Pink Slip Final Body

West Visayas State University
COLLEGE OF ARTS AND SCIENCES

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Chapter 1
Introduction to the Study
Chapter One consists of five parts: (1) Background and
Conceptual Framework of the Study, (2) Statement of the
Problem and Hypothesis, (3) Significance of the Study, (4)
Definition of Terms; and (5) Scope and Delimitation.
Part One, Background and Conceptual Framework of the
Study, justifies the need for conducting the study, and
provides the rationale for conducting the research.
Part Two, Statement of the Problem and Hypothesis,
states the specific questions which the study seeks to
answer. It also states what the researchers expect to find
out.
Part Three, Significance of Study, discusses the
importance of the study to the community.
Part Four, Definition of Terms, defines both conceptual
and operational definition of some terms mentioned in the
study.
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Part Five, Scope and Delimitation of the Study,
explains what information or subjects is being analyzed and
defines the limits and boundaries of the study.
Background of the Study
Seas and oceans represent a big store for beneficial
algae. It is a real fact that the importance of marine
organisms as a source of new substances is growing. With
marine species comprising approximately half of the total
global biodiversity, the sea offers an enormous resource for
novel compounds and it is classified as the largest
remaining reservoir of natural molecules to be evaluated for
drug activity (Rajasulochana, 2009).
According to Lalopua et al. (2012), seaweeds are one of
the most extensively used functional foods and medicinal
herbs with a long history in Asian countries. They are known
as functional food because of their richness in lipids,
minerals and certain vitamins, and also several bioactive
substances like polysaccharides, proteins, and polyphenols,
with potential medicinal uses against cancer, oxidative
stress, inflammation, allergy, diabetes, thrombosis,

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obesity, lipidemia, hypertensive, and other degenerative
diseases.
Hayashi et al. (2012) describes Kappaphycus alvarezii
synonymously known as red algae as one of the millions of
the diverse plants in the ocean. It is an important source
of a variety of commercial applications as gelling,
thickening, and stabilizing agents, especially food products
such as frozen desserts, chocolate milk, instant products,
yogurt, jellies, and in sauce preparation (Ranganayaki,
Susmitha and Vijayaraghavan, 2014).
Furthermore, it has been reported through the study of
Ferraces-Casais et al. (2011) and Senthil et al. (2011) that
it is a possible rich source of bioactive compounds such as
the presence of alkaloids, saponin, phenols, steroids,
protein, phytosterols, amino acids, sugars, reducing sugars,
flavonoids, tannins, high fiber and mineral content, with
significant amounts of protein, vitamins, and trace elements
and a wide range of secondary metabolites not found in other
organisms. Because of this additional uses as a natural
source of functional ingredients, this resource is
particularly attractive in the field of herbal medicine.

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In addition, a recent study conducted by Lau, Vittal
and Chew (2014) established a name for anti-inflammatory,
anti-bacterial, antioxidative diuretic, choleretic and
hemostatic properties and has been approved for food use in
South Eastern Asia in Japan, Korea, and Taiwan.
A phytochemical screening conducted by Rajasulochana,
Dhamotharan and Krishnamoorthy (2009) analyzed Kappaphycus
sp. for its primary phytochemical analysis. Chemical
composition was carried out for estimation of proteins,
fatty acids, B-carotene and sterol compounds. Their study
revealed that the Algae contains substantial amounts of
phytochemicals especially Beta-carotene. From the findings
of Guruvayoorappan & Kuttan (2007), they have concluded that
Beta -carotene exerts its anti-angiogenic effect by altering
the cytokine profile and could inhibit the activation and
nuclear translocation of transcription factors which are
significant in blood vessel formation.
Stimulated studies focusing on new potential uses of
this resource had ignited many researchers to conducts
studies. A brief review of the studies related to K.
alvarezii cultivation in southern and southeastern Brazil,
the latest discoveries in the world concerning

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pharmacological studies with this species and the advantages
of the use of carrageenan as a source of dietary fiber,
cholesterol reducer, and antioxidant, anti-viral and anti-
cancer compounds, as well as the effects in hemagglutination
activity (Bibiana et al., 2013).
In connection, angiogenesis on the other hand plays a
critical role in the growth and spread of the cancer.
Angiogenesis is one of cancer hallmarks that are required
for both cancer progression and metastasis (Sahib, 2007).
Kumaragu (2001) described angiogenesis as the formation of
new blood vessels wherein it is a process in a normal part
of growth and healing. It is also connected to the
development of several diseases, including cancer. Once a
tumor grows to a certain size, it requires nutrients and
oxygen found in the blood to help it grow, invade nearby
tissues, and spread, called metastasis. The tumor sends
chemical signals out that stimulate the growth of new blood
vessels that carry the blood to it. As a result, each part
of the angiogenesis process is a potential target for new
cancer treatments (Holmgren et al.,1995). The idea is that
if a drug can stop the tumor from receiving a blood supply,
the tumor will "starve" and die. Drugs that block

