CHAPTER 1

Introduction and Its Background

Introduction

Blood coagulation is a normal body process in humans that can

affect and impair the normal functioning of the different organs necessary

for metabolism. It is the process that involves the formation or presence of

one or more blood clots that may partially or completely block an artery or

vein. The clot that formed can break loose, especially when there is no

infection occurring and can be carried through the circulatory system until

it reaches a blood vessel that is too narrow to pass. There it lodges and

blocks the circulation that leads to the condition called embolism. Tissues

normally supplied by the vessel disintegrate and eventually die unless

their needs can be met by other nearby vessels. This abnormal clotting can

cause death especially in dangerous cases such as stroke and heart attack

induced by the blockage of the blood vessel leading to the brain, and the

coronary artery respectively. (Microsoft Encarta, 2007)

Anticoagulant drugs are needed for the short-term treatment of

arterial and venous thrombotic disorders and for the long-term prevention

of recurrences. Although heparin has been the mainstay of anticoagulant

treatment for acute thrombotic disorders for decades, this drug presents

some limitations related to its clinical application, such as inefficacy in

antithrombin deficient patients, bleeding complications, potential for the

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development of heparin-induced thrombocytopenia, immunosuppression

and osteoporotic effect with long-term application as side effects. So, the

search for new substances with anticoagulant and antithrombotic

activities is relevant. (Pallister, 2010)

According to WHO, fifteen million people suffer from stroke

worldwide each year. Of these, five million die and another five million

become permanently disabled (Reihardt E., 2005). Because of this there is

an increasing need for cheaper anti-coagulant in the market due to the

increased cases of heart attack and Stroke worldwide.

Researchers tried to give solution to this growing problem that

compelled to conduct the study of in vitro anticoagulant property of

Syzygium polycephaloides. Extracts from Lipoti leaves can be our nation’

s next source of alternative anti-coagulating substances cheaper than

commercial ones.

Background of the Study

The foundation of medicine has always been secured from plants,

even after synthetic drugs were made available, which makes the plant

kingdom indispensable in our daily lives. Herbal therapy has remained

popular among patients and physicians for centuries. Additionally, plant

derives medications are supported to be less toxic, simply accessible, and

less costly when compared to its synthetic equivalent. Phytoconstituents

which includes alkaloids, flavonoids, phenols, anthraquinones, glycosides,

vitamins, minerals and enzymes are responsible for the biological and
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specific chemical activities of plant material towards its various medicinal

properties like anti-inflammatory, antimicrobial, antioxidant and

anticoagulant activities.

Syzygium polycephaloides is known in many other common names

such as lipoti, igot, malig-ang, baligang, amhi, bulinayo, balasugan,

magtalulong and maigang. The use of lipoti depends on its constituents. A

widespread use of the different parts of Syzygium plants including the

leaves, bark and fruit as an anticoagulant has been validated. Therefore,

testing the effect of Lipoti leaves extract as anticoagulant in human blood

could be warranted following the Prothrombin Time Test and Thrombolytic

Activity. Lastly, individuals who suffers from heart problems and

hypertension has the risk of having blood coagulation and unfortunately,

some of the existing treatments including the surgeries can be rather

expensive which is why this study if proven to be effective, can significantly

benefit these patients especially those who simply cannot afford it.

Objectives of the Study

The “In Vitro Study on the Anticoagulant and Thrombolytic Activities of

Lipoti (Syzygium polycephaloides fam. Myrtaceae) Ethanolic Crude Extract

of leaves on Normal Human Blood Sample” specifically, it aims to:

1. determine the different constituents present in the ethanolic extract

of Syzygium polycephaloides leaves through phytochemical

screening.
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2. analyze the functional groups present in the ethanolic extract of

Syzygium polycephaloides leaves through Fourier Transform Infra-

Red Spectroscopy (FTIR).

3. identify the concentration of the ethanolic extract of Syzygium

polycephalides leaves for its anticoagulant and thrombolytic effect

based on:

a. Rate of clotting through PT test

b. Thrombolytic activity or Amount of reduction in clot

4. compare the anticoagulant and thrombolytic activity of the ethanolic

extract of Syzygium Polycephaloides leaves with EDTA and

Streptokinase, respectively.

Conceptual Framework

The leading cause of death today is Cardiovascular diseases. Heart

disease and stroke have been reported to become deadly that it killed 17

million persons every year (Reinhardt, E., 2005). According from the

Philippine Statistics Authority as of February 2018, the leading cause of

deaths is also Cardiovascular disease specifically ischemic heart failure.

So, the concept of this study to address this problem and sustain the

availability of cheaper and attainable medications for anticoagulants and

thrombolytic. The study was focused on the use of indigenous plants,

which was Lipoti (Syzygium polycephaloides Family Myrtaceae), to utilize

and spread the awareness of using herbal medicine. The use of medicinal
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has been there since ancient times and had showed efficiency especially

having less or without side effects compared to synthetic drugs.

