in vitro study of anti-thrombotic activity of lato (caulerpa lentillifera)

University of San Agustin COLLEGE OF PHARMACY AND MEDICAL TECHNOLOGY

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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,


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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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)

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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

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4

5

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8

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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

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60

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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.


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

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


23

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.


24

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


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% 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


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), 217-223.

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-Signal-Regulated 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-count-in-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/test-catalog/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

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)


46

APPENDIX C

RAW DATA


47

RAW DATA

Table 7

Prothrombin Time at Baseline


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


48

Table 8

Prothrombin Time in Treatment A (100%)


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


49

Table 9

Prothrombin Time in Treatment B (75%)


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


50

Table 10

Prothrombin Time in Treatment C (50%)


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


51

Table 11

Prothrombin Time in Positive Control (Aspirin)


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


52

Table 12

Prothrombin Time in Negative Control (Untreated Plasma)


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


53

Table 13

Activated Partial Thromboplastin Time at baseline


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


54

Table 14

Activated Partial Thromboplastin Time in Treatment A (100%)


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


55

Table 15

Activated Partial Thromboplastin Time in Treatment B (75%)


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


56

Table 16

Activated Partial Thromboplastin Time in Treatment C (50%)


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


57

Table 17

Activated Partial Thromboplastin Time in Positive Control (Aspirin)


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


58

Table 18

Activated Partial Thromboplastin Time in Negative Control (Untreated Plasma)


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


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: [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


66

NAME: Maria Gaye Margarette Cervantes

ADDRESS: San Juan Street, Poblacion, San Joaquin, Iloilo


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


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


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


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


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


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


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


in vitro study of anti-thrombotic activity of lato (caulerpa lentillifera)

Documents

medical technology committee

msmt dean college of

caulerpa species

positive control

negative control

msmt panelist panelist

prothrombin time pt

maria gaye margarette