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angiogenesis, which are called angiogenesis inhibitors have
become an important part of treatment for many types of
cancer (Sahib, 2007).
Due to the increasing cost of medicine, especially
drugs used to treat cancer, people and researchers alike are
trying to find alternative means to utilize various flora
and fauna on the environment.
Thus, this study aimed to determine the angiogenesis
inhibitory potential of K. alvarezii on the blood vessel
formation of the duck embryo using chorioallantoic membrane
(CAM) assay in the pursuit of educating community awareness
of the effects of the algae and its potential as an
alternative for antiangiogenic drugs.
Conceptual Framework of the Study
A phytochemical screening conducted by Rajasulochana,
Dhamotharan and Krishnamoorthy (2009) analyzed Kappaphycus
sp. for its primary phytochemical analysis. Their study
revealed that the algae contains substantial amounts of
phytochemicals especially beta-carotene. According to the
study of Guruvayoorappan and Kuttan (2007) beta -carotene
exerts its antiangiogenic effect by altering the cytokine

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profile and could inhibit the activation and nuclear
translocation of transcription factors which are significant
in blood vessel formation.
According to Niebauer et al. (2005), beta-carotene has
received a lot of attention as potential anti-cancer and
anti-aging phytochemical. Beta-carotene is a
powerful antioxidant, protecting the cells of the body from
damage caused by free radicals. Studies indicate that diets
low in beta-carotene can increase the body's susceptibility
to damage from free radicals, resulting in an increased risk
of chronic diseases like heart disease and cancers. Beta-
carotene supplements may help reduce sun induced skin
damage. Beta-carotene is one of the many carotenoids that
our body can convert into vitamin A (Weil, 2012).
Zeigler (1996) adds that beta-carotene acts as an anti-
cancer agent through its antioxidant property but it also
seems to stimulate cell to cell communication. Poor
communication between cells may eventually lead to cancer.
In this study, the duck embryo in in vivo CAM assay was
utilized, focusing primarily on screening the red seaweed
(K. alvarezii) ethanolic extract for its angiogenesis
inhibitory capacity. Algal samples were subjected to

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preliminary screening of its LD50. The LD50 study was
assessed by injecting the eggs with the arbitrary
concentrations using the method of Lorke (1983) and Carvalho
et al. (1978) where in it involves the determination of its
the toxicity range. The presence of avascular zone of
inhibition or lower count of blood vessel formation on the
duck embryo chorioallantoic membrane indicated that the
algal crude extract has an antiangiogenic activity.
Statement of the Problem
This study primarily aimed to determine the effect of
Kappaphycus alvarezii ethanolic extract on the blood vessel
formation using chorioallantoic membrane (CAM) assay on duck
embryo. Specifically, this study sought to answer the
following questions:
1. What is the level of effect of Kappaphycus alvarezii
ethanolic extract on the formation of blood vessel on the
duck embryo using chorioallantoic membrane (CAM) assay?
2. Is there a significant difference on the level of
angiogenic inhibitory activity on the duck embryo using
chorioallantoic membrane (CAM) assay using Kappaphycus
alvarezii ethanolic extract?

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Research Paradigm
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Figure 1 Shows the Research Paradigm of the study. The
independent variable consist of the 2.5% methotrexate
(Positive Control), 0.9 NaCl (Negative Control), and the K.
alvarezii extract. The dependent variable is the Degree of
angiogenic inhibitory activity observed on the CAM of the
duck embryo.
Independent Variables: Dependent Variable:
Positive Control
2.5% Methotrexate at 50µL
Negative Control Degree of angiogenic

0.9 % NaCl at 50µL inhibitory activity
on CAM of duck embryo
screened concentrations of
K. alvarezii extract using
LD50 in
0.2236 Mg/mL at 50µL
Figure 1. Research Paradigm showing the relationship
between the dependent and independent variables of the study.

