ANTI-ANGIOGENIC POTENTIAL OF Theobroma cacao L. (CACAO) SEED

EXTRACT ON Anas platyrhnchos (MALLARD DUCK) EMBRYO

Prepared and Submitted by:

Julienne S. Baldonado

Andrea G. Celis

Berna Indira Corinne O. Pontevedra

In partial fulfillment of the requirements in Research 2

Philippine Science High School

Southern Mindanao Campus

2008

ii

ABSTRACT

The study entitled ANTI-ANGIOGENIC POTENTIAL OF Theobroma cacao L.

(CACAO) SEED EXTRACT ON Anas platyrhnchos (MALLARD DUCK) EMBRYO

by Julienne S. Baldonado, Andrea G. Celis and Berna Indira Corinne O. Pontevedra was

conducted to evaluate the anti-angiogenic potential of T. cacao L. seed extract in terms of

the number of main vitelline vessels in A. platyrhynchos (mallard duck) embryo and

compare it with a commercial anti-angiogenic drug (cytoxan).

Two treatments for A. platyrhynchos were controlled namely T0 and T1. T0 was

not holed while T1 was holed on its egg shell. T2 and T3 were prepared with 0.25mL and

0.50 mL T. cacao seed extract in 25mL Phosphate Buffer Solution (PBS) respectively.

T4, a treatment of twenty five (25) mg of Cytoxan in fifty (50) mL PBS was also

prepared. Three days after the application of treatment, the eggs were opened. The

number of vitelline vessels in each egg was counted, recorded and analyzed using the

one-way Analysis of Varience (ANOVA) at 0.05 level of significance and Duncans

Multiple Range Test (DMRT).

The results of the study showed that T2 (5.33), T3 (3.00) and T4 (4.33) had

significantly lesser number of vitelline vessels compared to T0 (8.33) and T1 (6.67). This

result is attributed to quercetin which suppresses the eNOS activity that is responsible for

angiogenesis. Based on the findings of the study, it was concluded that T. cacao seed

extract can decrease the number of main vitelline vessels of A. playtyrhnchos embryo and

its efficacy is comparable to that of the anti-angiogenic drug cytoxan.

iii

CERTIFICATE OF APPROVAL

The research paper entitled ANTI-ANGIOGENIC POTENTIAL OF Theobroma

cacao L. (CACAO) SEED EXTRACT ON Anas platyrhynchos (MALLARD DUCK)

EMBRYO, prepared and submitted by Julienne S. Baldonado, Andrea G. Celis and

Berna Indira Corinne O. Pontevedra in partial fulfillment of the requirements in Research

2 is hereby accepted.

____________________

ARLYN A. DACANAY

Research 2 Adviser

Approved by the Committee on Oral Defense:

______________________ _____________________

SUZANNE G. MENDOZA SHARON M. DEJARME

Panel Member Panel Member

______________________________

ANDREA ANNE S. BALDONADO

Language Consultant

Accepted in Fulfillment of the Requirements in Research 2

____________________ _________________________

ARLYN A. DACANAY DR. ROSITA V. FUNDADOR

Research 2 Coordinator Director

iv

ACKNOWLEDGEMENT

This research study would not have been successful if it were not for the

Almighty Father who gave the proponents the wisdom, the knowledge, the patience and

determination to continue their work. Without Him, they would not have been able to do

their research study well. They thank God for being with them throughout the entire

research study.

This research study would not have been possible too if it were not for the help

and continued support they got from their research adviser, Mrs. Arlyn A. Dacanay, who

stood by and gave them suggestions to improve the quality of their work. They also thank

their parents who continued to support them especially on the financial side. Even though

the proponents came out demanding at times, they were still there to provide what the

proponents needed for this research.

They would also like to thank Lea Villanueva who provided them the camera to

document the data gathering. They thank her for being present during their data

gathering. The proponents would also like to thank Karla Cruzado, Miriam Edig, Ella

Gutierrez, Kim Lagrama, Mila Polinar and Jesryl Vicente who were with them during the

ups and downs they experienced during the research study. Lastly, the proponents thank

their friends and all those people who gave them the moral support to continue this

research study.

