treatments 17
Preparation of Respondents 18
Blood Collection 18
Calibration of Machine 18
APTT (Activated Partial Thromboplastin Time) 19
PT (Prothrombin Time) 20
Waste Disposal 20
Data Analysis Procedure 21
4. RESULTS AND DISCUSSION 22
5. SUMMARY OF FINDINGS, CONCLUSIONS, RECOMMENDATIONS
Summary of Findings 27
Conclusion 27
Recommendations 26
REFERENCES 29
APPENDICES
A. Relevant Communications 33
B. Procedural Flowchart 44
C. Raw Data 46
D. Documentation 59
CURRICULUM VITAE 64
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List of Tables
Table Page
1 Activated Partial Thromboplastin Time at Baseline 22
2 Prothrombin Time at Baseline 23
3 ANOVA Results on Activated Partial Thromboplastin Time 24
4 ANOVA Results on Prothrombin Time 24
5 Post Hoc Analysis of the Different Concentration versus
Positive Control (APTT)
25
6 Post Hoc Analysis of the Different Concentration versus
Positive Control (PT)
25
7 Prothrombin Time at Baseline 47
8 Prothrombin Time in Treatment A (100%) 48
9 Prothrombin Time in Treatment B (75%) 49
10 Prothrombin Time in Treatment C (50%) 50
11 Prothrombin Time in Positive Control (Aspirin) 51
12 Prothrombin Time in Negative Control (Untreated Plasma)
52
13 Activated Partial Thromboplastin Time at baseline 53
14 Activated Partial Thromboplastin Time in Treatment A (100%)
54
15 Activated Partial Thromboplastin Time in Treatment B (75%)
55
16 Activated Partial Thromboplastin Time in Treatment C (50%)
56
17 Activated Partial Thromboplastin Time in Positive Control
(Aspirin)
57
18 Activated Partial Thromboplastin Time in Negative Control
(Untreated Plasma)
58
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List of Figures
Figure Page
1 Conceptual Framework of the Study 4
2
3
4
5
6
7
8
9
10
Schematic Diagram of the Study
Caulerpa lentillifera After Washing with Distilled Water
Blending of Caulerpa lentillifera
Caulerpa lentillifera Soaked in Methanol
Filtration After Soaking for 48 Hours
Extraction using the Rotary Evaporator
Blood Extraction by a Registered Medical Technologist
Aspiration of Plasma
PT and APTT test
45
60
60
61
61
62
62
63
63
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CHAPTER 1
INTRODUCTION
Background and Rationale of the Study
As more than 70% of the worlds surface is covered by oceans, the
wide diversity
of marine organisms offer a rich source of natural products.
Marine environment
contains a source of functional materials, including
polyunsaturated fatty acids (PUFA),
polysaccharides, essential minerals, and vitamins, antioxidants,
enzymes and bioactive
peptides (Kim et al., 2010). Among marine organisms, marine
algae are rich sources of
structurally diverse bioactive compounds with various biological
activities. Recently, their
importance as a source of novel bioactive substances is growing
rapidly and researchers
have revealed that marine algal originated compounds exhibit
various biological
activities (Wijesekara et al., 2010).
Caulerpa lentillifera is abundant in the Visayas region of the
Philippine
archipelago. It is high in nutritional value and is also a
popular delicacy in the
Philippines. Few studies about the Caulerpa species have
reported that they contain
sulfated polysaccharides (SPs) that have the potential of
anti-thrombotic activity.
Sulfated polysaccharides also have a broad range of important
bioactivities comprising
antioxidant, antitumor, immunomodulatory, inflammation,
anticoagulant, antiviral,
antiprotozoan, antibacterial, and antilipemic activities (Bakyet
et. al., 2013). Hayakawa et
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al (2000) reported that SPs from C. okamurai and C. brachypus
are mainly composed of
galactose and have the specific heparin cofactor II dependent
thrombin inhibition activity.
Normally, blood flows through our arteries and veins smoothly
and efficiently, but
if a clot, or thrombus, blocks the smooth flow of blood, the
result - called thrombosis -
can be serious and even cause death. Diseases arising from clots
in blood vessels
include heart attack and stroke, among others. These disorders
collectively are the most
common cause of death in the Philippines. Due to the increasing
rates of death caused
by these diseases, the researchers decided to find an
alternative anti-thrombotic agent
using Caulerpa lentillifera extract.
Objectives of the Study
The main purpose of this study was to determine the In vitro
anti-thrombotic
activity of Caulerpa lentillifera in human blood.
Specifically, this study aimed to determine the:
1. Activated Partial Thromboplastin Time and Prothrombin Time
before
administration of Caulerpa lentillifera extract.
2. Activated Partial Thromboplastin Time and Prothrombin Time
after administration
of Caulerpa lentillifera extract using different concentrations
(100%, 75%, and
50%), positive control, and negative control.
3. Significant difference in the Activated Partial
Thromboplastin Time and
Prothrombin Time of human blood when treated with different
concentrations of
Caulerpa lentillifera extract:
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a. 100% C. lentillifera extract,
b. 75% C. lentillifera extract,
c. 50% C. lentillifera extract, and
d. Aspirin (Positive Control)
Hypothesis of the Study
There is no significant difference in the Activated Partial
Thromboplastin Time
and Prothrombin Time of human blood in vitro when treated with
different concentrations
of Caulerpa lentillifera extract before and after the
treatment:
a. 100% C. lentillifera extract,
b. 75% C. lentillifera extract,
c. 50% C. lentillifera extract, and
d. Aspirin (Positive Control)
Theoretical Framework
Studies about the Caulerpa species have reported that they
contain sulfated
polysaccharides (SPs) that have the potential of anti-thrombotic
activity. Sulfated
polysaccharides also have a broad range of important
bioactivities comprising
antioxidant, antitumor, immunomodulatory, inflammation,
anticoagulant, antiviral,
antiprotozoan, antibacterial, and antilipemic activities (Bakyet
et. al., 2013). Hayakawa et
al (2000) reported that SPs from C. okamurai and C. brachypus
are mainly composed of
galactose and have the specific heparin cofactor II dependent
thrombin inhibition activity.
