1

Chapter 1

The Problem and Its Setting

Introduction

The human body is a very complex system made up of a wide

variety of different and important parts that works together to

maintain homeostasis in able to sustain life. According to a recent

estimate published in July 5, 2013 in the "Annals of Human

Biology" by an international team of researchers, the human body

consists of approximately 37.2 trillion cells. The cells in our body

all have certain jobs to do. Normally, human cells undergo mitosis

to form new cells as the body needs them. When cells grow old, or

become damaged and worn out, they die through the process

called apoptosis and new cells take their place. This orderly

process stops when cancer cells develop and start to grow out of

control.

Cancer is the term given to a large group of diseases which

can greatly affect the immune system of an individual. It is an

abnormal cell that spreads rapidly to other organs of the body

through the blood and lymph leading to new serious medical

problems. This process is called metastasis and it is the major

cause of death from cancer. According to World Health

Organization (WHO), cancer is one of the leading causes of death

worldwide, accounting for 8.8 million deaths in 2015. Despite

 2

having many accounts and practices for many years on treating or

managing cancer, like surgery, chemotherapy, and radiotherapy,

prognosis of the disease has not greatly improved. There have been

issues regarding the toxicity, metastatic period, inherent and

acquired resistance to these treatments and practices.

Angiogenesis is the physiological process through which new

blood vessels form from pre-existing vessels. It is a normal and

vital process in growth development controlled by the chemical

signals in the body to help repair damaged blood vessels and the

formation of new ones. However, angiogenesis plays a critical role

in the growth and spread of cancer. In line with this present

medical situation, scientists continue to study on ways to treat

cancer and many novel cancer therapies have risen.

Many years ago, scientists were studying about the

relationship between angiogenesis, tumor growth and metastasis.

Until in 1971, Dr. Judah Folkman, pioneer of the study of

angiogenesis, published his research in the “New England Journal

of Medicine” states that in order to survive and grow, tumors

require blood vessels, and that by cutting off that blood supply, a

cancer could be starved into remission.

According to Jadhave, Thorat, Jamale, and Gonjari (2013),

angiogenesis normally helps in wound healing and embryonic

 3

development. However, persistent unregulated angiogenesis causes

chronic inflammation, tumor growth, and metastasis which further

leads to angiogenic diseases like, diabetic retinopathy and

rheumatoid arthritis. Since tumors cannot grow and spread

without a blood supply, the discovery of anti-angiogenic factors

would provide an important therapeutic value to block cancer

angiogenesis, with the idea that these molecules will prevent or

slow the growth of the disease.

Angiogenesis inhibitors, also known as anti-angiogenic

factors, are unique cancer-fighting agents because this therapy

does not necessarily kill tumors but instead they prevent tumors

from growing by inhibiting the growth of blood vessels. Anti-

angiogenic agents are now available in many countries but it is

more prevalent in first-world countries. At present, only synthetic

anti-angiogenic agents are available and it costs ranging from

$2,000-$49,000 or Php100,786.00-Php2,469,257.00.

In line with the cost-effectiveness of the synthetic anti-

angiogenic agent, the researchers of this study aim to provide a

low-cost yet effective natural anti-angiogenic agent which can be

readily available in the Philippines. The researchers chose an

animal duck embryo subject to chorioallantoic membrane (CAM)

assay as an in vivo model to study the effect of their natural anti-

 4

angiogenic. Commonly, mouse models are used for the in vivo

assessment of potential new therapeutic targets in cancer.

However, animal models are costly and time consuming. Other

models, such as the chorioallantoic membrane (CAM) assay, are

therefore an attractive alternative. Hence, this leads the

researchers to develop the study.

Background of the Study

Based on the recent demographics, three people die in the

Philippines every minute. In 24 hours, there is an estimate

mortality rate of 4,320. Most of their cause of death are diseases

which are difficult to cure. Usually, those are the diseases which

involve abnormal cells proliferating inside the body.

Cancer is a large group of diseases which may affect any part

of the body. It can either be malignant tumors and/or neoplasms.

Theoretically, this disease is caused by the rapid proliferation of

abnormal cells, which grow beyond their usual size and habitat.

These cells invade parts of the body and spread to other organs

through a process called metastasis. Cancer is one of the leading

causes of morbidity and mortality worldwide. According to the

World Health Organization, there had been 8.8 million deaths in

2015, all due to cancer. Common causes of cancer include cancers

of the lung, liver, colorectal, stomach, and breast.

 5

According to the American Society of Clinical Oncology,

angiogenesis is the growth of blood vessels from the existing

vasculature, unlike neovascularization, which is the formation of

entirely new blood vessels. Stimulation of angiogenesis can be

therapeutic in ischemic heart diseases, peripheral arterial disease,

and wound healing.

When a tumor occurs in the body, it is in need of nutrients

and oxygen to proliferate. These components are readily available

in the blood. These tumors send chemical signals to stimulate

blood vessel growth, and these blood vessels carry the blood to the

tumor. Angiogenesis inhibitors are drugs or chemical which block

angiogenesis, inhibiting it from nourishment in order to “starve” it.

Anti-angiogenesis can be therapeutic in cancer, ophthalmic

conditions, rheumatoid arthritis, and other diseases. It can be

used to treat multiple cancers alone, or with the aid of cytotoxic

drugs (Al-Husein, 2012).

Anti-angiogenic agents in the Philippines are quite

expensive. Which is why the researchers plan on having potential

anti-angiogenic agent that is organic, readily available in the

Philippines, and most importantly, cost efficient. Not only will the

inhibitor be able to help reduce the growth of cancerous tumors,

but it will also save the patients from spending too much for their

treatments.
 6

Ashitaba (Angelica keiskei) is a green, celery-like plant from

the Angelica genus family. It is known as the “Tomorrow Leaf”, for

its fast regenerative properties. The plant originated on the island

of Hachijo, Japan, where its medicinal properties were discovered.

It is known to grow for about 50-120cm in height, and can survive

through conditions with temperatures ranging from 12-22C (Noah,

2013). One of its unique characteristics is the ability to regenerate

a new sprout within 24 hours. When the leaf is cut, there would be

a new sprout growing the next day.

This plant has been used for its medicinal properties since

1600s. According to studies, Ashitaba has more nutrition than

broccoli, cabbage, asparagus, celery, and cucumbers. There had

been numerous scientific studies which confirmed its healing

properties. Locals have dubbed it as “Longevity herb” for it is

believed to extend the lifespan of humans (Eberle, 2015).

In traditional medicine, it is seen to be a strengthening tonic.

It is a powerful herb which is powerful against infectious diseases

such as smallpox. The sap is a sticky, yellowish juice that gushes

out from its stem. This sap can treat a variety of skin conditions

including boils, cysts, and pustules. Chlorophyll from the leaves

have anti-bacterial properties and can also help in the production

of new blood cells to heal wounds. Chalcone is a unique

 7

component only seen on the plant which can be used as a diuretic,

laxative, and metabolic rate booster (Eberle, 2015).

Xanthoangelol is a chalcone which can only be isolated in

the Ashitaba plant. Together with 4-hydroxyderricin, these

chalcones can induce apoptosis in cancer cells, antioxidant, anti-

inflammatory, anti-angiogenic, and antidiabetic properties. In some

studies, it had also demonstrated to inhibit platelet aggregation

(Maronpot, 2015).

Duck egg have excellent characteristics that make them an

excellent choice for poultry production in the Philippines. In the

Philippines, they are the most popular breed because they produce

high quality eggs. They have a great temperament and are

generally quiet in the cages. Duck production in Philippines have

various excellent characteristics such as excellent livability, high

number of eggs laid, good egg sizes as well as great egg quality.

Chorioallantoic membrane assay is the vascular membrane

found in eggs of birds and reptiles. It is the method widely used in

researches that involves blood vessel observations. The test is

composed of two parts: the allantois and chorion. These

components are relevant in the study because they both possess

different purposes which may help in nurturing the eggs.