J. S. B.

A. G. C.

B. I. C. O. P.

v

TABLE OF CONTENTS

Page

TITLE PAGEi

ABSTRACT....ii

CERTIFICATE OF APPROVAL..iii

ACKNOWLEDGEMENT......iv

LIST OF TABLES....vii

LIST OF FIGURES ........viii

LIST OF APPENDICES...ix

Chapter

1. INTRODUCTION

Background of the Study...1

Objectives of the Study..2

Significance of the Study...3

Scope and Limitations of the Study...3

Definition of Terms4

2. REVIEW OF RELATED LITERATURE

Cancer....5

Angiogenesis......6

Current Cancer Treatments................................................................................7

Cytoxan..................................................................................................8

Quercetin........................................................................................................9

Theobroma cacao L.....11

vi

Stages of Blood Vessel Development of

Anas platyrhynchos (mallard duck) Embryo.12

Related Studies on Theobroma cacao L. and Quercetin.....13

3. METHODOLOGY

Gathering of Materials.15

Sterilization of Materials..15

Preparation of Experimental Set-ups...16

Preparation of Phosphate Buffer Saline Solution16

Preparation of Cacao Seed Extract..16

Preparation of Treatments....17

Preparation of Anas platyrhynchos (mallard duck) Embryo....17

Application of Treatments...17

Incubation of Anas platyrhynchos (mallard duck) Eggs..18

Data Gathering.....18

Statistical Analysis...19

4. RESULTS AND DISCUSSION20

5. CONCLUSIONS...23

BIBLIOGRAPHY..............................................................................................................30

APPENDICES...24

vii

LIST OF TABLES

Table Page

1 Mean Number of Main Vitelline Vessels in A. platyrhynchos Embryo.....20

2 Tabulated Raw Data on the Number of Main Vitelline Blood Vessels.....25

3 Anova Table...26

4 DMRT Table......27

viii

LIST OF FIGURES

Figure Page

1 Angiogenesis in Tumor Formation6

2 Cytoxan..9

3 Molecular Structure of Cytoxan.9

4 Molecular Structure of Quercetin..10

5 Cacao.11

6 Female and male mallard duck..12

7 Anatomy of Duck Embryo.19

8 T0 (control, without hole)...28

9 T1 (control, with hole)........28

10 T2 (0.25mL T. cacao seed extract in 25mL PBS)..28

11 T3 (0.5mL T. cacao seed extract in 25mL PBS)............29

12 T4 (25mg of cytoxan in 50mL PBS).......29

ix

LIST OF APPENDICES

Appendix Page

A Flowchart of Methodology24

B Raw Data...25

C Statistical Analysis26

D Picture of the Set-up..........27

Chapter 1

INTRODUCTION

Background of the Study

Cancer has two main characteristics which are the uncontrolled growth of cells in

the human body known as benign and the ability of its cells to migrate from the original

site and spread to another part of the body known as malignant. When proliferation of

malignant tumor starts and it is not controlled, it can result to death (Carson-DeWitt,

2006).

In the year 2000, malignant tumor result for 12 percent of the estimated 56

million deaths worldwide from all causes. In many countries, more than a quarter of

deaths are attributed to cancer. In 2000, 5.3 million men and 4.7 million women;

developed a malignant tumor and altogether 6.2 million died from the disease (World

Health Organization, 2003). In the Philippines cancer is the third leading killer in the

country. Seventy-five thousand (75,000) new cancer cases are reported each year (Tacio,

2005). The statistics has increased from 166 deaths in 2004 to 243 in 2005 (Philippines

Information Agency, 2006).

Treatments for cancer include surgery, which removes tumors which are small

and confined in a specific area. Radiation ionization can kill and shrink tumors, while

chemotherapy uses anticancer drugs to destroy the cancer cells in the body. All of these

treatments lack the specificity to kill cancer cells without harming normal cells. They are

expensive and cannot not assure the absence of recurrences (Carson-Dewitt, 2006).

As with living things, even cancer cells need oxygen and nutrients to help them

grow and thrive. To get the fuel they need, tumors develop a network of new blood

2

vessels in a process called angiogenesis. Angiogenesis is an area of intense focus by

cancer researchers who hope that stopping angiogenesis could mean stopping cancer

from growing and spreading (Kerbel, 2006).

Tumor, a characteristic of cancer, is the product of the abnormal local increase in

growth of a tissue or an organ. A tumor at the size of about 1-2 mm has the angiogenic

factors that promote the growth of nearby blood vessels toward the tumor cells. The new

blood vessels deliver nutrients and oxygen, and remove their waste products thus

rendering them capable of unlimited growth (Karp, 1999).

Anti-angiogenic agents prohibit the tumor from receiving nutrients and oxygen by

cutting off its blood supply. It would stop cancer cells from metastasizing since no blood

vessel via the circulatory system could help them travel from one site to another. This

helps prevent new tumors from growing and may also help shrinking large tumor since

the blood supply is restricted (Karp, 1999).

