Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
Proponents:
Raganas, Savannah Zephorah
Paraon, James Francis
Delicano, Kaye Malou
Piala, Alliza
Ms. Yvonne Lynn V. Torres
Research Adviser
Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
ABSTRACT
Blood coagulating disorders such as haemophilia, poses a threat to people
afflicted with it around the world. The two major forms of haemophilia occur more in
males than in females. The proponents are trying to find a certain plant that can also aid
in blood clot and widen the range of potential medicinal plants that aids in these kinds of
disorders. Kalachuchi is a plant commonly found in the Philippine and within the tropics.
Its seed is considered to be hemostatic for it possesses the component called tannin.
There were 4 Set-ups labeled A, B, C and D. 3ml of fresh blood was obtained from
domestic pig using a syringe. The blood was instantaneously transferred to a sterilized
test tube. Each of the three test tubes in every set-up received blood to an equal
amount of 0.1ml.
Each test tube in every set-up received an equal amount of 0.1 ml blood.
Set-up A received 0.1ml pure extract of P. acuminata seed extract. Set-up B received
0.075ml pure extract of P. acuminata seed extract. Set-up C received 0.50ml of pure
extract of P. acuminata seed extract while Set-up D received none. Set-up D will be the
basis of the pig’s blood normal clotting time. Each set-up or treatments has 3 replicates.
The resulting mixture were observed within a certain period of time to determine the
effectiveness of the pure extract for blood coagulation by recording its clotting time.
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Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
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Table of contents
Chapter I
Background of the Study ………………………………………… Page 5
Significance of the Study…………………………………………. Page 6
Statement of the Objectives……………………………………... Page 7
Hypotheses………………………………………………………… Page 7
Scope and Limitations………………………………………..…... Page 8
Statement of the Objectives……………………………………… Page 9-12
Review of Related Literature…………………………………….. Page 13-19
Chapter II
Methodology……………………………………………………….. Page 20-21
Chapter III
Results and Discussion ………………………………………….. Page 22-24
Chapter IV
Conclusion ………………………………………………………… Page 25
Chapter V
Recommendations………………………………………………… Page 26
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Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
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Chapter VI
Acknowledgement………………………………………………… Page 27-28
Chapter VII
References …………………………………………………………Page 29-30
Chapter VII
Appendices………………………………………………………… Page 31-34
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Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
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Chapter I
BACKGROUND OF THE STUDY
Kalachuchi (Plumeria acuminata) is a small, deciduous tree, 3 to 7 meters high,
with a crooked trunk, smooth and shining stems, succulent, with abundant sticky, milky
latex. It is also used as medicinal plant native to Mexico, Central America, the
Caribbean and South America spread throughout the tropics. About 155 genera and
2000 species are distributed primarily in the tropical and subtropical region. Kalachuchi
has a variety of medicinal properties. The plant material is widely used as a purgative,
remedy for diarrhea and cure for itch. The milky juice is employed for the treatment of
inflammation and rheumatism. The bark has been reported to be useful in hard tumors,
diarrhea and gonorrhea.
Bleeding, or hemorrhage, is the name used to describe blood loss. It can refer to
blood loss inside the body, called internal bleeding. Or it can refer to blood loss outside
of the body, called external bleeding. Blood loss can occur in almost any area of the
body. Internal bleeding occurs when blood leaks out through a damaged blood vessel or
organ. External bleeding happens when blood exits through a break in the skin.
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Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
SIGNIFICANCE OF THE STUDY
For many years some people had suffered many blood coagulation disorders
such as hemophilia, a type of disorder in which people don’t blood clot immediately if
they have wound or hemorrhage a type of stroke in which people unable to clot the
certain part of the brain has a rupture of blood vessels enabling blood to escape which
is very dangerous if not treated properly and safely. If we didn't act immediately many
people will eventually did. So the proponents of this research think that maybe there is a
certain plant, a healthier way than undergoing a surgery or a synthetic drug that can
treat the disorder. Kalachuchi (Plumeria acuminate) seed has tannin, a type of
substance which helps the body clot. By extracting tannin extract from it and putting it in
the blood in the test tube this way is called vitro or testing something outside the body.
