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1
Anticoagulant Screening of Selected
Indigenous Plant Species
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.
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.
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
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.
5
Research Objectives
The study was intended to evaluate the potential of the plant materials in
their anticoagulant abilities on blood type O.
1.
2.
Hypothesis
There is no significant difference in the coagulation time of Hydrocotyl
asiatica, Euphorbia hirta, and Cymbopogon citratus.
6
Significance of the Study
7
Definition of Terms
8
The prothrombin time test was the method used in determining the rate of
clot formation.
9
Chapter 2
Review of Related Literature
10
Part Seven, Cympopogon citratus, describes the physical form, chemical
compounds and importance.
Part Four, Conceptual Framework of the Study.
11
Chapter 2
Review of Related Literature
Blood Coagulation
12
Anticoagulants
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
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
15
Euphorbia hirta
Cympopogon citratus
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)
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
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.
19
Purpose of the Study, Research Design and Layout
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
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.
22
to the plasma in order to counteract the sodium citrate and allow clotting to
proceed (Eldin et al., 2011).
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.
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.
25
Descriptive Data Analysis and Discussion
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. )
26
180
80
60
40
20
14
50
33
0
Control
C.citratus
E.hirta
H.asiatica
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
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
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
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
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.
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.
32
Summary of the Problem
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.
methanolic extracts of 80% Hydrocotyl asiatica, 80% Euphorbia hirta and 80%
Cymbopogon citratus.
34
Conclusions
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.
35
Recommendations
36
References
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
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
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
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
41
APPENDICES
42
APPENDIX A
Communication
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
44
April 3, 2013
MR. AUDIE SULADAY
Head, Central Science Laboratory
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 to allow me to use your laboratory and other
equipment and tools. 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
45
April 8, 2013
MRS. NENITA NACIONAL
Head, Department of Agriculture
Municipality of Ajuy
Ajuy, Iloilo
Madam:
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 identifying the needed plant materials. 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
46
March 8, 2013
MRS. RUBY LYN CHU
Medical Technologist
Barotac Viejo, Iloilo
Madam:
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 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.
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
47
APPENDIX B
List of Experts
48
List of Experts
49
APPENDIX C
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
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
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
53
Post Hoc Test
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
54
APPENDIX D
Observation Guide
55
Observation Guide
type O.
56
Conclusions
Recommendations
57
APPENDIX E
Procedural Flow
58
Gathering of plant
materials
59
APPENDIX F
Certificate of Editing
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):
Other remarks:
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________.
__________________________________
Golda A. Joseco
Research Adviser
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
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
63
APPENDIX G
Certificate of Participation
64
Awards this
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
65
APPENDIX H
Pictorials
66
In vacuo extraction
Filtering
67
68
APPENDIX J
Personal Information of the Researcher
69
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.