Quercetin is a known antioxidant and inhibitor of cell cycle progression. It

inhibits angiogenesis via a mechanism involving both suppression of endothelial nitric

oxide synthase (eNOS) and early M-phase cell cycle arrest (Jackson and Venema, 2007).

Cacao contains of 20.1mg/100mg of quercetin. The presence of quercetin in T.

cacao L. makes it a potential anti-angiogenic agent (Lakhanpal and Rai, 2007). Thus, the

proponents of this study decided to test the anti-angiogenic potentials of T. cacao L. on

the blood vessel formation of A. platyrhynchos (Mallard duck) embryo.

Objectives of the Study

The study aimed to determine the anti-angiogenic potential of T. cacao L. (cacao)

seed extract on A. platyrhynchos (Mallard duck) embryo.

3

Specifically the study aimed to:

1) Evaluate the anti-angiogenic potential of the following concentrations of T. cacao

L. seed extract in terms of the number of main vitelline vessels in A.

platyrhynchos (Mallard duck) embryo:

a.) 0.25mL of T. cacao L. (cacao) seed extract in 25mL PBS

b.) 0.5mL of T. cacao L. (cacao) seed extract in 25mL PBS

2) Compare the effects of the varying concentrations of T. cacao L. (cacao) seed

extract with that of cytoxan, a commercial anti-angiogenic drug, in terms of the

number of main vitelline vessels in A. platyrhynchos (Mallard duck) embryo.

Significance of the Study

The study is significant because it deals with the inhibition of cell angiogenesis

needed for tumor growth. The study would provide a natural way for fighting off a

disease which could then boost the economic growth of cacao. The study would also

provide a more accessible solution to excess angiogenesis as exhibited by tumors. Lastly,

the study would also provide baseline data for future research studies on the anti-

angiogenic potentials of T. cacao L.

Scope and Limitations of the Study

The study focused on the evaluation of anti-angiogenic potential of T. cacao L.

seed extract on ten-day old A. platyrhynchos embryo. The anti-angiogenic potential of T.

cacao L. seed extract limits to 0.25mL and 0.5mL each in 25mL phosphate buffer

solution (PBS). The positive control, cytoxan, is limited to 25mg in 50mL PBS. The

efficacy of the treatment was solely based on the number of main vitelline vessels.

4

The study was conducted under in vivo conditions in the Chemistry Laboratory of

Philippine Science High School Southern Mindanao Campus. This study was conducted

for two (2) weeks.

Definition of Terms

Anas platyrhynchos (Mallard duck). This refers to the species which was used as

experimental subject in determining the anti-angiogenic potential of T. cacao L. seed

extract.

Angiogenesis. This refers to the mechanism of the growth of new blood vessels

which was decreased by the use of natural T. cacao L. seed extract.

Cytoxan. This refers to the commercial anti-angiogenic drug used.

Number of Main Vitelline Vessels. This refers to the parameter in this study

which was used to determine the anti-angiogenic potentials of T. cacao L. crude seed

extract.

Theobroma cacao L.(cacao) seed extract. This refers to the substance that was

evaluated for its anti-angiogenic potential.

5

Chapter 2

REVIEW OF RELATED LITERATURE

Cancer

Cancer is a genetic disease because there will be alterations within specific genes,

but most of the time it is not inherited (Karp, 1999). Cancer requires a series of mutations

propelling towards an abnormal mass of cells, called a tumor. Normal cells can enter the

cell cycle for about fifty times, and then they die. Cancer cells can enter the cell cycle

repeatedly making them immortal (Mader, 2000).

Cancer has two main stages. In the first stage, initiation, a single mutated cell

begins to multiply abnormally. In progression, additional mutations occur and are

followed by rapidly growing cells increasing malignancy. When growth has reach beyond

the size of about a million cells, there is need for supply of nutrients and oxygen (Cooper,

2000).

A tumor is composed of rapidly dividing and growing cancer cells. Tumors

induce angiogenesis by secreting various growth factors that promote growth of blood

vessels towards them (Fig. 1), thus, there is availability of resources for survival and

reproduction (Mader, 2000).

6

Figure 1. Angiogenesis in Tumor formation

Source: National Cancer Institute, 2002

As shown in figure 1, the spreading of tumor occurs when single cancer cells can

break away from an established solid tumor, enter the blood vessel, and be carried to a

distant site, where they can implant and begin the growth of a secondary tumor

(Understanding Angiogenesis Organization, 2006).