The proponents can now determine the effects of the extract without putting any living
thing in danger.
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Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
STATEMENT OF THE OBJECTIVES
This study aims to extract tannins from the seed so it can be tested for its
effectiveness in blood coagulation so these are the objectives the proponents aim to
achieve:
To be able to test Kalachuchi seed extract and its hemostatic effect in vitro using
pig’s blood.
To present to the scientific community the medicinal ability of Kalachuchi seed
extract.
To find a potential remedy to hemophilia by conducting this study.
To find a safer way than undergoing a surgery or a synthetic drug.
HYPHOTHESIS
With the principal objective of this study, it is hypothesized that the clotting time
of pig's blood will be affected by the application of Plumeria acuminata's seed extract.
Analysis of variance (ANOVA) will be the statistical tool that will be used as the
determiner of the kalachuchi seed extract's effect on the clotting time.
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Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
SCOPE AND LIMITATIONS
The area of the study focuses solely in a certain species of Kalachuchi, which is
the Plumeria acuminata. There are other Kalachuchi specimen which require further
experimentation and study. Since this experiment is purely laboratory-based, the results
might differ from that of a live experimental subject. The study will cover what effect can
Plumeria acuminata induce when use as a hemostatic agent on pig’s blood.
Uncontrollable factors like a sick specimen or one that has a hidden disorder
might mess the outcome of the experimentation as well as using blood that is not fresh.
Phytochemical analysis of the nutrient component of the test plant are beyond
the scope of this study. Also, the study will not try to find out whether which condition of
the seeds, either dried or fresh, will yield more extract. Both dried and fresh leaves will
be utilized in the study for the reason of accumulating the appropriate amount needed in
order to be able to proceed to the experimentation.
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Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
DEFINITION OF TERMS
Aliquot
It is a portion of a larger whole, especially a sample taken for chemical analysis
or other treatment.
Bleeding
It is the name used to describe blood loss. It can refer to blood loss inside the
body, called internal bleeding. Or it can refer to blood loss outside of the body, called
external bleeding.
Blood clot
Scientifically called as thrombus, colloquially called a blood clot, is the final
product of the blood coagulation step in hemostasis. Blood clots stop bleeding, but they
can also form in the body when they're not needed.
Cheesecloth
A thin, loosely woven cloth of cotton, used originally for making and wrapping
cheese. It comes in seven grades, from open to extra-fine weave. The grade is
determined by the amount of threads per inch constructed in each direction.
Coagulation
Coagulation is a complex process by which the blood forms clots to block and
then heal a lesion/wound/cut and stop the bleeding. It is a crucial part of hemostasis -
stopping blood loss from damaged blood vessels.
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Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
Extract
Extract is any substance made by extracting a part of a raw material, often by
using a solvent such as ethanol or water. Extracts may be sold as tinctures or in powder
form.
Filtration
This is the process of separating suspended solid matter from a liquid, by
causing the latter to pass through the pores of some substance, called a filter.
Hemostatic
This pertains to a relationship with the suffix –tic. In simpler word hemostatic is
an agent to hemostasis, a process which causes bleeding to stop, meaning to keep
blood within a damaged blood vessel.
In Vitro
Made to occur in a laboratory vessel or other controlled experimental
environment rather than within a living organism or natural setting.
Plumeria
These are the tropical trees famous for their gorgeous flowers which are used to
make floral garlands. In the regions with cold winters, plumerias can be grown in
containers and brought indoors when the weather cools in autumn.
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Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
Pound
It means to strike repeatedly with great force, as with instrument, the fist, and etc.
To produce or effect by striking or thumping.
Seed Pod
It is a seed vessel or dehiscent fruit that splits when ripe. A carpe or pistil
enclosing the seeds of the plant, especially a flower plant.
