In Vitro Study of Anti-Thrombotic Activity of Lato (Caulerpa Lentillifera)

University of San Agustin
COLLEGE OF PHARMACY AND MEDICAL TECHNOLOGY
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

APPROVAL SHEET
This research entitled,
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
Panelist
MA. DEANNA B. JOLITO, RMT, MSMT

Panelist
Acknowledged:
ZESIL GAY E. GELLE, RMT, MSMT

Dean
College of Pharmacy and Medical Technology
ii

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
iii

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.
iv

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.

TABLE OF CONTENTS
Title
Page
CHAPTER
1. INTRODUCTION
Background and Rationale of the Study
Objectives of the Study
Hypothesis of the Study
Theoretical Framework
Conceptual Framework
Significance of the Study
Scope and Limitations
Definition of Terms
2. REVIEW OF RELATED LITERATURE

Introduction
Caulerpa species
Antithrombosis
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
vi

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
vii

List of Tables
Table
Page
Activated Partial Thromboplastin Time at Baseline
22
Prothrombin Time at Baseline
23
ANOVA Results on Activated Partial Thromboplastin Time
24
ANOVA Results on Prothrombin Time
24
Post Hoc Analysis of the Different Concentration versus

Positive Control (APTT)
25
Post Hoc Analysis of the Different Concentration versus

Positive Control (PT)
25
47
Prothrombin Time in Treatment A (100%)
48
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
viii

List of Figures
Figure
Page
Conceptual Framework of the Study
Schematic Diagram of the Study
45
Caulerpa lentillifera After Washing with Distilled Water
60
Blending of Caulerpa lentillifera
60
Caulerpa lentillifera Soaked in Methanol
61
Filtration After Soaking for 48 Hours
61
Extraction using the Rotary Evaporator
62
Blood Extraction by a Registered Medical Technologist
62
Aspiration of Plasma
63
10
PT and APTT test
63
ix

1
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

2
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:

3
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.

4
Conceptual Framework
Independent Variable
100% C. lentillifera extract

75% C. lentillifera extract
50% C. lentilliferae xtract
Aspirin (Positive Control)
Untreated Plasma (Negative
Control)
Dependent Variable
APTT and PT of human blood in

vitro
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.

5
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.

6
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.

7
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

8
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

9
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

10
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.

11
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).

12
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).

13
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

14
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.

15
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.

16
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).

17
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.

18
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.

19
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.

20
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

21
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.

22
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
28.05
0.93
30.35
1.01
24.7
0.82
25.9
0.86
30.2
1.01
27.2
0.91
27.2
0.91
25.1
0.84
28.8
0.96
10
28.2
0.94

23
Table 2 shows the clotting time using PT test before administration of Lato
extract.
Table 2
Patient
Clotting Time (s)
Prothrombin
Activity (%)
INR
13.5
101
1.00
13.2
108
1.02
13.3
105
0.98
12.9
116
0.95
13.6
98
1.01
13.6
98
1.01
13.0
114
0.96
13.6
98
1.01
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.

24
Table 3
ANOVA Results on the Activated Partial Thromboplastin Time
Between Groups
Within Groups
Total
Sum of Squares
df
Mean Square
Sig.
352390.13
70478.03
55.56
.000
68503.03
54
1268.58
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
Sum of Squares
df
Mean Square
Sig.
77.83
.000
Between Groups
16168.73
3233.75
Within Groups
2243.74
54
41.55
Total
18412.46
59
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

25
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%
Mean
Mean
Prob
Treatment Diff
Diff
Positive
90.12 0.00 74.31
*Mean Diff- Mean Difference
*Prob- Probability
75%
100%
Mean
Diff
Negative
Prob
Mean
Diff
Prob
Prob
Mean
Diff
Prob
0.00
22.97
0.195 -128.94 0.00
90.12
0.00
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%
Mean
Mean
Prob
Treatment Diff
Diff
43.97 0.00 42.76
Positive
*Mean Diff- Mean Difference
*Prob- Probability
75%
100%
Negative
Prob
Mean
Diff
Prob
Mean
Diff
Prob
Mean
Diff
Prob
0.00
41.48
0.00
21.93
0.00
43.97
0.00

26
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.

27
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.

28
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.

