GramarGroup 5 Antiangiogenic Chapter 12345

ANTIANGIOGENIC PROPERTY OF PEACOCK FLOWER (CAESALPINIA
PULCHERRIMA)
A Research Proposal Presented to the Faculty of the

SENIOR HIGHSCHOOL
Notre Dame of Dadiangas University
General Santos City
In Partial Fulfillment of the Requirement

for Research in Daily Life 1 & 2
Gumboc, Lei Pauline

Latorza, John Raeven
Magnabijon, Kate Lelu
Quintana, Jyrouz Abby
Sanchez, Seth Stephen
October 2019
iii
ACKNOWLEDGEMENT
The researchers would like to extend their heartfelt gratitude and appreciation to
the following persons who helped them bring this study into reality:
To their research adviser, Marianne Joyce V. Tubojan, whose expertise, guidance,
time and pieces of advice helped them bring this study into success;
To Joey Kevin D. Tiongco, for his valuable comments, suggestions, knowledge,
and for sharing his time to make this study more realistic;
To the Biology Laboratory Staff of Notre Dame of Dadiangas University,
especially John Mark Robles, for assisting the researchers in doing the experiment;
To the researchers' beloved parents, for their moral and financial support
throughout the process of completing this study;
To all the people who extended support and became part in the completion of the
study; and,
Most of all, to the Almighty Father, for giving the researchers His endless
blessings, wisdom and strength to make this research possible.
The Researchers
iv
ABSTRACT
Caesalpinia pulcherrima has a lot of potentials that have been proven by scientist
and other researchers which were used by the researchers of this study as reference in
conducting this investigation. In this paper, the researchers determined the antiangiogenic
property of C. pulcherrima leaf extract. It was hypothesized that there is no significant
difference in the number of blood vessels in the chorioallantoic membrane of mallard
embryos.
The researchers gathered nine eggs and collected two hundred and fifty grams of
C. pulcherrima prepared for the Chorioallantoic Membrane (CAM) assay. The findings
shows that C. pulcherrima, T3 has greater lengths than the rest of the treatments used in
this study. The leaf extract of C. pulcherrima was effective, and significant to be called as
a new replacement to the commercial Simvastatin, were the compound F- value was
higher than the tabular value. As a result, the null hypothesis was rejected and that there
was a significant difference in the number of branching points in the Chorioallantoic
membrane of mallard eggs after 48 hours using the different treatments.

v
TABLE OF CONTENTS
Title Page i
Approval Sheet ii
Acknowledgement iii
Abstract iv
Table of Contents v
List of Tables vii
List of Figures viii
Chapter I THE PROBLEM AND SETTING
Introduction 1
Statement of the Problem 2
Hypothesis 2
Scope and Delimitation 3
Significance of the Study 3
Definition of Terms 4
Chapter II REVIEW OF RELATED LITERATURE 6

vi
Chapter III METHODOLOGY
Research Design 13
Preparation of Materials 15
General Procedures 15
Data Gathering Techniques 16
Statistical Tools 16
Chapter IV PRESENTATION, ANALYSIS AND INTERPRETATION OF 17
DATA
Chapter V SUMMARY OF FINDINGS, CONCLUSIONS, AND
RECOMMENDATIONS
Summary of Findings 22
Conclusions 23
Recommendations 23
BIBLIOGRAPHY 24
APPENDIX 27
CURRICULUM VITAE 35
vii
LIST OF TABLES
Table Page
1 Number of Branching Points in the CAM after 48 hours 18
2 Summary Table for the One-way ANOVA of CAM Assay after 48 hours 19
3 CAM Vascularity of mallard eggs using the different treatments 20

viii
LIST OF FIGURES
Figure Page
1 Research Design 14
2 Chorioallantoic Membrane (CAM) after 48 hours 34

Chapter I
THE PROBLEM AND ITS SETTING
Introduction
Caelsalpinia pulcherrima could be an invasive bush or tiny tree within the family
Fabaceae. It’s listed as “Naturalized”, cultivation escape, and “weed” within the world
Compendium of Weeds (Randall, 2012). The species is taken into account native to Asia
and introduced to the archipelago and tropical regions round the world, because it has
been cultivated for its placing and colorful flowers (Acevedo-Rodriguez and Strong,
2012).
This species has been employed in varied cultures for medication functions.
Ailments treated embrace gastritis and internal organ inflammation, looseness of the
bowels and infectious disease (tea from leaves), flatulence, ulcers and liver disease
(leaves), and female internal reproductive organ pathology (bark and flowers), and from a
number of the earliest documentation of the species within the Amazon, as associate
degree agent and to induce abortions (Schiebinger, 2004). Plants with polyphenols and
trepans which have high antioxidants compounds are the ones that are most frequently
studied because it causes strong anti-oxidants effects.
A lot of people around the world are suffering from cancer, were the cells
multiply in an uncontrolled way, which is abnormal in the growth of cells. It is a group of
more than a hundred of diseases that can lead to death (Shiel, W, 2018). To prevent the
growth of new blood vessels, there is a process called antiangiogenesis were it is also to
prevent the cells to become a tumor. The agents’ angiostatin, endostatin, tetracyclines,
Antiangiogenic property of Caesalpinia pulcherrima 2
and paclitaxel plays a critical role in antiangiogenesis and are very useful in treating
cancer. (McGraw-Hill Concise Dictionary of Modern Medicine, 2002).
This study aimed to determine if the chemicals that in the leaf extracts of C.
pulcherrima can be used as antiangiogenics. Using the blood vessels present in the CAM
of Mallard embryos. It was conducted for the initial findings that it would serve a great
purpose and would lead to future investigation on the medicinal prospect of the plant.
Statement of the Problem
This study aimed to determine the antiangiogenic property of C. pulcherrima.
Specifically it answered the following questions:
1. Can the leaf extract of C. pulcherrima influence the growth of blood vessels?
2. Is there a significant difference developing blood vessels in the CAM of mallard
embryos using the following treatments:
2.1 Distilled Water;
2.2 Simvastatin; and
2.3 C. pulcherrima leaf extract?
Hypothesis
There is no significant difference in the number of blood vessels in the
chorioallantoic membrane of mallard embryos.