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Conceptual Framework
Independent Variable Dependent Variable
Figure 1
Conceptual Framework of the Study
Significance of the Study
In this study, the researchers evaluated the potential use of
Caulerpa lentillifera
extract as an in vitro anti-thrombotic agent in human blood.
In addition this study is also significant in the sense that it
maximized the use of
Lato not only in culinary material, but at the same time, in the
field of medicine.
The plant used, namely the Lato is much easier to obtain due to
its abundance
and availability in the locality; thus paving way as a cheaper
substitute to anticoagulant
drug.
It can provide a focused and standardized approach to the
medication of venous
thrombosis and pulmonary embolism.
100% C. lentillifera extract 75% C. lentillifera extract 50% C.
lentilliferae xtract Aspirin (Positive Control) Untreated Plasma
(Negative Control)
APTT and PT of human blood in
vitro
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Scope and Limitations
This study focused on the in vitro antithrombotic activity of
Caulerpa lentillifera
extract by measuring the PT and APTT of human plasma treated
with different
concentrations of the extract. There were ten respondents in the
study. They were
chosen according to the following criteria: female, age (19 to
20 years old); and with
normal body mass index (18.5-24.9), normal fasting blood sugar
(70-100 mg/dL) and
normal cholesterol levels (below 200 mg/dL). Ten replications
were done in measuring
the APTT and PT in one trial.
The laboratory procedures were conducted in the Research
Laboratory of the
University of San Agustin, Iloilo City.
Definition of Terms
For the purpose of clarity and understanding, the following
terms are given with
their conceptual and operational meaning.
Antithrombotic It is used against or tending to prevent
thrombosis (Merriam,
2015). In this study, this term refers to the property of the
algae to be tested.
APTT (Activated Partial Thromboplastin Time) It is a clot-based
test for
intrinsic coagulation (Rodak, 2012). In this study, this term
refers to one of the tests
performed to determine antithrombotic property.
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Aspirin A synthetic compound used medicinally to relieve mild or
chronic pain
and to reduce fever (Oxford Dictionaries, 2015). In this study,
this term refers to positive
control.
Extract To withdraw (as a juice or fraction) by physical or
chemical process
(Merriam-Webster, 2015). In this study, the extract refers to
Lato (Caulerpa lentillifera)
extracts that will be tested for antithrombotic activity in
vitro.
PT (Prothrombin Time) It is a test used to measure activity of
coagulation
factors which participate in the extrinsic and common pathways
of coagulation (Rodak,
2012). In this study, this term refers to one of the tests
performed to determine
antithrombotic property.
Thromoboanalyzer it is a machine that measures the ability of
blood to clot by
performing several types of tests and progress of clotting may
be monitored optically by
measuring the absorbance of a particular wavelength of light by
the sample and how it
changes over time. (Allied Health, 2009). In this study, this
term refers to the machine
used to perform the tests determining the antithrombotic
property.
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CHAPTER 2
REVIEW OF RELATED LITERATURE
The coagulation cascade is integral to the hemostatic process
and serves to limit
the amount of blood loss during trauma. However, derangements in
this process can
result in venous thrombosis and contribute to the development of
arterial
atherothrombotic disease. Indeed, in arterial thrombosis, the
effects of thrombin may
extend far beyond coagulation activation and play an important
role in activation of a
wide variety of cells and the inflammatory processes. Venous
thrombosis and arterial
thrombotic diseases have traditionally been thought of as
separate processes; however,
they share many similarities in pathophysiology and risk factors
(Davids, 2008).
Though aspirin is a well-established drug that provides
effective secondary
prevention of ischemic cardiovascular disorders, it produces
severe hemorrhagic events
and upper gastrointestinal bleeding. Recent studies indicate
that the mechanism of
aspirin may involve inhibition of pathways distinct from COX-1
(nonCOX-1 pathways).
In addition, aspirin is known to reduce thrombin generation, to
enhance fibrin clot
permeability and clot lysis, and to promote nitric oxide
production in platelets. Aspirin
also has anti-inflammatory properties that may enhance its
antithrombotic effect (Jagtap
et al., 2012).
In recent years, the medical potential of sulfated
polysaccharides (SPs) has
attracted the attention of the scientists. These anionic
polymers occur at high
concentrations in marine algae (Etcherla, 2014). Its
anticoagulant and antithrombotic
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actions are among the most widely studied (De Kunst, 2014).
However, SPs have been
mainly studied from red and brown seaweeds, and fewer from green
algae (Crain,
2008).
Caulerpa species
The Caulerpa species are widely distributed in tropical and
subtropical areas.
Growth rate in these places are high. Caulerpa species is
represented by benthonic
marine green algae, macroscopically featuring creeping thallus
formed by rhizomes that
expand along the substrate, fixed by structures known as
rhizoids. Studies report
important biological properties of their SPs, such as antiviral,
anticoagulant and
antitumor activities (Ji et al., 2008). Studies have shown that
by using sequential
extraction it is possible to identify new SPs with anticoagulant
activity in marine algae
(Rodrigues et al., 2010).
Caulerpa lentillifera is high in minerals, vitamin A, C, and
several essential
unsaturated fatty acids. It is also reported to have
antibacterial and antifungal
properties, and to be used to treat high blood pressure and
rheumatism. There are
many species of the genus Caulerpa, but Caulerpa lentillifera
and Caulerpa
racemosa are the two most popular edible ones. Both have a
grape-like appearance
and are used in fresh salads and as vegetables. Caulerpa
lentillifera is one of the most
popular edible species of Caulerpa because of its soft and
succulent texture. In the
Philippines, the seaweed is eaten fresh as a salad, or salted.