In order to dig deeper on angiogenesis and anti-angiogenesis,

the duck embryo for chorioallantoic membrane assay can be used

 8

for studying neovascularization. There had been many variations

developed in this assay. However, the basic assay is performed by

implanting the compound of interest on the chicken embryo, and

soon angiogenesis can be observed.

The researchers aim to investigate on the anti-angiogenic

activity of chalcone extract from Ashitaba (Angelica keiskei) roots

using CAM assay of duck eggs, through this, the researchers will

be able to gain more understanding with this topic, and contribute

in the advancement of cancer researches.

Statement of the Problem

The researchers aimed to determine the anti-angiogenic

activity of Chalcone extract from Ashitaba Roots (Angelica keiskei)

using Chorioallantoic Membrane Assay of Commercially Available

Philippine Duck eggs. The researchers specifically aimed to answer

the following:

1. Is the chalcone extract from local Ashitaba plant an effective

anti-angiogenic agent?

2. What concentration of chalcones extract can suppress the

arising of new blood vessel?

Hypothesis

This study tries to tell the null hypotheses as follows:

1. There will be no significant result between the treated and

untreated egg
 9

2. The treated egg will show progressive growth of blood

vessels.

3. Chalcone does not exhibit anti-angiogenic activity.

Assumption of the Study

The study is anchored with the assumption that the

chalcone content of the roots of the Ashitaba plant is an effective

anti- angiogenic agent. The embryo of duck is an effective assay in

observing the suppression of blood vessel growth through injection

of chalcone extract from the root of Ashitaba. The researchers also

assumed that there will a significant result between the treated

and untreated egg.

Significance of the study

Many believe that by maintaining a healthy diet and lifestyle

prevents a person from having cancer. However, for some, no

matter how careful they were, they still acquire it. This has been

the reason of emerging various studies regarding ways to treat and

prevent cancer cells. It can be an anti-tumor, anti-metastatic, anti-

angiogenic, chemopreventive and anti-proliferative. Nowadays,

these dietary agents were used to suppress angiogenesis and

believed to be an important step in the prevention and treatment of

all types of tumor for they possess prophylactic, reliable, available,

safe and affordable. (Hutnik, Li, and Chiou, 2012). Nonetheless,

not all drugs for cancer were said to be truly affordable and readily
 10

available especially for those people who belong to second class of

the society and less privileged.

This study would be significant for the researchers to

develop further ideas in preventing angiogenesis by using natural

products of the Philippines that will lessen the number of cancer

incidence in the country.

For the medical technologists, the researchers are

anticipating that this research will provide additional information

and knowledge about the concept of angiogenesis in histology and

immunology, and prevention of cancer by using the anti-angiogenic

property of chalcone extract through different concentrations.

For the Philippines, this study will help reduce the need to

import expensive anti-angiogenic drugs from other countries. The

researchers also anticipate that through this study, it will help in

lowering the mortality rate of cancer, which is one of the leading

cause of death in the Philippines. It will also be beneficial to the

environment since the variable to be used are naturally available in

the country.

Lastly, this study will benefit the Filipinos by promoting

cost-effective and natural anti-angiogenic treatments not just for

cancer but also for different conditions whereas excessive growth of

blood vessels may have been the main cause for the disease.
 11

Theoretical Framework

The term “angiogenesis” was created by Scottish surgeon Dr.

John Hunter who discovered the formation of new blood vessel

plays an important step in tissue development more than 2

centuries ago. Angiogenesis support tumor growth therefore can be

target for cancer therapy. “New England Journal of Medicine”

published in 1971 by Judah Folkman, pioneer in the study of

angiogenesis, termed and postulate the theory of anti-angiogenesis,

that to survive and develop tumor cells it requires nourishment

from a developing blood vessel and thus cutting its blood supply

stop the infestation of tumor cells that may lead to increased risk

of cancer, and could deplete the nutrient in reemission of

unwanted growth of vessels.

Today, theories have provided various insight on how

vascular network system works in a molecular level. Many

researchers have conducted studies accompanied the study of

angiogenesis. Arises of new study lead to development of not only

using natural health product to stop proliferation of tumor cell but

as well as development of new technologies that will help to

genetically prevent it.

The subject of the study Ashitaba (Angelika keiskei) derived

its Japanese name, ashita (tomorrow) and ba (leaf) was

conceptualize by its ability to regenerate new leaves after cutting.

 12

Sources of the study have of both crude and pure extract have

proven to inhibit tumor growth and ameliorate inflammation,

obesity, diabetics, hypertension, and ulcer. The extract has also

showed anti-thrombotic, anti-oxidative, anti-hyperlipidemic, anti-

viral, and anti-bacterial activities. Ashitaba have various chemical

constituents, one of the unique and rare compound of it is

Chalcones which use for cancer treatment has a derivative of

xanthoangelol and 4- hydroxydexydecirrin.

The study of the researcher will be focused on xanthoangelol

will be guided by previous researcher which the study result that a

major chalcone constituent of Angelika keiskei, Xanthoangelol

induces apoptosis in neuroblastoma and leukemia cells (Tabata

K1, 2005 Aug;28). And 4-hydroxyderricin from Ashitaba roots

induces caspase-dependenet apoptotic cell death in HLA 60

human leukemia cells. (Akihisa T1, 2011)

This study have a Day table as the procedure requires to

maintain the validity of result by firstly gathering enough ashitaba

plants that will be first subject for identification and verification in

Bureau of Plant and Industry in Manila and then proceed for

extraction with the use of chemicals as well as chromatograph and

further analyze the percent constituent that could mostly influence

the result of the experiment.

 13

Biological Analysis will be done with Chorioallantoic

Membrane Assay with the use of an eight day-old Philippine duck

egg to ensure fertilization, will be checked for defect by candling

the eggs and be incubated in day one, eggs would be separate into

six group. Three fertile 8-day duck eggs were used for each:

positive control, negative control, and the treatment.Eggs were

cleaned with the aid of 70% ethanol to remove dirt and other

debris that could infect the eggs when opened. All the eggs that

were used were incubated at 37 0C, The researchers will inoculate

the increasing treatment doses of the chalcone extract. Another

incubation will last for 24 hours. In day 4, researcher will measure

the CAM using stereomicroscope for visual examination and

quantification to evaluate the effect of treatment doses.

Conceptual Framework

This study will focus on the determination of the anti-

angiogenic property of Chalcone extract from Ashitaba Root

Extract (Angelica keiskei).

 14

Figure 1

Research methodology 1

Scope and Limitations of the Study

This study will focus on xanthoangelol extract of ashitaba

roots (Angelica keskei). The researchers aim to determine the

antiangiogenic activity of chalcone extract from ashitaba (Angelica

keiskei) roots using CAM assay of duck eggs. Xanthoangelol is a

chalcone which can only be isolated in the roots of Ashitaba plant.

Xanthoangelol together with 4-hydroxyderricin has been proven to

be anti-tumor and anti-metastatic in mice giving the reason for the

researchers to learn more and prove about the property

(Sumiyoshi, 2015).

However, the researchers will be using the embryo of duck

eggs instead of mice. Likewise, the researchers will only focus on

the compound xanthoangelol in order to further study its own

ability to be anti-angiogenic even without the presence of 4-

hydroxyderricin. The researchers aim to prove that xanthoangelol

can inhibit the growth of blood vessels and not just as anti-tumor
 15

and anti-metastatic. The researchers aim to acquire a purified

xanthoangelol extract by using several procedures and analyses.

With these, extraction of the xanthoangelol had been chosen.

Lastly, the researchers selected Chorioallantoic membrane

assay (CAM) as the biological assay for in vivo extraction of

xanthoangelol and duck eggs had been chosen as test samples to

be used in this assay for the determination of anti-angiogenic

activity

Definition of Terms

The following terms are defined according to the context in

which they will be used in the study for better understanding:

Angiogenesis. It is the physiological process through which

new blood vessels form from pre-existing vessels. This is distinct

from vasculogenesis, which is the de novo formation of endothelial

cells from mesoderm cell precursors.

