Antioxidant and Anti-Angiogenic Properties of the Crude Ethanolic Extract of

Saluyot (Corchorus olitorius) Leaves Utilizing Chorioallantoic Membrane (CAM)

Assay and DPPH Radical Scavenging Activity

A Research Paper Submitted to the Senior High School Department

College of Education
University of the East
Manila

In Partial Fulfillment of the Requirements for

Quantitative Research, Research Project, and
Immersion / Capstone Project

Agualin, Shanne Fondreea L.

Bungihan, Krystal Xyla E.
Calahi, Anne Margot C.
Gabriel, John Lorenze D.
Lazares, Kiana Ira C.
Lazo, Leigh Sofia T.

February 2020
 APPROVAL SHEET

This is to certify that this research study entitled “Antioxidant and Anti-

Angiogenic Properties of the Crude Ethanolic Extract of Saluyot (Corchorus

olitorius) Leaves Utilizing Chorioallantoic Membrane (CAM) Assay and DPPH

Radical Scavenging Activity,” submitted by Shanne Fondreea L. Agualin, Krystal

Xyla E. Bungihan, Anne Margot C. Calahi, John Lorenze D. Gabriel, Kiana Ira C.

Lazares, and Leigh Sofia T. Lazo, in partial fulfillment of the requirements for the

Quantitative Research, Research Project, and Immersion / Capstone Project, was

successfully defended and approved on February 2020.

_____________________________
Prof. Mark Anthony P. Lim
Research Project

_____________________________
Prof. Brann Ramos
Immersion / Capstone Project

_____________________________
Prof. Amielyn Alkathiri
Quantitative Research

i
 CERTIFICATE OF ORIGINALITY

This is to certify that the researchers on the study, “Antioxidant and Anti-Angiogenic
Properties of the Crude Ethanolic Extract of Saluyot (Corchorus olitorius) Leaves
Utilizing Chorioallantoic Membrane Assay and DPPH Radical Scavenging Activity,”
have completed and complied their research project with authenticity, reliability, and
relevance. They have observed proper academic guidelines necessary to the format,
structure, contents, mechanics, and overall standards/criteria set forth in writing with
application to their research output. Furthermore, they have successfully defended
their paper as part of their academic requirement for graduation.

Agualin, Shanne Fondreea L.

Bungihan, Krystal Xyla E.
Calahi, Anne Margot C.
Gabriel, John Lorenze D.
Lazares, Kiana Ira C.
Lazo, Leigh Sofia T.

ii
 ACKNOWLEDGMENT

The researchers glorify, praise, and thank the Lord God Almighty, who is the

ultimate source of knowledge and wisdom. He has been the guiding light and saving

grace all throughout this academic endeavor.

With gratitude and appreciation, the researchers would like to acknowledge

the following people for having extended help and guidance towards the completion

of this study:

To the researchers’ families, especially their parents, for providing them with

unconditional love, moral and financial support, and encouraging words in performing

their respective tasks on the accomplishment of their paper.

To their Research Adviser, Prof. Mark Anthony Lim, for patiently monitoring

on them as they progressively work on their research outputs. Also, for making time

for meaningful consultations and reliable suggestions.

To Dr. Melfei E. Bungihan, for unselfishly sharing her expertise in pursuing

this experimental study. She has shed light to the researchers by directing them in the

most difficult areas of their research.

Special thanks to Prof. Raynar Uybarreta for the constructive feedback and

suggestions, Prof. Brann Ramos for his additional information, and for his supervision

during laboratory hours with Prof. Amielyn Alkathiri.

iii
To our fellow students

iv
 ABSTRACT

Antioxidant and Anti-Angiogenic Properties of the Crude Ethanolic Extract of

Saluyot (Corchorus olitorius) Leaves Utilizing Chorioallantoic Membrane (CAM)
Assay and DPPH Radical Scavenging Activity

Shanne Fondreea L. Agualin, Krystal Xyla E. Bungihan, Anne Margot C.

Calahi,

John Lorenze D. Gabriel, Kiana Ira C. Lazares, Leigh Sofia T. Lazo

Senior High School Department, Basic Education, College of Education,

University of the East – Manila Campus, Claro M. Recto Avenue, Sampaloc,

Manila, 1008

The search for cheaper and effective antioxidant and antitumor sources from

our environment is now a trend in natural products discovery in order to address the

global burden of disease listed by World Health Organization. This study sought to

determine the antioxidant and anti-angiogenic properties of one of the common plants

in the Philippines, Corchorus olitorius, locally known as saluyot. The leaves of the

plant were soaked with ethanol to obtain the crude extracts. The crude ethanolic

extracts were subjected to DPPH radical scavenging activity assay to determine the

antioxidant property. Chorioallantoic membrane (CAM) assay was done to determine

the anti-angiogenic property of the different concentrations of the extract against a

negative control, distilled water. The ImageJ software was used for the counting of

vascularization in duck embryos. Results showed a 49.25% RSA of C. olitorius

compared to that of the positive control catechin with 87.36%. The anti-angiogenic

property of the different concentrations C. olitorius extract exhibited a decrease in

vascularity: T1 (VC=172), T2 (VC=168), T3 (VC=162) and T4 (VC=) compared to

the negative control, distilled water, (VC=167.3). Computed percent inhibition (PI)

v
were the following: T1 (6.58±5.65), T2 (19.13±4.51), T3 (11.54±9.79) and T4

(11.12±4.74). Analysis of variance showed no significant difference in the angiogenic

activities of the different treatment. Therefore, the crude ethanolic extract of C.

olitorius leaves shows antioxidant activity with a potential for anti-angiogenic

inhibition.

