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ABSTRACT Antigonon leptopus flower extract was tested for its anticoagulant effect on human blood. Five hundred (500.00) grams of A.leptopus flowers were macerated using a blender and the juice were squeezed to obtain the extract. Control and experimental set-ups with three replications each were prepared. Each replication in the control set-up was prepared by mixing 2.00 mL of distilled water with 2.00 mL of fresh blood in a vacuum tube while each replication in the experimental set-up was prepared by mixing 2.00 mL of the prepared extract with 2.00 mL of fresh blood in a vacuum tube. Microscopic observation and direct vacuum tube observations were done to note the time of blood to visibly coagulate in each trial in each set-up. In the microscopic observation the experimental and control set-ups had an average time elapsed for the presence of blood coagulation of 90.67 and 26.67 minutes respectively. The experimental and control set-ups had an average time elapsed for blood
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Page 1: Abstract

ABSTRACT

Antigonon leptopus flower extract was tested for its anticoagulant effect on

human blood. Five hundred (500.00) grams of A.leptopus flowers were macerated using a

blender and the juice were squeezed to obtain the extract. Control and experimental set-

ups with three replications each were prepared. Each replication in the control set-up was

prepared by mixing 2.00 mL of distilled water with 2.00 mL of fresh blood in a vacuum

tube while each replication in the experimental set-up was prepared by mixing 2.00 mL

of the prepared extract with 2.00 mL of fresh blood in a vacuum tube. Microscopic

observation and direct vacuum tube observations were done to note the time of blood to

visibly coagulate in each trial in each set-up. In the microscopic observation the

experimental and control set-ups had an average time elapsed for the presence of blood

coagulation of 90.67 and 26.67 minutes respectively. The experimental and control set-

ups had an average time elapsed for blood clots to be visible of 38.34 and 0.83 hours

respectively in the vacuum tube observation. Two-way ANOVA at 0.01 level of

significance, degrees of freedom: (2,5) for treatment showed that there is a significant

difference between the set-ups in both microscopic and vacuum tube observation in the

time elapsed for blood coagulation to be visible. Based on the results, the A.leptopus

flower extract can be used as an effective anticoagulant on human blood.

Page 2: Abstract

CADENA DE AMOR(Antigonon leptopus) FLOWER EXTRACT AS

BLOOD ANTICOAGULANT

Science Investigatory Project presented in partial fulfillment of the requirements for

graduation of the Revised Basic Education Curriculum (RBEC) to the panelists and

concerned staff of Bantayan Science High School, Bantayan, Cebu. Prepared under the

direction and guidance of Mr. Ulysis Tisado.

INTRODUCTION

Nature, Importance and Rationale of the Study

The shelf-life of human blood is just a short period of time due to the rapid

coagulation of the said body fluid. Certain medical purposes need a longer period of time

for the usage of blood (Cabatit, 1985). So, it is necessary to lengthen the shelf-life of

blood by delaying its coagulation or in other words applying a blood anticoagulant for

longer storage and time of usefulness. Anticoagulant decreases the normal range of

blood’s coagulation time; hence, blood can be stored at a longer time maintaining its

physical state and fluidity. Because of this, a study, which concerns about the possibility

of delaying the coagulation time of blood by making blood anticoagulants from plant

parts specifically A. leptopus flowers, was conducted.

A.leptopus locally known as Cadena de Amor is a climbing, perennial vine. Its

stems attain a length of 10 meters and leaves are alternate up to 10 cm long. Flowers are

white or pale to deep pink up to 2 cm long, with a 5-parted and persisted perianth. Fruit is

an ovoid achene and is about 1 cm long and broad at the base, narrowing towards the tip,

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and is loosely surrounded by the persistent lobes of the flower.

Problem and Objectives of the Study

The study aimed to answer the following problems:

1. What is the effect of A.leptopus flower extract to the coagulation time of blood as

compared to that of the distilled water?

2. Is A.leptopus flower extract a possible blood anticoagulant?

The study aimed to test if A.leptopus flower extract has a blood anticoagulant

effect. It target delaying the time of blood coagulation and lengthening the shelf-life of

blood samples. It focused on evaluating the effect of A.leptopus flower extract on the

coagulation time of blood as compared to that of the distilled water.