Anti-angiogenesis. It is the prevention or inhibition of

angiogenesis.

Apoptosis. Cell death.

Ashitaba. It is a green, celery-like plant from the Angelica

genus family. It is known as the “Tomorrow Leaf”, for its fast

regenerative properties.
 16

Blood Vessel. A small tube that carries blood to different

parts of a person or animal’s body.

Cancer. It is a term used for diseases in which abnormal

cells divide without control and are able to invade other tissues.

Chalcone. It is a unique component only seen on the plant

which can be used as a diuretic, laxative, and metabolic rate

booster.

Chorio-allantoic Membrane (CAM) Assay. It refers to the

vascular membrane found in eggs of some amniotes such as birds

and reptiles. It is formed by the fusion of mesodermal layers of two

development structures

Diabetes. It describes a group of metabolic diseases in which

the person has high blood glucose (blood sugar), either because

insulin production is inadequate, or because the body’s cells do

not respond properly to insulin or both.

Elute. Process of extracting an adsorbed substance by

washing with a solvent.

Extract. It is the pure liquid form of a substance.

In vacuo. In a vacuum.
 17

Neovascularization. Formation of entirely new blood

vessels.

Rheumatoid arthritis. It is a chronic progressive disease

causing inflammation in the joints and resulting in painful

deformity and immobility, especially in the fingers, wrists, feet and

ankles.

Suspend. To cause to be held in suspension in a fluid.

Tumor. It is a swelling of a part of the body, generally

without inflammation, caused by an abnormal growth of tissue,

whether benign or malignant.

 18

Chapter 2

Review of Related Literature and Studies

This chapter discusses the different literature and studies

which are used in the study. The researchers believe that through

these sources, readers would be able to gain further understanding

about the topic, and perhaps contribute to the advancement of this

research in the near future.

Review of Local Literature

Ashitaba (Angelica keiskei) is an herb that grows primarily in

Japan, but can also be found in the Philippines. Its root, leaf, and

stem are used to extract components which can be used for

medicinal purposes. According to other researches in the past, the

plant has various effects on cholesterol metabolism in a stroke-

prone spontaneously hypertensive group of subjects. Extracts also

showed beneficial effects of an active ingredient of the plant,

xanthoangelol, which is a type of chalcones that can inhibit certain

types of tumors and leukemia cells. This can be supported with the

article written by Ursulom (2017), which claims that the ashitaba

plant that was studied using animals and in test tubes has an

anti-oxidant, anti-cancer, anti-aging, anti-inflammatory,

antihypertensive, and anti-diabetic ability. In addition, this plant

 19

has a characteristic that can improve health and good for health

maintenance.

Review of Foreign Literature

According to American Cancer Society (2015), cancer is the

disease which harms the body when abnormal cells proliferate.

There are currently 100 types of cancer which are classified by the

type of cell that is initially affected.These cells usually proliferate

and create lumps or tumors which grow and interfere with the

digestive, nervous, and circulatory systems, and release hormones

that may alter body functions. These could either be benign or

malignant. This was supported by the World Health Organization

(WHO), adding that tumors send signals to the body which activate

the growth of blood vessels, also known as angiogenesis.

In order to stop the proliferation of tumors, anti-angiogenic

drugs must be administered. According to National Cancer

Institute (2015), These inhibitors stop the growth of new blood

vessels that branch out to give nourishment to existing tumors.

These drugs may also be called anti-angiogenics. Not only do these

drugs inhibit the growth of blood vessels, but they could also affect

the growth of the tumor itself. Some drugs include Axitinib,

Bevacizumab, Everolimus, Pazopaniv, and Sunitinib. Although

they could be beneficial, too much intake would also cause toxicity
 20

to the body. Therefore, the researchers plan on having an organic

alternative to inhibit angiogenesis.

Spironolactone is an aldosterone antagonist that is used as a

potassium-sparing diuretic. It is most commonly used in animals

in congestive heart failure or for those with ascites. This drug is

approved for use in human medicine and is marketed under the

brand name of Aldactone™. It may also serve as a vasodilator of

blood vessels. Research in both humans and in animals would

indicate improved survival when spironolactone is added to the

conventional congestive heart failure treatment. The most-common

side effects are due to dehydration and/or electrolyte imbalances

(hyperkalemia and hyponatremia), and may increase the effects of

neuromuscular blocking agents.In lined with this, heparin may

also be used as a vasodilator. The medicine is in a class of drugs

known as anticoagulants (blood thinners). It works by decreasing

the blood's ability to clot. Heparin is often given to people with

atrial fibrillation, deep vein thrombosis (DVT), pulmonary emboli,

certain blood clotting disorders, and to bed- or wheelchair-bound

people. It can also be used for patients with high blood pressure,

as it causes the vessels to relax. This medicine is given by injection

into a vein or under the skin, and shouldn't be injected into a

muscle.
 21

Phenylephrine is a nasal decongestant that provides relief

from nasal discomfort caused by colds, allergies, and hay fever. It

belongs to a class of drugs called vasopressors, and it works by

reducing swelling of the blood vessels in the nose. Though it

relieves most symptoms, it doesn’t treat the cause of the symptoms

or speed recovery. Patients should not use medicines that contain

phenylephrine if they have used a monoamine oxidase inhibitor

(MAOI) in the past 14 days because of a potentially dangerous drug

interaction. This drug is usually taken by patients with Heart

disease, High blood pressure, Diabetes, and Thyroid disorder.

Ashitaba (Angelica keiskei) is a naturally growing plant

found on coastal lands in Japan. It has been one of the most

popular plants in Japan for it has been used for decades.

According to legends, the Japanese used this plant generation-

after-generation with the knowledge of its outstanding health

properties based on hearsay and folklore. The plants contain

“chalcones” which are naturally occurring antioxidants that also

exhibit medicinal properties.

Chalcone is an aromatic ketone and an enone that forms the

central core for a variety of important biological compounds, which

are known collectively as chalcones or chalconoids. It has a density

of 1.07 g/cm³ They are naturally occurring compounds found in

various plant species like Angelica, Glycyrrhiza, Humulus and

 22

Scutellaria, which are widely used as traditional folk remedies.

They are rarely found anywhere in the natural world but are the

key factor in ashitaba (Angelica KeiskeiKoidzmi). Research has

shown that the unique properties of ashitaba are at least partly

due to these remarkable compounds. The chalcones that are in

ashitaba are known as Xanthoangelol, Xanthoangelol-E and 4-

Hydrooxyderricin and were discovered by Dr. Kimie Baba (MD,

Osaka University of Pharmacy). These are also associated with

pharmacological activities, which includes anti-tumor anti-

inflammatory, antimicrobial, and nitric oxide synthase inhibitors,

associated with diseases such as Alzheimer, Huntington, and

inflammatory arthritis.

Though chicken eggs are the most commonly used, duck

eggs are still catching on as effective alternatives for research

studies. Duck eggs are quite large compared to chicken eggs,

which makes them easily distinguishable. They have hard shells,

which provides them with stronger protection from trauma, and

gives them a considerably longer shelf life. An average of 100g of

egg will provide about 185 Kcal of energy, with a considerable

amount of carbohydrate content. These kinds of eggs are high in

selenium, manganese, zinc, copper, potassium, phosphorus, and

iron (Testessence.com, 2018).

 23

In the incubation of the egg, it is necessary to follow the

important precautions in order to raise a healthy egg. Ideally, eggs

should be stored in a dry place with a temperature of 37.5℃ and a

humidity of about 70%. Too much humidity prevents eggs from

drying out sufficiently; too little causes the contents to dry out too

quickly. In either case, eggs fail to hatch. Turning the eggs allows

free movement of the embryo and prevents the egg’s contents from

sticking to the shell. Eggs should be turned through an angle of

90°. If turning has to be performed manually, this should be done

at least three times a day. Automatic turning equipment should be

set to turn the eggs every hour. Eggs are candled by shining an

electric light through them so that their contents can be clearly

seen, and embryonic development determined. Eggs may be tested

for fertility on day 7 (Department of Primary Industries, 2018).

