Anticoagulant Screening of Selected Indigenous Plant Species

ILOILO NATIONAL HIGH SCHOOL
SPECIAL SCIENCE CLASS

La Paz, Iloilo City
1
Anticoagulant Screening of Selected
Indigenous Plant Species
Salve Regina Berco Babar
Chapter 1
Introduction
Chapter One is divided into five parts: (1) Background of the Study, (2)
Research Objectives and Hypothesis, (3) Significance of the Study, (4) Definition
of Terms, and (5) Delimitation of the Study.
Part One, Background of the Study, presents the introduction to the study,
and discusses the rationale for choosing the problem.
Part Two, Research Objectives and the Hypothesis, describes the
purpose in conducting the study and enumerates the specific objectives of the
research.
Part Three, Significance of the Study, states the benefits that could be
derived from the findings of the study.

La Paz, Iloilo City
2
Part Four, Definition of Terms, lists alphabetically and defines the
research terminologies for purpose of clarity and understanding.
Part Five, Delimitation of the Study, identifies the coverage of the
investigation, in terms of purpose, variables, subjects, research methods,
research instrument, descriptive and inferential statistical tool, and analytical
scheme.
Background of the Study and Theoretical Framework of the Study
Blood coagulation or blood clot is essential for the conservation of blood

and the maintenance of the circulation in all higher animals (University of
Michigan Gateway, 2013). Clot formation may either be a lifesaver in some
situations and a potential killer in others (University of Delaware, 2011).
Hypertension, cerebral hemorrhage, coronary thrombosis,
asterioscleriosis and congestive heart failures are some of the health problems of
most adults that are caused by a problem in the circulatory system as blood
clotting disorders, which constitute a serious medical problem. Non-steroidal anti
inflammatory drugs have been used as antithrobotic agents that are used in vitro

La Paz, Iloilo City
3
and in vivo that causes the inhibition of platelet aggregation and thromboxane
formation (Dey and Bhakta, 2012).
Coagulants and anticoagulants are substances that deal with blood.
Coagulants are used in means of blood loss prevention while the anticoagulant
prevents the blood flow packages. Coagulants are drugs that are designed to
help diseases like, hemophilia in which it helps to facilitate the clotting process. In
contrast, anticoagulants are used to prevent the thickening of blood, thus it is
applied to equipment that routinely holds or transports blood (Conjecture
Corporation, 2013).
In the Philippines, the Department of Health has advocated the availability
of locally available medicinal plants. The National Science Development Board,
had attested that the local plants and herbs in the Philippines backyard and field
have been found to be effective in the treatment of common ailments (Cuevas,
2010).
Research in medicinal plants and their development is crucial because
80% of the population in the developing countries uses traditional medicine for
healing. Plants are used alone or in combination in treating serious bleeding, a
major cause of maternal mortality worldwide (Dandjesso et al., 2012). The
exploration of the wound healing activities of plants in folklore had led to the

La Paz, Iloilo City
4
significant success in investigating it and confirming its acclaimed medicinal
properties. Records have it, that different parts of the plant may contain some
active components that are necessary for wound healing (Esimone et al., 2005).
Understanding the effects of the plant extracts and its effect in blood coagulation
will surely be a lifesaver not only in advance countries but also in under
developed communities.
The study is intended to evaluate the potential anti coagulant activity of
the Hydrocotyl asiatica, Euphorbia hirta and Cymbopogon citratus through an in
vitro assay using prothrombin time test. Also it aimed to determine the time of clot
formation of the plant materials and determine the significant difference in the
prothrombin time when the plasma is treated with plant extracts.

La Paz, Iloilo City
5
Research Objectives
The study was intended to evaluate the potential of the plant materials in
their anticoagulant abilities on blood type O.
1.
To determine the coagulation time of treated blood type O with 80%

concentration of Hydrocotyl asiatica, Euphorbia hirta , and Cymbopogon
citratus.
2.
To determine if there is a significant difference in the coagulation time of

Hydrocotyl asiatica, Euphorbia hirta, and Cymbopogon citratus.
Hypothesis
There is no significant difference in the coagulation time of Hydrocotyl
asiatica, Euphorbia hirta, and Cymbopogon citratus.

La Paz, Iloilo City
6
Significance of the Study
The study could benefit the following:

The local people. This study will be a significant endeavor for the locals to
acknowledge the available resources and knowing there right uses, based on the
findings of this study.
Pharmaceutical Companies. This study will help them recognize the
abundance and cheap sources of new medicinal herbs exhibiting coagulant and
anticoagulant activities. This will also provide sources of potent drug that can be
developed effectively.
The Patients. The results of this study is highly beneficial to stroke
patients, people who have deep vein thrombosis, astheriocleriosis, hemorrhage,
hypercoaguable state, pulmonary embolism, heart attack, coronary thrombosis,
hypertension, congestive heart failure, other illness involving abnormal clotting,
and used in major surgeries. It will also give them access to cheaper and more
effective medicine.
Future Researchers. Future researchers can embark on the isolation of
bioactives and evaluate these in vivo effects.

La Paz, Iloilo City
7
Definition of Terms
Blood clotting, or coagulation, is an important process that prevents

excessive bleeding when a blood vessel is injured. Platelets and proteins in
your plasma work together to stop the bleeding by forming a clot over the injury
(American Society of Hematology, 2012).
Coagulation in blood is essential in maintaining hemostasis in our body.
Blood coagulation disorder is one the primary cause of the many health problems
in adults. This study will test the coagulation time of the blood in vitro treated with
different plant extracts.
Anticoagulant medicines reduce the ability of the blood to clot (coagulation
means clotting). This is necessary if the blood clots too much, as blood clots can
block blood vessels and lead to conditions such as a stroke or a heart attack
(Anticoagulant Medicines, 2013).
Evaluation for potential anticoagulant activities of H. asiatica, E. hirta and
C. citratus was carried out in the study.
Prothrombin Time Test is a blood test that determines or measures the
time it takes plasma to clot. (U.S National Library for Medicine, 2013).