Sterilize
This is referring to any process that eliminates and kills all form of life and other
biological agents, including transmissible agents present in a specified region, such as a
surface, a volume of fluid, medication,or in a compound such as biological culture
media.
Tannins
These are astringent, polyphenolic biomolecule that binds to and precipitates
proteins and various other organic compounds including amino acids and alkaloid.
Tannins protect damaged tissue (skin), help stop bleeding (heavy menstrual flow) and
keep infection in check.
Thrombin
It is a serine protease that converts fibrinogen into fibrin in blood coagulation. In
addition to haemostasis, thrombin plays a major role in inflammation and wound
healing, and it is a potent mitogenic factor. Thrombin has anticoagulant effects by
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activating the protein C pathway and by releasing plasminogen activators from
endothelial cells, which promote fibrinolytic cascade.
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REVIEW OF RELATED LITERATURE
Origin of Kalachuchi
Plumerias, which are also called
frangipanis in Australia and some other
countries, originate from South America.
Early ocean travelers were attracted to
frangipani plants so much that they
transported them all round the world.
Frangipanis love warm climates so their
numbers have increased rapidly in tropical
Left: Kalachuchi seed; Right: Kalachuchi
countries. With their beautiful and wide
flower
range of flower colour combinations, pleasant fragrances and suitability to the climate,
frangipanis are a common selection for tropical holiday areas. They are so common in
tropical holiday destinations such as Hawaii and Fiji that most people associate
frangipanis with tropical islands more than South America.
Medicinal Uses of Kalachuchi
"The kalachuchi-based broad spectrum anti-fungal is safe, effective, without side-
effects and comparable to the standard drugs in the market today." -Dr. Gerard L.
Penecilla of the West Visayas State University (WVSU) in Iloilo City
Kalachuchi has a variety of medicinal properties. Its bark, which contains the
bitter glucoside plumierid, is used as a purgative, emmenagogue, and febrifuge when in
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Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
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decoction. In Mexico, the calcium salt-containing latex is given for toothache, and when
combined with coconut oil is used as a remedy for itching. Besides useful as ornamental
plants, frangipani flowers are also often used as a traditional medicine to address
several diseases. Among ulcers, gonorrhea (gonorrhea), swelling, warts, calluses,
smoothes the skin and yaws. The juice is rubefacient in rheumatic pains, and with
camphor, is also used for itching. A poultice of heated leaves is beneficial for swellings.
Decoction of leaves for cracks and eruptions of the soles of the feet. Infusion or extract
from leaves is used for asthma. In Mexico, decoction of flowers used in diabetes. Some
of its properties are to follow: Sweet tasting and neither warming nor cooling in effect,
aromatic, anti-inflammatory, antipyretic, diuretic, emmenagogue, febrifuge, purgative,
rubefacient and seeds are considered hemostatic.
Tannins and Its Uses
Tannins are polyphenolic compounds that contract and “astringe” tissues of the
body by binding with and precipitating proteins– hence their use to “tan” leather –
combining with them, and thus rendering them resistant to proteolytic enzymes.
Two distinct types of tannins have been identified on the basis of structure.
Condensed tannins are composed of many flavonoids or proanthocyanidins joined
together. However, the properties of condensed tannins are distinct from their building
blocks. Hydrolyzable tannins are composed of a glucose (or rarely, other
monosaccharide or polyol) core with several catechin derivatives attached. These
medium to large polymers are very widely distributed in the plant and fungus kingdoms.
Condensed and larger hydrolyzable tannins have most properties in common, although
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Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
hydrolyzable tannins are less stable (being susceptible to hydrolysis) and have greater
potential to cause toxicity.
Tannin-containing herbs are used to tighten up tissues (varicose veins), dry up
excessive watery secretions (diarrhea), protect damaged tissue (skin), and help to stop
bleeding (heavy menstrual flow) and to keep infection in check. These herbs have
astringent, hemostatic, antiseptic and toning properties.