Candling is a method used in embryology to study the

growth and development of an embryo inside an egg. A live embryo

is seen as a dark spot in the large end of the egg, near the air cell,

with blood vessels radiating from it. Infertile eggs are clear, and a

dead embryo shows as a dark spot stuck to the shell membrane. In

duck embryology, spider-like projections of blood vessels start to

appear on the first week of incubation. It is important to turn the

eggs during this week for about 3 times a day so that the

membrane would not stick on the egg shells. The body starts to
 24

grow on the second week, starting with the body, the head, and

webbed feet, which can be seen by day 9. As the days progress, the

egg sac expands, and movement from the embryo can be observed.

On day 15, big veins surround the entire egg, and there’s difficulty

in candling the eggs since the embryo has grown bigger and

stronger. On the third week of incubation, the air sac would be

about ¼ to ⅓ of the egg, and the embryo will look like a giant blob,

which is a good sign that it is developing properly. On day 28 of the

fourth week, the ducklings start to hatch from the egg (Steele,

2012).

The CAM is relatively simple, quick, and low-cost model that

is used for angiogenic and anti-angiogenic studies since the

changes in morphology of cancer cells arrested in the CAM

microcirculation can be readily observed by in vivo microscopy.

Some advantages of using this model include screening of a large

number of pharmacological samples in a short time; it does not

require administrative procedures for obtaining ethics committee

approval for animal experimentation; and results can be clearly

seen for a short span of testing days. Being naturally

immunodeficient, the egg embryo may receive transplantations

from different tissues and species, without immune responses. The

CAM model can be used as an experimental animal model of

metastasis since the chick embryo is naturally immunodeficient

 25

and can accept cancer cells regardless of their origin without

immune response. Moreover, cancer cells arrested in the CAM

microcirculation survive without significant cell damage, and

complete extravasation.

As stated by Ribatti (2016), Drug topically applied to the

CAM can reach the systemic circulation, after absorption through

the membrane and affect the development of the embryo. Toxicity

of drugs or carriers on embryos can be evaluated in terms of

embryo death and adverse effects on the CAM, including

inflammation and neovascularization. Drug delivery systems can

be topically applied on the CAM or injected into the amnion.

Staton (2014), made a systematic review about the current

methods used in the study of angiogenesis in vivo and in vitro. One

of the most widely used in vivo assay for studying angiogenesis is

the Chorioallantoic Membrane (CAM) assay. “The test substance is

prepared either in slow-release polymer pellets, absorbed by gelatin

sponges, or air-dried onto plastic discs” and these will be

implanted on CAM through careful making of window cut into the

eggshell. The parameter for the angiogenic activity of a particular

substance is measured by counting the blood vessels using

stereomicroscope. In addition, it is better and necessary to have

more than one in vivo assay to study the variation among

organisms being tested.

 26

Tecon (2015), studied about angiogenic property of ethanolic

extract from Manihot esculenta Crantz leaves on chorioallantoic

membrane of duck embryos. 56 one day-old eggs were incubated

for 10 days, and treatments were applied on the 10 day andth

incubated for two days. The CAM embryo were removed on the 12 th

day to manually count the blood vessels. The result showed an

angiogenic property of the ethanolic extract of M. esculenta leaves.

Azwanida (2015), reviewed the different extraction process

used in medicinal plants. He defined extraction as the process of

separating medicinally active constituents of plants through

standard procedures. The initial crude extracts using the following

methods contain alkaloids, glycosides, phenolics, terpenoids and

flavonoids. Maceration and Soxhlet extraction are traditional

methods used in medicinal plant research. The process of

maceration includes soaking of plant materials in a stoppered

container containing a solvent and allowed to dry at room

temperature for three days with agitation. The aim of this method

is to break the plant’s cell wall to release the phytochemicals

present. After three days, the mixture will undergo filtration. On

the other hand, decoction is done for hard plant parts such as

bark and roots and usually results to more oil-soluble compound.

Soxhlet extraction, in addition, uses a finely ground sample and

place it on the thimble of the apparatus. The extraction solvent is

 27

heated at bottom flask and vaporize it on the sample in the thimble

and undergoes condensation. When the liquid content reaches the

siphon arm, the liquids will be emptied in the bottom flask again,

and the process repeats.

Review of Local Studies

According to the study conducted by Camposano (2016),

angiogenesis is the biological process by which new blood vessels

are formed from pre-existing capillaries and post-capillary venules.

Some diseases have excessive angiogenesis, thus causing

abnormalities. These include liver cirrhosis, psoriasis, and cancer.

In order to prevent this, inhibiting angiogenesis may be the best

option. Some inhibitors readily available in the market are costly

and may sometimes cause resistance to anti-angiogenic therapy.

Therefore, the researchers in the study used herbal plants as

inhibitors to provide opportunities in the discovery of drugs under

this class. Moreover, since it is organic, it can be available in the

country and cost-friendly.

The study conducted by Gonzales (2015), used chitin

extracted from mussel tahong (Pernaviridis) shells as an anti-

angiogenic agent. The researchers used the chorioallantoic

membrane assay of duck eggs to confirm that chitin has

antiangiogenic properties. The extract had been through a series of

tests such as Phenyl hydrazine test, Biuret, Ninhydrin,

 28

Xanthoproteic, Hopkin’s Cole and Millon’s tests, just to prove that

it contains the calcites and amides, which are the active

components that may have antiangiogenic properties similar to its

control or standard. Upon further testing, it was confirmed that

the chorioallantoic membrane assay is an efficient tool in observing

angiogenesis, and that chitin exhibits anti-angiogenic properties.

Review of Foreign Studies

Increased blood flow, for instance by inducing vasodilatation

with prazosin, is known to induce angiogenesis in skeletal muscle

and increased shear stress is a critical mediator of vessel wall

thickness and composition. Increased blood flow initiates both

vessel growth and remodeling of angiogenic vessels. This is caused

by nitric oxide, which is a potent vasodilator. In the endothelium, it

is produced by catalysis of l-arginine by endothelial nitric oxide

synthase (eNOS), which is a calcium dependent enzyme involved in

growth factor mediated angiogenesis. The researchers aimed to test

the hypothesis that angiogenic blood vessels could be remodeled by

increased nitric oxide, presumably acting as a vasodilator to result

in arteriolar genesis (formation of muscular arterioles) as well as

angiogenesis (formation of new blood vessels, mainly capillaries).

To locally increase blood flow through the vessels around a

 29

mesenteric panel, eNOS was overexpressed to generate a

continuous gradient of NO.

According to Kimura (2013), the roots of Angelica keiskei

Koizumi have traditionally been used as a health food, with

diuretic, laxative, analeptic and galactagogic effects. It has been

thought that the roots and leaves of A. keiskei have preventive

effects against coronary heart disease, hypertension and cancer.

Sumiyoshi (2014), studied about the antitumor and antimetastatic

effects of xanthoangelol and 4-hydroxyderricin isolates from the

roots of Ashitaba plant (Angelica keiskei). The purpose of the study

was to inhibit the activation of M2 macrophage that have been

associated with tumor growth and metastasis. They test the effects

of chalcones showed that it can inhibit tumor growth and

metastasis to the lungs and liver with a concentration of 25 or 50

mg/kg, twice daily. They concluded that xanthoangelol and 4-

hydroxyderricin have potential in antitumor and antimetastatic

due to its ability to inhibit M2 macrophage differentiation and

activation.

This can be correlated with the study of Yasuda (2014),

which focused on the action of xanthoangelol and 4-

hydroxyderricin on lipopolysaccharide (LPS)-induced inflammatory

responses in RAW264 mouse macrophages. Xanthoangelol (5μM)

and 4-hydroxyderricin (10 μM) inhibited the production of nitric

 30

oxide (NO). In addition, they also inhibited tumor necrosis factor-

alpha(TNF-α), inducible nitric oxide synthase (iNOS) and

cyclooxygenase-2 (COX-2) which are known to induce

inflammation.