Keywords: Anti-angiogenesis, Antioxidant, Corchorus olitorius, Saluyot,

Chorioallantoic Membrane (CAM) Assay, DPPH Radical Scavenging Activity

vi
 TABLE OF CONTENTS

APPROVAL SHEET....................................................................................................i
ACKNOWLEDGMENT............................................................................................iii
ABSTRACT..................................................................................................................v
TABLE OF CONTENTS..........................................................................................vii
LIST OF FIGURES....................................................................................................ix
LIST OF TABLES.......................................................................................................x
LIST OF APPENDICES............................................................................................xi

CHAPTER I: INTRODUCTION...............................................................................1
A. Background of the Study.....................................................................................1
B. Statement of the Problem.......................................................................................2
C. Hypotheses..........................................................................................................3
D. Significance of the Study....................................................................................3
E. Conceptual Framework.......................................................................................4
F. Scope and Delimitation.......................................................................................6
G. Definition of Terms.............................................................................................7

CHAPTER II: REVIEW OF RELATED LITERATURES.....................................8

A. Corchorus olitorius.............................................................................................8
B. Phytochemical Analysis of Corchorus olitorius.................................................8
C. Cancer.................................................................................................................9
D. Antioxidant........................................................................................................10
E. Angiogenesis.....................................................................................................11
F. DPPH Radical Scavenging Activity..................................................................12
G. Chorioallantoic Membrane (CAM) Assay........................................................13

CHAPTER III: METHODOLOGY.........................................................................18

A. Research Design................................................................................................18
B. Saluyot Extract..................................................................................................18

vii
 C. Collection of Materials for CAM Assay...........................................................19
D. Anti-angiogenic Assay......................................................................................19
E. Antioxidant Assay.............................................................................................20
F. Data Collection..................................................................................................21
G. Framework of Methodology.............................................................................21
H. Statistical Analysis............................................................................................22

CHAPTER 4: RESULTS AND DISCUSSIONS.....................................................23

A. Antioxidant Activity of C. olitorius Extract.....................................................23
B. Anti-angiogenic Activity of C. olitorius Extract...............................................24

CHAPTER V: SUMMARY OF FINDINGS, CONCLUSIONS, AND

RECOMMENDATIONS...........................................................................................28
A. Summary of Findings........................................................................................28
B. Conclusions.......................................................................................................28
C. Recommendations.............................................................................................28

CHAPTER VI: BIBLIOGRAPHY...........................................................................30

CHAPTER VII: APPENDICES...............................................................................36

viii
 LIST OF FIGURES
Figure 1. Conceptual Framework ……………………………………………………. 4
Figure 2. Framework of Methodology ……………………………………………….
22
Figure 3. Plant Authentication ……………………………………………………….
36
Figure 4. Receipt of Plant Authentication ……………………………………………
36
Figure 5. Excuse Letter ……………………………………………………………...

37

ix
 LIST OF TABLES
Table 1. DPPH Radical Scavenging Activity of C. olitorius and Catechin (+control)
………………………………………………………………………………………. 23
Table 2. ImageJ Counts of Vascularization of Duck Embryos in CAM Assay and
Percent Inhibition of Angiogenesis ………………………………………………….
24
Table 3. Mean and Standard Deviation of PI of Treatment Groups ………………... 25
Table 4. Analysis of Variance of the Percent Inhibition Results …………………….
26

x
 LIST OF APPENDICES
Appendix 1. Plant Authentication ………………………………………………...…
36
Appendix 2. Plant Extract ...........................................................................................
37
Appendix 3. Antioxidant Assay ……………………………………………………..
37
Appendix 4. Anti-angiogenic Assay …………………………………………………
37

xi
 CHAPTER I: INTRODUCTION

A. Background of the Study

The World Health Organization records the global burden of diseases (GBD)

in 2016, along with the estimates of global mortality on the following years: 2000,

2005, 2010 and 2015. Of the top 20 leading causes of death in 2016, cardiovascular

diseases, cancers, diabetes mellitus, kidney diseases and respiratory infections are

listed. The number of deaths increased from years 2000-2016 (WHO, 2018).

Another problem which causes clamors from the public is the high risk of

oxidants and cancers nowadays. An update on the latest estimates on the global cancer

burden was reported by The International Agency for Research on Cancer (IARC) of

World Health Organization in 2018. On that same year, it is approximated that there is

an increase of 18.1 million new cases and 9.6 million deaths on global burden of

cancer. Statistics show that acquisition of cancer in men and women globally

throughout their lives is one in five and one in six respectively, while deaths in men

and women are one in eight and one in eleven respectively. It is also approximated

that 43.8 million of people having been diagnosed with cancer in span of five years

are alive (IARC, 2018). According to Philippine Statistics Authority (2018), the

second top leading cause of death in the Philippines in 2016 is neoplasms, which is

another term for cancer, with 60,470 or a percentage of 10.4 out of the total.

Neoplasm or cancer is the leading cause of death among women with 30,954 or

12.5%, while it is the third leading cause of death among men with 29,516 or 8.8% in

2016. With this growing number of deaths, there is a need to find for cure or

preventive measures. Anti-angiogenic drugs are those which can prevent the onset of

cancer while antitumor drugs are those that can kill cancer cells.

1
 According to Cancer Research UK (2019), people diagnosed with cancer often

use complementary and alternative therapies where in herbal medicine is their most

preferred. Most of the people who use herbal medicines in the 2011 study are more in

control. Furthermore, they find it more beneficial in terms of treatment management

and responsibility since there are no side effects with therapies.

With the previous and existing studies on herbal medicines, a primitive plant

from the tropics or most commonly found from Asia to Africa has been recognized

for its different properties and its strong fiber. The Corchorus olitorius – saluyot –

leaves possesses various micronutrients such as Calcium, Vitamin B, iron, Vitamins

A, E and C, thiamin, folate, riboflavin, niacin, protein and other dietary fibers. In

addition, medicinal uses of the said plant can be found. This includes treatment in

pregnant women with Gestational Diabetes (“Saluyot: Corchorus olitorius Herbal

Medicine”, 2013). There have been several studies on the utilization of saluyot for

many of the above-mentioned diseases, however, little has been known for the

potentiality of the saluyot with regard to cancer research for numerous studies have

been focused on the anti-diabetic potentiality of the plant.

B. Statement of the Problem

This study aims to identify the antioxidant and anti-angiogenic properties of

the crude ethanolic extract of the leaves of Corchorus olitorius, commonly known as

saluyot. Specifically, it aims to:

1. Determine the antioxidant capacity of the crude ethanolic extract of

saluyot leaves through DPPH scavenging activity.