Limitations of the Study

The study was limited only to the extraction of A. leptopus flower, the possibility

of the said extract as blood anticoagulant and the quantitative comparison of coagulation

time of blood under both microscopic and vacuum-tube observation of the extract and

distilled water.

Chemical tests and analysis of the extract was not conducted. A two-way

ANOVA was employed for the time elapsed for blood to coagulate to be visible under

both vacuum-tube and microscopic observations.

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REVIEW OF RELATED LITERATURE

Coagulation

Coagulation is a complex process by which blood forms clots. It involves a

cellular (platelet) and proteins (coagulation factors) (http://medical-

dictionary.thefreedictionary.com/Coagulation+Disorders). Blood coagulation is the

process of transformation of a liquid into a soft, semisolid, or a solid mass. Blood

coagulation is characterized by thrombosis-the formation, presence, or development of a

thrombus, a blood clot formed (Cabatit, 1985).

Coagulation Time

The coagulation time is the time by which the blood takes a clot. Normally in an

ordinary skin puncture, blood coagulation takes place in 2-6 minutes. It is affected by

such factors as temperature, size of the drop, and smoothness and cleanliness of the

instrument. But the normal coagulation time when blood is taken from a vein may reach

twenty minutes. (Cabatit, 1985)

Cadena de Amor

Chain of love, Antigonon leptopus, locally known as Cadena de Amor is a

climbing, perennial vine. Its stems attain a length of 10 meters and leaves are alternate up

to 10 cm long. Flowers are white or pale to deep pink up to 2 cm long, with a 5-parted

and persisted perianth. Fruit is an ovoid achene and is about 1 cm long and broad at the

base, narrowing towards the tip, and is loosely surrounded by the persistent lobes of the

flower. Studies have shown that it has anti-thrombin, analgesic, anti-inflammatory, ant-

diabetic and lipid peroxidation inhibitory properties. The anti-thrombin property was

observed under a methanol extract of the plant.

A.leptopus is found to be edible and practiced by some as medical remedies for

certain ailments and diseases. In some parts of the world, the tubers and flowers are

consumed as food; on the other hand, in Thailand, leaves are fried, coated with flour and

served with noodles. Ifugao in the Philippines, use Cadena de amor for wound closure. In

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Trinidad and Tobago, the plant is used for diabetes, low blood pressure, and as a heart

tonic. In Jamaica, decoction of flowers and aerial parts are used for cold remedies.

(http://stuartxchange.com/CadenaDeAmor.html)

Coagulation Components

There are many factors and substances that make up the coagulation process

namely platelets, platelet aggregators, and fibrin-formed from fibrinogen. The

coagulation system is comprised by coagulation factors: Factor XII, Factor XI, Factor X,

Factor VII and Factor V.

On the other hand, coagulation is prevented and delayed by application of blood

anticoagulants, vasoconstrictors, and platelet aggregator inhibitors. Blood anticoagulants

include heparin, coumadin and many more.

(http://www.naturdoctor.com/Chapters/Research/thrombosis_prevention.html)

Coagulation as a Complex Process

The sequence of events involving the process of coagulation (after tissue injury

and platelet destruction) are:

1. Platelet factors + Plasma factors →→→→→→→ Plasma thromboplastin

(AHG, PTC, PTA)

2. Plasma thromboplastin + (LF, SF, Ca) →→→→→→→ thromboplastin complex

3. Thromboplastin complex + Prothrombin →→→→→→→ thrombin

Platetelet acc. 1

4. Thrombin + fibrinogen →→→→→→→ Fibrin (insoluble clot)

Platelet acc.2

In the conversion of fibrinogen to fibrin, the 3 steps are:

1. proteolysis of fibrinogen

2. soft clot formation

3. hard clot formation

The clot formation is affected by the pH because the fibrin monomer aggregation of the

soft clot formation is affected by the strength of the hydrogen bonding and pH.

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(Cabatit, 1985)

Blood anticoagulants

Here are some inhibitors of blood coagulation or anticoagulants:

1. Heparin- a complex carbohydrate and is believed to be formed by the mast cells

and rapidly destroyed by heparinase. It inhibits activatiuon of prothrombin and

inactivates thrombin.