La Paz, Iloilo City
8
The prothrombin time test was the method used in determining the rate of
clot formation.
Delimitations of the Study
The study was conducted for determining the effectiveness of the

difference of the time when the selected plant extracts are joined to the blood
samples.
The subject of the study was the response of the blood when plant
extracts are set into it. The experimental research has two variables, the
independent and the dependent variable. The independent variables were the
plant extracts and distilled water, which is the control group, on the other hand,
the dependent variable was the rate of clot formation.
The study was conducted in Healthschure Laboratory, Barotac Viejo, Iloilo
on June 2013. Blood samples were obtained directly from a healthy respondent.
The respondent was chosen for the following criteria: normal prothrombin time,
not suffering any cardiovascular disease, using nonsteroidal anti inflammatory
drug, not obese, doesnt smoke and free from dyslipidemic disorder.

La Paz, Iloilo City
9
Chapter 2
Review of Related Literature
Chapter Two is divided into eight parts: (1) Blood Coagulation,

(2) Anticoagulants,(3) Prothrombin Time Test, (4) Medicinal Plants, (5) Hydrocotyl
asiatica, (6) Euphorbia hirta (7) Cympopogon citratus and (8) Conceptual
Framework.
Part One, Blood Coagulation, tells the definition, importance and the
cascade of blood coagulation.
Part Two, Anticoagulants, tells its definition and functions.
Part Three, Prothrombin time test, tells its uses and the process
undertaken.
Part Four, Medicinal Plants, explains the importance and economic value
of medicinal plants worldwide.
Part Five, Hydrocotyl asiatica, describes the physical form, chemical
compounds and importance.
Part Six, Euphorbia hirta, describes the physical form, chemical

La Paz, Iloilo City
10
Part Seven, Cympopogon citratus, describes the physical form, chemical
Part Four, Conceptual Framework of the Study.

La Paz, Iloilo City
11
Chapter 2
Review of Related Literature
Blood Coagulation
Blood coagulation is the process of clotting blood (Subbarao, 2012). It

includes two steps, the primary homeostasis or the platelet activation, in this step
the (Bose, 2011) platelets, act as vehicles to concentrate and potentiate
coagulation reactions on the damaged vessels (Bloom, 1990). Next is the
secondary homeostasis, it is the interaction of the clotting factors in a cascade of
reactions that results in the conversion of fibrinogen strands into fibrins strands.
Bose (2011) stated that the aim of this step is to produce thrombin, in which it is
required to cleave fibrinogen into fibrin that provides hemostatic lattice for the
aggregation of platelet and the formation of thrombus at the site of an injury (Lew
and Weaver, 2008).

La Paz, Iloilo City
12
Anticoagulants
Anticoagulants are a type of anti-clotting medicine used to prevent harmful

blood clots in the body (National Prescribing Service, 2013). It is a type of
medication that is used to eliminate or reduce the risk of blood clots. They are
often called blood thinners, but these medicines do not really thin the blood.
Instead, these medications help prevent or break up clots in blood vessels or
heart. It works by stopping the platelets to adhere with each other and clotting
proteins to bind (Healthline, 2013).
Prothrombin Time Test
Prothrombin time test is a useful screening test for the extrinsic

coagulation mechanism (Dey and Bhakta, 2012). The prothrombin time is a test
used to evaluate the ability of the blood to clot. Patients are often tested with the
prothrombin time test before surgery (Heisler, 2011).
In a prothrombin time test, the sample is mixed with tissue thromboplastin
to initiate coagulation. In a traditional prothrombine time test, the complete
activation is indicated when activated thrombin converts fibrinogen to fibrin and

La Paz, Iloilo City
13
extensive or localizes clots are detected (Abbott Point of Care Inc., 2010) This
test determines the cause of bruising or bleeding, and the effectiveness of blood
thinning therapy, and evaluates the functioning of the liver and detects the levels
of blood coagulation factors (MedicalHealthTests, 2009). Each prothrombin time
within the time range 11-15 seconds has a normal clotting time. Prolongation in
prothrombin time is considered abnormal (Dey and Bhakta, 2012).
Medicinal Plants
Research in medicinal plants and their development is crucial because

80% of the population in the developing countries uses traditional medicine for
healing. Plants are use alone or in combination in treating serious bleeding, a
major cause of maternal mortality worldwide (Dandjesso et al., 2012). More than
one third of the Americans use herbs for health purposes yet they, patients and
physicians, lack accurate information for the safety and efficacy of herbal
remedies. However, in Nigeria the local population uses many plants in the form
of ethanol and/or water extracts for treating various elements without
toxicological properties known. Interest in medicinal herbs has increased

La Paz, Iloilo City
14
scientific scrutiny of their therapeutic potential and safety in order to provide
physicians with data (Affone, 2000).
In the Philippines, the Department of Health has advocated the availability
of locally available medicinal plants. The National Science Development Board,
had attested that the local plants and herbs in the Philippines backyard and field
have been found to be effective in the treatment of common ailments (Cuevas,
2010).
Hydrocotyle asiatica
Gotu kola also known as Hydrocotyle asiatica which belongs to the

Apiaceae family (Seevaratnam et al., 2012) is a perennial, herbaceous creeper
with kidney shaped leaves (Thorne Research Inc., 2007). It contains triterpenoid
saponins, including asiaticoside, centelloside, madecassoside, and asiatic acid. It
also contains volatile oils, flavonoids, tannins phytosterols, and amino acids
(Thorne Research Inc., 2007). It has also exhibits potential antioxidant,
antimicrobial, cytotoxic, neuroprotective activities. It is distributed all throughout
the tropical and subtropical regions of the world (Seevaratnam et al., 2012).

