The Angiogenic Activity of Zea Mays Linne (Corn) Thesis

Liceo de Cagayan University College of Pharmacy | 2

Zea mays Linne (CORN) USING THE ANGIOGENIC ACTIVITY OF Zea CHORIOALLANTOIC MEMBRANE VASCULAR ASSAY ON FERTILIZED CHICKEN EGG

RAHIMA M. ADILAO KHIRA MAE U. DAGANATO BLEISHEEN M. DUHAYLUNGSOD DUHAYLUNGSOD AQUISAH L. GORONDAYA NIHAD R. MAPANDI

MARCH 2016 THE ANGIOGENIC ACTIVITY OF Zea Zea mays Linne (CORN) USING CHORIOALLANTOIC MEMBRANE VASCULAR ASSAY ON FERTILIZED CHICKEN EGG


A Thesis Presented to the Faculty of the College of Pharmacy Liceo de Cagayan University Cagayan de Oro City

In Partial Fulfillment of the Requirements for the Degree of Bachelor of Science Major in Pharmacy

RAHIMA M. ADILAO KHIRA MAE U. DAGANATO BLEISHEEN M. DUHAYLUNGSOD AQUISAH L. GORONDAYA NIHAD R. MAPANDI

MARCH 2016

Liceo de Cagayan University College of Pharmacy | 4 APPROVAL SHEET

This thesis entitled “The Angiogenic Activity of Zea mays Linne (Corn) Germ Using Chorioallantoic Membrane Vascular Assay on Fertilized Chicken Egg prepared and submitted by Rahima M. Adilao, Khira Mae U. Daganato, Bleisheen M. Duhaylungsod, Aquisah L. Gorondaya, and Nihad R. Mapandi in partial fulfillment of the requirements for the degree of Bachelor of Science in Pharmacy, has been examined and recommended for acceptance and approval for oral examination. ”

MERCEDITA S. AQUINO R.Ph., MSPharm., Ph.D Research Adviser

PANEL OF EXAMINERS

Approval by the committee on Oral Examination with a grade of___________

LESLEY CASAS LUBOS, Ph.D.

CHAIRMAN ______________________ MEMBER

_____________________ MEMBER

Acceptance and approved in partial fulfillment of the requirements for the degree of BACHELOR OF SCIENCE IN PHARMACY ___________________________ DATE OF FINAL DEFENSE MSPharm.,Ph. D Pharmacy

EDITOR

MERCEDITA S. AQUINO R.Ph., Dean, College of


DEDICATION

I would like to dedicate this thesis paper first and foremost to the Almighty Creator. You gave us the strength and knowledge we need in order to accomplish this and we are very thankful because without you we are not be able to stand on our own and have the courage to face all the challenges. To our beloved parents: Mr. &Mrs. Moumina-Abdul Rahim Adialao, NoraidaMohaymen Gorondaya, Yasmin-Omar Mapandi, Mr. & Mrs.Capistrano Daganato, Gerlita-Roland Duhaylungsod. Thank you so much for the undying support and love we receive from both of you. Again thank you and we love you to the fullest. To our teachers and adviser, You are one of the reasons why we are able to finish this thesis. Thank you for the advise and for the guidance. And last but not the least to our friends, whom we can always lean on when unnecessarily things happen. Thank you for the love and friendship.


ACKNOWLEDGEMENT

The researchers would like to extend their profound gratitude to the following individuals who have made this research paper a successful one:

Dr. Mercedita S. Aquino R.Ph., MSPharm., Ph.D. , Dean of the College of

Pharmacy and our research adviser, for unending support, valuable insights, motivation and encouragement; Dr. Lesley Casas Lubos, Ph.D. , Chairman of the Panel of Examiners, for the

recommendations and suggestions for the refinement of the study; Ms. Maria Eden K. Requina, Research Laboratory Instructor, for the willingness to

help us during our actual experiment The Faculty of the College of Pharmacy , for the unselfish molding of future

professional pharmacist; Mr. Bernard A. Guttierrez , for his expertise in statistics;

Most of all to the Heavenly Father , who has been the constant guide, to let us gain knowledge, and to develop and to benefit from all things-failures and success, sadness, and happiness.


ABSTRACT

Zea mays Linne, mostcommonly known as Corn, is rich in phytochemicals like the beta-sitosterol which is believed to have an angiogenic effect or the growth of new capillary blood vessels from pre-existing blood vessels in the body. This study aimed to evaluate the possible angiogenic property of Zea mays Linne germ extract using the chorioallantoic membrane vascular assay in chicken embryos. There were twelve pieces of one day old fertile chicken eggs used in the study. The ground Zea ground Zea mays may s Linne germ was macerated, extracted and confirmed by the presence of phytosterol which contains the beta-sitosterol as one of its component. The extract was divided into three to make different concentrations which was 50%, 75% and 100% that served as the treatment for the tenth day old incubated fertilized chicken eggs. The angiogenic effect of Zea of Zea mays Linne germ extract was observed by counting the number of branch points on the chorioallantoic membrane around the filter paper disc on the twelfth day of incubation. Result showed that there is no significant increase of blood vessel branch points indicated between the control and among the treatments used. Factor that may affect the results includes the presence of other components of phytosterol and the amount of betasitosterol.

