Anti-Angiogenic Potential of Theobroma cacao L. (Cacao) Seed Extract on Anas platyrhynchos (Mallard Duck) Embryo

eobroma oma cacao cacao L. (CACAO) SEED ANTI-ANGIOGENIC POTENTIAL OF Th eobr

EXTRACT ON An as platyrh nchos (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

ABSTRACT The study entitled “ANTI-ANGIOGENIC POTENTIAL OF Theobroma cacao L. (CACAO) SEED EXTRACT ON Anas ON  Anas platyrhnchos (MALLARD DUCK) EMBRYO” 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 significance and Duncan’s Multiple Range Test (DMRT). The results of the study showed that T 2 (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. of A. playtyrhnchos embryo and its efficacy is comparable to that of the anti-angiogenic drug cytoxan.

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ABSTRACT The study entitled “ANTI-ANGIOGENIC POTENTIAL OF Theobroma cacao L. (CACAO) SEED EXTRACT ON Anas ON  Anas platyrhnchos (MALLARD DUCK) EMBRYO” 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 significance and Duncan’s Multiple Range Test (DMRT). The results of the study showed that T 2 (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. of A. playtyrhnchos embryo and its efficacy is comparable to that of the anti-angiogenic drug cytoxan.

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CERTIFICATE OF APPROVAL The research paper entitled “ANTI-ANGIOGENIC “ANTI-ANGIOGENIC POTENTIAL OF Theobroma cacao L. (CACAO) SEED EXTRACT ON  Anas platyrhynchos (MALLARD DUCK) EMBRYO,” 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 Panel Member

_____________________ SHARON M. DEJARME Panel Member

 _________________________ _____  ______________________________ ANDREA ANNE S. BALDONADO Language Consultant

Accepted in Fulfillment of the Requirements in Research 2

 ____________________ ARLYN A. DACANAY Research 2 Coordinator

_________________________ DR. ROSITA V. FUNDADOR Director

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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.

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TABLE OF CONTENTS Page TITLE PAGE………………………………………………………………………………i ABSTRACT…………………………………………………………………………...….ii CERTIFICATE OF APPROVAL………………………………………………………..iii ACKNOWLEDGEMENT…………..…………………………………………………....iv LIST OF TABLES……………………………….…………….…………………….….vii LIST OF FIGUR ES ....………………………….……………………………………...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 Terms……………………………………………………………4 2. REVIEW OF RELATED LITERATURE Cancer ………………………………………………………………………....5 Angiogenesis………………………………………………………………......6 Current Cancer Treatments................................................................................7 Cytoxan………..................................................................................................8 Quercetin..................................................................................................…......9 Theobroma cacao L..………………………………………………………...11

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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 Buf fer Saline Solution……………………………16 Preparation of Cacao Seed Extract…………………………………………..16 Preparation of Treatments.…………………………………………………...17 Preparation of Anas platyrhynchos (mallard duck) Embryo………………....17 Application of Tr eatments…………………………………………………...17 Incubation of Anas platyrhynchos (mallard duck) Eggs……………………..18 Data Gathering…..…………………………………………………………...18 Statistical Analysis…………………………………………………………...19 4. RESULTS AND DISCUSSION…………………………………………………20 5. CONCLUSIONS.……………………………………………………………….. 23 BIBLIOGRAPHY..............................................................................................................30 APPENDICES…………………………………………………………………………...24

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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

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LIST OF FIGURES Figure

Page

1

Angiogenesis in Tumor Formation………………………………………………6

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

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LIST OF APPENDICES Appendix

Page

A

Flowchart of Methodology………………………………………………24

B

Raw Data………………………………………………………………...25

C

Statistical Analysis………………………………………………………26

D

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

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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, 19 99). 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 antiangiogenic 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).

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Angiogenesis

Tumor that can grow and spread

Signaling molecule

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 body’s 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

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 body’s own defense can no longer hold back its growth signals. When the tumor’s growth signals become stronger or make the body’s 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 cancer’s 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 C7H15C12 N2O2P·H20 and has a molecular weight of 279.1 as shown in figure 3. Cytoxan is soluble in water, saline or ethanol (RxList, 2008).

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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.

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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 Th eobroma 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=UTF8&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 female’s 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 37.5°C 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 Th eobroma 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-cance r 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 3

hardware store. One (1) 52 x 31 x 40 cm  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 120°C. After one hour, the materials were removed from the autoclave and were allowed to cool.

16 Preparation of Experimental Set-ups  3

An incubator was constructed out of the 52 x 31 x 40 cm  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 37.5°C. 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 ev ery 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 An as platyrh ynchos (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 co tton 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 An as platyrh ynchos (Mallard duck) Eggs

The incubator was allowed to stabilize a temperature of 37.5°C 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 37.5°C 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 Duncan’s 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. 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. 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 a (controlled; without hole) 8.33 T1 a (controlled; with hole) 6.67 T2  b (0.25mL Theobroma cacao L. (cacao) seed extract 5.33 in 25mL PBS) T3  b (0.50mL Theobroma cacao L. (cacao) seed extract 3.00 in 25mL PBS) T4  b (25mg cytoxan in 50mL PBS) 4.33  Note: Superscripts that are the same are not significantly different from each other. Based on ANOVA and DMRT, it was found that treatments T2, T 3 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 antiangiogenic 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

T0 R1 (no treatment R2 without hole) R3 T1 R1 (no treatment with R2 hole) R3 T2 R1 (0.25mL cacao R2  seed extract in R3 25mL PBS) T3 R1 (0.50mL of cacao R2  seed extract in R3 25mL PBS) T4 R1 (25mg of cytoxan R2 in 50 mL PBS) R3 * embryo was dead and did not develop

8 9 8 11 * 9 5 5

Mean Number of Main Vitelline Vessels 8.33

6.67

5.33

6 5 *

3.00

4 4 4 5

4.33

26 APPENDIX C STATISTICAL ANALYSIS Table 3. Anova Table Source of Variation

SS

df

MS

F

P-Value

F crit

Between

51.06667

4

12.76667

1.507874

0.272078

3.47805

84.66667

10

8.466667

-

-

-

135.7333

14

-

-

-

-

Groups Within Groups Total

Decision: Since f
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: T0 = x1 = T1 = x2= 9.67 T3 = x3 = 4.33

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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