2. Determine the anti-angiogenic property of the crude ethanolic extract of

saluyot leaves through chick embryo CAM as a model system.

2
 3. For the anti-angiogenic property, to determine the most effective

concentration of the crude ethanolic extract of saluyot leaves in terms of :

i. 25%

ii. 50%

iii. 75%

iv. 100%

4. Compare the significant difference between the different concentrations of

the crude ethanolic extract of saluyot leaves.

5. Find the significant difference of the control group – negative control:

Distilled water (DW) – and experimental groups – crude ethanolic extract

of saluyot leaves – on the chorioallantoic membrane assay.

C. Hypotheses

Ha: There is a significant difference in the means of the different

concentrations of the crude ethanolic extract of saluyot leaves.

Ha: There is a significant difference in the means of the control group and

experimental groups on the anti-angiogenic assay.

Ho: There is no significant difference in the means between the different

concentrations of the crude ethanolic extract of saluyot leaves.

Ho: There is no significant difference in the means of the control group and

experimental groups on the anti-angiogenic assay.

D. Significance of the Study

This study aims to identify the antioxidant and anti-angiogenic properties of

saluyot leaves. The identification of the antioxidant and anti-angiogenic properties of

saluyot leaves results to a greater cause for the future of medicine in cancer

3
prevention. In addition, with these properties being studied on the saluyot leaves,

discoveries that emerge as alternative medication is a reliable means at a much lower

price. This probability can aid numerous individuals, especially those who cannot

afford quality healthcare and expensive medical treatment.

Since the primary objective of this study is to determine the property of

saluyot leaves having an antioxidant and an anti-angiogenic agent, the probability of

reducing the number of people, diagnosed with malignant tumor, is attainable.

With regard to the botanical description of the saluyot, the outcome of the

study serves as a brief background on the prospective capacity of the saluyot leaves to

be used in future researches.

E. Conceptual Framework

Tumor

Benign Malignant

Cancer prevention Cancer

Anti-angiogenic Antioxidant

C. olitorius leaves crude

ethanolic extract

4
 A

Different concentrations 100% crude

Figure 1. Conceptual
of theFramework
crude ethanolic ethanolic extract of
extract of saluyot leaves: saluyot leaves
The conceptual
25%diagram presents the two general classifications of being benign
50%
and malignant. Malignant
75% tumor is described as a cancerous tumor whereas benign
100%
tumor is susceptible to cancer preventive measurements. In line with cancer

prevention lies different types of measurements, two of which are the antioxidant and

anti-angiogenic measurements. In this study,00a crude ethanolic extract of C. olitorius

DPPH Radical
was used wherein testing
CAMforAssay
its anti-angiogenic property required four different
Scavenging Activity
concentrations of the extract of 100%, 75%, 50%, and 25% whereas 100%
Data in
concentration was required Collection:
the antioxidant property Data
of theCollection:
extract. For the testing of
ImageJ Software UV-Vis Spectrophotometer
the anti-angiogenic property of the C. olitorius extract, the chorioallantoic membrane

(CAM) assay was utilized wherein

Data Analysisdata were collected Data
through the use of the ImageJ
Analysis

software. DPPH radical scavenging activity was used to test for the antioxidant
Statistical
property of the C. olitiorius Analysis:
extract. The data collection for the said assay utilized a
ANOVA
UV-Vis spectrophotometer. In analyzing the data, analysis of variance (ANOVA) was

used for the chorioallantoic membrane (CAM) assay (Figure 1).

F. Scope and Delimitation

The study, “Antioxidant and Anti-Angiogenic Properties of the Crude

Ethanolic Extract of Saluyot (Corchorus olitorius) Leaves Utilizing Chorioallantoic

Membrane Assay and DPPH Radical Scavenging Activity,” is limited in using the

crude ethanolic extract of leaves from the Corchorus olitorius – saluyot, thus does not

include other parts of the said plant. The saluyot leaves are only subjected to the

testing of their antioxidant and anti-angiogenic properties; testing for the different

plant properties such as antibacterial property and others are not included.

Since the goal of the study is to test for the antioxidant and anti-angiogenic

properties of the crude ethanolic extract of the saluyot leaves, a specific type of

5
testing was conducted for each property to be identified. The antioxidant property of

saluyot leaves was tested through the use of DPPH radical scavenging activity only;

while the anti-angiogenic property was tested through the use of chick embryo

chorioallantoic membrane assay (CAM) model system.

This study was conducted in the laboratory of the Qurino State University and

in the laboratory at the University of the East.

The time frame for this study was conducted within the span of three to five

months.

G. Definition of terms

For a better understanding of this study, the following terms are defined:

Angiogenesis - is a physiological process which refers to the growth of new

blood vessels that is vital for cancerous tumors.

Anti-Angiogenesis - is the prevention of new vessel sprout or the formation of

new blood vessels that are needed by tumors to grow, thereby suppressing or slowing

the growth or spread of tumors.

Antioxidant - is a medium for inhibiting or delaying the oxidation of

biologically relevant molecules either by specifically cleansing free radicals or by

chelation of redox metals.

Chick Embryo Chorioallantoic Membrane (CAM) Assay - is a method that

helps to facilitate the testing of different samples and the generation of dose – dilution

curves and has been used to distinguish almost all recognized angiogenetic variables.

Corchorus Olitorius - commonly known as jute or Jew’s Mallow and locally

termed as saluyot, is a green leafy vegetable plant popular in the northern part of

6
Luzon particularly in the Ilocos Region. It is classified under the kingdom Plantae and

a member of the family Malvaceae having a scientific genus name of Corchorus.

DPPH Radical Scavenging Activity - is a quick, easy, affordable, and widely

used method for measuring the potentiality of substances to act as free radical

scavengers.