Keywords: Zea mays Linne germ, beta-sitosterol, chorioallantoic membrane vascular

assay, fertile chicken embryo, angiogenesis


TABLE OF CONTENTS

Page i

COVER PAGE TITLE PAGE

ii

APPROVAL SHEET

iii

DEDICATION

iv

ACKNOWLEDGMENT

v

ABSTRACT TABLE OF CONTENTS

vii

LIST OF TABLES

x

LIST OF FIGURES

xi

LIST OF PLATES

xii

Chapter 1THE PROBLEM AND ITS SCOPE

Introduction

1

Objectives of the Study

3

Research Hypothesis

Liceo de Cagayan University College of Pharmacy | 9 Significance of the Study

4

Scope and Delimitations of the Study Definition of Terms

2REVIEW OF RELATED LITERATURE AND STUDIES

Foreign Literature and Studies Local Literature and Studies

3

MATERIALS AND METHODS

Research Setting

24

Research Design

24

Research Instrument

24

Research Protocol

25

Incubation Preparation

26

Collection and Preparation of Plant Samples

26

Extraction of Plant Material

26

Confirmatory Test of Phytosterol

26

Preparation of Test Subjects

Liceo de Cagayan University College of Pharmacy | Experimental Procedure

27

Statistical Techniques

28

Flow Chart

33

Appendix A- Letter of Consent

43

Appendix B- Figures and Plates

44

REFERENCES APPENDICES

Appendix C- Laboratory Results Appendix D- Time Format

53

Appendix E- Budget Proposal

55

Appendix F- Curriculum Vitae

56

Liceo de Cagayan University College of Pharmacy |

LIST OF TABLES TABLE

1

Page

The mean number of blood vessel branch points of

29

chicken eggs after application of corn extract 2

The significant difference in the mean number of

30

blood vessel branch points of fertilized chicken egg between control group and experimental group 3

Laboratory Results

52


LIST OF FIGURES FIGURE 1

Research Flow Chart

Page 33


LIST OF PLATES PLATE

Page

1.

Sampling size and collection of Corn (Zea mays) linn

44

2.

Soaking of corn kernels in distilled water

44

3.

Separation of germ from kernels

45

4.

Air drying of germs

5.

Grinding of germs

45

6.

Extraction with n-hexane

46

7.

Evaporating to small volume

46

8.

Confirmatory test for the presence of Phytosterol

9.

Crude extract partitioned using water

10.

Filter paper disc on extracts

45

46 47 47

Liceo de Cagayan University College of Pharmacy | 11.

Clean the eggs with the 70% Ethanol

48

12.

Incubation of eggs

48

13.

Removing of 2-3ml albumin from the egg

14.

Egg windowing

49

15.

Administration of treatment using the filter paper disc

50

16.

Covering the eggs with the Scotch tape

50

17.

Incubation of eggs

50

18.

Quantitation of blood vessel branch point

49

51


Chapter 1 THE PROBLEM AND ITS SCOPE

This chapter presents the introduction of the study, objectives of the study, hypothesis, significance of the study, scope and delimitations, and definition of terms. Introduction

Blood is an important body fluid as it carries oxygen and nutrients to all the parts of the body via vi a blood vessels like arteries and brings back ba ck the toxins and wastes from these peripheral p eripheral organs for purification via veins. When a new tissue was formed, it is vital that it has blood supply for its growth and sustenance. Formation of new tissues involves the formation of new blood vessels. For this function, angiogenesis is important (Mandal, 2014). Angiogenesis, the formation of new blood vessels in living humans and animals, is a beneficial natural process used for wounds by supplying nutrients from the blood capillaries to the site of damaged tissue for faster healing mechanism of the body. It occurs in the healthy body for healing wounds and for restoring blood flow to tissues after injury or insult. In females, angiogenesis also occurs during the monthly reproductive cycle to rebuild the uterus lining, to mature the egg during ovulation and during pregnancy to build the placenta, the circulation between mother and fetus (Zubar, 2015). Chick Chorioallantoic Membrane (CAM) assays have been widely used to study angiogenesis. The test requires fertilized and incubated chicken eggs which can be examined up to the eleventh day after the fertilization. The eggs should be used for testing purposes between sixth and tenth day of incubation to secure that the chick embryo does not suffer from pain since the embryonic nervous system development is not yet complete on its eleventh day. The CAM uses the capillary system connected to it for t esting procedures (Macfelda, 2015).