2. Antithrombin- present in small amount in plasma and inactivates thrombin.

3. Antithromboplastin- a lipid that inactivates thromboplastin.

4. Dicumarol- prevents the body from utilizing vitamin K: thus, it depresses the

production of proconvertin which is needed for blood coagulation.

5. Fibrinolytic substances- present in blood and tissues of normal persons. It limits

intravascular clotting.

Water as Amphoteric

At pH 7 water is neutral.But...water is amphoteric- it can act as an acid or a base

if the pH of 7 is disrupted. Its acidic or base like properties depend on whether it is

receiving or donating a proton. (acids-proton donors, bases-proton acceptors).

(http://wiki.answers.com/Q/Is_water_a_base_or_an_acid)

Statistical Parameter

A two-way Analysis of Variance is a statistical parameter to determine if there is

a significant difference between the existing treatments and replications. It is used if 2 or

more variables are involved.

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METHODOLOGY

Materials:

Blender

pH paper

wire gauze

125.00 mL Erlenmeyer flask

106.00 mL Distilled water

Dropper

12.00 mL Fresh blood samples

12 vaccuum tubes

3 microscopes

slides and cover slips

250.00 mL beaker

alcohol lamp

iron rings

iron stands

100.00 mL graduated cylinder

Cheese cloth

Triple beam balance

500.00 g A.leptopus flowers

GENERAL PROCEDURE:

*Collection of flowers

Pink-colored flowers of A.leptopus were collected and weighed. Before weighing

the fruits were separated from the flowers.

*Extraction A. leptopus flowers

Five hundred (500.00) grams of collected A.leptopus flowers were macerated with

100.00 mL of distilled water using a blender. The macerated flowers were decanted to a

cheese cloth and were squeezed to obtain the extract. The extract was transferred into an

e-flask. The pH, volume, color and odor of the extract were noted.

*Cleaning of vacuum tubes

The vacuum tubes were sterilized by subjecting them to boiling water placed in a

beaker.

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*Preparation of the Reagents

Three vacuum tubes were filled with 2 mL each of the prepared extract. This

composed the experimental set-up. Another three vacuum tubes were filled with 2 mL

each of distilled water, which composed the control set-up.

*Collection of Blood Samples

Blood was freshly extracted from two persons, labeled as A and B, by the Medical

Technician of Bantayan District Hospital. Six vacuum tubes were filled by 2 mL each of

the freshly extracted blood.

*Preparation of the Set-ups

Right after the blood was decanted into the vacuum tubes, they were immediately

mixed with the prepared reagents namely the A. leptopus flower extract and the distilled

water. The following set-ups were made:

Set-ups Trial 1 Trial 2 Trial 3

Experimental 2 mL blood(A) + 2

mL A. leptopus

flower extract

2 mL blood(B) + 2

mL A. leptopus

flower extract

2 mL blood(A) + 2

mL A. leptopus

flower extract

Control 2 mL blood(A) + 2

mL distilled water

2 mL blood(B) + 2

mL distilled water

2 mL blood(B) + 2

mL distilled water

*A and B are the source of the blood

*Microscopic Observation

The set-ups were continuously observed under the microscope (400x) by getting a

small amount of each sample using a dropper and putting them into the slides. After the

slide was used it was subjected to boiling water for sterilization for future use.

Supposedly, the observation interval must be one minute, but, considering that getting the

specimens, putting them into the slides, and focusing them clearly under the microscope

takes more than two minutes, a fixed interval was already neglected. Instead of fixing the

interval, an immediate continuous microscopic observation was made. It means that after

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a few minutes of observing under the microscope for determining the presence of blood

coagulation, new samples were immediately obtained and subjected to the said

observation. One researcher was responsible for taking the notes of every microscopic

observation finished and was the one taking the exact time for the said confirmation of

the presence of blood coagulation as viewed microscopically for every trial in each set-

up. Each time the blood coagulation was present and observed in a certain blood sample,

further microscopic observation to that certain sample was already being stopped.

*Vacuum tube observation

Simultaneous to the microscopic observation, a vacuum-tube observation was also

conducted by viewing from the image formed as being viewed at the outside of the

vacuum-tube if it had visibly coagulated already. The time and date for insoluble blood

clot to be visible under vacuum tube observation for each of the trials of every set-up was

noted.