Angiogenesis

Angiogenesis is the growth of new blood vessels in the body. It occurs both in

health and in diseases. They supply oxygen and nutrients (Karp, 1999). The bodys first

response to less blood flow to the heart is to grow tiny new collateral vessels to help

blood flow around the blockage. Some proteins in the body can help trigger new blood

vessel growth and so increase the oxygen supply to the ischemic tissue. Such angiogenic

proteins include the endothelial growth factor, vascular endothelial growth factor (VEGF)

and fibroblast growth factor (FGF) (American Heart Organization, 2007). It is a normal

process in growth and development, as well as in wound healing. However, this is also a

Tumor that can grow and spread Small localized tumor

Signaling molecule

Angiogenesis

Blood vessel

7

fundamental step in the transition of tumors from a dormant state to a malignant state

(Understanding Angiogenesis Organization, 2006).

Tumors that exhibit angiogenesis begin when the bodys own defense can no

longer hold back its growth signals. When the tumors growth signals become stronger or

make the bodys own defense weak it would result to what is called the angiogenic

switch. The angiogenic switch helps the tumor to change from a small, localized tumor

to a growing and spreading cancer (Mayo Clinic, 2006).

Once the angiogenic switch has been turned on, the tumor begins sending out

signals to the cells lining nearby blood vessels otherwise known as endothelial cells.

These signals cause the endothelial cells to grow and multiply. The endothelial cells

direct enzymes to clear a pathway to the tumor. The blood vessels form stems that reach

to the tumor. Once the blood vessel stems connect, the tumor uses the new blood vessels

to receive oxygen and nutrients. It also sends out cancer cells to spread elsewhere in the

body (Mayo Clinic, 2006).

Current Cancer Treatments

Nowadays, surgery is the method commonly undertaken by cancer patients to

remove visible tumors. It is only advisable when the tumor is still small and confined in a

specific area. Surgery may also entail the eradication of normal tissues surrounding the

tumor. Even with this precaution, surgery is still often followed by chemotheraphy and/or

radiation to ensure that there would be no recurrence (Carson-DeWitt, 2006). However

surgery have side effects such as pain, infection, loss of organ function, bleeding, blood

clots and altered bowel and bladder function (Mayo Clinic, 2005).

8

Chemotherapy is a treatment that uses anti-cancer drug or a combination of drugs.

Anti-cancer drugs will destroy cells or stop them from growing. Chemotherapy is a

known as a systemic therapy since it is administered through the blood stream and

travels throughout the body. Chemotherapy can be used to cure cancer, prevent cancer

from spreading, slow the cancers growth, or to kill cells that may have traveled from the

original tumor to other parts of the body or to relieve symptoms caused by cancer.

Chemotherapy can be done alone or it could be followed by surgery or by radiation

therapy. Chemotherapy have varied side effects such as nausea and vomiting, fatigue or

anemia, hair loss, infections due to reduction of white blood cell, slow blood clotting,

mouth or gum or throat problem, diarrhea and constipation (Jones, 1999).

Radiation therapy can be done in two ways. It could be done externally wherein a

machine directs a beam of radiation at the tumor. Another method is done internally by

implanting a radioactive material in a part of the body that is to be treated. It is commonly

followed up by surgery or chemotherapy. The side effects of radiation therapy are

fatigue, skin changes and loss of appetite (Parthun, 1999).

Cytoxan

Cyclophosphamide or commonly known as cytoxan as shown in figure 2, is a

synthetic antineoplastic drug. It has a molecular formula of C7H15C12N2O2PH20 and has

a molecular weight of 279.1 as shown in figure 3. Cytoxan is soluble in water, saline or

ethanol (RxList, 2008).

9

Figure 2. Cytoxan

Source:http://images.search.yahoo.com/search/images;_ylt=A0S0206XRpxHb0QAyzCJz

bkF?p=cytoxan&ei=UTF-8&fr=yfp-t-203&fp_ip=PH&x=wrt&js=1&ni=20

Cytoxan is an anti-angiogenic drug that cross-links with the nucleotides of DNA.

Once it is cross-linked with cytoxan, the DNA double helix is unable to unwind and DNA

replication does not occur and the tumor cells die (Marrs, 2003). However, cytoxan has

side effects which are nausea and vomiting, loss of appetite, diarrhea, mouth sores, hair

loss, low white blood cells, damage to bladder and fertility problems (Monson, 2007).