Scope and Delimitation
The main focus of this study was to determine the antiangiogenic activity of C.
pulcherrima. In addition to this, the study used three treatments: T1 is distilled water, T2 is
simvastatin and T3 is the C. pulcherrima.
The collection of data, through observation, validation and documentation was
conducted at the Biology Laboratory of Notre Dame of Dadiangas University Main
Campus.
Significance of the Study
The researcher intended to analyze the antiangiogenic property of C.
pulcherrima leaf extract. This study is beneficial to the medical professionals for they can
use this as the main ingredient to help mitigate angiogenesis that could cause cancer.
It can also help the Department of Health that this study can contribute to the
branch of studying medicinal plants and showing what C. pulcherrima can achieve.
For the future researchers and the future studies, which all the collected data
would be the basis of this study. This study can give other researchers further knowledge
and understanding of the conduct of the study.

Definition of Terms
This study used some terms which may be unfamiliar, thus, these words were
defined conceptually and operationally. Conceptual definitions are all that can be
found in any various journals.
Antiangiogenesis. This refers to the process where the cells multiply in an
uncontrolled way or the abnormal growth of cells. It is a group of more than a hundred
diseases that can lead to death. Operationally, this is the process that would stop the
growth of new blood vessels that can cause cancer.
Blood vessel. This refers to a cannula structure carrying blood through the tissue and
organs. Operationally, this is the primary subject of the study.
Caesalpinia pulcherrima. This refers to an upright rounded, generally sprawling,
multi-stemmed ligneous plant. It typically gets up to 10 feet tall, displaying characteristic
flowers with bright orange and yellow petals, and long red stamens. The flowers, which
might become old to 2 inches in diameter, bloom in terminal racemes at the dip of
branches with as several as forty flowers per flower cluster. Operationally, this refers to
where the extract that was needed came from and applied for this study.
Chorioallantoic Membrane (CAM). This refers to an extra embryonic that is wealthy
of blood vessels. Operationally, it refers to the location of development of
antiangiogenesis of C. pulcherrima on the blood vessels.
Inhibitors. This refers to limit, prevent, or block an action or function. Operationally,
this was the agent that interfered the angiogenic properties.

Replicate. This refers to the continual set of check conditions. Operationally, this can
be determined to possess a particular result. Three replicates in every treatment were
conducted within the experiment which was observed to have a clear result.
Simvastatin. This refers to the medication to decrease elevated supermolecule levels.
It is additionally used to decrease the risk of heart issue in those at high risk.
Operationally, this refers to a treatment used to stop the inhibition of tumors and cancer
cells.
Test Organism. This refers to the any living, ranging from microscopic bacteria to the
large African bush elephant and everything in between that is being studied.
Operationally, this refers to the organism studied within the research which is the duck
embryo and was used to confirm the antiangiogenic property of C. pulcherrima.