It has created waves in
the international food market because of its high nutritional
value. Caulerpa lentillifera is
a popular form of delicacy in Japan and Philippines, and is said
by some to be an
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upcoming popular product in the seafood industry (Seaweed
Industry Association,
2014).
Marine algae, including representatives of green algae
(Chlorophyta), to which
the genus Caulerpa belongs, are rich source of sterols that
differ in chemical structures
from cholesterol, the main sterol of higher animals. Steroids
are important biologically as
hormones, vitamins, and structural components of
biomembrane.
Polysaccharides are an important component of algae. The
heightened interests
in them is related to their broad spectrum of biological
activity. For example,
polysaccharides exhibiting anticoagulants, antitumor, and other
activities have been
isolated from green algae C. racemosa, C. brachypus, C.
okamurai, C. scapelliformis,
Chaeto morphacrassa, C. spiralis, Codium adherens, and Ulva
species (Shevchenko,
2013).
Antithrombosis
Cardiovascular diseases, including thrombosis, stroke, ischemic,
and coronary
heart diseases, are a leading cause of mortality, accounting for
around 30% of global
deaths especially thrombotic diseases constitute a major
cardiovascular complication
affecting a great number of patients. Thrombosis is closely
related to activated platelet
adhesion, aggregation, secretion functions, and activation of
intrinsic and extrinsic
coagulation systems, which cause blood coagulation and fibrin
formation. Most acute
coronary syndromes are caused by platelet aggregation and
subsequent thrombus
formation in areas of ruptured atheromatous plaques. Therefore,
inhibiting platelet
function represents a promising approach for preventing
thrombosis. Antiplatelet drugs
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have been developed to inhibit platelet activity in acute
thrombotic situations as well as
to prevent adverse events and treatment of atherothrombotic.
Aspirin and Clopidogrel for
oral administration and glycoprotein IIa/IIIb antagonists
(Abciximab, Eptifibatide,
Tirofiban, etc.) for injection are commonly used antiplatelet
drugs, but they have several
clinical disadvantages including gastrointestinal side-effects,
hemorrhage and
thrombocytopenia (Becker, 2011).
There are few reports of anticoagulant activity for SPs isolated
from green algae.
(Matsubara et al., 2000) isolated a highly sulfated
galactoarabinoglucan from the green
alga Codium pugniformis, with anticoagulant activity. A sulfated
galactan with
anticoagulant activity was also extracted from Codium
cylindricum (Matsubara et al.,
2001). Caulerpa racemosa contains SPs with anticoagulant and
antiviral activities
(Bakhubaira, 2013). Recently, anticoagulant SPs isolated from
marine green algae of the
Monostroma genus were reported by Mao et al. (2008) and Zhang et
al. (2008).
Cardiovascular disease is the leading cause of death worldwide.
The therapeutic
use of heparin, an SP isolated from pig intestines or cattle
lungs, is also limited due to its
side effects and other complications, such as the risk of
hemorrhage (Rhyu, 2014). In
this context, there is a great need for new compounds from
natural sources. In this
regard, marine green algae could be a promising potential
source. Caulerpa spp.,
belonging to the Caulerpaceae family, are commonly found along
the northeastern
Brazilian coast.
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Prothrombin Time Test
A prothrombin time test measures how quickly blood clots.
Sometimes called a
pro time test or PT test, a prothrombin time test uses a sample
of blood. Prothrombin is
a protein produced by the liver that helps blood to clot. When
there is bleeding, a series
of chemicals (clotting factors) activate in a stepwise fashion.
The end result is a clot
which stops the bleeding. One step in the process is prothrombin
turning into another
protein called thrombin. The prothrombin time test measures how
well the clotting
process works and how long it takes to occur (Mayo Clinic Staff,
2013).
The reference range for prothrombin time depends on the
analytical method
used, but is usually around 1213 seconds (results should always
be interpreted using
the reference range from the laboratory that performed the
test), and the INR in absence
of anticoagulation therapy is 0.8-1.2. The target range for INR
in anticoagulant use
(e.g. warfarin) is 2 to 3. In some cases, if more intense
anticoagulation is thought to be
required, the target range may be as high as 2.5-3.5 depending
on the indication for
anticoagulation. In The Netherlands, the target INR for 'low
intensity' is between 2.5 and
3.5 and for 'high intensity' between 3.0 and 4.0 (Doseren,
2014).
The prothrombin time is most commonly measured using blood
plasma. Blood is
drawn into a test tube containing liquid sodium citrate, which
acts as an anticoagulant by
binding the calcium in a sample. The blood is mixed, then
centrifuged to separate blood
cells from plasma. In newborns, a capillary whole blood specimen
is used (Fritsma,
2012).
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Most laboratories report PT results that have been adjusted to
the INR for people
on warfarin. These people should have an INR of 2.0 to 3.0 for
basic "blood-thinning"
needs. For some who have a high risk of clot formation, the INR
needs to be higher -
about 2.5 to 3.5. The doctor will use the INR to adjust a
person's drug dosage to get the
PT into the desired range that is right for the person and their
condition. The test result
for a PT depends on the method used, with results measured in
seconds and compared
to the normal range established and maintained by the laboratory
that performs the test.
This normal range represents an average value of healthy people
who live in that area
and will vary somewhat from region to region and may vary over
time. So someone who
is not taking warfarin would compare their PT test result to the
normal range provided
with the test result. A prolonged PT means that the blood is
taking too long to form a
clot. This may be caused by conditions such as liver disease,
vitamin K deficiency, or a
coagulation factor deficiency. The PT result is often
interpreted with that of the PTT in
determining what condition may be present (Perzborn, 2005).