*Data Processing

The data obtained on both microscopic and vacuum tube observation cannot be

used as a statistical data; hence, the obtained data were processed for presentation of

results and statistical use.

The time (in minutes) elapsed for the presence of blood coagulation under

microscopic observation was obtained by using the equation below.

(HH°MM’)f - (HH°MM’)i = HH° x + MM’

where:

(HH°MM’)f - noted time for presence of blood coagulation

(HH°MM’)i - time for blood sample to be freshly obtained

HH° x + MM’ – time elapsed in minutes

HH° - hour time

MM’ - minute time

To obtain the time elapsed, we subtract the noted time for the presence of blood

8

( )

60’

( )

60’

1°( )

( )

( )

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coagulation by the time for the blood sample to be obtained. The equation neglected the

date and time position (AM/ PM) because the data obtained had the same date, February

3, 2012, and time position, PM. The elapsed hour time was converted into minutes using

a conversion factor and then added with the elapsed minute time to obtain the time

elapsed in minutes for the presence of blood coagulation under microscopic observation.

The time (in hours) elapsed for insoluble blood clots to be visible was obtained by

using the equation below.

[ (DDf- DDt) x X + (HH°MM’)f] - (HH°MM’)i = HH° + MM’ x

where:

(HH°MM’)f - noted time for presence of blood coagulation

(HH°MM’)i - time for blood sample to be freshly obtained

HH° - hour time in 24- hour time format

MM’ - minute time

DDf - noted date for insoluble blood clot to be visible

DDt - date for blood sample to be freshly obtained

X - 12° if (HH°MM’)f is less than (HH°MM’)i

- 24° if (HH°MM’)f greater than (HH°MM’)i

[ (DDf- DDt) x X + (HH°MM’)f] – modified noted time for insoluble blood clot

to be visible

HH° + MM’ x - time elapsed in hours

To obtain the time elapsed, first, the date for blood sample to be freshly obtained

subtracted from the noted date for insoluble blood clot to be visible. And then, multiplied

the obtained value with the corresponding hour equivalent and added it to the noted time

for insoluble blood clots to be visible. The obtained value was already the modified noted

time. We subtract the modified noted time for insoluble blood clots to be visible by the

time for the blood sample to be obtained. The elapsed minute time was converted into

hours using a conversion factor and then added with the elapsed hour time to obtain the

time elapsed in hours for insoluble blood clot to be visible under vacuum tube

observation.

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60’( )

( ))1°

60’

( )

Page 11: Abstract

*Incineration of Blood Samples

After the blood samples were already observed by all the data and information

needed, they were incinerated and the vacuum tubes were disposed for safety and sanitary

purposes.

*Statistical Parameter and Analysis

Since the statistical parameter used is two-way ANOVA, we need to define our

hypotheses for replication and treatment to interpret our results in the given parameter.

The hypotheses for replication are the following:

Null hypothesis: There is no significant difference between the time (in minutes) elapsed

for blood to coagulate under microscopic observation or time (in hours) elapsed for

insoluble blood clots to be visible under vacuum tube observation in every replication or

trial; therefore,

Ho is R1 = R2 = R3 where: R- replication or trial

Alternative hypothesis: There is a significant difference between the time (in minutes)

elapsed for blood to coagulate under microscopic observation or time (in hours) elapsed

for insoluble blood clots to be visible under vacuum tube observation in every replication

or trial; therefore,

Hi is R1 ≠ R2 ≠ R3 where: R- replication or trial

The hypotheses for treatment are the following:

Null hypothesis: There is no significant difference between the time (in minutes) elapsed

for blood to coagulate under microscopic observation or time (in hours) elapsed for

insoluble blood clots to be visible under vacuum tube observation in every treatment-

blood with A. leptopus extract and blood with distilled water; therefore,

Ho is T1 = T2 where: T- treatment

Alternative hypothesis: There is a significant difference between the time (in minutes)

elapsed for blood to coagulate under microscopic observation or time (in hours) elapsed

for insoluble blood clots to be visible under vacuum tube observation in every treatment-

blood with A. leptopus extract and blood with distilled water; therefore,

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( )( )( )

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Hi is T1 ≠ T2 where: T- treatment

If the obtained F value is greater than or equal to the tabular value at 0.01 level of

significance, degrees of freedom: (2,5) for treatment and (1,5) for replication, null

hypothesis(Ho) was rejected and alternative hypothesis(Hi) was accepted.