Figure 3. Molecular Structure of Cytoxan

Source: RxList Incorporation

Quercetin

Quercetin is a flavonoid found in apples, onions, raspberries, red grapes, citrus

fruit, cherries, broccoli, cacao and leafy greens. It inhibits angiogenesis through multiple

mechanisms, including interaction with certain enzymes that inhibits cell proliferation

(Jackson and Venema. 2007). Its molecular structure is shown in figure 4.

10

Figure 4. Molecular Structure of Quercetin 9

Source: http://www.geocities.com/agnihotrimed/Paper05_Jul-Dec2007_.pdf

Quercetin provides many health promoting benefits, including improvement of

cardiovascular health, eye diseases, allergic disorders, arthritis, reducing risk for cancer

and many more (Lakhanpal, P. and Rai, D. K., 2007).

It is one of the most abundant in the human diet and is known as an antioxidant

and inhibitor of cancer cell cycle progression. It inhibits angiogenesis via a mechanism

involving the suppression of endothelial nitric oxide synthase (eNOS) and early M-phase

cell cycle arrest (Jackson and Venema, 2007).

In eNOS activity, nitric oxide is produced and is a stimulant of endothelial cell

proliferation. It is responsible for the growth and migration of endothelial cells that are

necessary for the initiation of angiogenesis. Therefore, the eNOS-derived nitric oxide is

an essential factor to the process of angiogenesis. Thus quercetin, an agent that limits

eNOS activity, is also efficacious as part of chemopreventive and chemotherapeutic

regimens (Jackson and Venema, 2007).

11

Theobroma cacao L.

Theobroma cacao L. (cacao) was named by Linnaeus. Its literal meaning is food

of the gods. The tree is twelve to sixteen feet high and its trunk is about five feet long. It

bears fruit which is yellowy red in color. As shown in figure 5, the texture of the fruit is

smooth. Its pulp is white and when the seeds are ripe they rattle in the capsule when

shaken. Each capsule contains an average of twenty-five seeds (Grieve, 2007).

Figure 5. Cacao

Source: http://images.search.yahoo.com/search/images?p=cacao&ei=UTF-

8&fp_ip=PH&js=1&ni=20&fr=yfp-t-203&b=21

Cacao is locally available in the Philippines. The total area devoted to cacao as of

1998 is 13,231 hectares. Mindanao accounted for 88.44% or 11,701.5 hectares, Luzon

6.83% or 903.5 hectares and Visayas 4.73% or 626 hectares (DA-Agricultural and

Marketing Assistance Service, 2006).

Cacao especially its beans are rich in polyphenols. Polyphenols in cacao have

antioxidant, antimutagenic and antitumor activities. Cacao beans have high phenolic

content of about 12-18% (dry weight) in unfermented beans (Othman, et al., 2005).

Phenolic compounds present in cacao are epicathechin, cathechin, procynadins and

flavonols specifically quercetin (Ding etal., 2006). Cacao powder (unsweetened) contains

about 20.1mg/100mg of quercetin (Lakhanpal and Rai, 2007). The phenolic compounds

12

mentioned above which is present in cacao are reported to have the ability to combat free

radicals, which are harmful to the body and food systems (Othman, 2005).

Stages of Blood Vessel Development of A. platyrhynchos (Mallard duck) Embryo

Malllard ducks are found in calm shallow sanctuaries. They can be found in

almost any body of freshwater across Asia, Europe and North America. They are also

found in saltwater and brackish water and are commonly found in wetlands (National

Geographic Society, 2008).

The common morphology on both sexes of Mallard duck is complemented with

shining blue speculum on the wings. As shown in figure 6, the notable characteristics of

the male mallard duck are the green iridescent plumage on the head and neck, and have

curled black feathers on the tail. The females plumage is drab brown (Rogers, 2001).

Figure 6. Female and male Mallard duck

Source: http://animals.nationalgeographic.com/animals/birds/mallard-duck.html

In duck embryo development, pear-like shaped blastodisc starts to be noticeable

during the second day. On this same day, blood islands that serve as the beginning of

vascular system start to appear. On the third day, the heart starts to be visible while the

body starts to turn on its left side. During the fourth day, the vascular zone of the yolk

circulation starts to form. The amnion starts to be visible and the amnion liquid forms on

13

the fifth day. During the eighth day, the allantois of the duck embryo could then be

distinguished (Smith, 2004).

