CHAPTER 1

THE PROBLEM AND ITS SETTING

Background of the Study

Anti-angiogenic drugs may show the spread of cancer by obstructing the delivery

of nutrients and oxygen to the tumor. Plants have long been used as a source of

treatment against a variety of diseases including cancer. The formation of new,

abnormal blood vessels during the complex process of cancer angiogenesis is what

causes tumor growth and metastasis. The constant release of pro-angiogenic factors by

cancer cells and cancer-associated stromal cells stimulates the angiogenic switch that

causes endothelial cells (ECs) to proliferate and migrate to form new blood vessels

during tumor growth (Lugano, Ramachandran, and Dimberg, 2019).

One factor that makes this disease deadly is its capability to metastasize in

one’s body. It is where cancer cells have the ability to grow outside of the place in the

body where they originated. The expansion of cancer cells to tissues and organs

outside of where the tumor originated, as well as the development of new tumors. It is

the leading cause of cancer treatment failure and cancer-related death. Even in tumors

that are radiotherapy or chemotherapy responsive, metastasis is often the cause of

treatment failure. Metastatic tumors not only diffuse localization. It normally occurs when

cancer cells break away from the main tumor and enter the bloodstream or lymphatic

system. Research evidence suggests that soluble factors secreted by the primary tumor

affect the establishment and development of angiogenesis(reference).

Clitoria ternatea L. (Butterfly Pea) is the most widely known species in its genus,

It has a widespread distribution in various natural habitats across the globe, including

Asia, Africa, and the Americas. Throughout history this species has been primarily
utilized in agricultural and medicinal fields. Recently, researches have proved that this

species exhibits antioxidant, hypolipidemic, hepatoprotective, antispasmolytic, analgesic,

estrogenic, cytotoxic, antibacterial, antifungal, hypotensive, and broncholytic effects. e

various parts of the plant has been scientically evaluated for various pharmacological

activities including antioxidant , anthelmintic , analgesic, anxiolytic, antidepressant,

anticonvulsant, hypoglycemic , larvicidal and cytotoxic activities.

Statement of the Problem

This study aims to determine the Anti-Angiogenic effect of Butterfly Pea leaves

(Clitoria ternatea) specifically to answer the following questions:

1. Determine if the Butterfly Pea Plant (Clitoria ternatea) possesses anti-angiogenic

properties.

2. What is the average blood vessel growth in the chorioallantoic membrane assay

in terms of the following:

● Treatment 1 (15% concentration of Butterfly Pea Plant leaf extract).

● Treatment 2 (30% concentration of Butterfly Pea Plant leaf extract).

● Treatment 3 (45% concentration of Butterfly Pea Plant leaf extract).

● Positive control group

● Negative control group

3. Is there a significant difference among the experimental treatments in terms of

blood vessel count?

4. Is there a significant difference between the experimental treatments and the

positive control group in terms of blood vessel count?

5. Is there a significant difference between the experimental treatments and the

negative control group in terms of blood vessel count?

 6. Is there a significant difference among the experimental treatments, positive,

and the negative control group in terms of blood vessel count.

Null Hypothesis

In terms of blood vessel count, there is no significant difference between

treatments.

Scope and Limitation

This study will primarily focus on the inhibition of the growth rate of blood vessel

growth of the Chorioallantoic membrane of duck eggs and the rate of survivability by

injecting the Clitoria ternatea extract into the Chorioallantoic membrane of the duck

eggs. It will also be conducted in ORCA Laboratories General Santos City within two

weeks of permission. This study aims to find out the Anti-Angiogenic activity of the

Clitoria ternatea extract in the duck eggs.

Significance of the study

Future Researchers. This study is significant to the future researchers who will

establish or dive into a research study which is similar or related to this study. This study

will be beneficial to the future researchers in a way that this study could be a sample

literature or the findings from this study will help the future researchers to achieve the

objectives of their study, thus also helping the future researchers to answer the problems

of their study by referring to this study as their guide or review of related studies (RRS).

Cancer Researchers. The advancement of cancer prevention knowledge can

help to ensure that cancer survivors live longer, higher-quality lives. Additionally,

research is shedding light on the origins of cancer and paving the way for more effective

diagnostic and therapeutic approaches which can aid other Cancer researchers.
 Medical Community. This is beneficial for those working in the medical field.

This research can assist in their search for novel treatments as well as ensuring that we

make the best use of those that already exist. Findings in this research can find answers

to things that are unknown, filling gaps in knowledge and changing the way that

healthcare professionals work.

Botanists. This research can assist botanists in learning more about the

characteristics and abilities of Clitoria ternatea (Butterfly Pea), which they can use to

increase their understanding of a particular plant. Findings from this study on plant

systems can also instruct us on how to approach issues with angiogenesis and

metastases matters.

Operational Definition of Terms

Angiogenesis - refers to migration, growth, differentiation of endothelial cells

Chorioallantoic membrane - is a highly vascular gas exchange membrane in

bird eggs lying just beneath the shell surface that is composed of the fused chorion and

wall of the allantois.