Chapter II
REVIEW OF RELATED LITERATURE
Antiangiogenics
These are drugs that try to stop, slow, or shrink the abnormal growth of blood
vessels that can lead to cancer. There are different types of antiangiogenic drugs such as
Bevacizumab, Sunitinib (Sutent), and Thalidomide and lenalidomide. Bevacizumab is a
monoclonal antibody drug that blocks the vascular endothelial growth factor (VEGF)
from attaching to the receptors on the cells that line the blood vessels, thus, stopping the
growth of blood vessels. It is also used as a treatment to several types of cancer.
Consequently, Sunitinib (Sutent) is a type of TKI or Tyrosine kinase inhibitors that block
the signaling within the cell. This stops the VEGF receptors from sending growth signals
into the blood vessel cells. It is also used for people with kidney cancer, gastrointestinal
stromal tumor, and neuroendocrine tumor of the pancreas. Lastly, Thalidomide and
lenalidomide (Revlimid) are drugs that affect signals between cells like which are used to
treat people with multiple myeloma (Jayson, et. al., 2016).
Mallard Embryo
Mallards are mostly found in places that are mostly wetlands, including
permanent wetlands like marshes, bogs, riverine floodplains, beaver ponds, reservoirs,
ponds, city parks, farms and estuaries. They are also seen in prairie potholes and
ephemeral wetlands and may be found feeding along roadside ditches, pastures, crop
lands, and rice fields (Drilling, N., 2002).
Caesalpinia pulcherimma
C. pulcherrima shrub or tiny tree is unarmed or armed with spines or curving
prickles on branches, leaf rachises, and typically on nerves. Leaves are bipinnately
compound, leaflets opposite or often alternate, stipules varied, giant and foliaceous or
minute. Flowers are caesalpiniaceous, good or sexual, in terminal and/or axillary racemes
that are usually aggregative into branced raceme inflorescences, bracts caduceus
bracteoles absent; plant organ short, 5 lobed, lower lobed usually covering the others,
hood like; petals five, imbricate, sub equal or the higher one smaller and with a lot of
developed claw; stamens ten, distinct, alternately longer and shorter; ovules typically 2-
10. Fruit pods are planar seldom cylindrical, dehiscent or indehiscent, winged on the
higher suture or unwinged. Seeds are 1-9, transversally organized, ellipsoid or sub
globose (Wagner and Lorence, 2014).
Caesalpinia pulcherimma Activity
The plant C. pulcherrima is a part of the Fabaceae family that was used as a
traditional medicine for different kinds of ailments in India. Internationally, traditional
medicines are used for treating cancer.

This study aimed to know the chemo modulatory potential of C. pulcherrima flowers in
breast cancer and explaining the possible mechanism in it. The cytotoxic potential of
ethyl acetate parts of C. pulcherrima (EAFCP) flower was tested in MCF-12A (normal
breast), MCF-7 (estrogen receptor [ER] positive), and MDA-MB-453 (human epidermal
growth factor receptor two positive) human breast cancer cells by sulforhodamine B
assay. Chemomodulatory potential was evaluated in vivo against N-methyl-N-nitrosourea
(MNU)-induced mammary carcinoma in female Sprague Dawley rats. The mechanism
for anticancer2 potential was screened by in vitro studies including Annexin V-FITC
assay (apoptosis), cell cycle patterns, intracellular reactive oxygen species, and
mitochondrial membrane potential measurement (FACS based) followed by docking
study on estrogen receptor-alpha (ER-α). The fractions showed perceptible cell growth
inhibition potency (IC50<50 μg/ml) in MCF-7 breast cancer cells. In MNU-treated
animals, antioxidant enzymes and histological examination showed statistically
significant (P < 0.001) changes. Treatment of MCF-7 cells with EAFCP reduced the rate
of cell growth by a mechanism associated with both apoptotic and necrotic cell death.
Molecular docking study further showed that rutin and catechin have a comparable
binding affinity for the ER-α. The researchers in this study affirmed that EAFCP was
most effective in reducing cell viability, scavenging physiological oxidant species, and
resulting mitochondria-mediated apoptosis and necroptosis in MCF-7 cell by selectively
modulating the functions of ER-α. (Sakle, Lokwani, Mokale, 2019). It is found out that
the plant C. pulcherrima have a flavonoid. Its compounds has antibacterial and
antiangiogenic activities and inhibit in vitro the growth of several microorganisms (Shim
et al., 2004).
Inhibitors
The findings from these results recommend that statin drug would possibly
probably be a potent drug for ATC treatment. Since isoprenylation is very important
for the perform of the many little GTPases (e.g., Ras, RhoA), that are crucial for the
cell proliferation and survival functions, it is seemingly that their discomposed
perform will directly or indirectly affect the energy standing and associated
metabolic processes. It is noteworthy that Mullen et al. urged the synergism of IGF-
1/AKT communication being crucial to take care of the conventional mitochondrial
perform and be free from simvastatin-induced harm in mitochondrial metastasis
chain. Within the study, they found that the accumulated phosphorylation level of
AKT is that the determinant to cut back mitochondrial susceptibleness to statin drug
impairment (Chen, 2017).
Blood Vessel
Blood vessels play a major role in the circulatory system whose main function
is to transport blood throughout our body. All of the blood vessels have the same
basic structure. The inner lining is called endothelium, and it is surrounded by
subendothelial connective tissue. Around this, there is a layer of vascular smooth
muscle, which is highly developed in arteries. Lastly, there is a further layer of
connective tissue known as the adventitia, which contains nerves that supply the
muscular layer, as well as nutrient capillaries in the larger blood vessels
(Anonymous, 2019).
It is a vessel found in humans or animals in which where the blood circulates. In
it, there is a vessel that carries blood away from the heart and is called arteries, and their
tiny branches are called arterioles. The tiny branches that collect the blood from the
various organs and parts are called venules, and they unite to form veins, which return the
blood to the heart. Capillaries are minute thin-walled vessels that are connected to the
arterioles and venules. It is through the capillaries that nutrients and wastes are
exchanged between the blood and body tissues (Rogers, 2019).
Simvastatin
Simvastatin, also known as statins, is one of the treatments used in the study. It
reduces the level of “bad” cholesterol (low-density lipoprotein or LDL) and triglycerides
in the blood while increasing the levels of “good” cholesterol (high-density lipoprotein or
HDL). Simvastatin is used to decrease cholesterol and triglycerides (types of fats) in the
blood. This treatment is also used to lower the risks of stroke, heart attack, and other
heart complications in people with diabetes, coronary heart diseases or other risk factors.
According to Dulak and Jockowicz (2005), statins are focused inhibitors of HMG-CoA
reductase (3-hydroxy-3methhyl-glutaryl-coenzyme A reductase), the compound which
catalyzes the rate-constraining stride in cholesterol synthesis, a four-electron reductive
deacylation of HMG-CoA to CoA and mevalonate. Statins restrain HMG-CoA reductase
reversibly by authoritative to the proteins dynamic site. The KM for statins is in the
nanomolar range, and they adequately dislodge HMG-CoA, the regular substrate which
ties at micromolar concentrations.