29
REFERENCES
Journals
Crain, E. J., Pinto, D. J., Wexler, P. Y. S., Wexler, R. R., Wong, P. C, Xin, B. (2008)
Apixaban, an oral, direct and highly selective factor Xa inhibitor: in vitro,
antithrombotic and antihemostatic studies. Journal of Thrombosis and
Haemostasis, 6, (5), 820829.
Davids, H. (2008). Antithrombotic/anticoagulant and anticancer activities of selected
medicinal plants from South Africa. African Journal of Biotechnology, 7 (3), 217223.
De Kunst, D. (2014). Federatie van NederlandseTrombosediensten.Retrieved July
1, 2014.
Etcherla, M. (2014).In Vitro Study of Antimicrobial Activity in Marine Algae
Caulerpataxifolia and CaulerpaRacemosa. International Journal of Applied
Biology and Pharmaceutical Technology, 5 (2), 57-60.
Jagtap, A. (2012). Antiplatelet and antithrombotic activity of ethanol extract of
Embeliaribes.Phytochem Pharmacology,2 (3), 150-156.
Ji, H.D. (2008). Antiplatelet Activity of Morusalba Leaves Extract, Mediated via Inhibiting
Granule Secretion and Blocking the Phosphorylation of Extracellular-SignalRegulated Kinase and Akt. Evidence-Based Complementary and Alternative
Medicine, 1 (3), 203-214.
Perzborn, E. (2005). In vitro and in vivo studies of the novel antithrombotic agent BAY
59-7939an oral, direct Factor Xa inhibitor. Journal of Thrombosis and
Haemostasis, 3, (3), 514521.
Rhyu, D.Y. (2014). Antiplatelet, Antithrombotic, and Fibrolytic Activities of Campomanesi
xanthocarpa. Pacific Journal of Tropical Biomedicine, 1 (4), 534-539.
Rodriguez et al. (2010). Antiplatelet, Antithrombotic, and Fibrinolytic Activities of C.
xanthocarpa
Zhang, T. (2008).Anticoagulant Activity of a Sulfated Polysaccharide Isolated from the
Green Seaweed Caulerpa cupressoides. Biological Sciences, 54 (4), 133-140.

30
Book Sources
Becker, R. (2011). Antithrombotic Therapy Paperback. New Jersey: Time-Life
Companies.
Daniels R, ed. (2009). Delmar's Guide to Laboratory and Diagnostic Tests. 2nd ed.
Delmar: Cengage Learning;
Fischbach, F.T. (2009). Manual of Laboratory and Diagnostic Tests. 8th ed.
Philadelphia: Lippincott Williams and Wilkins; chap 6.Overview of Chemistry
Studies.
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 Cardiovascular Disease.
Washington, DC: Author
Pagana K.D. (2010). Mosbys Manual of Diagnostic and Laboratory Tests. 4th ed. St. Louis: Mosby Elsevier; Chap 2. Blood Studies.
Rodak, B.F. (2012). Hematology: Clinical Principles and Applications. Washington
: Lippincott Williams and Wilkins; chap 6.Overview of Chemistry Studies.
Shevchenko, M.F. (2013). Harrisons of Internal Medicine. 16th ed. United States of
America: McGraw-Hill; part two: Cardinal Manifestation and Presentation Of
Diseases, section 10: Hematology Alteration, Bleeding And Thrombosis, p.373.
Online Sources
Allied Health. (2009). Equipments in the Hematology Laboratory. Retrieved July 1, 2015
from http://www.haemonetics.com/Products/Devices/Surgical%20%20Diagnostic%20Devices/TEG%205000.aspx
Bakhubaira, S. (2013). Automated versus Manual Platelet Count in Aden
Retrieved from http://omicsonline.org/automated-versus-manual-platelet-countin-aden-2161-0681-3-149.pdf
Bakyet, K.L. (2013). Caulerpa species. Retrieved June 4, 2015 from
http://www.marinebio.org/caulerpa/species
Dorseren, M.L. (2014). Coagulation, Thrombosis, and Applications.Retrieved from
http://www.anstaskforce.gov/Species%20plans/Amended%20Caulerpa%20Plan
%20w%20all%20Apps%20-%201-05.pdf

31
Fritsma, G. (2012). Evaluation of Hemostasis."Hematology: Clinical Principles and
Applications .Ed. Bernadette Rodak. W.B. Saunders Company: Philadelphia,
2002. 719-53. (American Association for Clinical Chemistry, May 24, 2012)
https://labtestsonline.org/understanding/analytes/pt/tab/test/
Hayakawa, N.F. (2000). Biochemical Compounds in Seaweeds. Retrieved April 24, 2015
from http://linkinghub.elsevier.com/retrieve/pii/S0753332209000432?via=sd&cc=y
Kim, et.al. (2010). Marine Environmental Research. Retrieved June 6, 2015 from
http://www.journals.elsevier.com/marine-environmental-research/
Mayo Medical Laboratories (2013).Prothrombin
Time.http://www.mayomedicallaboratories.com/testcatalog/Clinical+and+Interpretive/9236. Retrieved June 1, 2015
Seaweed Industry Association. (2014). Retrieved from
https://seaweedindustry.com/seaweed/type/caulerpa-lentillifera
Wijisekaraet. al. (2010).Marine life. Retrieved July 7, 2015 from
http://www.sciencedirect.com/science/journal/01411136