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Hypothesis
Based on the ongoing problem, the null hypothesis is
formulated:
There is no significant difference on the level of
angiogenic inhibitory activity on the duck embryo using
choroioallantoic membrane (CAM) assay using K. alvarezii
ethanolic extract.
Significance of the Study
The results of this study may give significant
contribution to the following:
Medicine. The study would greatly contribute on the
field by introducing K. alvarezii as a potential source
alternative for angiogenesis inhibitors and anti-cancer
treatment.
Community. People will be aware of the effects of the
plant and its potential as an alternative for anticancer
drugs. Also, its commercial demand on the market would
greatly increase therefore it would increase and sustain the
livelihood of local seaweed farmers.

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Future Researchers. The study would provide a
foundation for future researchers if they would like to
study again the efficacy of this plant in order to further
verify the claims of this study. This would give a
comprehensive knowledge and claims to the effectives of the
plant’s constituents. Furthermore, this will serve as basis
for other researchers to conduct various studies involving
the utilization of K. alvarezii to other potential
breakthroughs.
Definition of Terms
The following terms used in the study were defined
according to conceptual and operational definition for the
purpose of clarity.
Anti-angiogenesis – a process of inhibiting
angiogenesis of formation of new blood vessel from pre-
existing endothelial cell progenitors (National Cancer
Institute, 2015).
In this study, it refers to the angiogenesis inhibitory
activity of Kappaphycus alvarezii ethanolic extract tested.
Chorioallantoic membrane (CAM) assay - model system for
studying development, cancer behavior, properties of

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biomaterials, angiogenesis, and photodynamic therapy
(Ribatti, Nico, Vacca and Presta, 2006)
In this study, it was used as model system for studying
the effect of K. alvarezii ethanolic extract on the blood
vessel formation in the duck embryo.
Duck (Anas luzonica) - is a large dabbling duck of the
genus Anas. It has a black crown, nape and eye stripe, with
a cinnamon head and neck. Rest of body is greyish brown with
a bright green speculum. Its legs are greyish brown, and its
bill is blue grey (BirdLife International, 2012).
In the study, it refers to duck (Anas luzonica) eggs
used in determing the angiogenesis inhibitory activity of
K. alvarezii ethanolic extract.
Kappaphycus alvarezii- is a species of red alga. One
of the most important commercial sources of carrageenans, a
family of gel-forming, viscosifying polysaccharides (Hayashi
et al., 2012).
In this study, K. alvarezii extract was used as the
source of extracts tested for its angiogenesis inhibitory
activity on CAM of duck embryo.

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Scope and Delimitation of the study
This investigation was limited only to the
determination of the angiogenesis inhibitory activity of
Kappaphycus alverizii ethanolic extract on the angiogenesis
of duck embryo using chorioallantoic membrane (CAM) assay.
This study employed the experimental method of
investigation. The variables of the study were divided into
two: the independent variables and dependent variable. The
independent variables are the screened concentrations of the
Kappaphycus alvarezii ethanolic extract applied to the
chorioallantoic membrane of duck eggs (Anas luzonica). The
dependent variable is the degree of angiogenesis on the
chorioallantoic membrane of the duck embryo. The anti-
angiogenic efficiency was assessed by the degree of
angiogenesis after applying the different K. alvarezii
ethanolic extract treatments.
Duck eggs were ordered from Brgy. Trapiche, Oton. The
algae samples were collected at Brgy. Gogo,Estancia, Iloilo.
The study was conducted at West Visayas State University -
Central Science Laboratory from June-July, 2016.

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Chapter 2
Review of Related Literature
Chapter Two presents the related literature and studies
after the thorough and in-depth research done by the

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researchers in order to furnish a background necessary for
the interpretation of the results. It includes three topics:
(1) Kappaphycus alvarezii which tackles its geographic
distribution, uses, and Phytochemical composition.
Furthermore, it also includes related studies about the K.
alvarezii Usage and its medical applications. (2) Beta-
carotene is also discussed including its characteristics and
related Studies about its content and bioactivity. (3)
Angiogenesis is defined and discussed including its related
studies about role of angiogenesis inhibitors and the role
they play in tumor and cancer growth formation.
Kappaphycus alvarezii
Definition. According to Hayashi et al. (2010), it is a
species of red alga. It is one of the most important
commercial sources of carrageenans, a family of gel-forming,
viscosifying polysaccharides. Farming methods affect the
character of the carrageenan that can be extracted from the