Activated Partial Thromboplastin Time
In activated partial thromboplastin time (APTT), an activator is
added that speeds
up the clotting time and results in a narrower reference range.
The APTT is considered a
more sensitive version of the PTT and is used to monitor the
patients response to
heparin therapy (Pagana, et. al., 2010).
The reference range of the APTT is 30-40 seconds. Critical
values that should
prompt a clinical alert are as follows: APTT: More than 70
seconds (signifies
spontaneous bleeding) (Fischbach, et. al., 2009).
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The APTT test is used to measure and evaluate all the clotting
factors of the
intrinsic and common pathways of the clotting cascade by
measuring the time (in
seconds) it takes a clot to form after adding calcium and
phospholipid emulsion to a
plasma sample. The result is always compared to a control sample
of normal blood
(Pagana, et.al, 2010).
The APTT evaluates factors I (fibrinogen), II (prothrombin), V,
VIII, IX, X, XI and
XII (Fischbach, et.al, 2009).
When the APTT test is performed in conjunction with prothrombin
time (PT) test,
which is used to evaluate the extrinsic and common pathways of
the coagulation
cascade, a further clarification of coagulation defects is
possible. If, for example, both
the PT and aPTT are prolonged, the defect is probably in the
common clotting pathway,
and a deficiency of factor I, II, V, or X is suggested. A normal
PT with an abnormal APTT
means that the defect lies within the intrinsic pathway, and a
deficiency of factor VIII, IX,
X, or XIII is suggested. A normal APTT with an abnormal PT means
that the defect lies
within the extrinsic pathway and suggests a possible factor VII
deficiency (Daniels,
2009).
Summary of Review of Related Literature
Coagulation is the process by which blood changes from liquid to
a clot. It
potentially results in hemostasis, the cessation of blood loss
from a damaged vessel,
followed by repair. The mechanism of coagulation involves
activation, adhesion, and
aggregation of platelets along with deposition and maturation of
fibrin. Disorders of
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coagulation are disease states which can result in bleeding or
obstructive clotting also
known as thrombosis.
PT and PTT are the laboratory tests used to measure coagulation.
PT is used to
evaluate the extrinsic and common pathways of the coagulation
cascade while PTT on
the other hand, evaluates the intrinsic and common pathways of
the coagulation
cascade. They are essential in detecting coagulation disorders
and deficiencies.
Lato (Caulerpa lentillifera) is a green marine algae that has a
polysaccharide
component which is sulfated polysaccharide. This component has
been studied to have
a variety of biological activities such as anticoagulant and
antitumor activities which were
isolated from other Caulerpa species. Lato is also reported to
have antibacterial,
antidiabetic, antifungal and biostimulant property.
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CHAPTER 3
METHODOLOGY
Research Design and Purpose of the Study
The main purpose of this study is to determine the
anti-thrombotic activity of Lato
(Caulerpa lentilifera) in human blood in vitro.
This study is classified as an experimental design with three
experimental
groups, a positive control and a negative control in ten
replicates.
There are six treatment groups- Treatment A, Treatment B, and
Treatment C,
which were respectively administered as 100%, 75%, 50%
concentration of Lato extract;
Aspirin which serves as the positive control; and untreated
plasma for the negative
control. Each treatment group is composed of ten replicates in
one trial. PT and APTT
are done at baseline and after administration of Lato
extract.
Sampling and Sample Size Determination
Ten selected female Medical Laboratory Science 4 students of the
University of
San Agustin were the respondents of the study.
The researchers chose only female respondents to achieve a
homogeneous
sample; that is, a sample whose units share the same
characteristics or traits in terms of
age, gender, background, and others.
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The respondents were screened according to 3 different criteria.
The chosen
respondents of the study have a normal Body Mass Index (BMI)
which is 18.5-24.9,
normal fasting blood sugar for women which is 70-100 mg/dL, and
normal cholesterol
level which is below 200 mg/dL.
The researchers considered the three screening test due to the
reason that BMI
alters level of factors that affect coagulation and blood
clotting. Obesity changers the
hormones secreted by the adipose tissue or fat tissue. The major
hormones that are
affected are the adipokines that increase platelet activity
which leads to overproduction
of PAI-1 which inhibits clot breakdown or fibrinolysis promoting
clot formation.
Hyperglycemia exposes RBC to increase glucose concentration,
thus resulting in
glycation of prothrombin, fibrinogen, and other proteins
involved in clotting mechanism.
Glycation results in the incomplete activation and function of
clotting cascade. Glycation
of intrinsic and extrinsic clotting proteins will decrease the
availability of these proteins
which affect the clotting capacity.
Lastly, high levels of blood lipids have been associated with
high levels of
coagulation factors. Subjects with high triglyceride levels (200
mg/dL) showed shorter
PT values than those with lower triglyceride levels.
Data Gathering Procedures
Plant Identification
The identity of the plant was verified by a Marine Biologist at
Bureau of Fisheries
and Aquatic Resources (BFAR).
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Collection and Preparation of Plant Extract
Three kilograms of Caulerpa lentilifera was collected at
Dumangas, Iloilo. The
plant was washed with distilled water and dried under shade at
room temperature.
The plant was grounded using an electric blender. Then the
grounded plant was
soaked in 70% methanol for 48 hours in the mechanical shaker.
During that process, the
finely grounded plant materials were covered tightly in an
Erlenmeyer flask. After 48
hours, the plant materials were filtered using coarse filter
paper and were evaporated
using the rotary evaporator at 60 degrees Celsius at 120
revolutions per minute (rpm).
Finally, extracts were stored at 20 degrees Celsius using the
amber medicine bottles
until used.
Preparation of Treatments
One hundred (100) mL of treatment are used as the standard
volume in different
concentrations. The following solutions were the prepared:
A. Treatment 1 (100%) 100 mL Lato
B. Treatment 2 (75%) 75 mL Lato per 25 mL water
C. Treatment 3 (50%) 50 mL Lato per 50 mL water
D. Positive control (Aspirin) 150 mg tablet
E. Negative control (untreated plasma)
Treatments are administered in vitro.
