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 T
0
and T
1
. T
0
was
not holed while T
1
was holed on its egg shell. T
2
and T
3
were prepared with 0.25mL and
0.50 mL T. cacao seed extract in 25mL Phosphate Buffer Solution (PBS) respectively.
T
4
, 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 T
2
(5.33), T
3
(3.00) and T
4
(4.33) had
significantly lesser number of vitelline vessels compared to T
0
(8.33) and T
1
(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 T
0
(control, without hole)...28
9 T
1
(control, with hole)........28
10 T
2
(0.25mL T. cacao seed extract in 25mL PBS)..28
11 T
3
(0.5mL T. cacao seed extract in 25mL PBS)............29
12 T
4
(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 C
7
H
15
C
12
N
2
O
2
PH
2
0 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 cm
3
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:
T
0
: control, without hole
T
1
: control, with hole
T
2
: 0.25mL T. cacao seed extract in 25mL PBS
T
3
: 0.5mL T. cacao seed extract in 25mL PBS
T
4:
25mg cytoxan in 50mL PBS
No extracts were introduced to T
0
and T
1
. However, a hole was made on the flat
portion of the eggs in T
1
. The hole was then covered with sterile strips.
Treatment T
2
contained 0.25 mL of cacao seed extract in one hundred (100) mL
of phosphate buffer solution. Treatment T
3
contained 0.50 mL of the cacao seed extract in
one hundred (100) mL of phosphate buffer solution. Treatment T
4
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, T
0
had the largest mean count of 8.33 vitelline vessels. T
1

came next with a mean of 6.67 vitelline vessels. T
2
(0.25mL Theobroma cacao L. (cacao)
seed extract in 25mL PBS) followed with a mean of 5.33 while T
4
(25mg cytoxan in
50mL PBS) has a mean value of 4.33. T
3
(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
T
0
(controlled; without hole)

8.33
a
T
1
(controlled; with hole)

6.67
a
T
2
(0.25mL Theobroma cacao L. (cacao) seed extract
in 25mL PBS)

5.33
b
T
3
(0.50mL Theobroma cacao L. (cacao) seed extract
in 25mL PBS)

3.00
b
T
4
(25mg cytoxan in 50mL PBS)

4.33
b
Note: Superscripts that are the same are not significantly different from each other.
Based on ANOVA and DMRT, it was found that treatments T
2
, T
3
and T
4
are not
significantly different with each other. However, they are significantly different with T
0

and T
1
.
21
In T
2
and T
3
, 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
T
0

(no treatment
without hole)
R1 8
8.33 R2 9
R3 8
T
1
(no treatment with
hole)
R1 11
6.67 R2 *
R3 9
T
2
(0.25mL cacao
seed extract in
25mL PBS)

R1 5
5.33
R2 5
R3 6
T
3
(0.50mL of cacao
seed extract in
25mL PBS)
R1 5
3.00
R2 *
R3 4
T
4

(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<fcrit.accept H0
Conclusion:
There was a significant difference among the treatments.

Table 4. DMRT Table
P 2 3 4 5
r
p
3.151 3.293 3.376 3.430
R
p
1.243 1.2988 1.3315 1.3528

Let the mean of:
T
0
= x
1
= T
1
= x
2
= 9.67
T
3
= x
3
= 4.33
27
T
4
= x
4
= 3.67
T
5
= x
5
= 3.67
Conclusion: Treatments T
2
, T
3
and T
4
are not significantly different with each
other but are significantly different with T
0
and T
1
.



28
APPENDIX D
PICTURES OF THE SET-UP

Figure 8. 13-day old duck embryo control T
0
(without hole)

Figure 9. 13-day old duck embryo control T
1
(with hole)


Figure 10. 13-day old duck embryo applied with T
2
29

Figure 11. 13-day old duck embryo applied with T
3

Figure 12. 13-day old duck embryo applied with T
4




30

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