The results of this two-way ANOVA are favorable to the study if there is no

significant difference between the existing replications. And if there is a significant

difference between the treatments, since the comparison is between the A. leptopus

flower extract- the tested variable as blood anticoagulant- with the distilled water- an

unconsidered anticoagulant.

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RESULTS AND DISCUSSIONS

Physical Properties of the A. leptopus Flower Extract

Part of the study was the description of the A. leptopus flower extract’s physical

properties and characteristics. The macerated 500.00 g A. leptopus flowers yielded an

extract with the following physical characteristics:

Table 1.1. Physical properties of the A. leptopus flower extract

Characteristic Description

Color Pale-brown

Odor Strong, woody odor

pH 4

Volume 70 mL

The A. leptopus flower extract had a pale-brown color, strong, woody odor, and a

pH of 4. The obtained extract from the macerated flowers with 100.00 mL distilled water

was 70 mL. The extract was acidic based on its pH value. The acidic property of the

extract may possibly be caused by the organic materials and other substances contained

by the A. leptopus flowers. Those organic materials and substances were obtained by the

extract upon squeezing the macerated flowers. The low pH value or the acidic nature may

possibly affected the time of clot formation or blood coagulation as it is cited on the

Review of Related Literature (see Review; Coagulation as a Complex Process). The

extracted volume, 70 mL, was less than that of the volume of the added water, 100 mL,

may be attributed by: a. the minimal amount of water which remained in the flowers in

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the blender during decantation ( as it is observed that the A. leptopus flowers absorbed

water); b. the existing amount of water absorbed by the flowers in the residue after the

squeezing ( as it is considered that squeezing is not enough to extract the whole amount

of water that is contained by the macerated flowers).

Microscopic Observation

The presence of blood coagulation can be observed microscopically by viewing if

there is already a presence of cells that have already colonized or grouped or clumped,

and if viewed at technically high microscopes, there is already the presence of a clear

thrombus. The time by which this presence of coagulation occurs is important because it

will lead to the determination of the coagulation time of a certain blood sample. Table 2.1

below shows the elapsed time of blood for its coagulation as it was viewed

microscopically.

Table 2.1. Time (in minutes) elapsed for the presence of blood coagulation under

microscopic observation

Set-up Trial 1 Trial 2 Trial 3 Average

Experimental 98 88 86 90.67

Control 36 18 26 26.67

Table 2.1 shows the time difference of the time which the blood samples was

observed to have the presence of blood coagulation from the time by which the blood

sample was freshly obtained and was mixed by the reagents. The control set-up trial 2,

obtained the earliest time which is 18 minutes for blood coagulation to be observed,

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followed by control set-up trial 3 which coagulated 26 minutes after it was obtained, then,

followed by control set-up trial 1 which had a time elapsed of 36 minutes. The

experimental set-up trial 3 coagulated in the 86 th minute, and then experimental set-up

trial 2 coagulated after 88 minutes and lastly experimental set-up trial 1 was observed to

have the presence of blood coagulation 98 minutes after the blood was freshly obtained.

The coagulation time of the blood in control set-up trials 1 and 3, which were 36

and 26 respectively, were more than the normal time of coagulation, which was 20

minutes, as presented in the review. This few minutes of delay might be caused by the

presence of distilled water, which may sometimes act as an acid and sometimes as a base.

The nature of the distilled water of its basicity and acidity may also have effected the

coagulation time of blood. ( see Review; Water as Amphoteric).

*Statistical Analysis

The two –way Analysis of Variance (ANOVA) is essential to determine whether

or not there is a significant difference between the tested variables. We are also able to

determine if there is a significant difference between the replications as presented on the

data utilized. This two-way ANOVA will be able to lead us to an interpretation if the A.

leptopus flower extract can be used as a blood anticoagulant. Table 2.2 shows the two-

way ANOVA of the time elapsed for the presence of blood coagulation under the

microscope.