The study conducted by Li (2014), proved the inhibitory

effect of Angelica keiskei chalcone in microvessel density (MVD)

and vascular endothelial growth factor (VEGF) expression on mice

with hepatocarcinoma cells. Fifty mice were introduced to

hepatocarcinoma H22 cells and divided into five groups. Group one

to three were administered orally with chalcone concentrations of

5, 25 and 50 mg/kg/d. On the other hand, group four was given

Endostar 4mg/kg/d through intraperitoneal injection while group

five was given with normal saline and serves as a control group.

The experiment last for 10 days after administration of chalcone

and they metastasis of hepatocarcinoma cells were measured using

methyl tetrazolium (MTT) assay. Furthermore, the levels of MVD

and VEGF were determined through immunohistochemistry

method. The inhibitory effect of chalcones on tumors based on 5,

25, 50 mg/kg/d concentrations were 4.20%, 30.47% and 39.42%,

respectively. The average MVD count was 14.2, 11.2 and 8.5 (MVD

refers to the concentrations of small blood vessels on malignant

tumor) while the protein levels of VEGF in mice decreased. (VEGF

is a protein that signals in the formation of blood vessels).

 31

Based on the study by Deocaris (2005), is is stated that in

the Chorioallantoic Membrane Assay (CAM), two doses of the

extract were used, 500 and 100ug/egg. Heparin, an angiogenic

inducer, was given at 10ug/egg, and spironolactone, an

angiogenic-inhibitor was given at 200ug/egg. These were the ideal

concentrations given to each egg, which also served as controls.

 32

CHAPTER 3

Research Methodology

This chapter presents a comprehensive description of the

research design, research locales, research instrumentation,

validation of instruments, data gathering procedure, data

processing methods, statistical tools, and treatment.

Research Design

The researchers used experimental research design. The type

they used was a Parallel-group Design in which two or more

groups are used at the same time with only one single variable

changed. The researchers determined the cause and effect

relationships among the variable to be studied. It followed a

scientific approach done in the laboratory. In this type of research

design, the researchers had the ability to know the anti- angiogenic

activity of the chalcone extract of the Ashitaba (Angelica keiskei)

using chorioallantoic membrane assay of Duck eggs.

Research Locale

The study was conducted in Centro Escolar University-

Makati located at 25 Senator Gil Puyat Avenue, Makati City. This

place was selected for having the efficacy to provide the research

instruments and library references that the researchers will need,

and of the convenience in conducting the study among the

 33

researchers. The school’s laboratory helped the researchers obtain

accurate and reliable results that can be useful for future studies.

Figure 2

The Centro Escolar Makati GP campus map

Validation of Instrument

The researchers’ Head Professor in Laboratory and

Equipment of Centro Escolar University – Makati, Mr. Ronald V.

Mendoza, validated the procedure of this study. His corrections

and suggestions were incorporated in the final draft of the thesis.

The researchers also conducted a triplicate procedure in order to

provide precise and accurate results. Lastly, calibrations of the

 34

instrument were installed to ensure efficient data and information

in the study that were collected.

Validation of Ashitaba (Angelica keiskei)

The researchers collected Ashitaba (Angelica keiskei) in

Buendia, Makati, Philippines. The plant samples were submitted to

the Bureau of Plant Industry for authentication and identification.

Data Gathering and Procedure

1. Extraction

The dried parts of A. keiskei were extracted three times with

a mixture of 50% ethanol and were concentrated in vacuo to give a

dark greenish brown, viscous extract. A portion of the 50% ethanol

extract was suspended in distilled water and extracted with ethyl

acetate and petroleum ether that obtained the ethyl acetate-soluble

fraction.

2. Physicochemical Test

2.1 Physical Test

2.1.1 Organoleptic Test

The researchers detected the physical

appearance, color and odor of the chalcone extract.

 35

2.2 Chemical Test

2.2.1 Sulfuric Acid-Nitric acid Test

Nitric acid was added to the mixture of chalcone

and sulfuric acid. An orange to yellow color was the

positive test for chalcone.

2.2.2 Sulfuric Acid–Acetic anhydride Test

Addition of 2 drops of concentrated sulfuric acid

to a mixture of chalcone and acetic acid produced a

deep color (orange to purple) that indicated a positive

result.

Table 3.1 :Colours given by Various Chalcones with Different

Reagents

Chalcone Sulfuric Acid Sulfuric Acid Acetic

Nitric Acid Test Anhydride Test

4, 4′-Dihydroxy4, 4′- Reddish Dark

Dihydroxy-3-methoxy2- orange/Cherry yellow/Yellow/Wine

Chloro-4′-hydroxy4- red red

Hydroxy-4′-nitro /YellowRed

4-Hydroxy-3-methoxy- Purple-orange Violet

4′-nitro
 36

4-Dimethylamino-4′- Reddish orange Greenish yellow

nitro

4′-Amino-4-hydroxy Red Dark orange

4′-Amino-4-hydroxy-3- Violet- blood Brownish yellow

methoxy red/ Reddish

orange

4′-Amino-4- Yellow Dark orange

dimethylamine

4′-Amino-2-chloro Brick red Yellow

4-Hydroxy-3′-nitro Reddish orange Greenish yellow

4-Hydroxy-3-methoxy- Brick red Greenish brown

3′-nitro

4-Dimethylamino-3′- Purple-orange Greenish yellow

nitro

Interpretation:Sulfuric Acid-Nitric Acid Test yielded a color orange

to yellow and was positive for4′-Amino-4-dimethylamine. Sulfuric
Acid Acetic Anhydride Test yielded a color orange to purple and
was positive for 4-Hydroxy-3-methoxy-4′-nitro.
 37

3. Biological Analysis

3.1 Chorioallantoic Membrane (CAM) Assay

Preparation of Duck Eggs

Eight-day duck eggs were bought at R&M Balut

Industry located in Pateros. Eggs were cleaned with

the aid of 70% ethanol to remove dirt and other debris

that could infect the eggs when opened. All the eggs

that were used were incubated at 37C, Three fertile 8-

day duck eggs were used for each: positive control,

negative control, and the treatment. In day 2 of

incubation, a square window was open. The researcher

carefully removed the inner membrane using

dissecting kit ensuring the inner membrane was not

injured. The researchers removed 2-3 ml of albumin

then application of treatment with increasing

concentration were placed, cover the window with

sterile tape and reincubate for one day. In day 6, the

researchers evaluated the CAM using

stereomicroscope. The group were as follows:

Group 1 served as the untreated group or

the control group.

Group 2 served as negative control using

spironolactone which acts as inducer.

 38

Group 3 served as positive control using

phenylephrine(neozep) that acts as inhibitor and

spironolactone as inducer.

Group 4 received the chalcone extract with

a concentration of 67.0 ug/ml and

spironolactone.

Group 5 received the chalcone extract with

a concentration of 6.70 ug/ml and

spironolactone.

Group 6 received the chalcone extract with

a concentration of 0.67 ug/ml and

spironolactone.

3.2 AperioImageScope Software

Stereomicroscope was used to observe the anti-

angiogenic effect of chalcone extract by counting the

blood vessels growth on the egg. The

AperioImageScope Software was used to produce an

image with results of the count of blood vessels that

helped the researchers to evaluate the anti-angiogenic

effect of chalcone extract on CAM assay.

 39

4. Statistical treatment

One-way Analysis of Variance (ANOVA) and t-test was used

for existing significant difference of treated xanthoangelolashitaba

root extract of Angelica Keiskei in both treated and untreated

group of egg samples during the duration of study.