CHAPTER II: REVIEW OF RELATED LITERATURES

A. Corchorus olitorius

Saluyot (Corchorus olitorius) is a leafy vegetable that is known for its natural

fiber. It is identified with a number of names such as Jute, Jew’s mallow, bush okra

and Egyptian spinach. Nutrients such as protein, iron, calcium, riboflavin, thiamin,

folate, niacin and dietary fiber are highly contained in its leaves. Renowned for its

durable natural and weatherproof fibers, saluyot has been transformed to numerous

products such as bags, couture and furniture manufacturing. Another use involves

dried or fresh leaves, which are cooked, as a soup thickener. Furthermore, saluyot in

the Philippines can be classified into two kinds: ‘Pula’ or the ordinary purplish type

and ‘Puti’ which is the more prominent kind around Metro Manila. The Institute of

Plant Breeding published the ‘Sagisag’, a stopgap variety that resembles with ‘Pula’

when it comes to stems and leaves. (Garcia, J., 2012)

Saluyot (Corchorus olitorius) is a highly nutritional leafy vegetable because of

its vitamins A, C, and E, calcium, iron, and dietary fibers content. It aids as a function

to keep a strong immune system, have enough red blood cells, eliminate bad

cholesterols, prevent blood clots, and maintain good eyesight due to its beta-carotene

property. (Sobrena, K., 2019) Since then, saluyot as a household name, has been

providing numerous benefits in various ways.

7
B. Phytochemical Analysis of Corchorus olitorius

The phytochemical screening executed on the Corchorus olitorius - Jute leaf

extract shows that there are some active ingredients such as flavonoids, phenols,

saponins, cardiac glycosides, and alkaloids, which are studied to possess therapeutic

activity that justifies its utility as a traditional medicine. It has been documented that

these phytoconstituents has the potential for numerous pharmacological activities

such as wound healing, cholesterol lowering, and antidiabetic activity. (Sadat, A. et

al., 2017) The chemical constituents of the dicholoromethane extract of the stems of

Corchorus olitorious includes (1) oleanolic acid which exhibited anti-inflammatory,

hepatoprotective, gastroprotective, immunoregulatory and anti-ulcer activities, (2) 2-

hydroxyethyl benzoate, (3) chlorophyll a and its various derivatives that are used for

therapeutic and medicinal purposes, (4a) phytyl fatty acid esters, (4b) β-sitosteryl fatty

acid esters, (5a) βsitosterol which was observed to attenuate β-catenin and PCNA

expression, as well as quench the radical in-vitro, making it a potential anticancer

drug for colon carcinogenesis , and (5b) stigmasterol which showed cytostatic activity

against Hep-2 and McCoy cells, markedly inhibited tumour promotion in two stage

carcinogenesis experiments, and exhibited antimutagenic, topical anti-inflammatory,

antiosteoarthritic and antioxidant activities. (Ragasa, C. et al., 2016)

C. Cancer

When the modifications of cells result in their division and unexpected spread

in the body, cancer is developed. There are cancer cells that can either spread at a fast

pace while there are those that break slowly. There are cancer cells that are renewed

and are replaced by the instructions of dying. However, there are missing

8
compositions that block the division and dying of cells as described in the cancerous

cells. Tumors are developed from the cancerous cells. They destroy the body's

immunity and its normal functioning. (Nall, R., 2018)

Cancer may be mistaken to the term tumor which can cause confusion. If it

points out to the mass, this is a tumor or neoplasm. If it is a penetration or dispersal of

a new growth of cells over tissues that may kill a human life when not cured, this is a

cancer or the alarming type of tumor. A tumor may either be benign or malignant

growths. The non-cancerous tumor which is incapable of propagating to the rest of the

body is known as benign tumor. Despite being harmless, this type of tumor may turn

out to be cancerous if it continues to increase in size and yet neglected. On the other

hand, the cancerous growths which have the power to generate and recur in different

parts of the body are malignant tumors. There is a strong refusal to medications in

spite of being treatable in having malignant neoplasms, which is another term for

cancer. (What are Tumors? n.d.) With the on-going studies on cancer, the fight

against it would be worth saving lives of many cancer patients.

D. Antioxidant

Diseases and any living organisms are always found to be correlational with

one another for diseases are inevitable for any living organism. According to the book

Natural Antioxidants in Human Health and Disease (Keany & Balz, 1994), the effects

of the conception that antioxidants – having an oxidative damaging ability in being a

salient feature in the etiologic factors – possess abilities that may aid in the prevention

or slow the development of certain diseases. With the advancement in the field of

medicinal science, the effectiveness of antioxidants: vitamin C, vitamin E, and

carotenoids (Antioxidants in Human Health and Disease, 1994) as preventions to

9
diseases such as heart diseases, some types of cancer, cataract, injuries and some

neurological disorders (Antioxidants in Human Health and Disease, 1994). According

to Medical News Today, antioxidants – substances – possess the ability in the

prevention and retardation of damaging agents to cells that are caused by free radicals

– unstable molecules which encourage a certain reaction that are done to the body.

(Ware, M., 2018) Halliwell and Gutteridge (1995) defined antioxidants as varying

substances that significantly inhibit the oxidation of an oxidizable substrate when

present at a low concetration – in comparison to the substrate. From that time on,

oxidation was changed to oxidation damage. Most recently, according to Apak et al.

(2016), the natural or synthetic occurrence of substances that prevent damage of

oxidative cells as a result from physiological oxidants with a positive reduction

potential in which cover reactive nitrogen species (RNS) / reactive oxygen species

(ROS) and unstable molecules – free radicals. Generally, from the various definitions

and interpretations from multiple sources, antioxidants became widely known as

agents that aid in the prevention and retardation of certain diseases that do not go as

far in curing a certain disease. More so, antioxidants aid humans for the betterment of

their health.

E. Angiogenesis

Angiogenesis is the formation of blood vessel from the occurring blood

vessels. In this process, oxygen performs a leading role. It emerges when a degree of

oxygen deprivation was established by oxygen sensory mechanisms requiring the

formation of new blood vessels to meet the metabolic needs of parenchymal cells.

However, microvascular rarefraction is formed, if it is over oxygenated. Moreover,

the restructuring of established endothelial cell is only needed which makes it grow

10
rapidly, since it does not depend on the endothelial proliferation (Adair T, H. &

Montani, J, P., 2010). Therefore, according to Folkman (1971), the development of

tumor is dependent on it.

There are two types of angiogenesis, namely the intussusceptive angiogenesis

(Burri, P, H. & Tarek, M, R., 1990) and sprouting angiogenesis (Caduff, J,H., Fischer,

L,C., & Burri, P,H., 1986). Both could already exist starting from the womb.