CAM Assay - The CAM assay, or Chorioallantoic Membrane assay, is a widely

used experimental technique in biology and medicine. It involves studying the

development and behavior of blood vessels using the chorioallantoic membrane of a

developing chicken embryo.

Angioblast - also called endothelial progenitor cell, a mesenchymal cell derived

from hemangioblast that gives rise to blood vessels.

Angiopoietin 1 - a glycoprotein that activates its Tie2 receptor by inducing

tyrosine phosphorylation. It promotes vessel maturation and stability.

 Blood islands - clusters of angioblasts and hematopoietic precursor cells that

give rise to different parts of the circulatory system. Fusion of blood islands along with

lumina formation by angioblasts lead to primitive vascular networks.

Fibroblast growth factor 2 - (FGF2) also known as basic fibroblast growth

factor (bFGF) is a member of the fibroblast growth factor family that is bound to

basement membranes (BM) of blood vessels; its proangiogenic actions can be activated

by heparin sulfate-degrading enzymes, which causes it to be released from BMs.

 CHAPTER 2

REVIEW OF RELATED LITERATURE AND STUDIES

This chapter presents the related literature, studies, evidences, information, and

background about the present investigation.

Review of Related Literature

Angiogenesis

Regarding angiogenesis, Mandal (2017) stated that it is the formation of new

tissues involving the formation of new blood vessels. The quantitation of angiogenesis in

a biopsy specimen may help predict the risk of metastasis or recurrence. The

quantification of microvessel density in histologic specimens of invasive breast cancer,

for example, provides an indication for the risk of metastasis (Weidner, N., et al., 1991).

Angiogenesis is a complex, multi-step, tightly regulated process which is

balanced by regulators that act either to stimulate (e.g., VEGF) or to inhibit (e.g.,

endostatin) blood vessel growth. Inadequate angiogenesis results in insufficient vascular

growth that can cause ischemic diseases, while exaggerated vascular growth associated

with cancer and inflammatory ailments (Fiedler and Pellet-many, 2022).

Angiogenesis inhibitors, also called anti - angiogenics, block blood vessel growth

by blocking nutrients and oxygen from a tumor, the angiogenesis inhibitors "starve” the

tumor (Cancer.Net. 2022). Anti - angiogenics are a class of kinase inhibitors that bind

either angiogenic factors or their receptors; they act as agents that shape the irregular

and leaky vessels of cancer to create an almost normalized intratumoral vascular

network, at least transiently. Thus such improved vascular efficiency of cancer

microcirculation is mainly responsible for a more capable transport of chemotherapeutic

agents to cancer cells, and also the molecular background to the association of

antiangiogenic therapies with traditional chemotherapies (Quaresmini & Guida, 2022).

Clitoria ternatea

Clitoria ternatea, commonly known as butterfly pea, is a perennial herbaceous

plant from the Fabaceae family. It has recently attracted a lot of interest as it has

potential applications both in modern medicine and agriculture, and as a source of

natural food colorants and antioxidants. C. ternatea has long been cultivated as a forage

and fodder crop, and early studies assessed the plant for these purposes (Reid and

Sinclair, 1980; Barro and Ribeiro, 1983; Hall, 1985). The plant is reputed for its folkloric

uses in various diseases. The various parts of the plant have been scientifically

evaluated for various pharmacological activities including antioxidant , anthelmintic,

analgesic, anxiolytic, antidepressant, anticonvulsant , hypoglycemic , larvicidal and

cytotoxic activities. Its stem, roots, leaves, and flowers contain advantageous bioactive

chemicals that can be used as medicines. It can tolerate a broad range of temperatures,

precipitation levels, and altitudes. They can withstand heavy grazing, grow best in

heavier soil types, and function well in less tropical and environments where lucerne is

not acclimated. Due to how well-liked this plant is among people and the medical

community and how many health benefits it exhibits, it is one of the few hobbies that

gives cultivators a consistent income throughout the year, along with gardening. The

majority of butterfly peas are grown in backyard gardens for personal use and on farms

for local markets. (Gomez et al., 2003).

CAM Assay

The chick or duck embryo chorioallantoic membrane (CAM) model is an

extraembryonic membrane that is knowingly used in vivo to study either angiogenesis or

anti-angiogenesis or both. The CAM assay has become an extensively used tool for the

determination of angiogenesis and anti - angiogenesis property of various drugs

including herbal extracts. The accomplishment of anti-angiogenesis therapy for treating

cancer has led to an explosion in the field of research for potential anti-angiogenic

agents. As mentioned earlier, the Chorioallantoic Membrane (CAM) is a highly

vascularized avian tissue which performs multiple functions during embryonic

development. Throughout the past few decades, interest in the CAM as a robust

experiment platform to study blood 10 vessels is shared by specialists working in

bioengineering, development, morphology, biochemistry, transplant biology, cancer

research and drug development.