Normal closure of species at the sharp end of the egg usually takes place on the

fourteenth-fifteenth day of development. At this time, feathering of the lumbo-sacral area

begins. After the twenty-fifth to twenty-sixth day of development, the amount of amnion

and allantois liquid starts to decrease. On the twenty-ninth to thirtieth day of

development, the residual yolk sac starts to draw into the body and on the last day, the

embryo is then practically ready for hatching. The mean daily growth of the embryos was

1.37 g and the body length increased by 0.5 cm daily (Smith, 2004).

The incubation of duck eggs required a stabilized temperature of 37.5C with

relative humidity of 75%. The eggs should be turned 90 thrice a day to prevent the yolk

from touching the shell and injuring the embryo (Smith, 2007).

Related Studies on Theobroma cacao L. and Quercetin

The research paper titled Cocoa Has More Phenolic Phytochemicals and a

Higher Antioxidant Capacity than Teas and Red Wine, shows that cocoa teems with

antioxidants that prevent cancer, Cornell University food scientists say. Comparing the

chemical anti-cancer activity in beverages known to contain antioxidants, they have

found that cocoa has nearly twice the antioxidants of red wine and up to three times those

found in green tea (Cornell University, 2003).

A research study conducted by Hari Krishnan Nair, Kesava V. K. Rao, etal, found

that dietary bioflavonoid quercetin which are present in many plants are capable of

inhibiting the growth of prostate cells in vitro. Quercetin has anti-cancer effects which are

14

mediated by inhibition of the expression of various cell cycle genes and oncogenes and

enhances several tumor suppressor genes (Nair, et al., 2004).

15

Chapter 3

METHODOLOGY

Gathering of Materials

Fifteen (15) ten-day-old duck eggs were obtained from an egg dealer in Kabacan,

North Cotabato. Two (2) cacao fruits were bought from Bankerohan Public Market. One

(1) incandescent light bulb and one (1) light bulb socket were bought from NCCC

hardware store. One (1) 52 x 31 x 40 cm3 box was gathered in one of the proponents

residence. One hundred (100) grams of disodium hydrogen phosphate and one hundred

(100) grams of sodium dihydrogen phosphate were bought from Crimson Enterprises,

Agdao, Davao City. Three (3) masks and three (3) pairs of gloves were bought from

HBC. Fifteen (15) pieces of syringe and fifty (50) milligrams of cytoxan and one (1) pack

of sterile strips were bought from AMESCO Drugstore. Alcohol, cotton, sterile strips and

four (4) liters of distilled water were bought from the SM Supermarket. Ethanol solution,

autoclave, wash bottle, test tube with screw cap, knife, filter paper, Buchner filter,

pipette, rubber aspirator, electronic weighing scale, 250 mL Erlenmeyer flasks, 25 mL

volumetric flasks, beakers, mortar and pestle were borrowed from the Chemistry

Laboratory of Philippine Science High School Southern Mindanao Campus.

Sterilization of Materials

All glass wares and cotton were sterilized in an autoclave for one hour at 120C.

After one hour, the materials were removed from the autoclave and were allowed to cool.

16

Preparation of Experimental Set-ups

An incubator was constructed out of the 52 x 31 x 40 cm 3

box and one (1) light

bulb and socket. The light bulb and socket was positioned at the center of the box to

maintain a temperature of 37.5C. A basin filled with water was placed under the egg

trays to set a humidity of 75%. The temperature of the water was about the same as the

temperature of the incubator to avoid the eggs from being stressed. Humidity was

measured using a wet-thermometer and a dry- bulb thermometer placed inside the

incubator.

Fifteen (15) eggs were placed in egg trays and were placed about one half (1/2)

meter away from the light bulb. A thermostat was placed inside the incubator to make

sure that the temperature is regulated. The temperature is monitored every two hours.

Preparation of Phosphate Buffer Solution

In preparing for a 0.2 M Phosphate Buffer Stock Solution, two (2) grams of

monobasic sodium phosphate and dibasic sodium phosphate was dissolved in one

hundred (100) mL of distilled water. The two solutions was mixed and diluted with

distilled water in a five hundred (500) mL volumetric flask. This yields to a phosphate

buffer solution of pH 7.2.

Preparation of Cacao Seed Extract

The shells of the cacao fruit were cracked open and the seeds were removed. The

seeds were washed with water and air dried for three (3) days. The dried seeds were

crushed using mortar and pestle. Fifty (50) grams of cacao seed powder was soaked in

two hundred (200) mL of ethanol for three (3) days. After three (3) days the soaked cacao

17

seed powder was filtered using the Buchner funnel. The extract was then concentrated

using a rotary evaporator.