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Preparation of Respondents
Before respondents engaged in the conduct of the study, the
researchers asked
for an informed consent from the participants. The researchers
also included the ethical
considerations for the awareness of the respondents.
After written informed consent has been obtained, they were
chosen in this study
according to the following criteria: female, fourth year MLS
student; and normal body
mass index (BMI), fasting blood sugar levels and cholesterol
levels.
Blood Collection
Blood sample was collected by a Registered Medical Technologist
through
venipuncture from ten respondents.
Venous blood was obtained by clean venipuncture. Nine parts of
freshly collected
blood with 1 part tri-sodium citrate (0.11mol/L, 3.2%) was mixed
by inverting the tube.
The specimen was centrifuged at 3000 rpm for 15 minutes. Then,
the plasma was
transferred into a clean plastic tube.
Calibration of Machine
The machine was calibrated according to the manufacturers
instruction. Before
running the APTT and PT test, the machine used was calibrated
using control plasma.
Testing of control plasma was repeated several times until a
consistent pattern of results
within the reference range was achieved.
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APTT (Activated Partial Thromboplastin Time)
a. Baseline
The reagent and the plasma were prewarmed at 37C. A 100 L of
plasma
sample was aspirated and transferred in a cuvette. Then, 100 L
of APTT reagent
was also aspirated and transferred in the same cuvette where the
plasma sample
was placed. The tube was shaken briefly to mix the reagent and
plasma. Then, it
was incubated at 37C for 5 minutes. The cuvette was transferred
to the measuring
position. Optic key was activated. Then, 100 L of Calcium
Chloride was forcibly
added to the cuvette and Optic key was started simultaneously.
The result is
displayed in seconds and ratio.
b. Treated Plasma
The reagent and the plasma were prewarmed at 37C. A 100 L of
plasma
sample was aspirated and transferred in a cuvette. Then, 100 L
of APTT reagent
was also aspirated and 100 L of Lato extract was transferred in
the same cuvette
where the plasma sample was placed. The tube was shaken briefly
to mix the
reagent, plasma, and Lato extract. Then, it was incubated at 37C
for 5 minutes. The
cuvette was transferred to the measuring position. Optic key was
activated. Finally,
100 L of Calcium Chloride was forcibly added to the cuvette and
Optic key was
started simultaneously. The result is displayed in seconds and
ratio.
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PT (Prothrombin Time)
a. Baseline
The reagent and plasma were prewarmed separately at 37C. A 100 L
of
plasma sample was aspirated and transferred in a cuvette. It was
incubated at 37C
for 2 minutes. The cuvette was transferred to the measuring
position. Optic key was
activated. Then, 200 L of prewarmed thromboplastin reagent was
forcibly added to
the cuvette and Optic key was started simultaneously. The result
is displayed in
seconds, prothrombin activity, and INR.
b. Treated Plasma
The reagent and plasma were prewarmed separately at 37C. A 100 L
of
plasma sample was aspirated and transferred in a cuvette and 100
L of Lato extract
was transferred in the same cuvette where the plasma sample was
placed. It was
incubated at 37C for 2 minutes. The cuvette was transferred to
the measuring
position. Optic key was activated. Then, 200 L of prewarmed
thromboplastin
reagent was forcibly added to the cuvette and Optic key was
started
simultaneously. The result is displayed in seconds, prothrombin
activity, and INR.
Mrs. Cherry Rose Haro, RMT, Head of the Medical Technology
Laboratory,
validated the results obtained.
Waste Disposal
Properly labelled containers were provided for the proper
segregation and
disposal of wastes (infectious, non-infectious). Needles and
other sharps were
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disposed into the puncture-proof containers. They were
disinfected with 10%
Hypochlorite solution before disposal.
Data Analysis Procedure
The data obtained in this investigation was subjected to the
following descriptive
and inferential statistical treatments using IBM SPSS
software:
Arithmetic mean was used to compute the APTT and PT in ten
replicates.
Standard Deviation was used to measure the mean of dispersion of
the APTT
and PT value.
One-Way ANOVA was used to find out if there are any significant
difference in
using 50%, 75%, and 100% concentration of Lato extract, Aspirin
(positive control) and
untreated plasma (negative control).
Post Hoc (LSD Test) was used to find out if there is a
significant difference
existing between the different concentrations (50%, 75%, and
100%) and positive
control.
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CHAPTER 4
RESULTS AND DISCUSSION
After performing the coagulation assays, data were gathered and
tabulated
yielding results. Table 1 shows the clotting time using APTT
test before administration of
Lato extract.
Table 1 Activated Partial Thromboplastin Time at Baseline
Patient Clotting Time (s) Ratio
1 28.05 0.93
2 30.35 1.01
3 24.7 0.82
4 25.9 0.86
5 30.2 1.01
6 27.2 0.91
7 27.2 0.91
8 25.1 0.84
9 28.8 0.96
10 28.2 0.94
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Table 2 shows the clotting time using PT test before
administration of Lato
extract.
Table 2 Prothrombin Time at Baseline
Patient Clotting Time (s) Prothrombin
Activity (%) INR
1 13.5 101 1.00
2 13.2 108 1.02
3 13.3 105 0.98
4 12.9 116 0.95
5 13.6 98 1.01
6 13.6 98 1.01
7 13.0 114 0.96
8 13.6 98 1.01
9 13.1 111 0.97
10 12.3 136 0.91
Table 3 shows the significant difference in the Activated
Partial Thromboplastin
Time of human blood when treated with different concentrations
of Caulerpa lentillifera
extract, F(5,54) = 55.557, p=0.000. The difference in the
Activated Partial
Thromboplastin Time of human blood when treated with different
concentrations of
Caulerpa lentillifera extract varies.