Cancer Metastasis

Nearly half of all cancer patients present clinically detectable metastatic disease

(Martin et al., 2000), whereas a larger number of clinically diagnosed cancer patients

also have micrometastasis that is beyond detection limit of techniques currently

employed (Winter et al., 2015). The main route of spread to distant organs is the

circulatory system, 9 while lymphatic vessels provide a route to local lymph nodes, after

which metastasis also migrates through the blood. While the most common route tends

to be the circulatory system, the degree of lymphatic versus hematogenous spread

appears to depend on the origin and position of the primary tumor. Cancer is still the

world’s leading cause of death, resulting in approximately 8.2 million deaths in 2012

(Stewart and Wild, 2014). Cancer metastases account for the vast majority of cancer
patients’ morbidity and mortality and are associated with nearly 90% of all cancer-

associated deaths (Mehlen and Puisieux, 2006). The development of new tumors

(secondary and tertiary tumor nests) in tissues and organs away from the primary site of

tumor origin is known as cancer metastasis. Despite that, significant strides have been

made in understanding the mechanism of metastasis, crucial information on the process

of metastasis, the factors responsible for the cancer spread and establishment at distal

secondary locations are still relatively unknown (Hunter et al., 2008).

Prednisone

Prednisone provides relief for inflamed areas of the body. It is used to treat a

number of different conditions, such as inflammation (swelling), severe allergies, adrenal

problems, arthritis, asthma, blood or bone marrow problems, endocrine problems, eye or

vision problems, stomach or bowel problems, lupus, skin conditions, kidney problems,

ulcerative colitis, and flare-ups of multiple sclerosis. Prednisone is a corticosteroid

(cortisone-like medicine or steroid). It works on the immune system to help relieve

swelling, redness, itching, and allergic reactions. Prednisone is approved to be used to

reduce inflammation and suppress (lower) the body’s immune response. It is used with

other drugs to treat the following types of cancer: Acute lymphoblastic leukemia (ALL)

and acute myeloid leukemia (AML). It is used as palliative therapy in adults.

Prednisone decreases inflammation via suppression of the migration of

polymorphonuclear leukocytes and reversing increased capillary permeability. It also

suppresses the immune system by reducing the activity and the volume of the immune

system. The antineoplastic effects may correlate with the inhibition of glucose transport,

phosphorylation, or induction of cell death in immature lymphocytes. It may have

antiemetic effects by blocking the cerebral innervation of the emetic center via inhibition

of prostaglandin. After cell surface receptor attachment and entry into the cell,
prednisone enters the nucleus, binds, and activates specific nuclear receptors, resulting

in altered gene expression and inhibition of proinflammatory cytokine production. This

agent decreases the number of circulating lymphocytes, inducing cell differentiation, and

stimulates apoptosis in sensitive tumor cell populations. (Puckett et al., 2022)

Review of Related Studies

According to Plants in Anticancer Drug Discovery: From Molecular Mechanism to

Chemoprevention (Siddiqui et al., 2022), Cancer is one of the leading causes of death

worldwide, and the development of novel anticancer medications is the most pressing

demand in recent years. For more than 50 years, natural products have been recognized

as useful in the treatment of numerous ailments, including cancer. Plants and

microorganisms are the primary and potential sources of anti-cancer natural chemicals.

Furthermore, during the last several decades, research in the field of plant-based natural

chemicals has advanced to sophisticated and molecular level understandings, leading to

11 the discovery of effective anticancer medicines. Plants have also been recognized as

plentiful and profitable sources for the creation of innovative therapeutic compounds for

the treatment and prevention of many cancer types. Due to the high toxicity of some

cancer chemotherapy medications, as well as their unpleasant side effects and drug

resistance, natural chemicals are in high demand as potential anticancer therapies.

Facts and information about various medicinal plants, their bioactive compounds, and

their potent anticancer activities against various cancers have been gathered in this

detailed evidence-based mechanistic review, with a further approach to represent the

molecular mechanism behind the anticancer activity of these plants.

 Recent Advances in Anti-Metastatic Approaches of Herbal Medicines in 5 Major

Cancers: From Traditional Medicine to Modern Drug Discovery (Park et al., 2021).

Based on the recognition of these side effects, many studies have been conducted using

herbal medicine for metastatic cancer. Herbal medicine has been used in clinical

treatment for thousands of years in China, Japan, Korea, and other countries. Numerous

research exists that focus on the therapeutic effect of herbal medicine on metastatic

Cancer. Natural antioxidants are widely distributed in medicinal plants which exhibit a

wide range of biological effects, including anti-cancer, anti-inflammatory, anti-aging, and

anti atherosclerosis. reported phenolic content from extracts.