As said by Caro and Boyoc (2012), Simvastatin hinders the emission of MMP’s
(Matrix metalloproteinase) by macrophages in vitro also in vivo, Simvastatin stops
accentuations of MMP’s in the coronary course of an artery in a porcine copy of
hypercholesterolemia without any change of plasma cholesterol levels. Besides, statin
repress endothelial cell relocation perhaps by a diminishment of Rho
geranylgeranylation, At last, it can be finished up by Simvastatin that prevents vas
vasorum neovascularization in a test hypercholesterolemia autonomous of a plasma lipid
bringing down and showing the impacts of statins on one compound of the angiogenic
apparatus may convert into an angiogenic impact in vivo.
Simvastatin affects the antiangiogenesis because it stops the development of the
blood vessels, which continues to grow as “unwanted” vessel that may help tumor and
cancer cells. In the human vascular endothelium, statins reduce COX-2 and MMP-9
expression and activity. Through this mechanism, simvastatin exerts an anti-angiogenic
effect possibly contributing to the cholesterol-lowering-unrelated protective effects of
simvastatin against plaque inflammatory angiogenesis and rupture, and an indicator that it
is effective and being used through the study of antiangiogenesis as a positive control
(Massaro et al., 2010).
Duck Embryo as CAM
The duck embryo as a chorioallantoic membrane (CAM) is a preclinical model
that is widely used for vascular and anti-vascular effects of therapeutic agents in vivo. In
this study, The Chick or duck embryo chorioallantoic membrane (CAM): an alternative
predictive model in acute toxicological studies for anticancer drugs, they investigated the
probability of CAM as a predictive model for acute toxicology studies of drugs by
comparing it to conventional mouse and rat models for 10 FDA-approved anticancer
drugs (paclitaxel, carmustine, camptothecin, cyclophosphamide, vincristine, cisplatin,
aloin, mitomycin C, actinomycin-D, melphalan). Suitable formulations for intravenous
administration were determined before the average of median lethal dose (LD50) and
median survival dose (SD50) in the CAM were measured and calculated for these drugs.
The resultant ideal LD50 values were correlated to those reported in the literature using
Pearson’s correlation test for both intravenous and intraperitoneal routes of injection in
rodents. The results showed moderate correlations (r2=0.42 − 0.68, P<0.005–0.05)
between the ideal LD50 values gained using the CAM model with LD50 values from
mice and rats models for both intravenous and intraperitoneal administrations, suggesting
that the chick embryo can be a suitable alternative model for acute drug toxicity
screening before embarking on full toxicological investigations in rodents in development
of anticancer drugs (Kue, 2015).

Chapter III
METHODOLOGY
This section presents the research design of the study and discusses the general
procedures, materials and subjects of the study. It also presents the data gathering
techniques used in finding the connections among the variables under investigation.
Research Design
The study used an experimental design. Figure 1 on the next page shows the
research design which presents the variables, treatments and replicates that were used in
the study.
The study used three treatments for CAM assay. Three replicates was prepared for
each treatment where (T1) is distilled water, (T2) is the Simvastatin and (T3) is the C.
pulcherrima leaf extract.
The leaf extract from C. pulcherrima was subjected to the identification of
antiangiogenic property. Simvastatin was used as positive control and distilled water as
the negative control.

Antiangiogenic
Antiangiogenic Property
Agents
Number of branch points

R1 (blood vessels)
T1 R2
R3
R1
T2 R2
R3
LEGEND:
Rn – Replicates
R1
T1 – Distilled Water
T3 R2 T2 – Simvastatin
T3 –C. pulcherrima leaf extract