32
APPENDICES

33
APPENDIX A
RELEVANT COMMUNICATIONS

34

35

36

37

38

39

40

41

42

43

44
APPENDIX B
PROCEDURAL FLOWCHART

45
Procedural Flowchart
Gathering of
materials
Data analysis
Identification of Lato
APTT and PT of
respondents after
treatment
Extraction of Lato
Measurement of
baseline APTT and PT
of respondents
Preparation of different
concentrations of Lato
Screening tests for the

respondents (blood
sugar level, cholesterol
level, BMI)
Figure 2
Schematic Diagram of the Study

46
APPENDIX C
RAW DATA

47
RAW DATA
Table 7
Patient
Clotting Time (s)
Prothrombin
Activity (%)
INR
13.5
101
1.00
13.2
108
1.02
13.3
105
0.98
12.9
116
0.95
13.6
98
1.01
13.6
98
1.01
13.0
114
0.96
13.6
98
1.01
13.1
111
0.97
10
12.3
136
0.91

48
Table 8
Prothrombin Time in Treatment A (100%)
Patient
Clotting Time (s)
Prothrombin
Activity (%)
INR
42.8
12
3.28
36.3
15
2.77
41.1
13
3.15
27.2
24
2.06
41
13
3.14
41.4
13
3.17
28.5
22
2.16
42.8
12
3.28
29.2
22
2.21
10
22.2
33
1.67

49
Table 9
Prothrombin Time in Treatment B (75%)
Patient
Clotting Time (s)
Prothrombin
Activity (%)
INR
16.2
56
1.21
15.2
68
1.13
15.7
62
1.17
14.6
78
1.08
17
49
1.27
18.6
43
1.39
14.9
73
1.11
15.6
63
1.16
14.8
75
1.1
10
14.4
82
1.07

50
Table 10
Prothrombin Time in Treatment C (50%)
Patient
Clotting Time (s)
Prothrombin
Activity (%)
INR
14.8
75
1.1
14.4
82
1.07
14.8
75
1.1
14.2
85
1.05
14.4
82
1.07
14.6
78
1.08
14.3
83
1.06
14.4
82
1.01
14.3
83
1.06
10
14
89
1.04

51
Table 11
Prothrombin Time in Positive Control (Aspirin)
Patient
Clotting Time (s)
Prothrombin
Activity (%)
INR
71
5.53
49.1
10
3.78
52.6
4.06
42.8
12
3.28
70.2
5.46
82.6
6.46
51.3
3.96
62.4
4.84
48.4
10
3.73
10
41.4
13
3.17

52
Table 12
Prothrombin Time in Negative Control (Untreated Plasma)
Patient
Clotting Time (s)
Prothrombin
Activity (%)
INR
13.5
101
1.00
13.2
108
1.02
13.3
105
0.98
12.9
116
0.95
13.6
98
1.01
13.6
98
1.01
13.0
114
0.96
13.6
98
1.01
13.1
111
0.97
10
12.3
136
0.91

53
Table 13
Activated Partial Thromboplastin Time at baseline
Patient
Clotting Time (s)
Ratio
28.05
0.93
30.35
1.01
24.7
0.82
25.9
0.86
30.2
1.01
27.2
0.91
27.2
0.91
25.1
0.84
28.8
0.96
10
28.2
0.94

54
Table 14
Activated Partial Thromboplastin Time in Treatment A (100%)
Patient
Clotting Time (s)
Ratio
>300
>300
110.1
3.67
137.6
4.59
>300
>300
>300
118.6
>300
10
>300
3.95

55
Table 15
Activated Partial Thromboplastin Time in Treatment B (75%)
Patient
Clotting Time (s)
Ratio
89.8
2.99
112.2
3.74
85.3
2.84
87.2
2.9
104.5
3.89
88.8
1.96
92.2
3.07
81.2
2.7
102.4
3.42
10
103.6
3.46