 40

CHAPTER 4

Presentation, Analysis, and Interpretation of Data

This chapter deals with the presentation, analysis, and

interpretation of the results obtained by the researchers from the

semi-purified chalcone extract of Ashitaba plant (Angelica keiskei)

and its relevant findings in the study based from the results of the

organoleptic test, solubility test, phytochemical test, and the

instrumental analysis of the anti-angiogenic activity of chalcone

extract.

1. Percentage Yield

Ashitaba plant used in the experiment was purchased

during the month of Januaryand was brought to Mr. Manuel D.

Ching, OIC, Varietal Improvement Section of the Bureau of Plant

Industry, located at San Andres, Manila for its verification. The

plant availed and used in the study were fresh and in good

condition. The researchers separated the roots and was air-dried

until desired crispness was obtained and had undergone Soxhlet

procedure with 50% ethanol for 4 hours until it become dark

greenish brown viscous extract. The extract was then subjected to

the separatory funnel with distilled water, ethyl acetate and

petroleum ether. The ethyl acetate soluble fraction undergone

rotary evaporator and obtained an extractive yield result of 8.8%

(see Appendix B for percentage yield computation)

 41

2. Physicochemical Tests

2.1 Physical Test

2.1.1 Organoleptic Test. The semi-purified chalcone

extract was dark greenish brown viscous extract with an earthy

odor.

Table 4.1

Organoleptic Test of Semi-purified Chalcone Extract

PROPERTIES THEORETICAL ACTUAL

RESULT RESULT

Color Dark Greenish Brown Dark Greenish Brown

Odor Earthy odor Earthy odor

State Viscous liquid extract Viscous liquid extract

2.2 Chemical Test

2.2.1 Sulfuric acid-Nitric acid Test

Nitric acid was added to the mixture of chalcone and

sulfuric acid. An orange to yellow color was the positive test for

chalcone.

2.2.2 Sulfuric Acid–Acetic anhydride Test

Addition of 2 drops of concentrated sulfuric acid to a

mixture of chalcone and acetic acid produced a deep color

(orange to purple) that indicated a positive result.

 42

Table 4.2

Chemical Results and Interpretation for Presence of

Chalcone

PROPERTIES THEORETICAL ACTUAL INTERPRETATION

RESULT RESULT
Sulfuric Acid Orange to Yellow Indication for the
Nitric Acid Test Yellow Presence of
Chalcones
Sulfuric Acid Orange to Purple Indication of
Acetic Purple Presence of
Anhydride Test Chalcones
3. Biological Analysis

3.1 Macroscopic and Histologic Evaluation

The duck eggs were examined using stereomicroscopy at

Centro Escolar University – Makati Biological Sciences Laboratory.

In this assay, the macroscopic observation was done 2 days after

the administration of the chalcone extract to the CAM assay.

Inhibition of angiogenesis by chalcone results in a lack of new

blood vessel formation and the disappearance of pre-existing vessel

networks. Using Aperio ImageScope Software, the researchers

screened carefully the observable points in which the blood vessel

disappear. The Number of Strong Positive Pixels (%) is directly

proportional to the number of blood vessels and inversely

proportional to the area of inhibition. As shown in Figure 5.1

which represents Group 1 untreated group, the growth of blood

vessels was observable. Figure 5.2, Group 2 negative group,

 43

received spironolactone as angiogenic inducer of blood vessels.

Continuous development of blood vessels was observed. Figure 5.3,

Group 3 served as positive control using phenylephrine(neozep)

that acts as inhibitorand spironolactone as inducer. It was

observable that some of the blood vessels were suppressed. Figure

5.4, Group 4 served as the experimental group consisting of

spironolactone as angiogenesis inducer and 67.0 ug/mL of

chalcone extract as inhibitor. It was very evident that majority of

the blood vessels were inhibited, thus total inhibition was

observed. Figure 5.5, Group 5 served as the experimental group

consisting of spironolactone as angiogenesis inducer and 6.70

ug/mL of chalcone extract as inhibitor. No total inhibition had

been observed. Some of the blood vessels shrunk and disappeared.

Figure 5.6 Group 6 served as the experimental group consisting of

spironolactone as angiogenesis inducer and 0.67 ug/mL of

chalcone extract as inhibitor. Only few blood vessels disappeared.

No total inhibition had been observed.

 44

Figure 3

Evaluation of CAM Blood Vessels Using Stereomicroscopy

Figure 3.4
Figure 3.1
Group 4
Group 1
67.0 ug/mL +
Untreated
Spironolactone
Day 1 Day 3 Day 1 Day 3

Figure 3.2 Figure 3.5

Group 2 Group 5
Negative Control 6.70 ug/mL +
(Spironolactone only) Spironolactone
Day 1 Day 3 Day 1 Day 3

Figure 3.3
Figure 3.6
Group 3
Group 6
Positive Control
0.67 ug/mL +
(Spironolactone +
Spironolactone
Phenylephrine)
Day 1 Day 3 Day 1 Day 3
 45

3.2 Total percentage of CAM Blood Vessels

The researchers submitted all the images observed to

the Aperio ImageScope Software for the analysis of number

of blood vessels. The number of strong positive pixels (%) is

the percentage count of blood vessels in the CAM assay. It

means that high NSP(%) indicates numerous blood vessels

but low inhibition in the CAM assay. As shown in Figure 2,

the group 1 untreated group has 47.09%, group 2 negative

control with spironolactone as angiogenesis inducer has

44.67%, group 3 positive control with spironolactone as

angiogenesis inducer and phenylephrine as inhibitor has

12.85%, group 4 experimental group 67.0 ug/mL has 8.28%,

group 5 experimental group 6.70 ug/mL has 13.18%. Lastly,

group 6 experimental group 0.67 ug/mL has 16.06%.

 46

Table 4.3

Evaluation of CAM Blood Vessels Using Aperio

ImageScope

Untreated Group Negative Group Positive Group

No. of
Trials
A B C A B C A B C

Trial 1 55.14 43.80 42.33 44.78 46.93 42.30 12.23 15.93 10.40
Day 3 % % % % % % % % %
Average
47.09% 44.67% 12.85%
NSP(%)

67.0 ug/mL 6.70 ug/mL 0.67 ug/mL

No. of
Trials
A B C A B C A B C

Trial 1 14.17 11.06 16.98 15.73 17.42

9.51% 8.96% 7.63% 9.52%
Day 3 % % % % %
Average
8.70% 11.58% 16.71%
NSP(%)

A B C A B C A B C
Trial 2 15.40 13.87 15.07 17.94 16.52 15.67
9.86% 7.22% 8.32%
Day 3 % % % % % %
Average
8.47% 14.78% 16.71%
NSP(%)

A B C A B C A B C
Trial 3 14.11 13.48 11.95 14.51 13.68 16.05
7.21% 7.48% 8.32%
Day 3 % % % % % %
Average
7.67% 13.18% 14.75%
NSP(%)
TOTAL
AVERAG
8.28% 13.18% 16.06%
E OF
DAY 3
 47

4. Statistical Analysis

The significant differences in the different treatments of the

eggs were determined using the one-way analysis of variance

(ANOVA), general weighted mean, and standard deviation. The

post-ANOVA (t-test) was used to determine if there is a significant

difference on the anti-angiogenic property between the untreated

group and the negative group as well as with the experimental

groups. The null hypothesis states that there was no significant

difference between the untreated and the chalcone concentration

67.0ug/mL, 6.70ug/mL, and 0.67ug/mL.

4.1 One-way Analysis of Variance (ANOVA)

Table 4.4

Comparison of Treatments based on Number of Strong Positive

Pixels

Anti-angiogenic Activity in Number of Strong

Positive Pixels
GROUPS

G. MEAN ST. DEV. F- value

UNTREATED 25.90 23.64

NEGATIVE 31.70 14.43

3.4706
POSITIVE 14.43 2.84

0.67 ug/mL 16.96 1.36

 48

6.70 ug/mL 15.27 2.6

67.0 ug/mL 12.33 4.47

Based on the results shown in Table 5.1, it is evident that

the standard deviation of the negative control and the treatment

concentrations are close to each other, therefore the effect of the

treatments may be similar to the effects of the negative control.