R3
Figure 1. The Research Design

General Procedure
Preparation of Materials
The materials were obtained from the Biology Laboratory of Notre Dame of
Dadiangas University. The materials used were beakers, parafilm, petri dishes, filter
paper, and forceps.
Collection of Plant Samples and Preparation of Extracts
The plant samples were obtained from Doña Soledad Subdivision, Barangay
Labangal, General Santos City. 5 mL collected from C. pulcherrima was washed, air
dried, and grinded into smaller pieces. The leaves of the said plant were subjected to
Rotary Evaporation.
Chorioallantoic Membrane (CAM) Assay
The assay procedure was taken from Guevara (2005). For the preparation of the
CAM assay, the researchers prepared filter paper disc (approximately 6mm in diameter).
It was then sterilized in an autoclave for 15 minutes. The 50 mL distilled water, forceps
and other materials used were also sterilized.
The researchers used mallard eggs fertilized for 8 days. The eggs were cut to have
a small opening in the egg shell about 1x1 cm to expose the CAM to direct access for
experimental manipulation.
The 21 filter paper discs were submerged in 10 mL distilled water, another with
the simvastatin and the leaf extract of C. pulcherrima. The treated filter discs were placed
directly on the CAM. It was incubated for 48 hours after sealing the eggs with parafilm.
On the 10th day of the incubation, the eggs were opened and the hard shells were carefully
removed, leaving intact the soft membrane covering the embryo. The embryos were
transferred into the petri dish. Then, the CAM was examined for the number of major
blood vessels branching points by counting it manually.
Data Gathering Technique
For the chorioallantoic membrane assay, the CAM was examined at the site of
application. The blood vessel branching points were manually counted and CAM
vascularity was calculated using the formula according to Bernes et al. (2005):
CAM Vascularity:
No . of branching points(treated) – No . of branching points (control)x 100

No . of branching points (control)
Statistical Tools
The mean vascularity was obtained to identify a significant difference in the
number of branching points of the plant extract. One-Way Analysis of Variance
(ANOVA) was used for Complete Randomized Design (CRD) with equal replication as
statistical treatment for the study. The mean and the frequency count were also used in
the study.
Chapter IV
PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA
This chapter presents, analyzes and interprets the data gathered in the study using
the different treatments used in the CAM Assay. Data in this chapter were subjected to
different statistical tests to analyze them further and to find the relationships between the
variables under investigation.
Influence of C. pulcherrima on Angiogenesis
Table 1 on the next page presents the mean number of branching points in the
treated Chorioallantoic Membrane of the mallard eggs after 48 hours of incubation.
Forty-eight hours only of incubation were to partake in order for the treatment to soak in
and to have a significant result. T1 had the highest mean number of branching points
which was 15 followed by T3 which was the extract of C. pulcherrima, with 4.3 and T2
which was Simvastatin, the positive control having the lowest branching points with a
mean of 4.
As observed, the highest which was T1 (Distilled water) as the neutral control,
indicates that it helped and developed the formation of the blood vessels. On the contrary,
T2 (Simvastatin) as the positive control has a big difference to the neutral control; this is
where in fact that this treatment is a commercial agent that affects the development of
blood vessels. Meanwhile, the C. pulcherrima that has a higher mean branching point
than the positive control which surprisingly astonishes the result and it may be because of
the component tonic it possesses (Sakle, Lokwani, and Mokale, 2019).
Table 1. Number of Branching Points in the CAM after 48 hours
Number of Blood Vessel Branching Points

Treatment Mean
R1 R2 R3
T1
14 19 12 15
(Distilled Water)
T2
5 5 2 4
(Simvastatin)
T3
(C. pulcherrima 4 5 4 4.3
leaf extract)
It shows that the leaf extract of C. pulcherrima, T3 has greater lengths than the
positive control, Simvastatin. However, there are many factors why this had been the
result though, such as the temperature and the humidity of the incubator, the maturity of
the egg and the chemicals present inside the C. pulcherrima.
As stated in the Review of Related Literature that Caesalpinia pulcherrima has a
lot of potentials that have been proven by scientist and other researchers, it was observed
to have some qualities that may be helpful such as tonic properties. According to Medical
research of the Department of Pharmacology and the Department of Medicinal Chemistry
(2019), in the study of the chemo modulatory potential of the C. pulcherrima (EAFCP) in
breast cancer and explaining its possible mechanism, results showed that EAFCP was
most effective in reducing cell viability, scavenging physiological oxidant species, and
causing mitochondria-mediated apoptosis and necroptosis in MCF-7 cell by selectively
modulating the functions of ER-α, but it can also inhibit the development of blood vessels
from growing new one resulting the lowest mean number shown in the table is C.
pulcherrima.
The data for the mean number of blood vessels were subjected to the One-way
Analysis of Variance to determine if the mean differed significantly with each other. The
results are presented in the Table 2 below.
Table 2. Summary Table for the One-way ANOVA of CAM Assay after 48 hours
________________________________________________________________________
Source of Degrees of Sum of Mean Computed F- Tabular F-
Variation Freedom Squares Squares value at 1% value at 1%
Treatment 2 234.8888889 117.4444445 21.57 10.92
Error 6 32.6666667 5.44444445
Total 8 267.56
**-significant at 1%
The computed F- value is 21.57, which is significant at 1% level of significance.
This value is higher than the tabular value with df (2,6), which is 10.92. Meaning, the
null hypothesis is rejected and that there is a significant difference in the number of
branching points in the Chorioallantoic membrane of the mallard eggs after 48 hours
using the different treatments.
Pieces of evidence supporting the result of the One- Way ANOVA are shown in
Table 3 on page 21 with images of the Chorioallantoic membrane after treatment