La Paz, Iloilo City
15
Euphorbia hirta
E. hirta belongs to the plant family Euphorbiaceae and genus Euphorbia. It

is a slender- stemmed, annual hairy plant, spreading up to 40 cm in height,
reddish or purplish in color. Leaves are opposite, elliptic - oblong to oblonglanceolate, acute or subacute, dark green. The fruits are yellow, three- celled,
hairy, keeled capsules, 1-2 mm in diameter, containing three brown, four-sided,
angular, wrinkled seeds. E. Hirta contain alkanes, triterpenes, phytosterols,
tannins, polyphenols, and flavanoids (Kumar et al., 2010).
Cympopogon citratus
Cympopogon citratus are indigenous in tropical and semi tropical areas in

Asia cultivated in South and Central America, Africa and other tropical countries
(Kumar et al., 2010). This tropical grass grows in dense clumps that can grow to
6 ft (1.8 m) in height and about 4 ft (1.2 m) in width, with a short rhizome. The
strap like leaves are 0.5-1 in wide about 3 ft long. The evergreen leaves are
bright bluish green that releases a citrus aroma. This plant does not produce
flowers. The Cymbopogon citratus is used a flavoring and fragrance and in folk

La Paz, Iloilo City
16
medicine. The compounds identified in the plant are terpenes, alcohols, ketones,
aldehyde and esters. Some of the reported phytoconstituents are essential oils
that contain Citral , Citral , Nerol Geraniol, Citronellal, Terpinolene, Geranyl
acetate, Myrecene and Terpinol Methylheptenone. It also contains flavonoids and
phenolic compounds (Shah et al.,2011)

La Paz, Iloilo City
17
Conceptual Framework
seconds
Independent Variables
Plant Extracts
80% Methanolic Extract Of:
Dependent Variable
Hydrocotyle asiatica
Prothrombin time in
seconds
Euphorbia hirta
Cympopogon citratus
Controlled Variable
Distilled water
Figure 1. The different variables used in the study.

La Paz, Iloilo City
18
Chapter 3
Methodology
Chapter 3 is divided into five parts: (1) Purpose of the Study, Research
Design and Lay-out (2) The Subject/Respondents of the Study (3) Research
Instrument (4) Procedure and (5) Statistical Data Analysis Procedure.
Part One, Purpose of the study, Research Design and Lay-out, states the
purpose of the study, describes the research design and the variables used in
the study, and shows the lay-out of the experiment.
Part Two, Materials and Equipment, enumerates the materials with their
costs and specification of quantity used in the experiment.
Part Three, Procedure, describes the subject of the study and discusses
the details of the steps of procedures to be undertaken in the conduct of the
research. It includes laboratory or field procedures that were employed in the
study.
Part Four, Data and Data Gathering Procedure, enumerates the different
data to be gathered and describes the process of data gathering.
Part Five, Statistical Analysis Procedures, discusses the different
descriptive and inferential statistical tools used in testing the study.

La Paz, Iloilo City
19
Purpose of the Study, Research Design and Layout
Anticoagulant evaluation of Hydrocotyle asiatica, Cympopogon citratus

and Euphorbia hirta was carried out in the study using prothrombin time test.
Experimental research had two variables, the independent and the
dependent variables. The independent variables comprised the selected
indigenous plant species found in the province of Iloilo. The dependent variable
was the prothrombin time of the blood.
The following treatments were used in the study:
Methanolic Extract of:

Treatment A: 80% Hydrocotyle asiatica
Treatment B: 80% Euphorbia hirta
Treatment C: 80% Cymbopogon citratus
The study was organized using the Completely Randomized Design in

three replications. This design was used in order for each unit to receive equal
share in receiving the same specified treatments. For the Completely
Randomized Design, any difference among experimental units receiving the

La Paz, Iloilo City
20
same treatment was considered an experimental error. Completely Randomized
Design is appropriate for experiments with homogeneous experimental units
such as laboratory experiments where environmental effects are relatively easy
to control.
The Procedure
A. Plant Material
Fresh Hydrocotyle asiatica, Euphorbia hirta, Cymbopogon citratus, were
gathered in Ajuy, Iloilo. The botanical identity of the plant species were confirmed
by Mrs. Nenita Nacional, Head of The Department of Agriculture, Municipality of
Ajuy.
B. Preparations of Extracts
Fresh and recently cropped Hydrocotyle asiatica, Euphorbia hirta and
Cympopogon citratus were washed with fresh water and plants having
mechanical injury were disposed. Plant materials were air dried at room
temperature for several weeks, until it attained a crispy state. After complete
drying, the slices were grinded into a fine powder. The powder was weighed
using sensitive balance and then suspended in methanol solution in a conical

La Paz, Iloilo City
21
flask with continuous shaking for 48hours. The supernatant of Hydrocotyle
asiatica, Euphorbia hirta and Cympopogon citratus extract were filtrated using
filter paper. The filtrates were then directed for rotary evaporation until solid
paste. The attained solid paste was then diluted in distilled water to prepare 80%
concentration.
C. Collection of the Blood Samples