56
Table 16
Activated Partial Thromboplastin Time in Treatment C (50%)
Patient
Clotting Time (s)
Ratio
38.7
1.29
52.6
1.75
37
1.23
36.8
1.23
53.7
1.79
46.7
1.56
44.4
1.48
35.8
1.19
44.9
1.5
10
43.2
1.44

57
Table 17
Activated Partial Thromboplastin Time in Positive Control (Aspirin)
Patient
Clotting Time (s)
Ratio
120.4
4.02
107.5
3.59
118.8
3.96
122.2
4.08
116.4
3.88
124.3
4.16
115.6
3.86
110.7
3.69
116.8
3.9
10
124.2
4.15

58
Table 18
Activated Partial Thromboplastin Time in Negative Control (Untreated Plasma)
Patient
Clotting Time (s)
Ratio
28.05
0.93
30.35
1.01
24.7
0.82
25.9
0.86
30.2
1.01
27.2
0.91
27.2
0.91
25.1
0.84
28.8
0.96
10
28.2
0.94

59
APPENDIX D
DOCUMENTATION

60
Figure 3
Caulerpa lentillifera after washing with distilled water
Figure 4
Blending of Caulerpa lentillifera

61
Figure 5
Caulerpa lentillifera soaked in methanol
Figure 6
Filtration after soaking for 48 hours

62
Figure 7
Extraction using the rotary evaporator
Figure 8
Blood extraction by a Registered Phlebotomist

63
Figure 9
Aspiration of plasma
Figure 10
PT and APTT Test

64
CURRICULUM VITAE

65
NAME: Melvin Lloyd Casamayor

ADDRESS: Casamayor Street, Passi City, Iloilo
E-MAIL ADDRESS: melvinx44_rules@yahoo.com
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

66
NAME: Maria Gaye Margarette Cervantes

ADDRESS: San Juan Street, Poblacion, San Joaquin, Iloilo
E-MAIL ADDRESS: minniecervantes@gmail.com
MOTHERS NAME: Gazel Cervantes
FATHERS NAME: Ronnan Cervantes
PRIMARY: San Joaquin Central Elementary School
SECONDARY: Philippine Science High School - Western Visayas Campus

67
NAME: Freysie Kate S. Cham

ADDRESS: Lopez Jaena Street, Oton, Iloilo
E-MAIL ADDRESS: freysiekatecham@gmail.com
MOTHERS NAME: Elsie S. Cham
FATHERS NAME: Godfrey P. Cham
PRIMARY: Oton Central Elementary School
SECONDARY: Oton National High School

68
NAME: Alyssa Jame M. Chang

ADDRESS: Block 36 Lot 10 Florvel Homes Subdivision, Oton, Iloilo
E-MAIL ADDRESS: lysjamechang@icloud.com
MOTHERS NAME: Fredelisa M. Chang
FATHERS NAME: James M. Chang
PRIMARY: Immaculate Conception Parochial School
SECONDARY: Iloilo National High School - Special Science Class

69
NAME: Mary Elizabeth Chua

ADDRESS: 24 Sta. Isabel Street, Jaro, Iloilo City
E-MAIL ADDRESS: marychua127@yahoo.com
MOTHERS NAME: Merlyn M. Chua
FATHERS NAME: Alejandro U. Chua
PRIMARY: Iloilo Scholastic Academy
SECONDARY: Iloilo Scholastic Academy

70
NAME: Edrylle Gomez Cofreros

ADDRESS: Lopez Jaena Street, Oton, Iloilo
E-MAIL ADDRESS: ejaecofreros@yahoo.com
MOTHERS NAME: Jhamil Cofreros
FATHERS NAME: Edren Cofreros
PRIMARY: Oton Central Elementary School
SECONDARY: Oton National High School

71
NAME: Lovely Therese C. Coronel

ADDRESS: Pinto Street, Villa Caridad Subdivision, La Carlota City, Negros Occidental
E-MAIL ADDRESS: therese_hyuuga@yahoo.com
MOTHERS NAME: Evelyn C. Coronel
FATHERS NAME: Melgar B. Coronel
PRIMARY: La Carlota South Elementary School II
SECONDARY: Doa Hortencia Salas Benedicto National High School

72
NAME: Angelica Florence B. Fabiaa

ADDRESS: Lot 3, Block 3, Don Francisco Village, Jaro, Iloilo City
E-MAIL ADDRESS: angelica_fabiana@yahoo.com
MOTHERS NAME: Ma. Rena B. Fabiaa
FATHERS NAME: Eugene S. Fabiaa
PRIMARY: West Visayas State University - Integrated Laboratory School
SECONDARY: West Visayas State University - Integrated Laboratory School

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