Anti-Angiogenic Therapy: Current Challenges and Future Perspectives

(LopesCoelho et al., 2021). Anti-angiogenic therapy is an old cancer treatment strategy

that tries to stop the delivery of nutrients and oxygen to tumor cells by shrinking the

vascular network and preventing the development of new blood vessels. Because VEGF

signaling is thought to be the primary angiogenesis promoter, the majority of anti-

angiogenic 12 medicines licensed for cancer treatment target VEGF actions. In addition

to controlling angiogenesis, these medicines can enhance immunological treatment

because VEGF has immunosuppressive properties. Despite the molecular rationale that

firmly supports the effectiveness of medications in preventing cancer progression, they

have been shown to be insufficient in the majority of cases. Rehabilitating outdated

medications that interfere with angiogenesis pathways associated with tumor

microenvironment could be a promising method. In this review, we delved deeper into

the molecular mechanisms underpinning anti-angiogenic techniques and their failure, as

well as the alternate pathways influencing angiogenesis.

 Medicinal Plants Extracts with Antiangiogenic Activity: Where Is the Link?

(Hoseinkhani et al., 2020). Plants contain several active chemical compounds

simultaneously and unlike chemical drugs, they can have synergistic effects and

therefore, influence different aspects of disease pathology at the same time. In other

words, plant extracts rich in biologically active compounds can slow down the growth of

cancer cells and induce apoptosis in them at the same time which leads to tumor

eradication by hindering angiogenesis and therefore, metastasis. Interactions of the

active ingredients in plant extracts with tumors can give this opportunity to the immune

system to identify and respond to the tumor cell. Herbal components have less negative

effects than chemicals and anti-cancer treatments. However, there are a number of

negative side effects associated with the use of crude plant extract in vivo. The usage of

therapeutic herbs should be standardized because improper use can result in serious

poisoning, allergic responses, hemorrhage, and death. Herbal medicines may inhibit the

absorption of 13 of certain essential nutrients. Other types can potentially boost or

decrease the effect of a medicine while causing substantial side effects.

Chorioallantoic Membrane (CAM) Assay of Different Extracts of Rhizome and

Inflorescence of Heliconia rostrata (Dhara et al., 2018). According to the Botanical Drug

development Guidance, the knowledge of the carcinogenicity of a botanical drug product

is one of the key steps in its development as a drug for clinical use. Many in vivo studies

are present that allow the preliminary investigation of carcinogenic potential of a new

molecule or drug product without being directly entered into the studies on rodents. One

of them is Chicken embryo chorioallantoic membrane (CAM) assay. CAM assay has

been widely used as an in vivo model to study angiogenic effect, tumor cell invasion and

metastasis of many new molecules and herbal preparations. CAM assay is an in vivo

preclinical model used for testing the vascular and anti-vascular effects of health care
agents. This technique of studying therapeutic agents is not only easy but also is

economical in terms of cost and time.

The Anti-Angiogenic Effect of Nelumbo nucifera Leaf Extracts in Human

Umbilical Vein Endothelial Cells. According to the study of Lee et al. (2015). A process

of angiogenesis completes following several major steps, including proliferation,

sprouting, elongation, and migration of endothelial cells. However, abnormal and

unregulated process of pathological angiogenesis may result in various pathogenesis

diseases such as diabetic retinopathy, myocardial infarction, and chronic inflammation.

Hence, to control or inhibit the angiogenesis could be considered an important strategy

in anti-cancer therapy. Recently, investigations into new sources of natural antioxidants

have become very important to improve human health. Plants are considered rich

sources of polyphenol compounds such as flavonoids which exert potent antioxidant

ability. A number of plant based polyphenols including flavonoids exhibit significant

biological potential in terms of their remarkable antioxidant activities, as well as

scavenge ROS and inhibit metal chelation and lipid peroxidation.

Chorioallantoic Membrane Assay as Model for Angiogenesis in Tissue

Engineering (Focus on Stem Cells | Tissue Engineering Part B: Reviews, 2021). The

highly vascularized CAM has been utilized by scientists as an in vivo model to examine

tumor biology and angiogenesis. The very first described study dated from 1911, which

successfully employed the extraembryonic membrane to grow implanted chicken

sarcoma tumors. Two years later, it was demonstrated that the CAM is also suitable for

the transplantation of xenogeneic tissues without significant impact on their intrinsic

morphological and biological characteristics. Screening for the anti-angiogenic activity of

selected Philippine medicinal plants using chorioallantoic membrane assay. According to

Camposano, the anti-angiogenesis research utilizes the chorioallantoic membrane

(CAM) of the avian embryo. The CAM model has many advantages including its low

cost, ease of use, high reproducibility, reliability and simplicity. The CAM assay, in

comparison with other animal models, is a closed system, which promotes a longer half-

life on many compounds because excretion of such compounds is eliminated. This, in

turn, allows the use of minute amounts of experimental compounds. 15 Studies on the

anti-angiogenic effect of Marsdenia tenacissima extract in vitro and in vivo.