The blood samples were placed separately in containers containing trisodium citrate to prevent the clotting process. Centrifugation (15 minutes at rate
3000 rpm) was carried out to separate the blood cells from plasma in order to
obtain pure platelet plasma (ppp) for prothrombin time test. The obtained plasma
sample of each individual was poured separately in plane containers using
automatic pipette and stored at room temperature.
D. In vitro anticoagulant test of the plant materials

0.2 ml plasma, 0.1 ml of crude extract of different concentration and
calcium thrombosplastin were added together in a clean fusion tube and pre
incubated at 37C in water bath. For control experiment extract solution was
replaced by same volume of distilled water. Thromboplastin reagent was added

La Paz, Iloilo City
22
to the plasma in order to counteract the sodium citrate and allow clotting to
proceed (Eldin et al., 2011).
E. Determination of Prothrombin Time (PT)

Two glasses of hemolysis tubes, one for testing and one for the witness
were used. 100 l of plasma was put in each tube. 10 l of extract solution to
10% was added in the test tube and 10 l of distilled water was added to the
control tube. The mixture was incubated for 1 minute at 37C. Added to each
tube was 200 l of calcium thromboplastin,pre incubated for 10 mins at 37 C .
The clotting time was measured by tilting the tubes at an angle of 45 every 30
seconds to recognize the presence or absence of coagulum. For each sample,
the test was repeated five times and the average time was calculated
(Dandjesso, 2012).

La Paz, Iloilo City
23
Statistical Data Analysis Procedure
The data obtained from the study were subjected to the following
descriptive and inferential statistical treatments using the Statistical Package for
Social Sciences (SPSS) Software. The statistical tools were the following:
Mean. The obtained mean was use to determine pro-coagulant activities
of the selected plant species.
Standard Deviation. Determines the dispersion of the means, standard
deviation was used.
Duncans Multiple Range Test. DMRT was used to determine if there is a
significance in the filtration rates of the Lampirong.
One Way Analysis of Variance. Analysis of Variance was one of the
statistical tools mostly used in analyzing experimental data. The One Way
Analysis of Variance was used in determining the variance.
All statistical computations were computer-processed using the Statistical
Package for Social Sciences (SPSS) software set at 0.05 level of significance.

La Paz, Iloilo City
24
Chapter 4
Results and Discussions
Chapter 4 is divided into two parts: (1) Descriptive Data Analysis and
Discussion, and (2) Inferential Data Analysis and Discussion.
Part One, Descriptive Data Analysis, provides the results of the mean and
standard deviation.
Part Two, Inferential Data Analysis and Discussion, provides the statistical
computation in ANOVA and discusses the significant differences on the
prothrombin time test when blood is treated with different extracts.

La Paz, Iloilo City
25
Descriptive Data Analysis and Discussion
The anti-coagulant activities of the methanolic extracts of 80%

Hydrocotyle asiatica, 80% Cympopogon citratus and 80 % Euphorbia hirta were
carried out through prothrombine time test. The table below shows the mean and
the standard deviation.
Table 1
Prothrombin time test using of the extracts.
Prothrombin Time in seconds
Sample
Mean*
SD
R1
R2
R3
Control
14.1
13.6
13
13.56 d
0.550757
C. citratus
29
33.5
36
32.83 c
3.547299
E. hirta
180
180
180
180.00 a
H. asiatica
45
45
13
13.56 b
0.550757
*Column mean with common letter superscripts are not significantly different at
=0.05 by One-Way ANOVA and Duncan Test (See Appendix p. )

La Paz, Iloilo City
26
Average Prothrombin Time

180
160
140
120
100
180
80
60
40
20
14
50
33
0
Control
C.citratus
E.hirta
H.asiatica
Figure 2. Average Prothrombin Time
Human blood was treated with the methanolic extracts of 80%

Hydrocotyl asiatica, 80% Euphorbia hirta and 80% Cymbopogon citratus and was
carried out through prothrombin time test. The results showed that Treatment 3
showed the longest prolongation time at 180 s for the clotting and Treatment 1
showed the shortest.
Statistical analysis by One-Way ANOVA revealed that the p value,
0.000 is less than =0.05. Therefore there is a significant difference in the

La Paz, Iloilo City
27
clotting time of the methanolic extracts of 80% Hydrocotyl asiatica, 80%
Euphorbia hirta and 80% Cymbopogon citratus.
Table 2.
ANOVA Test Results
Sum of
Mean of
Df
Squares
Sig
565.207
.000
Squares
Between
51385.777
17128.592
242.440
30.305
51628.217
11
Groups
Within
Groups
Total

La Paz, Iloilo City
28
The Duncans test revealed that E.hirta having the longest prothrombin
time differs significantly in the treatment means.
Table 3.
Duncans Post Hoc Test Result
Subset for alpha = 0.05

S
N
Control
1
3
13.5667
Cymbopogon
3
32.8333
citratus
Hydrocotyl
3
48.3333
asiatica
Euphorbia
3
180.0000
hirta
Sig.
1.000
1.000
1.000
1.000

La Paz, Iloilo City
29
Discussion
One good tool in preventing conditions such as strokes and heart attacks
is reducing the bloods ability to clot. People who have history of these kinds of
conditions or who are at risk of developing them are given anticoagulants
(Webvision,2009). This study had used in vitro, prothrombin time test that is used
to measure how long it takes for blood to clot. It is also used in checking whether
the medicine used to prevent blood clots is working (WebMD, 2010). The study
had used 80% concentration of Hydrocotyl asiatica, Euphorbia hirta and
Cymbopogon citratus instead of using an anticoagulant medicine in the blood.
The normal prothrombin time of a person who doesnt take anticoagulant
medicines is in the range of 11-13.5 seconds (U.S National Library for
Medicine,2013). However, the results showed that Euphorbia hirta possessed
the greatest anticoagulant properties having a terminated average time of 180
seconds, Hydrocotyl asiatica 50 seconds, Cymbopogon citratus 32 seconds and
the quickest time was the controlled variable, distilled water 14 seconds.
According to the literature gathered, plant materials are used worldwide for
medicinal purposes and the National Science Development Board had also
attested that local plants have been found effective on the treatment of common

La Paz, Iloilo City
30
ailments. The selected plant materials are also popular because of its wound
healing properties and the wide use of it in medicinal folklores.
The isolation, purification and evaluation of the bioactives with
anticoagulating properties for drug development is deliberated.