application. Treatments 2 and 3, which were categorized with antiangiogenic properties,
had caused a drastic effect on the CAM. In some replicates, there was a complete
suppression of the blood vessel formation.
Table 3. CAM Vascularity of mallard eggs using the different treatments
Treatments Compared CAM Vascularity (%)
T1 and T3 -71.11
T2 and T3 8.33
CAM Vascularity is a measure of how effective the experimental treatment
compared to the control is. It is a form of analysis used for pair comparison expressed in
percentage difference. Table 3 on the next page shows the comparison of both the
positive and the neutral control to C. pulcherrima leaf extract. The intensity of the
difference between T1 and T3 with CAM Vascularity is -71.11, since it was expressed
negatively which is also an indication of the degree of difference between blood vessel
development of the mallard eggs in distilled water (T1) and C. pulcherrima leaf extract
(T2). The finding means that the degree of suppression of blood vessel development is
greater in C. pulcherrima since the lowest CAM Vascularity was observed between T1
and T3. T1, which is distilled water, shows the normal blood vessel development because
water enhances and grows the branching points of mallard embryos, so it is expected that
it is the lowest at CAM Vascularity as it also represents the neutral control. Furthermore,
it means that the antiangiogenesis is highest if C. pulcherrima is compared to the neutral

control which was not biased and only helped to develop or may represent the normal
path of developing branching points of Mallard embryo.
Meanwhile, in comparison with Simvastatin, T3, which is the leaf extract of C.
pulcherrima, has the result of 8.33 and is expressed positively. This comparison may
mean that the leaf extract in terms of prominence to the positive control may be an
antiangiogenic property. The table above also shows that it has a significant difference in
the mallard embryo. Simvastatin, as stated in Review of Related Literature that
suppresses and reduces bad cholesterol and triglycerides, lowers the risk of stroke, heart
attack and other risk factors giving it the essential to be called as the positive control. It
affects the antiangiogenesis by suppressing the unwanted blood vessels to get nutrients in
the central parts in the body, to sum it up, having the power to suppress new blood vessel
in tumors. C. pulcherrima have higher branching point of the mallard embryo than the
Simvastatin, hence, because of the said properties discussed in Review of Related
Literature in the sub-topic Caesalpinia pulcherrima might have a stand on anticancer,
and as shown in the ANOVA that the capability is significant to inhibit the
Chorioallantoic membrane. It is significant in the CAM Model for it may have exceeded
the Simvastatin due to its compound and may reuse it as a new replacement comparing to
the positive control. Everything is because the computed F has a higher value than the
tabular value, which does not support the hypothesis which was rejected.
Chapter V
SUMMARY. CONCLUSIONS AND RECOMMENDATIONS
This chapter includes the summary of findings, recommendations and conclusions
that were formulated based from the results gathered on the CAM Assay using the
different treatments.
Summary of Findings
This study aimed to determine the antiangiogenic activity of the C.
pulcherrima leaf extract using the CAM Assay.
The CAM Assay used three treatments: Distilled water (T1), Simvastatin (T2)
and C. pulcherrima leaf extract (T3). The test subject for the experiment was the Mallard
eggs. Forty-eight hours of incubation was allotted to determine the CAM Vascularity and
number of blood vessels branching points.
In CAM Assay, it showed that distilled water could not inhibit antiangiogenesis,
but in fact, it helped to nurture the branching points of the mallard embryo. The leaf
extract of C. pulcherrima was useful and is significant as a new replacement to the
commercial Simvastatin. Results showed that T1, which the distilled water having 15
mean followed by the T3, which was the leaf extract of C. pulcherrima having 4.3 mean
and lastly, the Simvastatin having a mean of 4. Nevertheless, the computed value which
is 21.57 is higher than the tabular F-value, which is 10.92, that resulted in the rejection of
the null hypothesis.

Conclusion
After the researchers conducted the study on the antiangiogenic activity of the C.
pulcherrima leaf extract, the following conclusions were made:
1. The C. pulcherrima leaf extract can reduce the growth of blood vessels.
2. There is a significant difference in developing blood vessels in the CAM of
mallard embryos using the treatments distilled water, simvastatin, and C.
pulcherrima leaf extract.
Recommendations
Based on the results of the study, the researchers give the following
recommendations:
1. To conduct studies on the angiogenic activity of the C. pulcherrima leaf extract;
2. To conduct further research using other parts of C. pulcherrima such as roots,
stems and flower used in this study;
3. The essential oils from the plant used should be subjected to phytochemical
screening.
BIBLIOGRAPHY
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origsite=gscholar&cbi=636322
APPENDIX A
LETTER
Notre Dame of Dadiangas University