According to the study HUANG et al. (2013), the Studies on the anti-angiogenic

effect of Marsdenia tenacissima extract in vitro and in vivo. Angiogenesis is important in

the development of cancer. The main features of angiogenesis are increased

vasculature and overexpression of vascular endothelial growth factor (VEGF). In the

present study, the effects of M. tenacissima extract (MTE) on human umbilical vein

endothelial cell (HUVEC) proliferation, migration and capillary-like tube formation were

investigated in vitro and using the chick embryo chorioallantoic membrane (CAM) assay

in vivo.

Medicinal Plants and Cancer Chemoprevention (Desai et al., 2008). The

anticancer properties of plants have been recognized for centuries. Isolation of

podophyllotoxin and several other compounds (known as lignans) from the common

mayapple (Podophyllum peltatum) ultimately led to the development of drugs used to

treat testicular and small cell lung cancer. The National Cancer Institute (NCI) has

screened approximately 35,000 plant species for potential anticancer activities. Among

them, about 3,000 plant species have demonstrated reproducible anticancer activity.

Many studies have focused on the chemoprotective properties of plants such as

anticarcinogenic properties of Abrus precatorius on Yoshida sarcoma in rats,

fibrosarcoma in mice and ascites tumor cells. Similarly, Dhar et al. have examined the
anticancer properties of Albizzia lebbeck on sarcoma in mice and Alstonia scholaris on

benzo[a]pyrene-induced forestomach carcinoma in humans.

VEGF as a key mediator of angiogenesis in cancer (Carmeliet, 2005). Vascular

endothelial growth factor (VEGF) is a homodimeric glycoprotein with a molecular weight

of approximately 45 kDa. It is the key mediator of angiogenesis (the formation of new

blood vessels), and binds two VEGF receptors (VEGF receptor-1 and VEGF receptor-2),

which are expressed on vascular endothelial cells. In healthy humans, VEGF promotes

angiogenesis in embryonic development and is important in wound healing in adults.

VEGF is the key mediator of angiogenesis in cancer, in which it is up-regulated by

oncogene expression, a variety of growth factors and also hypoxia. Angiogenesis is

essential for cancer development and growth: before a tumor can grow beyond 1-2 mm,

it requires blood vessels for nutrients and oxygen. The production of VEGF and other

growth factors by the tumor results in the 'angiogenic switch', where new vasculature is

formed in and around the tumor, allowing it to grow exponentially. Tumor vasculature

formed under the influence of VEGF is structurally and functionally abnormal. Blood

vessels are irregularly shaped, tortuous, have dead ends and are not organized into

venules, arterioles and capillaries. They are also leaky and hemorrhagic, which leads to

high interstitial pressure. These characteristics mean that tumor blood flow is

suboptimal, resulting in hypoxia and further VEGF production. This central role of VEGF

in the production of tumor vasculature makes it a rational target for anticancer therapy.
 CHAPTER III

MATERIALS AND METHODS

This chapter presents and describes the research methods and procedures that

the researchers will use for this study. This chapter includes the research design,

procedure, flowchart, preparation of materials, and statistical analysis.

Research Design

The researchers in this study will utilize a quantitative method of research to

determine which of the following solutions: 15% of Clitoria ternatea extract, 30% of

Clitoria ternatea extract, 45% of Clitoria ternatea extract, will efficiently induce the

angiogenic activity in the Chorioallantoic Membrane (CAM) Assay.

Fig. 1 Research Design

Procedures Flowchart

The following flowchart shows the process of the experiment that the research

will conduct.

Fig. 2 General Procedure Flowchart

Collection of plant specimens

The researchers utilized the leaves of Clitoria ternatea that was sourced locally

within the city of General Santos. The researchers collected approximately one

kilogram of leaves, the leaf samples were washed using distilled water. The leaves were

air dried without exposure in the sun for 3-5 days. After air drying, the dried leaves are

grinded in a low speed blender until they reach a powder-like texture and are stored in

an air-tight container and placed at room temperature away from direct sunlight until

further use (Kanchana & Balakrishnan, 2011).

Preparation of ethanolic leaf extracts

The dried and powdered leaves were soaked in 95% ethanol solution for 24

hours and were subjected to extraction with 500 ml of 96% ethanol at room temperature

in three cycles of 72 h each. After each cycle, the extract was filtered through filter paper

and the solvent was removed by vacuum distillation under reduced pressure with a

rotary evaporator (38 °C). To prepare the stock solutions, the extract and active
substance were dissolved in dimethyl sulfoxide (DMSO). The mixtures were then filtered

and sterilized using a 0.22 µm filter, and kept frozen until analyzed. Serial dilutions of the

EEA and ART was freshly prepared from stock solution before use by dissolving them

into the cell culture media. These extracts were then diluted in various quantities using

distilled water, then they were weighed and stored until further use (Muresan, et al.,

2013). The treatment group will consist of the following concentrations:

Treatment 1: 15% of Clitoria ternatea leaf extract

Treatment 2: 30% of Clitoria ternatea leaf extract

Treatment 3: 45% of Clitoria ternatea leaf extract

Phytochemical screening for leaf extracts

Angiogenic Studies:

Angiogenesis is the formation of new blood vessels out of pre-existing capillaries.