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Table 3 ANOVA Results on the Activated Partial Thromboplastin
Time
Sum of Squares df Mean Square F Sig.
Between Groups 352390.13 5 70478.03 55.56 .000
Within Groups 68503.03 54 1268.58
Total 420893.16 59
Table 4 shows that there is a significant difference in the
Prothrombin Time of
human blood when treated with different concentrations of
Caulerpa lentillifera extract,
F(5,54) = 77.827, p=0.000. The difference in the Prothrombin
Time of human blood
when treated with different concentrations of Caulerpa
lentillifera extract varies.
Table 4 ANOVA Results on Prothrombin Time
Table 5 shows the Post hoc results that a significant difference
exist between
positive control and baseline, positive control and 50% Lato
extract, positive control and
100% Lato extract, positive control and negative control. This
implies that the Activated
Partial Thromboplastin Time of human blood when treated with 50%
and 100% Lato
differ significantly with positive control. However, no
significant difference exist between
Sum of Squares df Mean Square F Sig.
Between Groups 16168.73 5 3233.75 77.83 .000
Within Groups 2243.74 54 41.55
Total 18412.46 59
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the positive control and 75% Lato extract. This implies that the
Activated Partial
Thromboplastin Time of human blood when treated with 75% Lato
extract is perceived to
be comparable with positive control.
Table 5 Post Hoc Analysis of the Different Concentration versus
Positive Control (APTT)
Baseline 50% 75% 100% Negative
Treatment
Mean
Diff Prob
Mean
Diff Prob
Mean
Diff Prob
Mean
Diff Prob
Mean
Diff Prob
Positive
90.12
0.00
74.31
0.00
22.97
0.195
-128.94
0.00
90.12
0.00
*Mean Diff- Mean Difference *Prob- Probability
Table 6 shows the Post hoc results that a significant difference
exist between
positive control and baseline, positive control and negative
control, positive control and
50% Lato extract, positive control and 75% Lato extract,
positive control and 100% Lato
extract. This implies that the Prothrombin Time of human blood
when treated with 50%,
75% and 100% Lato extracts differ significantly with positive
control.
Table 6 Post Hoc Analysis of the Different Concentration versus
Positive Control (PT)
Baseline 50% 75% 100% Negative
Treatment
Mean
Diff Prob
Mean
Diff Prob
Mean
Diff Prob
Mean
Diff Prob
Mean
Diff Prob
Positive 43.97 0.00 42.76 0.00 41.48 0.00 21.93 0.00 43.97
0.00
*Mean Diff- Mean Difference *Prob- Probability
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As seen in the result of the Post Hoc Analysis, in the APTT
test, a significant
difference exist between the positive control and 100% and 50%
Lato extract; however
75% Lato extract is comparable with the positive control
(Aspirin).
Meanwhile, in PT test, all concentrations of Lato extract differ
significantly with
the positive control.
According to Klafke, da Silva, Rossato et al., 2012, the result
of their study show
that C. xanthocarpa did not have an anticlotting effect when
examined by PT test while
an anticoagulant effect is portrayed by APTT test, which
indicates that the C.
xanthocarpa might not inhibit a factor or factors in the
intrinsic pathway of blood
coagulation.
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CHAPTER 5
SUMMARY OF FINDINGS, CONCLUSION AND RECOMMENDATIONS
Summary of Findings
This study aimed to determine the in vitro antithrombotic
activity of Caulerpa
lentillifera in human blood. Results of this study showed that
Activated Partial
Thromboplastin Time and Prothrombin Time are within normal range
before
administration of Caulerpa lentillifera extracts. APTT and PT
were prolonged after
administration of the different concentrations of Lato extract
and positive control. In the
APTT test, a significant difference exist between the positive
control and 100% and 50%
Lato extract; however 75% Lato extract is comparable with the
positive control (Aspirin).
Meanwhile in PT test, all concentration of Lato extract differ
significantly with the positive
control.
Conclusion
Based on the results of the study, Caulerpa lentillifera extract
has antithrombotic
activity due to prolonged clotting time. The results in APTT
test show that a significant
difference exist between the positive control and different
concentrations of Lato extract
(50% and 100%); the extracts have greater effect on the
intrinsic pathway of
coagulation. However 75% of Lato extract is comparable with the
positive control. In PT
test, all concentrations of Lato extract differ significantly
with the positive control.
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Recommendations
Based on the results and conclusion the researchers recommend
the following
for future studies:
1. For future references, it would help the study to have more
trials and replicates to
guarantee that Caulerpa lentillifera extract has anti-thrombotic
activity.
2. It would also be recommended to try performing the experiment
in vivo to ensure
that the body will have a participation in the coagulation
process.
3. Also, it would be beneficial to test the different species of
Caulerpa to know if its
antithrombotic activity is applicable to its entire species.
4. To try using the pure Lato juice if it has a more potent
effect as an antithrombotic
agent compare to the different concentrations prepared.
5. It is also suggested to explore the different potentials of
Lato as a bioactive
agent.
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REFERENCES
Journals
Crain, E. J., Pinto, D. J., Wexler, P. Y. S., Wexler, R. R.,
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antihemostatic studies. Journal of Thrombosis and Haemostasis, 6,
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Davids, H. (2008). Antithrombotic/anticoagulant and anticancer
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De Kunst, D. (2014). Federatie van
NederlandseTrombosediensten.Retrieved July 1, 2014.