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seaweed. This alga grows to two meters long and is green or
yellow in color. It is very fast-growing, known to double
its biomass in 15 days.
Description. Lenget (2000) describes the thallus of
Kappaphycus alvarezii ranges from 24 to 48 cm. The branches
are cartilaginous and pliable, ranging from 8 to 12 cm in
length with unilateral to irregular branching type. Branch
diameter ranges from a few mm at the branch tips to greater
than 1 cm in older tissue. Generally, the branches are
smooth and the thallus can be short with many branches to
much larger with long smooth branches. The diameter of the
different types of vegetative cells are as follows: 2-4 µm
outer cortex, 30-240 µm inner cortex and, 25-40 µm medulla.
Thylles of the medulla are present but rhizoids are absent.
Geographic Distribution. Kappaphycus alvarezii is
found in the upper part of the sublittoral zone, from just
below the low tide line, of reef areas on sandy-corally to
rocky substrates where water flow is slow to moderate
(Hayashi et al., 2010).
Bulboa and De Paula (2005) reports that Kappaphycus has
been recorded from the Philippines, Malaysia, Cuba, China,
Vietnam, Micronesia, Fiji, Hawaii and Tahiti. However, many

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of these locations are not part of the native distribution
but are rather the result of aquaculture introductions.
Uses. According to Lau et al. (1990), they are used in
a variety of commercial applications as gelling, thickening,
and stabilizing agents, especially in food products such as
frozen desserts, chocolate milk, cottage cheese, whipped
cream, instant products, yogurt, jellies, pet foods, and
sauces. Aside from these functions, Lau, Vittal and Chew
(2014) added that carrageenans are used in pharmaceutical
formulations, cosmetics, and industrial applications such as
mining.
Phytochemical Composition. According to Rajasulochana,
Dhamotharan and Krishnamoorthy (2009), Kappaphycus sp. is an
edible seaweed from the sea coast of Rameswaram,India. They
analyzed for its primary phytochemical analysis. Chemical
composition was carried out for estimation of proteins,
fatty acids, β-carotene and sterol compounds. B-carotene was
estimated through high performance liquid chromatography
whereas fatty acids and sterol compounds were determined
using gas chromatography technique. From the standard graph,
the protein was estimated as 0.169 gm/ml indicating that the
protein content is quite high in red algae. Sterols were

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estimated on the basis of chromatographic peak areas for
each with respect to total sterol peak area. The predominant
sterol identified is cholesterol. From the qualitative
analysis β-Carotene, it was observed that one compound is
present in large besides other impurities. Results of their
study suggests for the utilization of Kappaphycus sp. for
various nutritional products for use as health food or
nutraceutical supplement.
Kiruba, Pradeep, and Juliana (2015) analyzed K.
alvarezii for its chemical composition and found that this
species is rich in protein (16.2% w/w), fiber (29.4% w/w)
and carbohydrates (27.4% w/w), with a high proportion of
unsaturated fatty acids (44.5% of the total; 11.0% oleic
acid, 13.5% cisheptadecenoic acid, 2.3% linoleic acid) and
saturated fatty acids (37.0%, composed mainly of
heptadecanoic acid). According to them, this species is also
a good source of minerals, containing 0.16% calcium, 0.033%
iron and 0.016% zinc. The bioavailability of iron, tested
via in vitro assays, exhibited higher efficiency under
intestinal conditions (pH 7.5) than under stomach conditions
(pH 1.35).
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Further studies by Kiruba, Pradeep and Juliana (2015)
tested the antimicrobial activity of Kappaphycus alvarezii
and studied its bioactive compounds. The compounds present
were confirmed using high performance liquid chromatography
(HPLC) and the functional groups were identified using
Fourier Transform-Infrared spectroscopy. Phytochemical
analysis tested positive for the presence of flavonoids,
cardiac glycosides, sterols and quinones. Fourier Transform-
Infrared spectroscopy analysis of the Ethanolic and
Chloroformic extracts of Kappaphycus alvarezii showed
similar peaks corresponding to functional groups such as
amines (-NH2), alcohols (-OH) and carboxyl (-C=O) groups.
The qualitative HPLC fingerprint profile of both extracts
showed prospective peaks at lower Rf values indicating the
major presence of quinones.
Thus, the results showed that the presence of bioactive
quinones, sterols and isoflavonoids, lead to active
inhibition of microbial growth in a dose-dependent manner.
Thus suggesting a wide array of bioactive chemicals found in
the plant (Kiruba, Pradeep & Juliana, 2015).
K. alvarezii Usage and Medical Applications