INTEGRATED BASIC EDUCATION DEPARTMENT
Espina Campus, Labangal, General Santos City
July 26, 2019
Ms. Imelda Casicas
Biology Laboratory-In-Charge
NOTRE DAME OF DADIANGAS UNIVERSITY
Marist Avenue, General Santos City
Dear Ma’am;
Greetings of Peace and Love from St. Marcellin!
We, the Grade 12 STEM students of NDDU – IBED - Espina Campus is currently conducting the study
Antiangiogenic Property of Peacock Flower (Caesalpinia pulcherrima).
In line with this, we would like to ask your permission to allow us to have access to your laboratory for
experiment. Furthermore, we would like to ask a consent from your office to allow us to have an access in
different laboratory equipment and apparatus to properly execute our research study.
You may contact us in the mobile number 0926 428 3608 or email us at [email protected] for more
details and questions. Thank you and we look forward for a positive response for this request.
Respectfully yours,
Kate Lelu F. Magnabijon
Research Representative
Noted by:
MARIANNE JOYCE V. TUBOJAN EVELYN N. DEMAPE M.A
Research Advicer Principal
APPENDIX B
Raw Data
T1R2 T1R3 T3R1
T3R2 T2R2 T2R1
T2R3 T1R1 T1R3
Antiangiogenic Assay
Complete Randomized Design
APPENDIX C
Statistical Analysis
One-Way Analysis of Variance
Table 1. Mean number of major blood vessels formed
Treatment R1 R2 R3 ∑ Mean
T1 14 19 12 45 15
T2 5 5 2 12 4
T3 4 5 4 13 4.3
Step 1: Identify the Problem and Hypothesis

Problem: Is there a significant difference in the number of blood vessels in the
chorioallantoic membrane of mallard embryos?
Ho: There is no significant difference in the number of blood vessels in the
chorioallantoic membrane of mallard embryos.
Step 2: Statistical Tool
One-way ANOVA for Complete Randomized Design (CRD)
Step 3: Significance Level
α = 0.01 or 1%
Step 4: Sampling Distribution
N=9
Total df = rt – 1
= 3(3-1)
= 9-1
=8
Treatment df = t-1
= 3-1
=2
Error df = t (r – 1)
= 3(3-1)
= 3(2)
=6
Step 5: Rejection Region

The null hypothesis will be rejected if the Computed F-value is equal to our
greater than the tabular value of the t-test at 0.01 at level of significance with
(2,6).
Step 6: Computation
2
G
a . CF=
N
2
70
¿
9
4900
¿
9
¿ 544.4444444
n
b. Total SS = (∑ xi −CF ¿ ¿
2
i=1
= (142+192+122+52+52+22+42+52+42) – 544.4444444
= 812- 544.4444444
= 267.5555556
c. Treatment SS = (∑i=1 Ti2/r) – CF
= [( 452+ 122+ 132)/3] – 544.4444444
= (2338/3) – 544.4444444
= 779.3333333 – 544.4444444
= 234.8888889
d. Error SS = Total SS – Treatment SS
= 267.5555556 – 234.8888889
= 32.6666667
Treatment SS
e. Treatment MS =
t−1
234.8888889
¿
(3−1)
¿ 117.4444445
Error SS
f. Error MS =
t (r −1)
32.6666667
¿
[3 ( 3−1 )]
32.6666667
¿
6
¿ 5.44444445
Treatment MS
g. F- value =
Error MS
117.4444445
¿
5.44444445
¿ 21.57
h. Tabular F- value = 10.9
Step 7: The computed F- value is 21.57 which is significant at 1% level of significance.
This value is higher than the tabular F- value with df (2,6) which is 10.9. This indicates
that there is a significant difference in the branching points of the blood vessel in the
chorioallantoic membrane of the mallard eggs using the different treatment, thus the
hypothesis is rejected.
CAM Vascularity
No . of branch points ( treated )−No . of branch points (control)

¿ x 100
No . of branch points(control)
Treatment 1 and Treatment 3
13−45
¿ x 100
45
¿−0.7111 x 100
¿−71.11%
Treatment 2 and Treatment 3
13−12
¿ x 100
12
¿ 0.0833 x 100
¿ 8.33 %
APPENDIX D
Documentations
Figure 2: Chorioallantoic Membrane (CAM) after 48 hours