Tannins are used to interfere in alkaloids and heavy metals absorption, due to its
coagulant properties. The tannins have the property of coagulate proteins and mucosal
tissues, by creating an insulating and protective layer that soothes irritation and pain on
the skin. Herbal preparations containing tannins are used for stop local small
hemorrhages, sore mouth, bronchitis, burns, scars of the skin, wounds and many
others.
Hemorrhage
Hemorrhage is defined as bleeding or
the abnormal flow of blood. It is excessive or
uncontrollable bleeding, often caused by
trauma, surgical or obstetrical complications,
or the advanced stages of certain illnesses,
such as cirrhosis and peptic ulcer disease. A
hemorrhage may be "external" and visible on
Image. 2: Left: Hemorrhage Classification;
the outside of the body or "internal," where
Right: Subarachnoid Hemorrhage
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Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
there is no sign of bleeding outside the body. Bleeding from a cut on the face is an
external hemorrhage. Bleeding into the spleen or liver are examples of internal
hemorrhage.
Severe hemorrhage, if untreated, is a very dangerous condition. The normal
compensating responses eventually fail, the sympathetic nerves cease to fire, and
activity in the vagus nerve to the heart may increase. This results in fainting. If very low
blood pressure and low flow persist they are likely to cause irreversible changes so that
even replacement of all the lost blood does not restore blood pressure The term
"hemorrhagic" comes from the Greek "haima," blood + rhegnumai," to break forth; a free
and forceful escape of blood.
Sus domesticus
Pigs are mammals belonging the Suidae
family, which includes eight genera and 16
species. They have stocky bodies, flat snouts
and large ears. Pigs are cognitively complex
like that of animals we consider as smart such
as dogs and primates. But this does not say
that their intelligence is of equivalent value to
that of the primates.
Image 3: Sus domesticus
These smart animals usually weigh between 300 and 700 lbs. (140 and 300
kilograms). Domestic pigs are often bred to be heavier, according to the San Diego
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Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
Zoo.Pigs are omnivores and can consume a wide range of food, similar to humans. Pigs
can harbor a range of parasites and diseases that can be transmitted to humans.
Because of the similarities between pigs and humans, pigs are used for human medical
research.
ANOVA (Analysis of Variance)
ANOVA is a statistical technique that assesses potential differences in a scale-
level dependent variable by a nominal-level variable having 2 or more categories. For
example, an ANOVA can examine potential differences in IQ scores by Country (US vs.
Canada vs. Italy vs. Spain). The ANOVA, developed by Ronald Fisher in 1918,
extends the t and the z test which have the problem of only allowing the nominal level
variable to have just two categories. This test is also called the Fisher analysis of
variance.
General Purpose of ANOVA
Researchers and students use ANOVA in many ways. The use of ANOVA
depends on the research design. Commonly, ANOVAs are used in three ways: one-
way ANOVA, two-way ANOVA, and N-way Multivariate ANOVA.
One-Way ANOVA
A one-way ANOVA refers to the number of independent variables--not the
number of categories in each variables. A one-way ANOVA has just one independent
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Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
variable. For example, difference in IQ can be assessed by Country, and County can
have 2, 20, or more different Countries in that variable.
Two-Way ANOVA
A two-way ANOVA refers to an ANOVA using 2 independent variable.
Expanding the example above, a 2-way ANOVA can examine differences in IQ scores
(the dependent variable) by Country (independent variable 1) and Gender (independent
variable 2). Two-way ANOVA’s can be used to examine the INTERACTION between
the two independent variables. Interactions indicate that differences are not uniform
across all categories of the independent variables. For example, females may have
higher IQ scores overall compared to males, and are much much greater in European
Countries compared to North American Countries.
Two-way ANOVAs are also called factorial ANOVA. Factorial ANOVAs can be
balanced (have the same number of participants in each group) or unbalanced (having
different number of participants in each group). Not having equal size groups can make
it appear that there is an effect when this may not be the case. There are several
procedures a researcher can do in order to solve this problem.