Intussusceptive angiogenesis involves the creation of blood vessels by a separating

mechanism in which interstitial tissue elements penetrate existing vessels, creating

transvascular tissue pillars that extend. On the other side, sprouting angiogenesis

simply means a new blood vessel made up of endothelial cells is developed from pre-

existing vessels. Blood vessels can thus attach to tissue parts previously devoid of

blood vessels by germinating in angiogenesis. (Adair T, H. & Montani, J, P., 2010)

F. DPPH Radical Scavenging Activity

Testing for the antioxidants property of a substance requires a specific type of

analysis that mainly focuses on the oxidative stress due to the free radicals found in

the body. According to Jessie Szalay (2016), oxidative stress; where free radicals are

the occurrence of unpaired oxygen in the body wherein the unpaired electrons

scavenge the body to search for electrons to pair up with. The result of which causes

damage to different structural organelles, cells, and genetic materials.

Molyneux, P. (2004) stated that DPPH radical scavenging activity is defined

where delocalization of spare electron is characterized as a stable free radical over the

whole molecule in which molecules do not form a union of two – which is the case

with most free radicals. Holtz (2009) defined the results of having the presence of

either a hydrogen donating or electron transfer with regard DPPH, most formally

11
known as 2,2-diphenyl-1-picrylhydrazyl, where it is being discolored - which

originally gives off a color of a deep violet. The testing for antioxidant property of a

substance requires for the DPPH radical scavenging activity; solidified by Villaño, D.,

et al. (2006), that even though there are artificial radicals in situations that are vivo,

the use of DPPH is effective in determining the antioxidant activity in a substance.

More so, Holtz R. W. (2009) stated that after the introduction of a test material, the

difference in the absorbance of the DPPH is used as an index for the material’s

antioxidant capacity. Thus, proves the ability of the test in determining antioxidant

activity among substances.

G. Chorioallantoic Membrane (CAM) Assay

Chick Embryo Chorioallantoic Membrane (CAM) Assay has been used to

distinguish almost all recognized angiogenetic variables by means of facilitating the

test of different samples. It has become a model to study neovascularization since the

early 1970’s when it was adopted by Folkman et al. CAM Assay is relatively easy,

fast, and affordable system that enables a significant number of pharmacological

specimens to be tested in a short period of time and is not necessarily needed to have

bureaucratic requirements to receive ethics committee permission for animal research.

(Ribatti, 2016). This assay has been widely utilized in providing a convenient and

versatile model that allows the study of tumor formation, growth and metastasis,

angiogenic and anti-angiogenic molecules. (The Enzymes, 2019) There have been

reports regarding the range of compounds that stimulate and inhibit angiogenesis by

using the Chick Embryo Chorioallantoic Membrane (CAM). These include growth

factors, hormones, biological chemicals, anti-cancer agents, gases, metallic organic

compounds, pro-angiogenic molecules, antibiotics, antibodies, and small artificial

12
molecules. Usually, the testing agent is being introduced as a small filter disks or

polymerized material such as methylcellulose, alginate, gelatin sponge, or any other

biological inter synthetic polymer. Assessment of angiogenesis description were being

manipulated using all the quantitative, semiquantitative and qualitative methods such

as vascular morphology, density, diameter, blood vessel length, vessel branch points,

and total area of the CAM. In the studies of angiogenesis inhibitors, two different

approaches may be used; one evaluating the inhibition of the basal angiogenesis, and

the other evaluating the inhibition of an angiogenic stimulus initially applied to the

CAM. This system has become the center of interest of every specialist of different

fields in studying the blood vessel and morphological function of in vivo angiogenesis

and evaluating the effectiveness and mechanism of pro-angiogenic and anti-

angiogenic molecules over the last 20 years. (Ribatti et al., 2016)

Nowak-Sliwinska et al. (2014) demonstrated that CAM model is basically a

highly vascularized extraembryonic membrane which, during embryonic

development, performs multiple functions. Without any immune responses, the chick

embryo may receive cancer cells or transplantations from different tissues and species

because of its being naturally immunodeficient. This is also stated by Chambers et al.

(1992) where he enumerated the major advantages of the CAM model as an

experimental animal model of metastasis: (a) the chick embryo is naturally

immunodeficient and can accept cancer cells regardless of their origin without

immune response; (b) the changes in morphology of cancer cells arrested in the CAM

microcirculation can be readily observed by in vivo microscopy; (c) most cancer cells

arrested in the CAM microcirculation survive without significant cell damage, and

complete extravasation. Lokman (2012) also confirmed key benefits of CAM model

such as its high vascularization properties, high reproducibility, simplicity, and cost

13
effectiveness which sums up the model’s main advantages. CAM’s tissue structure

and functionality for experimental manipulation makes it an appealing in-vivo

preclinical model for drug screening and vascular growth, Nowak-Sliwinska et al.

also stated.

Multilayer epithelium is the primary composition of Chick Embryo

Chorioallantoic Membrane (CAM); with ectoderm at the air interface, and mesoderm

(or stroma) and endoderm at the allantoic sac interface (Valdes et al., 2002). Another

composition of CAM includes extracellular matrix proteins (ECM) such as

fibronectin, laminin, collagen type I and integrin ανβ3 (Giannopoulou, 2001). The

physiological cancer cell environment was being simulated by the existence of these

proteins in the extracellular matrix. (Lokman, 2012) By the incubation of the

membrane or coverslip through a hole cut in the egg shell, CAM angiogenesis assay is

being conducted. This membrane or coverslip consists the compound of interest on

the chick embryo chorioallantoic membrane. With the use of this assay, Fett et al.

confirmed the angiogenic activity of angiogenin, a potent simulator of new blood

vessels through the process of angiogenesis. The experiment has been conducted and

the hypothesis has been verified by the implantation of angiogenin into the cornea of a

rabbit eye (Fett et al., 1985). Langer and Folkman (1976) had made use of this

method that showed the evidence of the angiogenic activity of angiogenin using CAM

assay. A window is cut into the eggshell, and the potential angiogenic substance is

deposited on the surface of the exposed membrane. The appearance of blood vessels

in response to the test substance is measured after several days. The implantation of

an angiogenic material into the cornea of a rabbit eye, where the second eye serves as

the control, is an alternative but more sophisticated method. The assay will then

demonstrate the angiogenic potentiality of angiogenin when 35 fmol of angiogenin

14
was added per egg. 3.5 pmol of angiogenin was required to induce extensive blood

vessel growth in the rabbit cornea. (Langer and Folkman, 1976) Knighton et al.