Preparation of Treatments

There were five (5) treatments with seven (7) replicates each. The following are:

T0: control, without hole

T1: control, with hole

T2: 0.25mL T. cacao seed extract in 25mL PBS

T3: 0.5mL T. cacao seed extract in 25mL PBS

T4: 25mg cytoxan in 50mL PBS

No extracts were introduced to T0 and T1. However, a hole was made on the flat

portion of the eggs in T1. The hole was then covered with sterile strips.

Treatment T2 contained 0.25 mL of cacao seed extract in one hundred (100) mL

of phosphate buffer solution. Treatment T3 contained 0.50 mL of the cacao seed extract in

one hundred (100) mL of phosphate buffer solution. Treatment T4 contained 0.50 mL of

cytoxan in one hundred (100) mL of phosphate buffer saline solution.

Preparation of Anas platyrhynchos (Mallard duck) Embryo

Fifteen ten-day-old duck eggs were cleaned by lightly rubbing them with fine

wool to remove dry mud and manure. The eggs were wiped with cotton damped with

distilled water. After which, the eggs were wiped with cotton damped with alcohol.

Application of Treatments

The ten day-old-duck embryos were given different concentrations of the extract

prepared in 0.05 M phosphate buffered saline solution with a pH 7.2. A small hole was

18

created in the flat bottom part of the egg using a knife. The flat bottom part of the egg

was previously cleaned with distilled water and alcohol. About 0.2 mL of the extract was

introduced using a syringe. The flat bottom part of the egg has an empty space and the

extract was allowed to diffuse slowly through the membrane. The holes were sealed with

sterile strips and incubated. The numbers of main vitelline vessels in the embryos were

observed after three days.

Incubation of Anas platyrhynchos (Mallard duck) Eggs

The incubator was allowed to stabilize a temperature of 37.5C a day before the

actual incubation. The eggs placed in the egg trays were positioned in its pointed down

end. The incubator was then set to 37.5C with a relative humidity of 75% (Smith, 2007).

The temperature was maintained throughout the incubation process. The eggs were

manually turned at an angle of 90 for at least three times a day during the incubation

period. Turning the eggs allowed the embryo to move freely and prevented the eggs

content from sticking to its shell.

Data Gathering

The number of main vitelline blood vessels was observed, counted and recorded

after two and three days. The efficacy of T. cacao as an anti-angiogenic agent was based

on the number of main vitelline vessels in the duck embryo. The main vitelline vessels

are shown in figure 7.

19

Figure 7. Anatomy of a 7- day old duck embryo

Source: http://www.ext.vt.edu/resources/4h/4hpubs/pdfs/408-029.pdf

Statistical Analysis

The gathered data will be analyzed using One-way Analysis of Variance

(ANOVA) and Duncans Multiple Range Test (DMRT) at 5% level of significance.

20

Chapter 4

RESULTS AND DISCUSSION

The study evaluated the anti-angiogenic potential of T. cacao L. (cacao) crude

seed extract through the inhibition of vitelline vessel formation in A. platyrhynchos

(Mallard duck) embryo. The anti-angiogenic property of cacao seed extract was

determined based on the number of main vitelline vessels of the duck embryo after

treatments were applied.

As shown in Table 1, T0 had the largest mean count of 8.33 vitelline vessels. T1

came next with a mean of 6.67 vitelline vessels. T2 (0.25mL Theobroma cacao L. (cacao)

seed extract in 25mL PBS) followed with a mean of 5.33 while T4 (25mg cytoxan in

50mL PBS) has a mean value of 4.33. T3 (0.50mL Theobroma cacao L. (cacao) seed

extract in 25mL PBS) comes last with a mean of 3.00

Table 1. Mean Number of Main Vitelline Vessels in A. platyrhynchos Embryo

Treatments Mean No. of Vitelline Vessels

T0 (controlled; without hole)

8.33a

T1 (controlled; with hole)

6.67a

T2 (0.25mL Theobroma cacao L. (cacao) seed extract

in 25mL PBS)

5.33b

T3 (0.50mL Theobroma cacao L. (cacao) seed extract

in 25mL PBS)

3.00b

T4 (25mg cytoxan in 50mL PBS)

4.33b

Note: Superscripts that are the same are not significantly different from each other.

Based on ANOVA and DMRT, it was found that treatments T2, T3 and T4 are not

significantly different with each other. However, they are significantly different with T0

and T1.