Soxhlet Extraction Method
This method is applicable to the isolation and concentration of water-insoluble
and slightly water soluble organics in preparation for a variety of chromatographic
procedures. This method is restricted to use by or under the supervision of trained
analysts. Each analyst must demonstrate the ability to generate acceptable results with
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Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
this method. The solid sample is mixed with anhydrous sodium sulfate, placed in an
extraction thimble, and extracted using an appropriate solvent in a Soxhlet extractor.
The extract is then dried, concentrated (if necessary), and, as necessary,
exchanged into a solvent compatible with the cleanup or determinative step being
employed. Soxhlet method is applicable to the extraction of nonvolatile and semi-volatile
organic compounds from solids such as soils, relatively dry sludges, and solid wastes.
Soxhlet extraction uses relatively inexpensive glassware, once loaded requires no
hands on manipulation, provides efficient extraction, but is rather lengthy (16 to 20
hours) and uses fairly large volumes of solvent. It is considered a rugged extraction
method because there are very few variables that can adversely affect extraction
efficiency.
This method is also used to analyze potentially hazardous samples, and uses
potentially hazardous reagents. Use of a respirator is advised if a hood is not adequate
or feasible. Splash guard goggles should be used when acid rinsing, solvent rinsing,
transferring samples, or when working with solvents under pressure.
Prior to performing this analysis, review the MSDS for all standards and reagents
to be used. Observe the recommended safety precautions. Protective clothing and
safety glasses should be worn when handling samples or reagents, and all
manipulations should be done in a hood. Maintain a clean and uncluttered workspace.
Return all chemicals, reagents and resultant wastes to their designated storage area at
the completion of the test.
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Chapter II
METHODOLOGY
Collection of Kalachuchi Seeds
Kalachuchi seeds were obtained in the lower areas of Busay, churches of Laray
and Inayawan. The pods were washed thoroughly and the damaged parts were
removed. After washing the pods thoroughly and discarding the damaged ones, the
seeds were separated from their pods.
Extraction of the Kalachuchi Seeds
The project is focused mainly on using the seed extract of Kalachuchi. Before
carrying out the experimentation, at least 500 grams of kalachuchi seeds were collected
beforehand. The kalachuchi seeds were sent to the laboratory to undergo Soxhlet
Extraction method.
Blood Collection and Preparation
One hundred ml of blood was obtained from a private licensed slaughterhouse
located at Pardo, Cebu City. It was assured that the blood is fresh because it was
immediately obtained right before slicing open the pig which was to be butchered.
For the transfer of blood, test tubes will be sterilized and warmed in boiling water
(100°C). The twelve test tubes were labeled accordingly based on which set-up they
belong. The set-ups were labeled for no extract (Set-up D), for 0.05ml extract (Set-up
C), for 0.075 ml (Set-up B) extract and 0.1ml extract (Set-up A).
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Clotting Time Determination
The collected blood was divided among the twelve test tubes in an approximate
amount of 0.1 ml per test tube. Instantaneously, designated amount of the extract
solutions were added into the respective tubes. (Set-up A: 0.1 ml, Set-up B: 0.05ml,
Set-up C: 0.075ml, Set-up D: No extract added) Stopwatch was started once the extract
solutions have been added. Clotting time determination was carried out by fully inverting
the test tubes every 30 seconds until it can inverted with no blood dripping from the top
part of the test tube, which means that blood has completely clotted. Quantitative data
was jotted every 30 seconds and were be compared and tabulated to get results.
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Chapter III
RESULTS AND DISCUSSION
The potential hemostasis-induced properties of the Plumeria acuminata seed
extract was evaluated in vitro on pig's blood (Sus domesticus) as being mixed with pure
extract. There were three appropriate units of pure extract tested on treatments: 0.05ml,
0.75ml, and 0.1ml pure extract. Quantitative Data on the blood clotting time at different
Set-up are shown below.