Etcherla, M. (2014).In Vitro Study of Antimicrobial Activity in
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Jagtap, A. (2012). Antiplatelet and antithrombotic activity of
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Ji, H.D. (2008). Antiplatelet Activity of Morusalba Leaves
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Perzborn, E. (2005). In vitro and in vivo studies of the novel
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Rhyu, D.Y. (2014). Antiplatelet, Antithrombotic, and Fibrolytic
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Rodriguez et al. (2010). Antiplatelet, Antithrombotic, and
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xanthocarpa
Zhang, T. (2008).Anticoagulant Activity of a Sulfated
Polysaccharide Isolated from the Green Seaweed Caulerpa
cupressoides. Biological Sciences, 54 (4), 133-140.
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Book Sources
Becker, R. (2011). Antithrombotic Therapy Paperback. New Jersey:
Time-Life Companies.
Daniels R, ed. (2009). Delmar's Guide to Laboratory and
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Fischbach, F.T. (2009). Manual of Laboratory and Diagnostic
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Mao, D. J. (2008).Clinical Guide to the Use of Antithrombotic
Drugs in Coronary Artery mDisease. Washington, DC: Elsevier
Matsubara, T.H. (2000). Antithrombotic Drug Therapy in
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Pagana K.D. (2010). Mosbys Manual of Diagnostic and Laboratory
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Rodak, B.F. (2012). Hematology: Clinical Principles and
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Shevchenko, M.F. (2013). Harrisons of Internal Medicine. 16th
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Bakhubaira, S. (2013). Automated versus Manual Platelet Count in
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Bakyet, K.L. (2013). Caulerpa species. Retrieved June 4, 2015
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Dorseren, M.L. (2014). Coagulation, Thrombosis, and
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Fritsma, G. (2012). Evaluation of Hemostasis."Hematology:
Clinical Principles and
Applications .Ed. Bernadette Rodak. W.B. Saunders Company:
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Hayakawa, N.F. (2000). Biochemical Compounds in Seaweeds.
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Mayo Medical Laboratories (2013).Prothrombin
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Wijisekaraet. al. (2010).Marine life. Retrieved July 7, 2015
from
http://www.sciencedirect.com/science/journal/01411136
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APPENDICES
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APPENDIX A
RELEVANT COMMUNICATIONS
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APPENDIX B
PROCEDURAL FLOWCHART
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Procedural Flowchart
Figure 2 Schematic Diagram of the Study
Gathering of materials
Data analysis
Measurement of
baseline APTT and PT
of respondents
APTT and PT of
respondents after
treatment
Identification of Lato
Extraction of Lato
Preparation of different
concentrations of Lato
Screening tests for the
respondents (blood
sugar level, cholesterol
level, BMI)
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APPENDIX C
RAW DATA
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RAW DATA
Table 7
Prothrombin Time at Baseline
Patient Clotting Time (s) Prothrombin
Activity (%) INR
1 13.5 101 1.00
2 13.2 108 1.02
3 13.3 105 0.98
4 12.9 116 0.95
5 13.6 98 1.01
6 13.6 98 1.01
7 13.0 114 0.96
8 13.6 98 1.01
9 13.1 111 0.97
10 12.3 136 0.91
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Table 8
Prothrombin Time in Treatment A (100%)
Patient Clotting Time (s) Prothrombin
Activity (%) INR
1 42.8 12 3.28
2 36.3 15 2.77
3 41.1 13 3.15
4 27.2 24 2.06
5 41 13 3.14
6 41.4 13 3.17
7 28.5 22 2.16
8 42.8 12 3.28
9 29.2 22 2.21
10 22.2 33 1.67
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Table 9
Prothrombin Time in Treatment B (75%)
Patient Clotting Time (s) Prothrombin
Activity (%) INR
1 16.2 56 1.21
2 15.2 68 1.13
3 15.7 62 1.17
4 14.6 78 1.08
5 17 49 1.27
6 18.6 43 1.39
7 14.9 73 1.11
8 15.6 63 1.16
9 14.8 75 1.1
10 14.4 82 1.07
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Table 10
Prothrombin Time in Treatment C (50%)
Patient Clotting Time (s) Prothrombin
Activity (%) INR
1 14.8 75 1.1
2 14.4 82 1.07
3 14.8 75 1.1
4 14.2 85 1.05
5 14.4 82 1.07
6 14.6 78 1.08
7 14.3 83 1.06
8 14.4 82 1.01
9 14.3 83 1.06
10 14 89 1.04
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Table 11
Prothrombin Time in Positive Control (Aspirin)
Patient Clotting Time (s) Prothrombin
Activity (%) INR
1 71 6 5.53
2 49.1 10 3.78
3 52.6 9 4.06
4 42.8 12 3.28
5 70.2 6 5.46
6 82.6 5 6.46
7 51.3 9 3.96
8 62.4 7 4.84
9 48.4 10 3.73
10 41.4 13 3.17
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Table 12
Prothrombin Time in Negative Control (Untreated Plasma)
Patient Clotting Time (s) Prothrombin
Activity (%) INR
1 13.5 101 1.00
2 13.2 108 1.02
3 13.3 105 0.98
4 12.9 116 0.95
5 13.6 98 1.01
6 13.6 98 1.01
7 13.0 114 0.96
8 13.6 98 1.01
9 13.1 111 0.97
10 12.3 136 0.91
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Table 13
Activated Partial Thromboplastin Time at baseline
Patient Clotting Time (s) Ratio
1 28.05 0.93
2 30.35 1.01
3 24.7 0.82
4 25.9 0.86
5 30.2 1.01
6 27.2 0.91
7 27.2 0.91
8 25.1 0.84
9 28.8 0.96
10 28.2 0.94
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Table 14
Activated Partial Thromboplastin Time in Treatment A (100%)
Patient Clotting Time (s) Ratio
1 >300
2 >300
3 110.1 3.67
4 137.6 4.59
5 >300
6 >300
7 >300
8 118.6 3.95
9 >300
10 >300
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