4.2 Post-ANOVA (T-test)

Table 4.5

Comparison of Treated Groups with Untreated Group

GROUPS SIGNIFICANCE REMARKS

UNTREATED vs. NEGATIVE 0.0165 < 0.05 Significant

UNTREATED vs. 67.0 ug/mL 0.2256 > 0.05 Not Significant

UNTREATED vs. 6.70 ug/mL 0.3235 > 0.05 Not Significant

UNTREATED vs. 0.67 ug/mL 0.2834 > 0.05 Not Significant

The untreated group had a significant difference with the

negative group All of the remaining concentrations (670ug/mL,

6.70ug/mL, and 0.67ug/mL) had no significant difference with the

untreated group because it is greater than the level of significance

 49

which is 0.05. This proves that all concentrations have the ability

to inhibit the proliferation of blood vessels.

4.3 Summary of Results in Post-ANOVA (T-test)

Post-ANOVA t-test was used to determine the

significant differences between the groups. Based on the number of

strong positive pixels, only the untreated vs. negative group have

significant means. On the other hand, the untreated group vs. all

the other concentrations have no significant differences, therefore

chalcone extract from local Ashitaba plant is an effective inhibitor

of angiogenesis.
 50

CHAPTER 5

Summary, Conclusions and Recommendations

This chapter contains the summary of data obtained by the

researchers regarding the anti-angiogenic activity of chalcone

extract of Ashitaba (Angelica keiskei) roots. Furthermore,

conclusions about the effectivity of the study as well as the

recommendations are also provided.

Summary of Findings

It determined the anti-angiogenic property of chalcone

extract from Ashitaba (Angelica keiskei) roots, by determining the:

1. Percentage Yield

2. Characterization of chalcone extract

The obtained chalcone was dark greenish brown, viscous in

form, and had an earthy odor. The chemical tests done were

Sulfuric acid-Nitric acid test and Sulfuric acid anhydride test. The

result obtained for the chemical tests all showed expected results.

3. Evaluation of Anti-angiogenic Effect of Chalcone

3.1 Biological Analysis

The result obtained by the researchers, vessel density

percentage of duck embryos treated with 67.0ug/mL, 6.70ug/mL

and 0.67ug/ml of the semi purified chalcone extract had shown

that more production of the blood vessel can still arise in lower

concentration than in the higher concentration. It was also

 51

observed that 67.0ug/ml of semi purified chalcone extract had

been able to greatly reduce the vascularity in chorioallantoic

membrane in duck embryos as compared with the untreated

group.

Conclusion

The researchers concluded that chalcone extract from local

Ashitaba Plant is an effective anti-angiogenic agent because it was

able to inhibit the growth of blood vessels using Chorioallantoic

Membrane Assay. The most effective concentration that yield total

inhibition of blood vessels is 6.70 ug/mL. Lastly, the researchers

proved that CAM Assay is an efficient tool for observing the growth

of blood vessels upon administration of the chalcone extract other

than the use of laboratory mice.

Recommendations:

Based on the scope and limitations, summary of the findings

and conclusions, the researchers recommend the following:

1. To research more about the other properties of Ashitaba

2. To conduct an experiment regarding the purification of

chalcones of the Ashitaba extract

3. To further specify toxic doses of chalcone

4. To subject CAMs assay analysis to a more professional image

analyzer to lessen workload of researchers

 52

5. To the next researchers who will conduct the same study, we

recommend to use two kinds of software where it counts the

blood vessel density and the area of inhibition

6. To modify the experiment by adding human cancer graft cell

in the CAM assay

7. To try using Ex ovo method of CAM assay

 53

REFERENCES

American Cancer Society. (2015). What is Cancer. Retrieved from

(https://www.cancer.org/cancer/cancer-basics/what-is-
cancer.html)
(https://www.ncbi.nlm.nih.gov/books/NBK21590/)

Azwanida, N. (2015). A Review on the Extraction Methods Use in

Medicinal Plants, Principle, Strength and Limitation. Retrieved
from https://www.omicsonline.org/open-access/a-review-
on-the-extraction-methods-use-in-medicinal-plants-principle
-strength-and-limitation-2167-0412-000196.php?aid=58448

Benest, A. (2008). Arteriolar Genesis and Angiogenesis Induced by

Endothelial Nitric Oxide Synthase Overexpression Results in a
Mature Vasculature. Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/
PMC2566940/

Camposano, J. (2016). Screening for the anti-angiogenic activity of

selected Philippine medicinal plants using chorioallantoic
membrane assay. Retrieved from
http://www.pharmacy.mahidol.ac.th/journal/_files/
2016-43-4_2.pdf

Castillo, R. (2015). Revisiting the Ashitaba and other anticancer

herbs. Retrieved from http://business.inquirer.net/200549/
revisiting-the-ashitaba-and-other-anticancer-herbs

Chavan, B. (2016). Synthesis and Medicinal Significance of

Chalcones- A Review. Retrieved from
https://www.alliedacademies.org/articles/
synthesis-and-medicinal-significance-of-chalcones-a-
review.pdf

Department of Primary Insustries. (2018). Duck egg production,

lighting, and incubation. Retrieved from
https://www.dpi.nsw.gov.au/animals-and-livestock
/poultry-and-birds/species/duck-raising/egg-production
 54

Eberle, N. (2016). The Amazing Health Benefits of Ashitaba.

Retrieved from http://www.ashitabaplant.com/2016/01/
ashitaba-benefits.html

Everyday Health.com, (2018). What is Heparin?. Retrieved from

https://www.everydayhealth.com/drugs/heparin

Everyday Health.com, (2018). What is Phenylephrine?. Retrieved

from https://www.everydayhealth.com/drugs/
phenylephrine

Forney, B. (2017). Spironolactone for veterinary use. Retrieved from

https://www.wedgewoodpetrx.com/learning-
center/professional-monographs/spironolactone-for-
veterinary-use.html

Kimura, Y. (2013). Antitumor and antimetastatic activities of

Angelica keiskei roots, part 1: Isolation of an active substance,
xanthoangelol. Retrieved from https://www.ncbi.nlm
.nih.gov/pmc/articles/PMC3652162/

Li, Z. (2014). Angelica keiskei Chalcone and its Effect on Microvessel

Density and VEGF Expression of Mice Hepatocarcinoma Cells.
Retrieved from https://www.researchgate.net/publication
/286789807_iAngelica_keiskei_Chalcone_and_its_Effect_on_
Microvessel_Density_and_VEGF_Expression_of_Mice_Hepato
carcinoma_Cells

National Cancer Institute. (2015). Angiogenesis Inhibitors.

Retrieved from https://www.cancer.gov/about-
cancer/treatment/types/immunotherapy/angiogenesis-
inhibitors-fact-sheet

Ribatti, C. (2016). The chick embryo chorioallantoic membrane

(CAM). A multifaceted experimental model. Retrieved from
https://www.sciencedirect.com/science/article/pii/S09254
77316300363
Simon, S. (2018). Facts & Figures 2018: Rate of Deaths From
Cancer Continues Decline. Retrieved from https://www
 55

.cancer.org/latest-news/facts- and-figures-2018-rate-of
-deaths-from-cancer-continues-decline.html)

Staton, C. (2014). Current methods for assaying angiogenesis in

vitro and in vivo. Retrieved from https://www.onlinelibrary
.wiley.com/doi/10.1111/j.0959-9673.2004.00396.x/full

Steele, L. (2012). The Great Eggscape Too - Hatching Duck Eggs.