21

In T2 and T3, Theobroma cacao L. (cacao) crude seed extract were used since it

was found that cacao seeds are rich in quercetin (Raintree Nutrition, Inc., 1996). T. cacao

L. contains 10 grams per 100 grams of antioxidant flavonoids (Health Product

Distributors, Inc., 2004). Quercetin is a dietary-derived flavonoid that was found to

inhibit several important steps in angiogenesis. This implies that quercetin has an anti-

angiogenic potential (Tan, et al., 2003). According to Indap, et al. (2006), quercetin

exerts antiproliferative effect and anti-angiogenic properties. It is also said that the

natural polyphenolic quercetin has a definite potential to be a chemotherapeutic agent for

human cancer.

The antiangiogenic mechanism of quercetin involves deactivation of endothelial

nitric oxide synthase (eNOS). The eNOS produces nitric oxide which functions as the

downstream effector of vascular endothelial growth factor (VEGF). The VEGF is a

crucial factor in one of the steps of angiogenesis. The nitric oxide is also responsible for

the mobility of the endothelial cells which is a required step in the angiogenic process

(Jackson and Venema, 2007).

This mechanism is also similar to the mechanism of cyclophosphamide or

commonly known as cytoxan. Cyclophosphamide causes prevention of cell division

primarily by cross-linking DNA strands. It is transformed to active alkylating

metabolites, the most common of which are the phosphoramide mustard and acrolein.

These metabolites are the ones responsible for the hindrance of endothelial cell division

(Canadian Pharmacists Association, 2006).

Therefore, the data gathered from this study shows that Theobroma cacao L.

(cacao) crude seed extract has anti-angiogenic properties due to the presence of quercetin.

22

The findings of the study can help boost the cacao industry here in the Philippines and

may add more opportunities to grow cacao locally. The findings will also add to the

ongoing studies about angiogenesis and its natural inhibitors.

23

Chapter 5

CONCLUSIONS

Based on the study conducted it is concluded that Theobroma cacao L. (Cacao)

seed extract exhibits anti-angiogenic potential on Anas platyrhynchos (Mallard duck)

embryo.

Specifically, it is concluded that:

1) Different concentration of T. cacao L. seed extract: 0.25 mL seed extract in 25

mL PBS and 0.50 mL seed extract in 25 mL PBS are effective anti-angiogenic

agents in terms of the number of main vitelline vessels on duck embryo; and,

2) T. cacao L. seed extract has a comparable effect to that of cytoxan, a

commercial anti-angiogenic drug.

24

APPENDIX A

FLOWCHART OF THE METHODOLOGY

Gathering of Materials

Sterilization of Materials

Preparation of Experimental Set-ups

Preparation of Phosphate Buffer

Solution

Preparation of Cacao Seed Extract

Preparation of Treatments

Preparation of Anas platyrhynchos

(Mallard duck) Embryo

Application of Treatments

Incubation of Duck Eggs

Data Gathering

Statistical Analysis

25

APPENDIX B

RAW DATA

Table 2. Number of Main Vitelline Blood Vessels on Embryo at Different Treatments

Treatments Replicates Number of Main

Vitelline Vessels

Mean Number of

Main Vitelline

Vessels

T0

(no treatment

without hole)

R1 8

8.33 R2 9

R3 8

T1 (no treatment with

hole)

R1 11

6.67 R2 *

R3 9

T2 (0.25mL cacao

seed extract in

25mL PBS)

R1 5

5.33 R2 5

R3 6

T3 (0.50mL of cacao

seed extract in

25mL PBS)

R1 5

3.00 R2 *

R3 4

T4

(25mg of cytoxan

in 50 mL PBS)

R1 4

4.33 R2 4

R3 5

* embryo was dead and did not develop

26

APPENDIX C

STATISTICAL ANALYSIS

Table 3. Anova Table

Source of

Variation

SS

df

MS

F

P-Value

F crit

Between

Groups

51.06667 4 12.76667 1.507874 0.272078 3.47805

Within

Groups

84.66667 10 8.466667 - - -

Total 135.7333 14 - - - -

Decision:

Since f

27

T4 = x4 = 3.67

T5 = x5 = 3.67

Conclusion: Treatments T2, T3 and T4 are not significantly different with each

other but are significantly different with T0 and T1.

28

APPENDIX D

PICTURES OF THE SET-UP

Figure 8. 13-day old duck embryo control T0 (without hole)

Figure 9. 13-day old duck embryo control T1 (with hole)

Figure 10. 13-day old duck embryo applied with T2

29

Figure 11. 13-day old duck embryo applied with T3

Figure 12. 13-day old duck embryo applied with T4

30

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