Set-up B Set-up C
Set-up A (0.75ml pure (0.05ml pure Set-up D
(0.1 ml pure extract) extract) extract) (control)
Mins. Mins. Mins. Mins.
T1 25.35 T1 20.50 T1 18.36 T1 4.48
T2 28.45 T2 21.32 T2 18.59 T2 5.16
T3 27.55 T3 22.46 T3 18.45 T3 5.58
Table 1. Quantitative Data on the blood clotting time
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Table 2 shows the hemostatic effects of the Plumeria acuminata seed extract on
the normal blood clotting time of pig’s blood using one-way ANOVA.
SS df MS
Source of (Sum of (Degree of (Mean F P
Variation squares) freedom) squares) (F-values) (P-values)
Between
Group 786.563 3 262.188 273.557 0.000
Within
Group 7.668 8 0.958
Total: 794.231 11
Table 2. One-Way Analysis of Variance (ANOVA)
Results revealed that the change in the mean scores between different
treatments on pig’s blood clotting time is significant for it defines Kalachuchi seed
extract’s effect on the clotting time. The average of the clotting time of blood in set-up A
is 27.117 minutes. The results above show that the coagulation of pig’s blood in set-up
A, which received the highest amount of extract -0.1ml, prolonged the most among the
four set-ups. While blood in set-up B, which received 0.075ml of pure extract,
coagulated in lesser time than set-up A with a mean of 21.427 minutes. Set-up C
coagulated in lesser time than set-up B with a mean score of 18.467 minutes. Above all,
the standard control set-up, which is set-up D, coagulated the fastest with a mean score
of 5.073 minutes.
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Standard
Set-up Mean N
Deviation
0.1ml Pure Extract 27.117 3 1.595
0.075ml Pure Extract 21.427 3 0.984
0.05ml Pure Extract 18.467 3 0.116
Control Group 5.073 3 0.555
AVERAGE: 18.021 3 0.8125
Table 3. Mean Values and Standard Deviation on effect of Plumeria acuminata on pig’s
blood
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Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
Chapter IV
CONCLUSION
Based on the conducted experiment and the generated results, the proponents
have arrived at the following conclusions:
Kalachuchi seed extract induces prolongation on the clotting time of pig’s blood
when investigated in vitro.
Higher amounts of Kalachuchi seed extract added on pig’s blood causes longer
clotting time.
The quality of used blood affects the speed of coagulation based on results
yielded from the experimentation.
Thereby, the proponents conclude that Plumeria acuminata seed extract induces
prolongation of pig’s blood coagulation as shown by the continuous increase of the
mean values of Set-ups A, B, and C in respect with the amount of seed extract added.
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Chapter V
RECOMMENDATIONS
With the results of this study, the proponents came up with these recommendations:
1. Obtain the blood, which is going to be used in the experiment, with the help of a
professional to secure its freshness and quality.
2. Increase the number of treatments to increase the level of accuracy of the data.
3. Utilize more statistical tools in computing the data collected such as Duncan’s
Multiple Range Test (DMRT).
4. Conduct an even deeper study on the properties and chemicals present in the
Kalachuchi seed extract by means of phytochemical analysis and other appropriate
tests.
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Chapter VI
ACKNOWLEDGEMENTS
The proponents wish to extend their humble gratitude for the guidance and
assistance of these individuals for without the help of these individuals and
organizations, this study wouldn’t be able to reach its conclusions.
To our parents, for their undying support by sacrificing their time and giving their
best efforts to help us.
To Ms. Yvonne Lyn Torres, our present Research teacher, for giving us constant
reminders for the importance and progress of this study and for motivating us
throughout the duration of this study.
To Ms. Irene Lopez, our first Research teacher, for endowing us with her
knowledge on making investigatory projects to the best of her capabilities and for
allowing us to do this study.