CURRICULUM VITAE
Curriculum Vitae
PERSONAL DATA:
NAME : Kate Lelu F. Magnabijon
ADDRESS : Phase 3B Blk 12 Lot 24 Doña Soledad Subd.
Labangal, General Santos City
DATE OF BIRTH : May 26, 2001
PLACE OF BIRTH : General Santos City
AGE : 18 years old
RELIGION : Roman Catholic
FATHER’S NAME : Lowie L. Magnabijon
MOTHER’S NAME : Rhodora F. Magnabijon
EDUCATIONAL BACKGROUND:
ELEMENTARY : NDDU – IBED Espina Campus
SY: 2008 - 2014
JUNIOR HIGH SCHOOL : NDDU – IBED Espina Campus
SY: 2014 - 2018
SENIOR HIGH SCHOOL : NDDU – IBED Espina Campus
SY: 2018 - 2020
STRAND : STEM (Science Technology Engineering
Mathematics)
CURRICULUM VITAE
Curriculum Vitae
PERSONAL DATA:
NAME : Lei Pauline C. Gumboc
DATE OF BIRTH : October 10, 2001
AGE : 17 years old
FATHER’S NAME : Napoleon D. Gumboc
MOTHER’S NAME : Lualhati C. Gumboc
SY: 2009 - 2014
SY: 2014 - 2018
SY: 2018 - 2020
Mathematics)
CURRICULUM VITAE
Curriculum Vitae
PERSONAL DATA:
NAME : John Raeven V. Latorza
DATE OF BIRTH : November 14, 2001
AGE : 17 years old
RELIGION : Protestant
FATHER’S NAME : Ranny L. Latorza
MOTHER’S NAME : Josephine V. Latorza
ELEMENTARY : P. Kindat Elementary School
SY: 2008 – 2014
SY: 2014 - 2018
SY: 2018 - 2020
Mathematics)
CURRICULUM VITAE
Curriculum Vitae
PERSONAL DATA:
NAME : Jyrouz Abby U. Quintana
ADDRESS : Phase 1 Blk 7 Lot 28 Doña Soledad Subd.
DATE OF BIRTH : September 29, 2001
PLACE OF BIRTH : Calumpang San Miguel General Santos City
AGE : 17 years old
RELIGION : Protestant
FATHER’S NAME : Jypril L. Quintana
MOTHER’S NAME : Abba U. Quintana
SY: 2008 – 2014
SY: 2014 – 2018
SY: 2018 – 2020
Mathematics)
CURRICULUM VITAE
Curriculum Vitae
PERSONAL DATA:
NAME : Seth Stephen A. Sanchez
DATE OF BIRTH : December 10, 2001
PLACE OF BIRTH : Osaka, Japan
AGE : 17 years old
FATHER’S NAME : Somer C. Sanchez
MOTHER’S NAME : Ruchel A. Sanchez
SY: 2010 – 2014
SY: 2014 – 2018
SY: 2018 – 2020
Mathematics)

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ANTIANGIOGENIC PROPERTY OF PEACOCK FLOWER (CAESALPINIA

A Research Proposal Presented to the Faculty of the

In Partial Fulfillment of the Requirement

Gumboc, Lei Pauline

To their research adviser, Marianne Joyce V. Tubojan, whose expertise, guidance,

To Joey Kevin D. Tiongco, for his valuable comments, suggestions, knowledge,

To the Biology Laboratory Staff of Notre Dame of Dadiangas University,

throughout the process of completing this study;

blessings, wisdom and strength to make this research possible.

property of C. pulcherrima leaf extract. It was hypothesized that there is no significant

difference in the number of blood vessels in the chorioallantoic membrane of mallard

was a significant difference in the number of branching points in the Chorioallantoic

membrane of mallard eggs after 48 hours using the different treatments.

List of Tables vii

List of Figures viii

Chapter I THE PROBLEM AND SETTING

Statement of the Problem 2

Scope and Delimitation 3

Significance of the Study 3

Chapter II REVIEW OF RELATED LITERATURE 6

Chapter III METHODOLOGY

Data Gathering Techniques 16

Chapter IV PRESENTATION, ANALYSIS AND INTERPRETATION OF 17

Chapter V SUMMARY OF FINDINGS, CONCLUSIONS, AND

1 Number of Branching Points in the CAM after 48 hours 18

3 CAM Vascularity of mallard eggs using the different treatments 20

2 Chorioallantoic Membrane (CAM) after 48 hours 34

studied because it causes strong anti-oxidants effects.

multiply in an uncontrolled way, which is abnormal in the growth of cells. It is a group of

cancer. (McGraw-Hill Concise Dictionary of Modern Medicine, 2002).

Statement of the Problem

This study aimed to determine the antiangiogenic property of C. pulcherrima.

Specifically it answered the following questions:

2. Is there a significant difference developing blood vessels in the CAM of mallard

embryos using the following treatments:

2.1 Distilled Water;

2.2 Simvastatin; and

2.3 C. pulcherrima leaf extract?

There is no significant difference in the number of blood vessels in the

chorioallantoic membrane of mallard embryos.

Scope and Delimitation

simvastatin and T3 is the C. pulcherrima.

The collection of data, through observation, validation and documentation was

conducted at the Biology Laboratory of Notre Dame of Dadiangas University Main

Significance of the Study

The researcher intended to analyze the antiangiogenic property of C.

and understanding of the conduct of the study.

found in any various journals.

Antiangiogenesis. This refers to the process where the cells multiply in an

growth of new blood vessels that can cause cancer.

organs. Operationally, this is the primary subject of the study.

Caesalpinia pulcherrima. This refers to an upright rounded, generally sprawling,

multi-stemmed ligneous plant. It typically gets up to 10 feet tall, displaying characteristic

Chorioallantoic Membrane (CAM). This refers to an extra embryonic that is wealthy

of blood vessels. Operationally, it refers to the location of development of

antiangiogenesis of C. pulcherrima on the blood vessels.

Inhibitors. This refers to limit, prevent, or block an action or function. Operationally,

this was the agent that interfered the angiogenic properties.

be determined to possess a particular result. Three replicates in every treatment were

Simvastatin. This refers to the medication to decrease elevated supermolecule levels.