(1977) claimed that the chicken egg’s CAM is a common effective tool to study the

development of blood vessels. A study by Tufan & Satiroglu-Tufan (2005) provided

different information on the variation of the chorioallantoic membrane assay. Tufan &

Satiroglu-Tufan (2005) stated two different types of CAM assay: in ovo – in the shell

– and ex ovo – outside the shell – models. The use of the in ovo CAM model

provides a more suitable environment for the developing chick, however, this method

limits the use and observation of the CAM area (Tufan & Satiroglu-Tufan, 2005).

There are two main methods that are usually used in preparing the CAM; first is a

CAM assay that involves the extraction of 2 – 3mL of albumen from the egg wherein

the extraction of the albumen will result to CAM of the fertilized egg being separated

from the top of the shell – a more successful approach. Another method is known as

the dropped CAM, Tufan & Satiroglu-Tufan (2005) described the process as eggs

with an age from 10 – 14 days of embryonic development wherein the chorioallantoic

membrane of said eggs are then ‘dropped’ through the use of albumin aspiration on

top of the interested area, and air from the naturally occurring air sac. The ex ovo

CAM assay involves the materials inside the egg being placed in a container outside

of that said egg shell. After 4 -7 days a CAM will form on the surface of the egg.

The use of the Chorioallantoic Membrane Assay as a model system has been

widely used for determining angiogenic and anti-angiogenic activities. A study by Li,

M. et al. (2015) utilized an in ovo CAM assay for determining the efficiency as a

xenograft model of Hepatocellular Carcinoma. They utilized 8 day old eggs which are

then placed in an incubator for 2 days with a 36 °C temperature a and 50% humidity.

This study demonstrated the use of a dropped CAM assay model wherein tumors are

15
also added to determine their efficiency as a new model for Hepatocellular Carcinoma

in terms of the xenograft model. The results of the study show the relation between

the CAM assay and the studies with regard to cancer researches; as described by Li,

M. et al (2015) where the chorioallantoic membrane is a natural implant for tumor due

to its naturally immunodeficient and its high level of vascularity. Another study by

Lokman, Elder, Ricciardelli, & Oehler (2012) where they utilized an in vivo model in

order to the effect of newly identified molecules on ovarian cancer invasion and

metastasis. The study incorporated both an in ovo and ex ovo CAM assay wherein the

ex ovo CAM assay had a low survival rate 10%, while the in ovo CAM assay

exhibited a much higher survival rate of 70%. Both were evaluated up until the 14th

day. In conducting this study, the shells were broken off or detached from the CAM

on the 3rd day of the chick embryonic development. Then ovarian cancer cells (9 ×

105 cells) were mixed with matrigel which is then placed on top of the CAM. The

evaluation of this study determined the effectivity of the CAM assay in studying

ovarian cancer cell metastasis.

16
 CHAPTER III: METHODOLOGY

A. Research Design

This study will be performed through the use of an experimental design in

order to test for the antioxidant and anti-angiogenic properties of saluyot leaves. The

variables present in this study are the different concentrations of the crude ethanolic

extract of saluyot leaves, which are the experimental variables in testing the

antioxidant and anti-angiogenic properties of saluyot leaves. In order to obtain the

desired results, CAM assay and DPPH Radical Scavenging activity shall be used in

garnering the desired data. The following assays to be used will be done in triplicate.

The CAM assay will require the use of fifteen (15) duck eggs for testing in which

three duck eggs shall be used as a control – since the experimentation shall be done in

triplicate. The CAM Assay shall be using a negative control: Distilled water (DW),

while the DPPH shall be requiring catechin as the positive control. After obtaining the

required data, analysis of variance (ANOVA) shall be used to analyze the data

mathematically.

17
B. Saluyot extract

1. Collection of materials for saluyot extract

The saluyot plant samples were collected at Bayombong, Nueva

Vizcaya. The collected saluyot samples – with leaves that were not wilted –

were placed in clean plastic bags and was transported to the University of the

Philippines for authentication.

For the preparation of the crude ethanolic extract of the saluyot leaves,

dried saluyot leaves were transported to the laboratory of Quirino State

University.

The 95% ethanol was garnered from the Quirino State University

Laboratory.

2. Preparation of saluyot extract

Saluyot leaves will be air-dried and powdered using a laboratory blender.

One kilogram (1 kg) leaves were soaked in 500mL of 95% ethanol for 24 hours.

The crude ethanolic extracts of the saluyot leaves were placed on a water bath at

40 – 45 oC until they will become syrupy and all solvents have evaporated. The

crude ethanolic extract of the saluyot leaves were then weighed for the four (4)

different concentrations containing: 25%, 50%, 75% and 100%. With the use of

a graduated cylinder and a digital scale, the crude ethanolic extract of the

saluyot leaves were prepared using percent by mass/volume. The concentrations

were then stored inside a house refrigerator until ready for use in the anti-

angiogenic assay and antioxidant assay.

18
C. Collection of materials for CAM Assay

Fifteen (15) one day old duck eggs were bought at local duck store in Pateros.

Then a dissecting needle was needed for the puncturing of a hole in the duck eggs.

For the incubation part of the CAM Assay, a manual incubator with the

capacity to hold thirty-five (35) eggs shall be bought and delivered to Santolan, Pasig

City.

D. Anti-angiogenic Assay

Chick embryo will be used as model organisms for the anti-angiogenic assay.