The researchers had applied for Institutional Ethics Review

Committee (IERC) for the study will involve human volunteers for the

extraction of blood. Institutional Ethics Review Committee (IERC) focuses

on the ethical issue on researches that involves human participation.

Figure 1 depicts the overall proposition of this study. The main

purpose of this study is to conduct an in vitro study on anticoagulant and

thrombolytic activity of lipoti and will show significant effectivity that will

be a potential use in the future. The paradigm shows that through

conducting phytochemical screening and Fourier transform infrared

spectroscopy will assess the constituents present in the plant that may be

the cause of its anticoagulant or thrombolytic activity. Through

Prothrombin Time test and the Clot reduction, it will determine how well

the plant sample will produce an anticoagulant and thrombolytic property

as compared to the positive controls used, such as EDTA and

Streptokinase, respectively.

Significance of The Study

The findings of this research will be beneficial to:

Patient. As stroke increasingly affects 15 millions of people around

the world (Reinhardt, E., 2005) it will help through contributing a new way
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of treating clots through anticoagulant and thrombolytic activity of

Syzygium polycephaloides leaves.

Researchers. This study will serve as a foundation in their future

researches of enhancing and exploring the other use of lipoti.

>Plant sample: >Collection and Drying >Phytochemical

INPUT

PROCESS

OUTPUT
Syzygium of Plant Sample Screening results and
polycephaloides leaves analysis
>Authentication of Plant
>Healthy Human Blood Sample >Confirmation of the
sample constituents present in
>Crude extract ethanolic extract of
>Procurement of Extraction lipoti leaves
Chemicals/Reagent
>Phytochemical >Anticoagulant and
>Procurement of Screening Thrombolytic Activity of
Streptokinase as a Syzygium
positive control >FTIR of the crude
extract polycephaloides
>Budget >Concentration of
>Approval for IERC form
Syzygium
>Prothrombin Time Test polycephaloides leaves
>Thrombolytic Activity extract that is significant
Test to the anticoagulant and
thrombolytic activity

Figure 1.

Conceptual Framework

Hypothesis of the Study

The study attempts to test the null hypothesis of the anticoagulant

and thrombolytic activity of Syzygium polycephaloides leaves ethanolic

extract on normal human blood sample.

Ha: Syzygium polycephaloides will produce an anticoagulant and

thrombolytic activity on normal human blood sample. The concentration

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of the ethanolic extracts in the leaves has a significant value in

determining the anticoagulant and thrombolytic effect of the plant.

Scope and Limitation

This study focused on testing the anticoagulant and thrombolytic effect

of the ethanolic extract of lipoti leaves through in vitro.

The research confines the following:

1. Phytochemical screening of Syzygium Polycephaloides leaves.

2. Maceration was done with the use of 80% ethanol solvent.

3. In each extract, 62mg/mL, 125mg/mL, 250mg/mL, and 500

mg/ml with dimethyl sulfoxide concentrations were prepared.

4. Normal Human blood sample was used in the testing of

anticoagulant activity.

5. Streptokinase and EDTA as the reference drug for

Thrombolytic test and Anticoagulant test respectively.

6. Comparison between EDTA and Lipoti leaf extract’s

anticoagulant property.

7. Comparison between Streptokinase and Lipoti leaf extract’s

thrombolytic property.

8. Prothombin Time Test was done to determine the rate of

clotting.

9. Reduction of Clot was executed to know the percentage of

clot lysis.
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10. Analysis of the functional groups present in the ethanolic

extract using FTIR.

The researchers limited the study in determining;

1. Only the ethanolic extract was used for the study of

anticoagulant and thrombolytic effect.

Definition of Terms

To understand and clarify the terms used in the study, below are

their meanings:

Anticoagulant- ability to inhibit or to slow down the formation of

blood clots by suppressing synthesis of various clotting factors.

Antithrombotic - agents that prevents platelets from clumping and

also prevent clots from forming and growing (American Society of

Hematology, 2008).

EDTA (ethylenediaminetetraacetic acid)- A chemical that binds

certain metal ions. It is used in medicine to prevent blood samples from

clotting (National Cancer Institute).

Ethanolic Crude Extract- A preparation used to separate the active

ingredients in the lipoti using the ethanol solvent.

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FTIR (Fourier Transform Infrared Spectroscopy)- used to identify

organic, polymeric, inorganic materials that uses infrared light to scan test

samples (RTI Laboratories).

Human Blood Sample- The blood extracted used in the testing of

anticoagulant and thrombolytic activity of lipoti leaves.

In-Vitro- A process that is performed outside the living organism. (In

a test tube or cultured dish)

Partial Thromboplastin Time - this test measures on how fast a clot

forms.

Syzygium polycephaloides- the scientific name of a species in the

genus Syzgium (family Myrtaceae) which is commonly known as “Lipoti”

(Philippine Medicinal Plants).

Thrombolytic Test- used to detect dissolving clots in blood vessels.