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Food Application. Hayashi and Reis (2012) described the
preparation of natural polymers with well-defined
properties, such as swelling ability, pH, thermal, and
degradation ability, which could be utilized in specific
food applications, as well as in applications as polymeric
carriers or supports. They prepared a hydrogel that
exhibited stability in the wide range of pH 1-12 by cross-
linking a blend of agar and kappa-carrageenan hydrocolloids
with the naturally occurring cross-linker genipin. Hayashi
and Reis (2012) also added that the ratio of the
hydrocolloids and genipin optimized in the blend as well as
in the cross-linked product. Genipin has been used in herbal
medicine for its anti-inflammatory, diuretic, choleretic and
hemostatic properties and has been approved for food use in
Southeastern Asia in Japan, Korea, and Taiwan.
In a study published by Alwarsamy and Ravichandran
(2011), revealed that 90% methanolic, 70% acetonic and
aqueous extracts from K. alvarezii (strains Crocodile, Giant
and Brown) and K. striatum were used to inhibit the growth
of HeLa cell lines. MTS assay was carried out to determine
the proliferation of HeLa cells in the presence of different
seaweed extracts. Both 500 μg/mL of aqueous and methanolic

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extracts from K. striatum demonstrated highest anti-
proliferative activity against HeLa cells with cell growth
inhibition of 53.5 and 43.7%, respectively. Treatment with
the aqueous extracts from three strains of K. alvarezii did
not show any growth inhibition against HeLa cell lines. The
acetonic extract of Kappaphycus seaweeds exhibited a very
poor cell growth inhibition with inhibitory activity
observed under the treatment of 300 to 500 μg/mL of K.
alvarezii strain Brown only.
Beta-carotene
β-carotene Characteristics. is a strongly colored red-
orange pigment abundant in plants and fruits. It is an
organic compound and chemically is classified as a
hydrocarbon and specifically as a terpenoid (isoprenoid),
reflecting its derivation from isoprene units. Β-carotene is
biosynthesized from geranylgeranyl pyrophosphate. It is a
member of the carotenes, which are tetraterpenes,
synthesized biochemically from eight isoprene units and thus
having 40 carbons (Meschino, 2013).
In addition, Meschino (2013) stressed that among this
general class of carotenes, β-carotene is distinguished by
having beta-rings at both ends of the molecule. Absorption

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of β-carotene is enhanced if eaten with fats, as carotenes
are fat soluble.
Weil (2012) explains that the splitting of beta-
carotene (and other carotenes) into retinol within
intestinal cells is well regulated to help guard against
Vitamin A toxicity. The retinol that is formed from beta-
carotene enters the chylomicron and is metabolized from that
point forward as preformed Vitamin A. Chylomicrons primarily
deliver Beta-Carotene to the liver, where they are
repackaged within another lipoprotein carrier system known
as the very-low-density lipoprotein.
Furthermore, Weil (2012) adds that the beta-carotene
(and other carotenoids) enters the bloodstream from the
liver and is transported to peripheral tissue by very-low
density lipoproteins and low-density lipoproteins (VLDL and
LDL, which is the remnant particle of VLDL after
triglycerides are removed by fat cells, muscle fibers and
other tissues). In contrast, Vitamin A is transported form
the liver attached to retinal-binding protein (RBP).
According to Meschino (2013), beta-carotene is stored
in fat tissues, and the adrenal glands, testes, ovaries,
rather than the liver and is responsible for the yellowish

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tinge to the skin when large amounts are stored
(carotenodermia). However, carotenodermia is considered to
be a nonpathological, reversible condition; not associated
with any health risks.
Bioactivity of Beta-carotene on Angiogenesis and
Cancer. A study published by Guruvayoorappan and Kuttan
(2007) focused evaluating the antiangiogenic effect of beta-
carotene using in vivo and in vitro models. Male C57BL/6
mice as well as B16F-10 cells were used for the experimental
study. The in vivo study includes the inhibitory effect of
beta-carotene on the formation of tumor-directed
capillaries. Rat aortic ring assay, human umbilical vein
endothelial cell proliferation, migration, and tube
formation are used for assessing the in vitro antiangiogenic
effect of beta-carotene. The differential regulation of
proinflammatory cytokines as well as the inhibitory effect
of beta-carotene on the activation and nuclear translocation
of transcription factors are also assessed. Beta-carotene
treatment significantly reduces the number of tumor-directed
capillaries accompanied by altered serum cytokine levels.
Beta-carotene is able to inhibit proliferation, migration,
and tube formation of endothelial cells. Beta-carotene