An in ovo chorioallantoic membrane assay model shall be used in this study. Fifteen

(15), one day old duck eggs were incubated for nine days in a manual incubator. After

the ninth day, the egg shells were then perforated with a 1.5-2 cm hole with the use of

a dissecting needle to carefully remove it so as not to destroy the egg membrane. In

perforating the eggs, a light source was used in order to find the hollow part of each

egg, then a pencil was used to line out the part where the embryo is not in contact

with the egg shell. Prior to this, twelve (12) 6-mm Whatmann paper discs were soaked

for twenty-four hours in the different concentrations of the saluyot crude ethanolic

extract while three (3) disk were soaked in distilled water. After the punctured shells

have been removed, one (1) disc were placed on top of the membrane of each egg

after which the eggs were sealed with parafilm. The eggs were placed back in the

incubator. After twenty-four (24) hours, the effect of the extract were evaluated by

counting the veins or the vascularization found in each chick embryo. In the

evaluation of the assay, a light source and Image J software were used in counting the

veins of the chick embryo.

19
E. Antioxidant Assay

To determine the antioxidant potential of the crude ethanolic extract of the

saluyot leaves, the radical scavenging activity will be determined. It will be subjected

to DPPH radical scavenging activity assay as described in Eskandarighadikolaii,

Bungihan and dela Cruz (2015). In this assay, the crude ethanolic extract of the

saluyot leaves will be dissolved in ethanol to a final concentration of 500 ppm. A

0.1mM DPPH solution in ethanol was also freshly prepared by diluting 1 mL DPPH

stock solution (3.49 mg DPPH in 1 mL ethanol) to 100 mL ethanol. One mL of each

of the crude extract and 4 mL of the DPPH solution were mixed and incubated in the

dark at 37 ˚C for 30 minutes. The reaction will be done in triplicate for each crude

culture extracts. The absorbance reading will be monitored at 515 nm using UV-Vis

spectrophotometer (Spectronic-200, Thermofisher). Finally, the ability to scavenge

the DPPH radical will be calculated using the equation below:

DPPH scavenging effect = [(A control –A sample)/A control] x 100

Where Acontrol is the absorbance of the control (DPPH solution without the

crude culture extract), and A sample is the absorbance of the test sample containing the

mixture of DPPH and the crude culture extract. Catechin will be used as positive

control.

The DPPH radical scavenging activity of the crude ethanolic extract of the

saluyot leaves shall be conducted at the Quirino State University.

F. Data Collection

The data for the anti-angiogenic property of the saluyot leaves were observed

and collected through ImageJ software with regard to the CAM assay. In terms of the

20
data in relation to the antioxidant property, they were gathered through the use of

DPPH radical scavenging activity with the use of an equation indicated above.

G. Framework of Methodology

Collection of materials
for the Saluyot Extracts

Preparation of the
Saluyot Extracts

21
 Figure 2. Framework of Methodology

H. Statistical Analysis

SPSS software will be used in statistical analyses on the different experiments

done. Values will be statistically evaluated using analysis of variance (ANOVA).

CHAPTER 4: RESULTS AND DISCUSSIONS

A. Antioxidant Activity of C. olitorius Extract

Testing for the antioxidant property was done in triplicate through the use of

DPPH radical scavenging activity. Table 1 shows the absorbance values of the

positive control, catechin and that of C. oiltorius. From the mean absorbance values,

the %RSA was calculated by interpolating the values from the absorbance of the

DPPH standard used. Results showed that the %RSA of C. olitorius (49.25%) is lower

than that of the standard control catechin (87.36%).

Table 1. DPPH Radical Scavenging Activity of C. olitorius and Catechin (+control)

Absorbance Mean SD Radical scavenging activity (%)

Sample
1 2 3
Catechin
0.144 0.147 0.145 0.145 0.00158 87.36
(+control)
C. olitorius
0.591 0.582 0.578 0.584 0.00667 49.25
(extract)

22
 Although the radical scavenging activity of the plant material is lower than the

positive control, it is still noteworthy that the 49.25%RSA is comparable to some

other plant materials studied like Ricinus communis butanol extract with 61.49% and

in chloroform extract with 22.37% (Iqbal et al., 2012). The different parts of eggplant

also exhibited RSA with values ranging from 0.5 to 26%RSA (Jung et al., 2011).

Also, in the study of Mohammed (2016) in Sudan, the RSA of C. olitorius ethyl

acetate extract was 23% and methanol extract was 17%. In this study, the C. olitorius

leaves used were still crude compared to that of the catechin which is already a pure

compound. It can be deduced that C. olitorius in the Philippines has a high antioxidant

capacity and can be used as a cell protecting agent even when eaten as a whole.

B. Anti-angiogenic Activity of C. olitorius Extract

Angiogenesis is the formation of new blood vessels from the occurring blood

vessels. In this process, oxygen performs a leading role. It emerges when a degree of

oxygen deprivation was established by oxygen sensory mechanisms requiring the

formation of new blood vessels to meet the metabolic needs of parenchymal cells

(Adair and Montani, 2010). According to Folkman (1971), the development of tumor

is dependent on it.

In this study, the anti-angiogenic property of C. olitorius was established

concurrent to its antioxidant or cell protecting potential which is important for

studying the antitumor activity of the plant. The in ovo chorioallantoic membrane

(CAM) assay was done to assess the anti-angiogenic activities of the different

treatments.

Table 2. ImageJ Counts of Vascularization of Duck Embryos in CAM Assay and

Percent Inhibition of Angiogenesis

23
 Treatment Vascular Count Percent Inhibition (PI)
DW
1 172
2 168
3 162
Mean Count: 167.3
100%
1 143 14.52
2 162 3.17
3 164 1.97
75%
1 130 22.30
2 130 22.30
3 146 12.73
50%
1 157 6.16
2 162 3.17
3 125 25.28
25%
1 151 9.74
2 157 6.16
3 138 17.51

Table 2 shows the ImageJ counts of the vascularizations of the 10-day old

duck embryos. Results show that compared to the negative control, the treated groups

have lesser vascularization counts. Calculation of the percent inhibition of

angiogenesis was done by taking the difference of each vascularization for every trial

to the mean count of the control (DW) which is 167.3 using the formula:

Percent Inhibition = (Vcontrol-Vtreated)/Vcontrol x 100,

where Vcontrol is the vascularization count of DW and Vtreated is the vascularization count
of the treated group.