La Paz, Iloilo City
31
Chapter 5
Summary, Conclusions, Implications, and Recommendations
Chapter Five is divided into three parts: (1) Summary of the Problem,
Methods and Findings, (2) Conclusions and Implications and (3)
Recommendations.
Part One, Summary of the Problem, Methods and Findings, states the
points and findings of the study.
Part Two, Conclusions and Implications, presents the interference drawn
from the results of the study. It also clarifies the relationship between the findings
of the study and the theories that were presented previously.
Part Three, Recommendations, offers suggestions in terms of findings,
conclusions, and implications.

La Paz, Iloilo City
32
Summary of the Problem
The ability of the methanolic extracts of 80% Hydrocotyl asiatica, 80%

Euphorbia hirta and 80% Cymbopogon citratus to act as anticoagulant was
tested using the prothrombin time test. The plant materials were gathered in
Ajuy, Iloilo last May 2013. Extractions of plant materials were done on May 2013
at West Visayas State University under the supervision of Dr. Gerard Penicillia
and Mr. Audie Saluday. The plant extracts were prepared using methanol
solutions and distilled water for the controlled variable. The methanolic solutions
were then prepared to 80% concentrations. Determination of prothrombin time
test was carried out in Healthschure Laboratory, Barotac Viejo, Iloilo on June
2013. It was supervised by Mrs. Ruby Lyn Chu, Medical Technologist.
Treatments were laid out in a Completely Randomized Design (CRD) with three
treatments having three replications each. Exprerimental group were identified
as: 80% Hydrocotyl asiatica, 80% Euphorbia hirta and 80% Cymbopogon
citratus. Distilled water was used as the control group.

La Paz, Iloilo City
33
Specifically, it sought to:
Determine the prothrombin time of the 80% methanolic extracts of
Cymbopogon citratus, Euphorbia hirta and Hydrocotyl asiatica compared to the
control treated with EDTA.
Determine significant differences in the prothrombin time in relation to the
treatment used.
Findings
1.
The results showed that Euphorbia hirta possessed the greatest
anticoagulant properties having a terminated average time of 180 seconds,

Hydrocotyl asiatica 50 seconds, Cymbopogon citratus 32 seconds and the
quickest time was the controlled variable, distilled water 14 seconds. This means
that the evaluated plant materials possess anticoagulant properties which can be
developed into a medicinal drug.
2.
There is a significant difference in the clotting time of the
methanolic extracts of 80% Hydrocotyl asiatica, 80% Euphorbia hirta and 80%
Cymbopogon citratus.

La Paz, Iloilo City
34
Conclusions
Methanolic extracts of 80% Hydrocotyl asiatica, 80% Euphorbia hirta, and

80% Cymbopogon citratus exhibited anticoagulant activities. Based on their
prothrombin time, the 80% Euphorbia hirta showed the longest prolongation of
blood coagulation and Cymbopogon citratus showed the least. Nevertheless, all
showed a significant difference in prolonging the blood coagulation thus the
selected plant material can be used as anticoagulant.
For Theory
The presence of bioactives like saponins, flavonoids, tannins, phytoserols,
polyphenols, triterpenes and alkanes.
For Practice
Based upon the gathered results, the selected plant materials exhibited a
significant difference in prolonging the blood coagulation. Thus it can be
concluded that the selected indigenous plant materials is an efficient
anticoagulant.

La Paz, Iloilo City
35
Recommendations
Based on the findings and conclusions, the following recommendations are

formulated:
1. Use different concentrations of the extracts
2. Elucidation, isolation and purification of the bioactive compounds that
exhibits anticoagulant activity for drug development.

La Paz, Iloilo City
36
References
Abbott Point of Care Inc. [A.P.C.I.].(2010). Prothrombin Time Test. Retrieved on

February 24,2013 from http://www.medicalhealthtests.com/blog
/coagulation-studies/coagulation-studies.html
Affone.(2000).Richters Herb Letter. Retrieved on December 7,2012 from
http://www.richters.com/show.cgi?page=HL/20020507-1.html
American Society of Hematology [A.S.H.].(2012). Blood Clots. Retrieved on
February 25,2013 from http://www.hematology.org/patients/blooddisorders/blood-clots/5233.aspx
Anticoagulant Medicines.(2013).Retrieved on December 7,2012 from
http://www.nhs.uk/conditions/anticoagulantmedicines/Pages/Introduction.aspx
Bloom, A. (1990). Physiology of Blood Coagulation. Retrieved on December
6,2012 from http://www.ncbi.nlm.nih.gov/pubmed/2083865
Bose, D. (2011). Blood Clotting Process. Retrieved on December 6,2012 from
http://www.buzzle.com/articles/blood-clotting-process.html