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Table 15
Activated Partial Thromboplastin Time in Treatment B (75%)
Patient Clotting Time (s) Ratio
1 89.8 2.99
2 112.2 3.74
3 85.3 2.84
4 87.2 2.9
5 104.5 3.89
6 88.8 1.96
7 92.2 3.07
8 81.2 2.7
9 102.4 3.42
10 103.6 3.46
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
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Table 16
Activated Partial Thromboplastin Time in Treatment C (50%)
Patient Clotting Time (s) Ratio
1 38.7 1.29
2 52.6 1.75
3 37 1.23
4 36.8 1.23
5 53.7 1.79
6 46.7 1.56
7 44.4 1.48
8 35.8 1.19
9 44.9 1.5
10 43.2 1.44
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
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Table 17
Activated Partial Thromboplastin Time in Positive Control
(Aspirin)
Patient Clotting Time (s) Ratio
1 120.4 4.02
2 107.5 3.59
3 118.8 3.96
4 122.2 4.08
5 116.4 3.88
6 124.3 4.16
7 115.6 3.86
8 110.7 3.69
9 116.8 3.9
10 124.2 4.15
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
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Table 18
Activated Partial Thromboplastin Time in Negative Control
(Untreated Plasma)
Patient Clotting Time (s) Ratio
1 28.05 0.93
2 30.35 1.01
3 24.7 0.82
4 25.9 0.86
5 30.2 1.01
6 27.2 0.91
7 27.2 0.91
8 25.1 0.84
9 28.8 0.96
10 28.2 0.94
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
59
APPENDIX D
DOCUMENTATION
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
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Figure 3 Caulerpa lentillifera after washing with distilled
water
Figure 4
Blending of Caulerpa lentillifera
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
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Figure 5
Caulerpa lentillifera soaked in methanol
Figure 6 Filtration after soaking for 48 hours
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
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Figure 7 Extraction using the rotary evaporator
Figure 8 Blood extraction by a Registered Phlebotomist
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
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Figure 9 Aspiration of plasma
Figure 10 PT and APTT Test
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
64
CURRICULUM VITAE
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
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NAME: Melvin Lloyd Casamayor
ADDRESS: Casamayor Street, Passi City, Iloilo
E-MAIL ADDRESS: [email protected]
MOTHERS NAME: Ma. Nilma E. Casamayor
FATHERS NAME: Leo D. Casamayor
EDUCATIONAL BACKGROUND:
PRIMARY: Assumption School Passi City
SECONDARY: Passi National High School Special Science Class
TERTIARY: University of San Agustin
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
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NAME: Maria Gaye Margarette Cervantes
ADDRESS: San Juan Street, Poblacion, San Joaquin, Iloilo
E-MAIL ADDRESS: [email protected]
MOTHERS NAME: Gazel Cervantes
FATHERS NAME: Ronnan Cervantes
EDUCATIONAL BACKGROUND:
PRIMARY: San Joaquin Central Elementary School
SECONDARY: Philippine Science High School - Western Visayas
Campus
TERTIARY: University of San Agustin
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
67
NAME: Freysie Kate S. Cham
ADDRESS: Lopez Jaena Street, Oton, Iloilo
E-MAIL ADDRESS: [email protected]
MOTHERS NAME: Elsie S. Cham
FATHERS NAME: Godfrey P. Cham
EDUCATIONAL BACKGROUND:
PRIMARY: Oton Central Elementary School
SECONDARY: Oton National High School
TERTIARY: University of San Agustin
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
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NAME: Alyssa Jame M. Chang
ADDRESS: Block 36 Lot 10 Florvel Homes Subdivision, Oton,
Iloilo
E-MAIL ADDRESS: [email protected]
MOTHERS NAME: Fredelisa M. Chang
FATHERS NAME: James M. Chang
EDUCATIONAL BACKGROUND:
PRIMARY: Immaculate Conception Parochial School
SECONDARY: Iloilo National High School - Special Science
Class
TERTIARY: University of San Agustin
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
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NAME: Mary Elizabeth Chua
ADDRESS: 24 Sta. Isabel Street, Jaro, Iloilo City
E-MAIL ADDRESS: [email protected]
MOTHERS NAME: Merlyn M. Chua
FATHERS NAME: Alejandro U. Chua
EDUCATIONAL BACKGROUND:
PRIMARY: Iloilo Scholastic Academy
SECONDARY: Iloilo Scholastic Academy
TERTIARY: University of San Agustin
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
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NAME: Edrylle Gomez Cofreros
ADDRESS: Lopez Jaena Street, Oton, Iloilo
E-MAIL ADDRESS: [email protected]
MOTHERS NAME: Jhamil Cofreros
FATHERS NAME: Edren Cofreros
EDUCATIONAL BACKGROUND:
PRIMARY: Oton Central Elementary School
SECONDARY: Oton National High School
TERTIARY: University of San Agustin
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
71
NAME: Lovely Therese C. Coronel
ADDRESS: Pinto Street, Villa Caridad Subdivision, La Carlota
City, Negros Occidental
E-MAIL ADDRESS: [email protected]
MOTHERS NAME: Evelyn C. Coronel
FATHERS NAME: Melgar B. Coronel
EDUCATIONAL BACKGROUND:
PRIMARY: La Carlota South Elementary School II
SECONDARY: Doa Hortencia Salas Benedicto National High
School
TERTIARY: University of San Agustin
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University of San Agustin COLLEGE OF PHARMACY AND MEDICAL
TECHNOLOGY
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NAME: Angelica Florence B. Fabiaa
ADDRESS: Lot 3, Block 3, Don Francisco Village, Jaro, Iloilo
City
E-MAIL ADDRESS: [email protected]
MOTHERS NAME: Ma. Rena B. Fabiaa
FATHERS NAME: Eugene S. Fabiaa
EDUCATIONAL BACKGROUND:
PRIMARY: West Visayas State University - Integrated Laboratory
School
SECONDARY: West Visayas State University - Integrated Laboratory
School
TERTIARY: University of San Agustin