Liceo de Cagayan University College of Pharmacy This method uses the CAM of a 10-day-old egg. The primary CAM methods being |

evaluated in Europe use 9-10-day-old eggs because older eggs are considered live animals; using 10-day-old eggs is possible to be more globally acceptable as a non-animal test (Bagley et al., 1994). The main advantages of CAM model are its low cost, simplicity, reproducibility, and reliability. Zea mays Linne (Corn) belongs to the family of Poaceae. It is the second most important crop in the Philippines which is a rich source of vitamins A, B, E and many minerals that provide protection against some chronic diseases such as controlling diabetes, prevention of heart ailments, and lowering hypertension. It is also rich in phytochemicals like the beta-sitosterol which is believed to have an angiogenic effect. Chemists call it a “ plant sterol ester. ” It is one of many sterols that come from plants (phytosterols) and have a structure like the cholesterol produced in the body (Bradford et al., 2013). It stimulates HUVEC motility; enhances expression of von Willebrand factor, VEGF, VEGF receptor Flk-1 and laminin (Fan, 2006). Marathon runners sometimes use beta-sitosterol to reduce pain and swelling after a run while some people apply beta-sitosterol to the skin for treating wounds and burns. As BS Pharmacy students, the researchers were interested in developing a non-animal test to determine the angiogenetic activity of Zea of Zea mays Linne to help the community.

Objectives of the study

The study shall investigate the potential angiogenic property of the Zea mays Linne(Corn) in the fertilized chicken eggs using Chorioallantoic Membrane Vascular Assay (CAMVA) method. Specifically, it will aim to attain the following objectives:

Liceo de Cagayan University College of Pharmacy 1. To determine the mean number of blood vessel branch points of the chicken eggs after |

the plant extract is applied both the experimental group and the control: 1.1 Control group (T0) 1.2 Experimental Group 1 (T1) 1.3 Experimental Group 2 (T2) 1.4 Experimental Group 3 (T3) 2. To compare the significant difference in the mean number of blood vessel branch points of the fertilized chicken egg applied with the plant extract in the treatment of: 2.1 Control Group vs. Experimental Group

Hypothesis

H0: There is no significant difference in the mean number of blood vessel branch points of the fertilized chicken eggs applied with the plant extract among the experimental groups (T1, T2, and T3) and the control group.

Significance of Study

The results of this conducted research may be advantageous to the following: Pharmaceutical Industry

The result of the study will provide additional information about the health benefits of Zea of Zea mays Linne (corn) for the development of o f new drugs. Academe

This study may develop insights into the expansion of the University’s Research and Extension activities to include follow-up studies regarding this area. Students

Liceo de Cagayan University College of Pharmacy The results of this investigation is usefulas a source for future researches. This study may also |

serve as an additional reference to support new researches in relation to this topic.

Health providers

The result of the study may consider the components of Zea of Zea mays Linne (Corn) in addition to the health treatment providing system as another option for medication therapy. Community

The society may utilize the Zea mays Linne (Corn) to stimulate angiogenesis promoting neovascularization and tissue repair which is primarily important in wound healing. National Development

The study may contribute to the national development by providing information to the entire public of the role of angiogenesis in our body’s homeostasis.

Scope and Delimitation of the Study

The study focused only on determining the angiogenic effect of the Zea mays Linne(Corn) using the chick Chorioallantoic Membrane (CAM) Assay on the fertilized 20 homogenous 10-day old chicken eggs. The treatment administration and data collection were conducted within a 12 to13-day period only. Limitations of the study include the limited expertise, time and finances of the researchers in conducting the assay. The Material and equipment available in Cagayan de Oro City limits the accuracy of experimental results.

Definition of terms Angiogenesis.

This term refers refers to the process of formation of new blood vessels from prepre-

existing vessels or endothelial cell progenitors. It plays a crucial role in tumor growth and

Liceo de Cagayan University College of Pharmacy metastasis. |

Beta-sitosterol .

This term refers

to

the

most

abundant phytosterol and they are syrupy liquids soluble in most organic solvents but insoluble in water, and contain one alcohol functional group. refers to a very unique membrane covering the Chorioallantoic Membrane (CAM ). This term refers embryo where blood capillaries are located delivering the nutrients to the developing embryo. Chorioallantoic Membrane (CAM) Assay . This term refers to a developed alternative assay to

the Draize Rabbit Eye Test. It uses the vascularized membrane of fertile chicken eggs to assess a test material's potential to cause vascular changes (hemorrhaging, capillary injection, ghost vessels).

Chapter2 Review of Related Literatures and Studies

This chapter presents the review of foreign related literatures and the foreign and local related studies. Foreign Literature

The word angiogenesis comes from the two words angio- and -genesis which means blood vessels and creation. Literally means creation of new blood vessels according to Dr. Dr.

The Angiogenic Activity of Zea Mays Linne (Corn) Thesis

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