La Paz, Iloilo City
37
Conjecture Corporation.(2013). What Is The Difference Between Coagulants And
Anticoagulants?.Retrieved on January 5,2013 from
http://www.wisegeek.com/what-is-the-difference-between-coagulants-andanticoagulants.htm
Cuevas.(2010). Herbal Medicines in the Philippines. Retrieved on January
19,2013 from http://www.scribd.com/doc/33687052/Herbal-Medicines-inthe-Philippines
Dandjesso, C.; Kloto, J.; Dougnon, T.V.; Sgbo, J.; Atgbo, M.; Gbaguidi, F.;
Fah, L.; Fanou; B.; Loko, F. and Dramane, K.(2012). Phytochemistry and
hemostatic properties of some medicinal plants sold as anti-hemorrhagic
in Cotonou markets (Benin). Retrieved on February 24, 2013 from
http://www.indjst.org/archive/Aug-12/4-2170-dandjesso-aug12.pdf.
Dey, P. and Bhakta, T. (2012). Evaluation of in vitro anticoagulant activity of
Molineria recurpata leaf extract. Retrieved on January 19,2013 from
http://scholarsresearchlibrary.com/JNPPR-vol2-iss6/JNPPR-2012-2-6685-688.pdf

La Paz, Iloilo City
38
Esimone,C.; Ibezim, E. and Chah, K. (2005) The Wound Healing Effect Of Herbal
Ointments Formulated Wilh Napoleona Imperialis. Retrieved on January
19,2013 from http://indexmedic
us.afro.who.int/iah/fulltesxt/jophas3.7esimone.pdf
Healthline.(2013). Anticoagulant and Antiplatelet Drugs. Retrieved on February
24, 2013 from http://www.healthline.com/health/anticoagulant-andantiplatelet-drugs
Heisler, A. (2011). Prothrombin Time Test. Retrieved on January 6,2013 from
http://www.heisler.medlineplus/ency/article/003689/heisler.htm
Kumar, S.; Kumar, D. and Malhotra R. (2010). Euphorbia hirta: Its chemistry,
traditional and medicinal uses, and pharmacological activities. Retrieved
on Decemeber 05, 2012 from http://www.ncbi .nlm.nih.go v/pmc/art
icles/PMC3249903/
Lew, W. and Weaver, F. (2008). Clinical use of topical thrombin as a surgical
hemostat. Retrieved on February 23,2013 from
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2727895/
Medical Health Tests.(2009). What Does Coagulation Studies Include? Retrieved
on February 23,2013 from http://www.medicalhealthtests.com /blog/coagu
lation-studies/coagulation-studies.html

La Paz, Iloilo City
39
National Prescribing Service [N.P.S.].(2013). Anticoagulant Medicines. Retrieved
on February 24,2013 from http://www.nps.org.au/medicines/heart-bloodand-blood-vessels/anti-clotting-medicines/for-individuals/anticoagulantmedicines
Seevaratnam, V.; Bahumathi, P.; Premalatha, M.R.; Sundaram, S.P. and
Arumugam, T. (2012). Functional Properties of Centella asiatica (L.): A
Review. Retrieved on December 8, 2012 from http://www.ijppsjournal.com
/Vol4Suppl5/4798.pdf
Shah, G.; Shri, R.; Panchal, V.; Sharma, N.; Singh, B. and Mann, A. (2011).
Scientific basis for the therapeutic use of Cymbopogon citratus, stapf
(Lemon grass). Retrieved on December 5, 2012 from
http://www.ncbi.nlm.n ih.gov/pmc/articles/PMC3217679/
Subbarao, A. (2012).Coagulation Cascade. Retrieved on February 24, 2013 from
http://www.doctorshangout.com/profiles/blogs/coagulation-cascade-thephysiology-of-blood-clotting
Thorne Research Inc. [T.R.I.]. (2007). Centella asiatica. Retrieved on December
7,2012 from http://www.anaturalh ealingcenter.com/docume
nts/Thorne/mono s/Centell a-asiat ica-mono_12.1.pdf

La Paz, Iloilo City
40
U.S National Library of Medicines. (2013). Prothrombin time (PT). Retrieved on
February 04,2013 from http://www.nlm.nih.gov/medlineplus
/ency/article/003652.htm
University of Delaware [U.D.].(2011). Prof Presents Blood-Clotting Studies at
Gordon Research Conference. Retrieved on January 19,2013 from
http://www.udel.edu/udaily/2011/mar/naik-blood-clotting-032211.html
University of Michigan Gateway [U.M.G.].(2013). Molecular Genetics of Blood
Clotting. Retrieved on January 19,2013 from http://www.lsi.umich.ed
u/ginsburg-lab/ginsburg-research-block
WebMD.(2013). Information and Resources: Prothrombin Time Test. Retrieved
on February 1,2013 from http://www.webmd.com/a-to-zguides/prothrombin-time
Webvision.(2009). Anticoagulant Medicines. Retrieved on August 15, 2013 from
http://www.nursingtimes.net/whats-new-in-nursing/anticoagulantmedicines/1985118.article

La Paz, Iloilo City
41
APPENDICES

La Paz, Iloilo City
42
APPENDIX A
Communication

La Paz, Iloilo City
43
April 3, 2013
DR. GERARD PENICILLA
Chair, Biological Science Department
West Visayas State University
La Paz, Iloilo City
Sir:
Greetings of peace and prosperity!
The undersigned is a Fourth Year High School student of Iloilo National High- School Special
Science Class currently conducting a research on Anticoagulant Screening of Selected
Indigenous Plant Species, in partial fulfilment of the requirements for graduation.
In this regards, may I request your permission in extracting the plant materials in your laboratory
under your supervision. Approval to this will greatly contribute to the completion of the study.
Thank you very much and God bless.
Respectfully yours,
SALVE REGINA B.BABAR
Researcher
Noted:
GOLDA A. JOSECO
Research Adviser
EDWIN F. MOSURA
Department Head, Research
Recommending Approval:
NORDY D. SIASON JR., Ed. D; C.E.S.E
Principal IV
Conforme:
_______________________________
ROSE R. UMADHAY
Program Head, Special Science Class