The study of the angiogenic process has gained popularity in the study of tumor

development where the growth of solid tumors are dependent on the induction of an

adequate blood supply. The chorioallantoic membrane (CAM) assay is an established

screening assay for potential pro- and anti-angiogenic agents. The chorioallantoic

membrane of the chick or duck embryo develops over the first 11 days of the 21 days

gestation period. Capillaries appear in the yolk sac of fertilized eggs at 28 hours and

grow rapidly over the next 6-8 days. This is the right time to introduce the substance to

be tested and observe its effect on the 12th day.

Preparation of test organism

The test organisms that the researchers will be using for this study are fertilized

duck eggs that will be sourced locally at an available distributor at Barangay Baluan,

General Santos City, Philippines, the researchers will be using ten (10) days old eggs,
using a flashlight each egg will be checked manually if it’s fertilized or not. The gathered

fertilized duck eggs will be manually washed in order to remove feathers, dirt, excrement

and other foreign debris and it will be disinfected using 70% ethanol solution. The

researchers will place the eggs under a flashlight and the researchers will then proceed

to identify the location of the air sac inside the egg, after it is identified the researchers

will use a pencil to mark the air sac location, the researchers will also identify where the

embryo is and place a square mark using the pencil. After the marking process the eggs

will be carefully placed in an egg tray and stored inside an insulated box or an icebox.

Preparation of equipment

The researchers will be using the following equipment: surgical gloves, surgical

tape, 5cc sized syringe, syringe filter, comedone extractor, hair nets, face masks,

scissors, and incubator. Each of the equipment mentioned will be thoroughly sterilized

under UV light and the room where the researchers will perform the procedure will also

be disinfected and sanitized.

Shell Drilling

Using the air sac mark as a guide, the researchers will use the comedone

extractor to poke a tiny hole in the egg shell, this will act as a window for the syringe

needle to be able to fit and inject the chorioallantoic membrane with the prepared

treatments.

Application of treatments on chorioallantoic membrane

At Day 10, a treatment application was injected into each embryo. The

researchers ensured that only viable embryos with evident heartbeats, movements and

blood vessels were used. The test organisms were divided into five groups with five

replications for each treatment group. For each of the different extract concentrations,
10ml was taken using a calibrated automatic pipettor and was applied to the center of

the embryo’s blastodisc.

Window Sealing

Using the sterilized surgical tape, the researchers will seal the created window to

allow the growth of the new blood vessels, this is a crucial step since leaving the shell

open will cause severe heat inside the surface of the fertilized duck egg.

Incubation

After the sealing of the window created in the egg, the researchers will place the

eggs in the incubator, and the eggs will undergo the incubation process with the

temperature set at 37 degrees Celsius . To fully observe the growth and formation of

new blood vessels in the fertilized duck eggs treated with the Clitoria ternatea leaf

extract the incubation process will take twenty-four (24) hours.

Visual assessment and photography

At Day 12, the eggs were photographed and numerical measurements were

done on these photographs. Only viable embryos with well-developed blood vessels

were used for observation. Each treatment group contained at least three replications.

Statistical Analysis

The growth of blood vessels in CAM assay was analyzed by the researchers by

utilizing mean and percentage. The data obtained from the blood vessel count were

statistically analyzed using One-Way Analysis of Variance (ANOVA) with Tukey’s

multiple comparison post hoc tests to compare the level of significance between control

and experimental groups to determine which solution of Clitoria ternatea leaf extract will

best regulate angiogenesis in the chorioallantoic membrane (CAM) assay. The values of
p<0.05 were considered as significant for each group determining if results were

significantly different from one another or if they were relatively the same. Independent

sample t-test was used to determine the differences between experimental, positive and

control groups.
 CHAPTER IV

PRESENTATION, ANALYSIS, AND INTERPRETATION OF DATA

This chapter presents, analyzes, and interprets the data gathered in this

study. The results presented in the succeeding tables with corresponding

discussions and explanations of the different parts of the instruments used. It

also answers specific problems given in the first chapter.

Table 4.1

The Result of Phytochemical Analysis of the Ethanolic Extract of

Butterfly Pea (Clitoria ternatea).

Indications
Class of
Observations
Compounds
Leaf

Reddish-brown precipitate was

Alkaloids +
observed

No formation of red color was

Flavonoids -
observed

Saponins Froth formed +

Formation of blackish-green color

Tannins +
was observed

Phenols No white precipitate was observed -

 The table presented above shows details of the bioactive compounds

found from the phytochemical analysis conducted on the Clitoria ternatea or

Butterfly Pea.