It can be gleaned from the table above that the treated groups have lesser

vascularization compared to the untreated group. The percent inhibition of

angiogenesis was varied from treatment to treatment. Table 3 shows the mean and

standard deviation of the PI values of the different treatments.

24
 Table 3. Mean and Standard Deviation of PI of Treatment Groups

Treatment Mean PI Standard Deviation

T1(100%) 6.58 5.65
T2 (75%) 19.13 4.51
T3(50%) 11.54 9.79
T4(25%) 11.12 4.74

It can be shown from the table that the highest PI was that of Treatment 2

(75%) which has angiogenic inhibition of 19.13% and the lowest was Treatment 1

(100%) with only 6.58% inhibition. The trend in the percent inhibition was linear,

however, the 100% concentration of crude extract can be seen to be lower. This might

be attributed to some factors like “masking” of the effectiveness of the bioactive

component of the plant. Meaning, at lower concentration, the effect of the

components that mask the biological activities is also lower. It is not recommended to

take high concentration of the plant because its potency is better at lower

concentration. However, due to the high standard deviations of the measurements in

each treatment, there is a need to check for the statistical significance of the

differences in the means.

Statistical analysis of the anti-angiogenic properties of the different treatments

was done using one-way analysis of variance (ANOVA). Table 4 shows the ANOVA

table of results.

Table 4. Analysis of Variance of the Percent Inhibition Results

Sum of Mean
Squares df Square F Sig.
Between Groups 243.450 3 81.150 1.268 .349
Within Groups 512.104 8 64.013
Total 755.554 11

25
 From the results of the ANOVA, it can be seen that there is no significant

difference between the treatment groups at (α=0.05). The Sig. 0.349 is higher than

0.05, so the means are comparable to each other. Statistically, all concentrations have

the same anti-angiogenic potency. Since in the ANOVA, there is no significant

difference shown, the Post-Hoc analysis is no longer needed. This is because all the

treatment groups will belong to only one sub-group. Overall, there is an anti-

angiogenic potential of the C. olitorius extract, which means that it has the property of

being anti-tumorigenic. According to Salas and Totaan (2015), the decrease in

vascularity also means a decrease in metastasis and formation of tumor cells. This is

because the action of the vascular endothelial growth factor (VEGF) is suppressed

(Kim, 2003).

Other studies show that some other indigenous Philippine plants showed

decreased vascularity in CAM assay compared to the negative control, distilled water

(VC=189.93). These plants are, Sabungai (VC=43±29.86), Tsaang-gubat

(201.17±15.05) and Pandan (VC=28.53±25.31) (Salas and Totaan, 2015). They

attributed these anti-angiogenic properties of the plants to the presence of compounds

like flavonoids.

26
 CHAPTER V: SUMMARY OF FINDINGS, CONCLUSIONS, AND
RECOMMENDATIONS

The study was conducted at the laboratory of University of the East – Manila.

The primary purpose of this study was to find the antioxidant and anti-angiogenic

properties of C. olitorius. The assays used in this study were DPPH radical

scavenging activity for the antioxidant property and duck embryo chorioallantoic

membrane assay for the anti-angiogenic property.

A. Summary of Findings

Results showed that C. olitorius has an antioxidant capacity with 49.25%

radical scavenging activity. Also, it has an anti-angiogenic capacity as shown

by the decrease in vascularization in the different treatments. Statistics showed

27
that there is no significant difference in the anti-angiogenic potential of the

different treatments.

B. Conclusion

Based on the foregoing results of the study, it can be concluded that

crude ethanolic extract of the C. olitorius leaves possesses antioxidant and

anti-angiogenic properties. Thus, it can be a good source of natural cell –

protective and anti-tumor agents.

C. Recommendations

For the future development of studies in relation to the antioxidant and

anti-angiogenic property of saluyot, the researchers would like to recommend

the analysis of other parts of the said plant. Also in the angiogenesis of the

saluyot plant, utilization of the ex ovo CAM assay would be a good addition in

comparing the differences of vascularity among the methods. Furthermore,

studies on the saluyot plants should also intend to seek other beneficial

properties that the said plant possess.

Based on the study conducted, in terms of the various equipment to be

used, the researchers would like to recommend to use an actual laboratory

incubator for incubating the duck eggs. Also, in the utilization of the ImageJ

software, a camera with 5 glass lenses over the regular lenses should be used

for capturing the images with a much higher quality in order to achieve

accurate results in the usage of the ImageJ software. Lastly, the researchers

highly recommends to conduct the entirety of the experimentation in a

laboratory with the available, adequate, and accessible materials necessary to

28
 achieve a constant control of the temperature, humidity, wind resistance and

other variables that may cause an intervention to the experimentation,

resulting to inconsistencies.

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Properties of Fungal Endophytes Associated with the Three Medicinal Plants

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Garcia, R. (2012). The Healthiest Fruit- Vegetable Drink in the Philippines. Retrieved

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corchorus-capsularis/

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sciences/antioxidant

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Holtz, R. W. (2009). Skin aging handbook. Retrieved from

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vE0ZQJolpeKh0HH40ROkZ0sejE

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2018. Retrieved from https://www.who.int/cancer/PRGlobocanFinal.pdf

Jung, E.J., Bae, M.S., Jo, E.K., Jo, Y.H., & Lee, S.C. (2011). Antioxidant activity of

different parts of eggplant. Journal of Medicinal Plants, 4610-4615.

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Kunz P, Schenker A, Sähr H, Lehner B, Fellenberg J (2019). Optimization of the

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experiments: JoVE, 52411(104). doi:10.3791/52411

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of_Corchorus_olitorius_L

Ribatti, D. (2016). The Chick Embryo Chorioallantoic Membrane (CAM). A

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Angiogenesis in Vivo. The International Journal of Biological Markers, 14(4),

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34
 tW5f6KN2jBsGqlmG-S4A#v=onepage&q=dpph%20scavenging

%20activity&f=false

CHAPTER VII: APPENDICES

Appendix 1: Plant Authentication

35
Figure 3: Plant authentication

Figure 4: Receipt of plant authentication

Appendix 2. Plant Extract

Appendix 3. Antioxidant Assay

36
Appendix 4. Anti-angiogenic Assay

Figure 5. Excuse letter

37