La Paz, Iloilo City
44
April 3, 2013
MR. AUDIE SULADAY
Head, Central Science Laboratory
West Visayas State University
La Paz, Iloilo City
Sir:
In this regards, may I request your permission to allow me to use your laboratory and other
equipment and tools. Approval to this will greatly contribute to the completion of the study.
Respectfully yours,
Researcher
Noted:
GOLDA A. JOSECO
Research Adviser
EDWIN F. MOSURA
Principal IV
Conforme:
_______________________________
ROSE R. UMADHAY

La Paz, Iloilo City
45
April 8, 2013
MRS. NENITA NACIONAL
Head, Department of Agriculture
Municipality of Ajuy
Ajuy, Iloilo
Madam:
In this regards, may I request your permission in identifying the needed plant materials. Approval
to this will greatly contribute to the completion of the study.
Respectfully yours,
Researcher
Noted:
GOLDA A. JOSECO
Research Adviser
EDWIN F. MOSURA
Principal IV
Conforme:
_______________________________
ROSE R. UMADHAY

La Paz, Iloilo City
46
March 8, 2013
MRS. RUBY LYN CHU
Medical Technologist
Barotac Viejo, Iloilo
Madam:
In this regards, may I request your permission for your approval to supervise me in conducting the
prothrombin time test and to use your laboratory. Approval to this will greatly contribute to the
completion of the study.
Respectfully yours,
Researcher
Noted:
GOLDA A. JOSECO
Research Adviser
EDWIN F. MOSURA
Principal IV
Conforme:
_______________________________
ROSE R. UMADHAY

La Paz, Iloilo City
47
APPENDIX B
List of Experts

La Paz, Iloilo City
48
List of Experts
Dr. Gerard Penicillia
Mr. Audie Saluday
Mrs. Ruby Lyn Chu R.M.T
Mrs. Nenita Nacianal

La Paz, Iloilo City
49
APPENDIX C
Raw Data and Statistical Computations

La Paz, Iloilo City
50
Raw Data
Plant
Control
Concentratio
n
Plasm
a
Amt.
of
Extrac
t
Ca
Thromboplasti
n
Prothrombi
n Time
(L)
(L)
(L)
(s)
100
10
200
14.1
100
10
200
13.6
100
10
200
13
(L)
(L)
(L)
(s)
80
100
10
200
29
80
100
10
200
33.5
80
100
10
200
36
(L)
(L)
(L)
(s)
80
100
10
200
180
80
100
10
200
180
80
100
10
200
180
(L)
(L)
(L)
(s)
80
100
10
200
45
80
100
10
200
40
80
100
10
200
60
Cymbopogo
n citratus
Euphorbia
Hirta
Hydrocotyl
asiatica
Amt. of

La Paz, Iloilo City
51
Common
Scientific Name
Container
Quantity
Solvent
Lemon Grass
2.1
50g
Methanol
Euphorbia hirta
Tawa-tawa
1.2
50g
Methanol
Hydrocotyl
Yahong1.3
50g
Methanol
Name
Cymbopogon
Citratus
asiatica
yahong
Weight of
Weight of
Concentration
Sample(g)
Water(g)
(%)
0.2933
0.068
80
1.0052
0.26
80
1.8931
0.473275
80
Sample
Cymbopogon
citratus
Euphorbia hirta
Hydrocotyl
asiatica

La Paz, Iloilo City
52
Statistical Computations
One Way ANOVA
Sum of
Mean of
Df
Squares
Sig
565.207
.000
Squares
Between
51385.777
17128.592
242.440
30.305
51628.217
11
Groups
Within
Groups
Total
Ona Way ANOVA showed that p value 0.000 =0.05. Therefore

there is a signifant difference in the clotting time of the methanolic extracts of
80% Hydrocotyl asiatica, 80% Euphorbia hirta and 80% Cymbopogon citratus.

La Paz, Iloilo City
53
Post Hoc Test
Subset for alpha = 0.05

S
N
Control
1
3
13.5667
Cymbopogon
3
32.8333
citratus
Hydrocotyl
3
48.3333
asiatica
Euphorbia
3
180.0000
hirta
Sig.
1.000
1.000
1.000
1.000
Means for groups in homogeneous subsets are displayed
a. Uses Harmonic Mean Sample Size = 3.000

The Duncans test revealed that E.hirta having the longest prothrombin
time differs significantly in the treatment means.

La Paz, Iloilo City
54
APPENDIX D
Observation Guide

La Paz, Iloilo City
55
Observation Guide
Pro and Anti Coagulant Screening of Selected Indigenous Plant Species
Objectives and Problem of the Study
The study is intended to evaluate the

potential of the plant materials in
their anticoagulant abilities on blood
To evaluate the
type O.
anticoagulant activity of the 80%

methanolic extract Cymbopogon
To determine the coagulation in
citratus, 80% Euphorbia hirta
time of treated blood type O with
and 80% Cymbopogon citratus.
80% concentration of Hydrocotyl

asiatica, Euphorbia hirta , and
To determine if there is a
significant difference in the
coagulation time of Hydrocotyl
asiatica, Euphorbia hirta, and

La Paz, Iloilo City
56
Results and Discussion
Methanolic extract of 80% Euphorbia hirta exhibited the longest time in

prolonging blood coagulation while the methanolic extract of 80%
Cymbopogon citratus exhibited the least. Nevertheless, all showed a
significant difference in prolonging the time it takes for blood to clot.
Conclusions
Methanolic extracts of 80% Hydrocotyl asiatica, 80% Euphorbia hirta, and

80% Cymbopogon citratus exhibited excellent anticoagulant activities. Thus
the selected plant materials can be used as an anticoagulant and can be
developed into a potential anticoagulant drug.
Recommendations
1. Use different concentrations of the extracts.