To our dear mentors, for being with us through thick and thin throughout our
study. Thank you for providing us with information which we really needed to know and
for boosting our morale whenever we needed it.
To Ms. Nancy Mila, licensed chemist, for lending us her valuable time, effort and
knowledge for the fulfillment of our study and for facilitating our needs in Techno Lab.
To the associations and staffs of the former Inayawan St. Augustine Church and
of Laray’s Our Lady of Consolacion Parish Church, a thousand thanks to you! For
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Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
allowing our group to collect and use much of your resources especially your
Kalachuchi seeds.
Above all, to the Almighty God, Thank you for giving us the strength and burning
goal to pursue and accomplish this study. We glorify you, Lord.
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Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
Chapter VII
REFERENCES
About Frangipanis: History (2016). Retrieved from://www.allthingsfrangipani.com
ANOVA (Analysis of Variance) (August 22, 2016) Retrieved from:
http://www.encyclopedia.com/
Arnold T. (2010). Duncan’s Multiple Range Test. SAS/STAT(R) 9.2 User's Guide,
Second Edition. Retrieved from support.sas.com
"haemorrhage." World Encyclopedia. (November 12, 2016). Retrieved from:
http://www.encyclopedia.com/
Health Benefits of Plants: Frangipani Flower Benefits as Antibiotics (2013).
Retrieved from http://plantsandbenefits.blogspot.com
Introduction to Blood Coagulation (1993-2016). Retrieved from:
http://themedicalbiochemistrypage.org
Marciano, Dr. Marisa (August 22, 2016). The Naturopathic Herbalist: Tannins.
Retrieved from: http://www.encyclopedia.com/
Nordqvist C. (2016). All about Hemophilia/Haemophila. Retrieved from
http://www.medicalnewstoday.com
Omar N. (2013). Hemostatic Effects of Peperomia pellucida Plant Crude Extract, in
vitro An Undergraduate Thesis Presented to In Partial Fulfilment of the
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Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
Requirement for the Degree Bachelor of Science in Biology. Retrieved from
academia.edu
Stuart G. U. & Santiago A. S. (2013). Philippine Medicinal Plants. Retrieved from
http://www.stuartxchange.com
Stuart, Godofredo (May 5, 2016) Kalachuchi. Retrieved from:
http://www.stuartxchange.com/
World Health Organization. (2010).WHO guidelines on drawing blood: best in
phlebotomy. Retrieved from http://apps.who.int
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Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
Chapter VIII
APPENDICES
PHOTO DOCOMENTATION
I) Collection of Kalachuchi Seeds
Hand-Picking the Kalachuchi seed The proponent used a long stick to
pods. knock down the seed pod.
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Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
The proponents washed the pods Most of the collected seed pods.
thoroughly.
II) Extraction of the Kalachuchi Seeds
Ms. Nancy Mira, licensed chemist, Ms. Mira measures the weight of
III)
lets the seeds be dried. the seeds contained in the thimble.
IV)
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Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
Actual process of Soxhlet Extraction
Method.
III.) Blood Collection and Preparation
Sterilization of laboratory apparatus in
preparation for the transfer of blood.
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Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
V) Clotting Time Determination
Ms. Mira measures the weight of
the seeds contained in the thimble.
Transferring blood from the test tube into the syringe. Amount of blood placed in
each test tube is 0.1ml.
Transferring blood from the test tube into the syringe. Amount of blood placed in
each test tube is 0.1ml.
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Hemostatic Effects of Kalachuchi (Plumeria acuminata) Seed Extract in Vitro
Raganas, S. Z., Paraon, J. F., Delicano, K.M., Piala, A.
Inverting the test tubes every after 30 seconds until the blood fully coagulates.
Set-up A with 0.1ml Set-up B with 0.075ml Set-up C with 0.05ml Set-up D with 0.1ml
pure extract and pure extract and pure extract and blood but no extract.
blood. 0.1ml blood. 0.1ml blood.
35