Table 4.2

Counting of Vessel Growth in Treated Duck Chorioallantoic Membrane

(CAM)

Treatment Replication Replication Replication Total Average

1 2 3

(15%
Concentrate
d leaf
extract)

(30%
Concentrate
d leaf
extract)

(45%
Concentrate
d leaf
extract)

(Prednisone
Solution)

(Sterile
water)
 Table 4.2 presents the result acquired through the CAM assay. The

counting and comparing of all the blood vessels from the replicates of the

experimental groups and the control groups, the results showed that the blood

vessel growth in the experimental group was lower than the result of the blood

vessel growth in the control group, henceforth the angiogenic property of the

Butterfly Pea leaf extracts in terms of blood vessel count is anti-angiogenic.

The ethanolic extracts Clitoria ternatea were used as experimental

treatments to determine the percentage decrease in the 8-day old Duck

Chorioallantoic Membrane (CAM) Assay. The primary and secondary blood

vessels were counted. The regarded veins were subjected through the

comparison between the control groups; thus, the total number of the remaining

blood vessels will be the basis for percentage decrease.

The control groups consisted of Prednisone applied or the positive control

group, and the sterile water applied or the negative control group. For the

positive control, it was ensured that the commercialized treatment is

active on inhibiting an anti-angiogenic factor for it to be comparable to the

effects performed by the ethanolic extract of Clitoria ternatea.

The positive control (P1) has an average vessel growth of (), which means

that the Prednisone solution affected the normal growth of blood vessels and

thus caused a blockage in the natural growth of blood vessels. The negative

control (N1) sterile water did not affect the growth of blood vessels.
 Figure 4. Number of Blood Vessels in each variable

Table 4.3

The percentage Decrease of Blood Vessels using the Ethanolic Extracts of

Butterfly Pea (Clitoria ternatea) leaves.

EXPERIMENTAL AVERAGE PERCENTAGE DECREASE

TREATMENTS

-
+

T1

T2
 T3

The table above details the percentage decrease between the

experimental treatments of the ethanolic extract of Butterfly Pea (Clitoria

ternatea) leaves. For the researchers to obtain the percentage decrease, the

ratio between each replication and the average blood vessel growth of each

treatment was computed first.

A normal embryo with stable Chick Chorioallantoic Membrane is only able

to exhibit approximately 43 secondary blood vessel growth in a single strand of

vein within 24 hours. The first treatment which is the 15% concentration of the

ethanolic extract of Clitoria ternatea was revealed to be to be effective in

inhibiting the Angiogenic activity in the duck embryo since its average vessel

growth was (_____), giving a (_________) percentage decrease in blood vessel

formation. In comparison to the positive control, its effect on the Chick

Chorioallantoic Membrane has better performance with vessel increase of

(________). This indicated that the 15% concentration of the ethanolic extracts of

Clitoria ternatea was active in inhibiting blood vessel growth and formation, and

thus angiogenesis and vascular endothelial growth factor.

Nonetheless, on the second treatment, the 30% concentration of the

ethanolic extract of Clitoria ternatea showed more significant effects in the

decrease of vessel formation. It exhibited an average vessel growth of (___),

displaying a (____) percentage increase when compared to the positive control,

and (____) percentage increase compared to the negative control group. The

results indicated that the 30% concentration of the ethanolic extracts of Clitoria

ternatea revealed a higher efficacy rate and less active blood vessels and

showed anti-angiogenic effect. Finally, the third treatment, the 45% concentration

of the ethanolic extracts of Clitoria ternatea has an average vessel growth of

(_____), and revealed a (_____) percentage decrease compared to the positive

control and a (______) percentage decrease compared to the negative control.

The result revealed that the 45% concentration of the ethanolic extract of Clitoria

ternatea has the highest efficacy rate and was very active in inhibiting the blood

vessel growth formation, and thus angiogenesis and vascular endothelial growth

factor

Table 4.4

One-Way Analysis of Variance (ANOVA) Comparing experimental and

positive control group

Source of SS df MS F-value P-value F crit

Variation

Between

Groups
Within

Groups

Total

The table above demonstrates the data result using ANOVA comparing the

positive control group ( Prednisone solution ) and the experimental group. In

order to determine whether or not there is a significant difference in the blood

vessel growth among the different treatments, two aspects obtained from the

One-way ANOVA are to be considered. The first aspect to be considered is that

the F-value must be greater than the F-critical value. With this, the researchers

can determine whether there is a significant difference between the variables.

The computed F-value, which is (________), is greater than the computed F-

critical value resulting in (_____), rejecting the null hypothesis. Thus, there is a

significant difference in the blood vessel growth among the treatments based on

the F-value. The second aspect to be considered is the P-value; the P-value

must be less than 0.05 in order for the null hypothesis to be rejected. The

computed P-value is 0.0282, which is less than 0.05, thus the hypothesis is

rejected. Hence, there is a significant difference between the experimental and

the positive control group.