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Table 2.2. Two-way Analysis of Variance (ANOVA) for the time (in minutes) elapsed

for the presence of blood coagulation under microscopic observation

Source of

Variance

Degrees of

freedom

Sum of

Squares

Mean

SquaresF value

Table value

(0.01)

Replication 2 217.33 108.67 15.52* 18.00

`Treatment 1 6144 6144 877.71** 21.20

Error 4 28 7 N/A N/A

**-significant *- not significant

As stated above in the table, there are three sources of variance: the replication,

treatment and error. The degrees of freedom of replication and treatment, 2 and 1

respectively, were obtained from the total number of replication of treatments decreased

by 1. The degrees of freedom of error, 4, was from the sum of the total number of

replications and treatments decreased by 1. The sum of squares for replication, treatment,

error were 217.33, 6144, and 28 respectively. The mean squares for replication,

treatment, error were 108.87, 6144, and 7 respectively obtained by dividing the sum of

squares by their respective degrees of freedom. The F value for replication and treatment,

15.52 and 877.71 respectively, were obtained by dividing each mean square by the mean

square of error. The obtained F value for replication, 15.52, is less than the critical value

which is 18.00 at 0.01 level of significance; therefore, the Ho is accepted which states

that there is no significant difference between the time (in minutes) elapsed for the

presence of blood coagulation under microscopic observation in the three trials or

replications. It can then be interpreted that the 3 trials or replications has obtained

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acceptable data from the accepted Ho that the trials have insignificant differences with

each other; hence the three trials had a just and fair data based from the statistical result.

The F value for treatment, 877.71, is greater than the critical value, 21.20, at 0.01

level of significance. So, there is a significant difference between the the time (in

minutes) elapsed for the presence of blood coagulation under microscopic observation of

the experimental set-up, the blood with A. leptopus flower extract, and the control set-up,

the blood with distilled water. It can then be concluded that the A. leptopus flower extract

has an effect on the delay in the the time (in minutes) elapsed for the presence of blood

coagulation under microscopic observation.

Vacuum tube Observation

The naked eye observes and detects the presence of blood clot as the blood

sample is seen on a transparent container like a vacuum tube. The time of the presence

the blood clot had been observed will identify the time by which the blood sample

produces an observable, insoluble blood clot, which is a product of blood coagulation.

Table 3.1 shows the time (in hours) elapsed for insoluble blood clots to be observed as

observed from the outside of the vacuum tube.

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Table 3.1. Time (in hours) elapsed for insoluble blood clots to be visible under vacuum

tube observation

Set-up Trial 1 Trial 2 Trial 3 Average

Experimental 49.18 49.15 16.70 38.34

Control 0.85 0.82 0.82 0.83

Table 3.1 shows the time difference of the time which the blood samples was

observed to have the presence of blood coagulation from the time by which the blood

sample was freshly obtained and was mixed by the reagents. The control set-up trials 2

and 3 obtained the earliest time elapsed which is 0.82 hours for blood clots to be

observed. The remaining trial, trial 3, of the control set-up obtained the next time

difference with 0.85 hours. The trials of the experimental set-up garnered the longest time

that elapsed. Trial 3 of the said set-up had a 16.7 time elapsed in hours. Next with 49.15

hours of elapsed time, trial 2 was observed with blood clots. And lastly, trial 1 had the

longest time difference with 49.18 hours of elapsed time for insoluble blood clots to be

observed.

The very early coagulation of trial 3 of the experimental set-up as compared to the

other trials in the same set-up may possibly be caused by the following events which

occurred during the experiment:

In taking samples for the blood to be used for microscopic observation, a dropper

was just utilized. Because the instrument is not that appropriate for taking blood

specimen, spills, decantation errors and other wasteful scenarios had occurred. So, after

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the microscopic observation was already done, trial 3 was observed to have the least

volume remaining in the tube as compared with the other trials. Since the extract was less

dense than the blood (as it is observed that the extract floats over the blood in the mixing

process), it was more prone to waste and spills. Since the A. leptopus extract is one of the

independent variables, which causes the change in the dependent variable- the time for

blood clots to be visible under vacuum tube observation- it is possible that the less

volume of the extract that remained in the trial caused the early delay of blood clots to be

visible as compared to that of the other trials in the experimental set-up.

* Statistical Analysis

The two –way Analysis of Variance (ANOVA) is essential to determine whether

or not there is a significant difference between the tested variables. We are also able to

determine if there is a significant difference between the replications as presented on the

data utilized. This two-way ANOVA will be able to lead us to an interpretation if the

blood with A. leptopus extract can delay the time elapsed for blood clots to be visible.