2. Elucidation, isolation and purification of the bioactive compounds that
exhibits anticoagulant activity for drug development.

La Paz, Iloilo City
57
APPENDIX E
Procedural Flow

La Paz, Iloilo City
58
Gathering of plant
materials
In vacuo extraction of the plant

materials.
Preparation of blood samples

for Prothrombin time test.
Record the prothrombin

time

La Paz, Iloilo City
59
APPENDIX F
Certificate of Editing

La Paz, Iloilo City
60
CERTIFICATION
This is to certify that I have checked and edited the manuscript of Salve
Regina Berco Babar last ________________ and found it to be (Please check):
________________Complete and ready for final printing
________________Complete but needs revision
________________Incomplete and needs revision
Other remarks:
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________.
__________________________________
Golda A. Joseco
Research Adviser

La Paz, Iloilo City
61
CERTIFICATION
This is to certify that I have checked and edited the manuscript of Salve
Regina Berco Babar last ________________ and found it to be (Please check):
Other remarks:
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________.
__________________________________
Cynthia P. Superficial
Grammarian

La Paz, Iloilo City
62
CERTIFICATION
This is to certify that I have checked and edited the manuscript of Salve Regina
Berco Babar last ________________ and found it to be (Please check):
Other remarks:
________________________________________________________________
________________________________________________________________
________________________________________________________________
_______________________________________________________________.
__________________________________
Portia J. Estorque
Statisticain

La Paz, Iloilo City
63
APPENDIX G
Certificate of Participation

La Paz, Iloilo City
64
Republic of the Philippines

Department of Education
Region VI Western Visayas
Duran St., Iloilo City
Awards this
to the entry entitled

Anticoagulant Screening of Selected
Indigenous Plant Species.
Submitted by
Salve Regina Berco Babar
to the
on September 26-28, 2013.
Given this 28th day of September 2013
at the Iloilo National High School
La Paz, Iloilo City.
JOHN ARNOLD S. SIENA
Schools Division Superintendent
Officer-In-Charge, Office of the Director
Regional Office VI

La Paz, Iloilo City
65
APPENDIX H
Pictorials

La Paz, Iloilo City
66
Air drying of the plant
Pounding of the plant materials
materials until crispy dry.
Soaking for 48 hrs in methanol solvent
In vacuo extraction
Filtering
Dilution of the solid paste

La Paz, Iloilo City
67
80% methanolic extracts

Centrifugated blood. Plasma is used in
the study
Mixing the plasma and the extract.
Tilting the plasma at a; 45 angle every 30

seconds to recognize the presence or
absence of a coagulum. Recorded time is
the prothrombin time.

La Paz, Iloilo City
68
APPENDIX J
Personal Information of the Researcher

La Paz, Iloilo City
69
Research Title: Anticoagulant Screening of Selected Indigenous Plant Species

Name: Salve Regina Berco Babar
Birth date: August 9, 1997
Address: Real St., Ajuy, Iloilo
Father: Melquiades Bacabac Babar
Occupation: Mechanical Engineer, Councilor
Mother: Rita Palomata Berco
Occupation: Physician

La Paz, Iloilo City
70
Educational Background:
Elementary: Ajuy Christian Development Academy
High School: Iloilo National High School Special Science Class
25 years from now, Ill be walking in my Louis Vuitton stilletos in
Manhattan where my Architectural-Engineering Empire is located. Having multimillion contracts both wih the private and public sector including with the United
Nations. My company is also the largest company to have built free
hospitalization and housing in third world countries also one of the advocates for
world peace.
I know with Jesus Christ, nothing is impossible.

Anticoagulant Screening of Selected Indigenous Plant Species

Încărcat de

Informații document

Drepturi de autor

Formate disponibile

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Formate disponibile

Anticoagulant Screening of Selected Indigenous Plant Species

Încărcat de

Drepturi de autor:

Formate disponibile

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

Salve Regina Berco Babar

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

Background of the Study and Theoretical Framework of the Study

Blood coagulation or blood clot is essential for the conservation of blood

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

To determine the coagulation time of treated blood type O with 80%

To determine if there is a significant difference in the coagulation time of

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

The study could benefit the following:

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

Blood clotting, or coagulation, is an important process that prevents

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

Delimitations of the Study

The study was conducted for determining the effectiveness of the

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

Chapter Two is divided into eight parts: (1) Blood Coagulation,

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

Blood coagulation is the process of clotting blood (Subbarao, 2012). It

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

Anticoagulants are a type of anti-clotting medicine used to prevent harmful

Prothrombin Time Test

Prothrombin time test is a useful screening test for the extrinsic

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

Research in medicinal plants and their development is crucial because

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

Gotu kola also known as Hydrocotyle asiatica which belongs to the

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

E. hirta belongs to the plant family Euphorbiaceae and genus Euphorbia. It

Cympopogon citratus are indigenous in tropical and semi tropical areas in

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

Figure 1. The different variables used in the study.

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

Anticoagulant evaluation of Hydrocotyle asiatica, Cympopogon citratus

Methanolic Extract of:

The study was organized using the Completely Randomized Design in

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS

ILOILO NATIONAL HIGH SCHOOL

SPECIAL SCIENCE CLASS