Table 4.5

One-Way Analysis of Variance (ANOVA) Comparing experimental and

negative control group

Source of SS df MS F-value P-value F crit

Variation

Between

Groups

Within

Groups

Total

Table 4.6

One-Way Analysis of Variance (ANOVA) Comparing all Concentrations

Source of SS df MS F-value P-value F crit

Variation

Between

Groups

Within

Groups

Total

Table 4.7
 One-Way Analysis of Variance (ANOVA) Comparing all the treatments

Source of SS df MS F-value P-value F crit

Variation

Between

Groups

Within

Groups

Total
 CHAPTER V

SUMMARY, FINDINGS, CONCLUSION AND RECOMMENDATIONS

This chapter presents the summary of the findings, conclusion, and

recommendations based upon the data analyzed in the previous chapter. The

first section contributes to an overall summary of the study followed by the

summary of the findings and their conclusions. Subsequent to this includes the

recommendations in lieu of the Anti-angiogenic effect of Clitoria ternatea leaf

extract through the application of CAM-Assay.

Summary of Study

The main objective of this study was to investigate if the leaf extract of

Clitoria ternatea displayed Anti-angiogenic effects. Based on the results

presented in the previous chapter it was concluded that the higher the

concentration percentage of the leaf extract, the higher the percentage decrease

in the blood vessel formation in the CAM Assay. In addition, the results

demonstrate that the highest concentration of the extracts leads to a greater

disruption of veins.

Findings

The following are the findings based on the data gathered. For Research

Question 1 the findings are as follows:

 a) Based on the result acquired using CAM assay, the replicates of

the control groups and the experimental groups the results show

that there are less blood vessels in the experimental groups than

the control groups, thus the anti-angiogenic property of the Butterfly

Pea in terms of the Butterfly Pea leaf extracts and control groups in

terms of blood vessel count is anti-angiogenic.

Research Question 2:

Based on the result using ANOVA comparing the positive

control group (Prednisone) and the experimental group. The

computed F-value, which is (_____), is greater than the computed

F-critical value resulting in (_____), rejecting the null hypothesis.

The computed P-value is (_____), which is less than 0.05, thus the

hypothesis is rejected. Hence, there is a significant difference

between the experimental and the positive control group.

Research Question 3:

a) Based on the result using ANOVA comparing the negative control

group (sterile water) and the experimental group. The computed F-

value, which is (______), is less than the F- critical value, and the

P-value is (_____) which is greater than 0.05, thus there is no

significant difference between the experimental group and the

negative control group.

Research Question 4:

a) Based on the result using ANOVA comparing all the concentration

(15%, 30%, 45% concentration). The computed F- value, which is

(___), is less than the F- critical value, thus the hypothesis is not

rejected. The computed P- value is (_____), which is greater than

0.05 therefore there is no significant difference among the

concentration.

Research Question 5:

a) Based on the results acquired using One-Way Analysis of Variance

comparing all the treatments used in the study. In order to

determine whether or not there is a significant difference in the

blood vessel growth among the different treatments, two aspects

obtained from the One-way ANOVA are to be considered. The first

aspect to be considered is that the F-value must be greater than

the F-critical value. With this, the researchers can determine

whether there is a significant difference between the variables. The

computed F-value, which is (______), is greater than the computed

F-critical value resulting in (_____), rejecting the null hypothesis.

Thus, there is a significant difference in the blood vessel growth

among the treatments based on the F-value. The second aspect to

be considered is the P-value; the P-value must be less than 0.05 in

order for the null hypothesis to be rejected. The computed P-value

 resulted in (_______)) rejecting the null hypothesis. Hence, there is

a significant difference among the treatments.

Conclusions

The current findings demonstrate that the extract derived from the leaves of

Clitoria ternatea possessed considerable anti-angiogenic effects in a

concentration-dependent manner. These findings exhibits the potential to be an

effective inhibitor of abnormal angiogenesis and other disorders caused by

abnormal rapid vasculogenesis, such as certain variants of cancer.

Recommendations for Further Studies

This study has contributed to the understanding of the Anti-angiogenic effect

of the leaf extract of Clitoria ternatea through the utilization of Chorioallantoic

Membrane Assay. As the study progressed, a few areas surfaced as suggested

areas for future studies. The recommendations are as follows:

a) For a more precise evaluation of the results, future researchers

may opt to increase the number of replications for each treatment.

b) It is also encouraged for future researchers to try either lower or

slightly higher concentrations of the Clitoria ternatea extract.

c) To highlight accuracy and to add more information, future studies

may also explore further the phytochemical analysis of the leaf

 extract and study the exact percentages of the chemical

components it possesses.

d) Future researchers may also resort in pursuing the other parts of

the Butterfly Pea plant such as the stem, roots, and seeds. They

may also perform a toxicity study or a phytochemical analysis on

the other parts of the plant.

e) Trying other varieties of the same family of Clitoria ternatea is also

highly suggested as an area to explore for future studies.

f) For future studies, attempting to utilize other parameters of

methods instead of the Chorioallantoic Membrane Assay is also

highly recommended.

g) For future studies, to identify the percentage Clitoria ternatea’s

phytochemical properties and its benefits to various diseases.