Retrieved from https://www.fresheggsdaily.com/2012/05/
great-eggscape-too-hatching-duck-eggs.html

Sumiyoshi, M. (2014). Antitumor and antimetastatic actions of

xanthoangelol and 4-hydroxyderricin isolated from Angelica
keiskei roots through the inhibited activation and
differentiation of M2 macrophages. Retrieved from
http://www.sciencedirect.com/science/article/pii/S094471
1315001178

Tecon, S. (2015). Angiosuppressive Potential of Manihot Esculenta

Crantz Leaves Ethanolic Extract Using Chorioallantoic
Membrane Assay in Duck Embryos. Retrieved from https://
www.ejournals.ph/article.php?id=10379

Testessence.com. (2018). Duck eggs vs. chicken eggs. Retrieved

From https://tastessence.com/duck-eggs-vs-chicken-eggs

Ursolom, F. (2017). Beneficial Effects of Ashitaba (Gynura

nepalensis): Users’ Testimonies”. Retrieved from
https://webcache.googleusercontent.com/search?q=cache:/
SHfgSvujeU0J:https://ejournals.ph/article.php%3Fid
%3D3212+&cd=2&hl=en&ct=clnk&gl=ph

Yasuda, M. (2014). Inhibitory effects of 4-hydroxyderricin and

xanthoangelol on lipopolysaccharide-induced inflammatory
responses in RAW264 macrophages. Retrieved from
https://www.ncbi.nlm.nih.gov/pubmed/24369884
 56

APPENDIX A

Certificate of Authenticatication
 57

APPENDIX B

Physical Test Results

1.1. Organoleptic test

1.1.1.Appearance – Viscous

1.1.2.Color – Dark Greenish Brown

1.1.3.Odor – Earthy odor

 58

APPENDIX C

Chemical Tests Results

1.3.1 Alcoholic Ferric Chloride

Theoretical Result: Variety of colors

Actual Result: Yellow

POSITIVE RESULT: Yellow

1.3.2 Sulfuric Acid – Nitric Acid

Test

Theoretical Result: Orange to Yellow

Actual Result: Yellow

POSITIVE RESULT: Yellow

1.3.3 Sulfuric Acid – Acetic anhydride Test

Theoretical Result:

Orange to Purple:

Actual Result: Purple

POSITIVE RESULT: Purple

 59

APPENDIX D

Percentage Yield of Chalcone Extract from Ashitaba Roots:

Data and Computations

Weight of the Sample ( roots of Ashitaba) = 500 g

Weight of evaporating dish = 42.46 g

Weight of evaporating dish with extract = 86.46 g

Weight of extract alone = 44 g

% yield = 8.80 %
 60

MARIA BIANCA S. BERNALES

City Address: Block 8 lot 2 Surigao corner
Davao St. Macaria Village, Binan City, Laguna
Mobile: 09268354135
Email: mariabiancabernales@gmail.com

CAREER OBJECTIVE

To be able to enhance my skills and character for the

betterment of my abilities.To show my competence in the field of
medical technology I seek to leverage my technical and professional
expertise to contribute to the society.

EDUCATIONAL ATTAINMENT

2015 to present Centro Escolar University

Sen. Gil Puyat Ave., Makati
Bachelor of Science in Medical Technology
Honor/Awards Received:
Dean’s lister : SY 2015-2016
Dean’s lister : SY 2016-2017

2011-2015 Colegio san Agustin Biñan

San Francisco, Biñan City, Laguna
Secondary Education

2010-2011 Colegio san Agustin Biñan

San Francisco, Biñan City, Laguna
Elementary Education

SEMINARS ATTENDED

 PAMET Research forum and Poster Presentation

(National Medical technology Week celebration 2017)
 Singing and beyond: “Road to the International Stage”
 “Inspiring and Empowering Future medical technologists
through ciencia y virtud”

CHARACTER REFERENCES

Available upon request

 61

JUSTINE MONICA GRACE CAYETANO

City Address: 47E E. Santos St., San Joaquin,
Pasig City
Mobile: 09266147251
Email: icaicayetano97@gmail.com

CAREER OBJECTIVE

To strive for excellence, to work in such an environment that

will enhance my knowledge and skills, wherein I can perform my
tasks according to my strong caliber and efficiency, and provide
medical care to the society.

EDUCATIONAL ATTAINMENT

2015 to present Centro Escolar University

Sen. Gil Puyat Ave., Makati
Bachelor of Science in Medical Technology

2011-2015 St. Paul College Makati

Poblacion, Makati City
Secondary Education

2010-2011 Holy Word Academy

Caniogan St., Pinagbuhatan, Pasig City
Elementary Education

SEMINARS ATTENDED

 Red Cross Youth Seminar

 Leader in Me Leadership Training: The 7 Habits of Highly
Innovative People
 Dolljoy Entrepeneurship Seminar
 Basic Caregiving Service Training

CHARACTER REFERENCES

Available upon request

 62

JOANNA ROSE P. CORTEZANO

City Address: 2169 A Rd 4 Granate St.,
San Andres Bukid, Manila
Mobile: 09177167717
Email: joannacortezano@gmail.com

CAREER OBJECTIVE
Highly motivated Medical Technologist aspiring to work in a
challenging environment to practice exceptional organizational
skills in performing clinical laboratory assays with high quality
precision and accuracy in order to promote good health in general

EDUCATIONAL ATTAINMENT
2015 to present Centro Escolar University
Sen. Gil Puyat Ave., Makati
Bachelor of Science in Medical Technology
Honors/Awards Received: President’s
Lister (School Year 2015-2017)

2011-2015 Paco Catholic School

1521 Paz St., Paco, Manila
Secondary Education
Honors/Awards Received: Dux
Leadership Awardee. First Honorable
Mention. Loyalty Awardee. Best in
Conduct.

2010-2011 Paco Catholic School

1521 Paz St., Paco, Manila
Elementary Education
Honors/Awards Received: First Honor
Awardee. Best in Conduct.

SEMINARS ATTENDED
 Red Cross Youth Seminar – Basic Life Support
 Leader in Me Leadership Training: The 7 Habits of Highly
Innovative People
 Basic Caregiving Service Training
 PAMET Research forum and Poster Presentation
(National Medical technology Week celebration 2017)

CHARACTER REFERENCES
Available upon request
 63

joan
 64

RACHELLE C. GABUAT
City Address: Blk 40 Lot 1A Tecson corner
Gabrielle St. BF Resort Village Las Piñas City
Mobile: +639171163862
Email: rachgabuat@gmail.com

CAREER OBJECTIVE

To succeed in a stimulating and challenging environment

that will provide me with advancement opportunities.

EDUCATIONAL ATTAINMENT

2015 to present Centro Escolar University

Sen. Gil Puyat Ave., Makati
Bachelor of Science in Medical Technology
Honors/Awards Received: Dean’s Lister
(School Year 2015-2016) and Quiz Bee in
Math (1st Place)

2011-2015 University of Perpetual Help System

Dalta
Alabang-Zapote Road, Pamplona, Las
Piñas City
Secondary Education
Honors/Awards Received: Loyalty Award
and Service Award

2010-2011 University of Perpetual Help System

Dalta
Alabang-Zapote Road, Pamplona, Las
Piñas City
Elementary Education
Honors/Awards Received: Third
Honorable Mention and Loyalty Award

CHARACTER REFERENCES

Available upon request.

 65

SAMUEL S. PASCO
City Address: 16 Masthead St. Fourth Estate
Subdivision, Sucat, Parañaque City
Mobile: +639454938744
Email: iamsamuelpasco@gmail.com

CAREER OBJECTIVE

To perform all the knowledge and skills of a Medical

Technologist in order to provide a nation with high quality
standard of laboratory diagnosis and promote good health in
general

EDUCATIONAL ATTAINMENT

2015 to present Centro Escolar University

Sen. Gil Puyat Ave., Makati
Bachelor of Science in Medical Technology

2011-2015 St. Theresa de Avila School of

Parañaque
Sucat, Parañaque
Secondary Education
Honors/Awards Received: Outstanding
Award and Loyalty Award

2010-2011 St. Theresa de Avila School of

Parañaque
Sucat, Parañaque
Elementary Education
Honors/Awards Received: Outstanding
Award

CHARACTER REFERENCES

Available upon request.