Table 3.2 shows the two-way ANOVA of the time elapsed for the presence of blood

coagulation as it is observed in the vacuum tube.

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Table 3.2.Two-way Analysis of Variance (ANOVA) for the time (in hours) elapsed for

insoluble of blood clots to be visible under microscopic observation

Source of

Variance

Degrees of

freedom

Sum of

Squares

Mean

SquaresF value

Table value

(0.01)

Replication 2 351.65 175.83 2.00* 18.00

`Treatment 1 2110.87 2110.87 24.06** 21.20

Error 4 351 87.75 N/A N/A

**-significant *- not significant

As stated above in the table, there are three sources of variance: the replication,

treatment and error. The degrees of freedom of replication and treatment, 2 and 1

respectively, were obtained from the total number of replication of treatments decreased

by 1. The degrees of freedom of error, 4, was from the sum of the total number of

replications and treatments decreased by 1. The sums of squares for replication,

treatment, and error were 351.65, 2110.87, and 351 respectively. The mean squares for

replication, treatment, and error were 108.87, 6144, and 7 respectively obtained by

dividing the sum of squares by their respective degrees of freedom. The F value for

replication and treatment, 2.00 and 24.06 respectively, were obtained by dividing each

mean square by the mean square of error. The obtained F value for replication, 2.00, is

less than the critical value which is 18.00 at 0.01 level of significance; therefore, the Ho

is accepted which states that there is no significant difference between the time (in hours)

elapsed for blood to coagulate under vacuum tube observation in the three trials or

replications. It can

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then be interpreted that the 3 trials or replications had obtained acceptable data from the

accepted Ho that the trials have insignificant differences with each other; hence the three

trials had a just and fair data based from the statistical results.

The F value for treatment, 877.71, is greater than the critical value, 21.20, at 0.01

level of significance. So, there is a significant difference between the the time (in hours)

elapsed for blood to coagulate under vacuum tube observation of the experimental set-up,

the blood with A. leptopus flower extract, and the control set-up, the blood with distilled

water. It can then be concluded that the A. leptopus flower extract has an effect on the

delay in the time (in hours) elapsed for blood to coagulate under vacuum tube

observation..

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CONCLUSIONS AND RECOMMENDATIONS

CONCLUSIONS

Based from the interpretations of the statistical parameter on both time elapsed for

visible blood coagulation under microscopic and vacuum tube observations, there is a

delay in the coagulation perion of blood with A. leptopus flower extract as compared to

that of the distilled water. So, by delaying the coagulation time , it will lengthen the shelf-

life of blood. In summary, Cadena de Amor (Antigonon leptopus) flower extract can be

used as a blood anticoagulant.

RECOMMENDATIONS

Conduct a chemical test on the A. leptopus flower extract to know what

component of the extract causes the anticoagulant activity on human blood

Compare the effectiveness of the extract as blood anticoagulant with existing and

commonly used blood anticoagulants

Let the treated blood undergo in a centrifuge machine to clearly view the size and

presence of the insoluble blood clots

Utilize a more appropriate device or instrument for taking the specimen to be used

in microscopic observation for better accuracy and fairness to each trial

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Conduct a study if the A. leptopus flower extract has an effect to the physical and

chemical characteristics and composition of blood other than coagulation time

which may affect the needed blood components for medical and other purposes

Use a blood sample from one person only throughout the experiment for a more

just experiment

Utilize a more highly powered and technically high microscopes for easy and fast

viewing of blood coagulation; in this case, the microscopic determination would

be easier and faster; thus, it would take a little time and a fixed small time interval

can be utilized in the microscopic observation, which produces a more accurate

result

Conduct another study which focuses on the effectiveness of A. leptopus flower

extract with respect to the ratio of the volume of the extract and the volume of the

blood sample treated.

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BIBLIOGRAPHY

Cabatit (Espino, Belen). Biochemistry. Quezon City: NA, 1985

http://medical-dictionary.thefreedictionary.com/Coagulation+Disorders

http://stuartxchange.com/CadenaDeAmor.html

http://www.naturdoctor.com/Chapters/Research/thrombosis_prevention.html

http://wiki.answers.com/Q/Is_water_a_base_or_an_acid

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