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treatment downregulates the expression of matrix
metalloproteinase (MMP)-2, MMP-9, prolyl hydroxylase, and
lysyl oxidase gene expression and upregulates the expression
of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-
2.
The study of Guruvayoorappan and Kuttan (2007) revealed
that beta-carotene treatment could alter proinflammatory
cytokine production and could inhibit the activation and
nuclear translocation of p65, p50, c-Rel subunits of nuclear
factor-kappa B, and other transcription factors such as c-
fos, activated transcription factor-2, and cyclic adenosine
monophosphate response element-binding protein in B16F-10
melanoma cells. Observations showed that beta -carotene
exerts its antiangiogenic effect by altering the cytokine
profile and could inhibit the activation and nuclear
translocation of transcription factors.
Another study by Upadhyaya, Radha, and Madhyastha
(2007) reports the efficacy of beta-carotene towards
differentiation and apoptosis of leukemia cells. Dose (20
microM) and time dependence (12 h) tests of beta- carotene
showed a higher magnitude of decrease (significance p <
0.05) in cell numbers and cell viability in HL-60 cells than

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U937 cells but not normal cell like peripheral blood
mononuclear cell (PBMC). Microscopical observation of beta-
carotene treated cells showed a distinct pattern of
morphological abnormalities with inclusion of apoptotic
bodies in both leukemia cell lines. When cells were treated
with 20 microM of beta-carotene, total genomic DNA showed a
fragmentation pattern and this pattern was clear in HL-60
than U937 cells. Both the cell lines, on treatment with
beta- carotene, showed a clear shift in G (1) phase of the
cell cycle.
In addition, the study of Upadhyaya, Radha and
Madhyastha (2007) also revealed anti-oxidant properties of
beta-carotene since there was reduction in relative
fluorescent when treated than the control at lower
concentration. Collectively, the study shows the dual
phenomenon of apoptosis and differentiation of leukemia
cells on treatment with beta-carotene.
Angiogenesis
Definition. Angiogenesis is the growth of blood vessels
from the existing vasculature. It occurs throughout life in
both health and disease (Coultas et al., 2008). Kumaragu
(2001) connotes that no metabolically active tissue in the

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body is more than a few hundred micrometers from a blood
capillary, which is formed by the process of angiogenesis.
Capillaries are needed in all tissues for diffusion exchange
of nutrients and metabolites. Changes in metabolic activity
lead to proportional changes in angiogenesis and, hence,
proportional changes in capillarity. Oxygen plays a pivotal
role in this regulation. Meanwhile, Hemodynamic factors
according to Coultas et al. (2008) are critical for survival
of vascular networks and for structural adaptations of
vessel walls.
As been noted by Kumaragu (2001), decreasing or
inhibiting angiogenesis can be therapeutic in cancer,
ophthalmic conditions, rheumatoid arthritis, and other
diseases.Capillaries grow and regress in healthy tissues
according to functional demands.
Role of Angiogenesis Inhibitors in Tumor and Cancer
growth. According to Steiner (1992), Angiogenesis requires
the binding of signaling molecules, such as vascular
endothelial growth factor (VEGF), to receptors on the
surface of normal endothelial cells. When VEGF and other
endothelial growth factors bind to their receptors on
endothelial cells, signals within these cells are initiated

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that promote the growth and survival of new blood vessels
for tumor metastasis. Angiogenesis inhibitors bind to
receptors on the surface of endothelial cells or to other
proteins in the downstream signaling pathways, blocking
these activities.
Nyberg, Xie, and Kalluri (2005) point out that when a
tumor stimulates the growth of new vessels, it is said to
have undergone an 'angiogenic switch'. The principal
stimulus for this angiogenic switch appears to be oxygen
deprivation, although other stimuli such as inflammation,
oncogenic mutations and mechanical stress may also play a
role. Furthermore, Nyberg, Xie, and Kalluri (2005) add that
his ‘angiogenic switch’ leads to tumor expression of pro-
angiogenic factors and increased tumor vascularization.
Specifically, tumor cells release various pro-angiogenic
paracrine factors (including angiogenin, vascular
endothelial growth factor (VEGF), fibroblast growth factor
(FGF), and transforming growth factor-β (TGF-β) cells to the
extracellular matrix.
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Chapter 3
Research Design and Methodology
Chapter Three is composed of four parts: (1) Purpose of
the Study and Research Design (2) Method (3) Procedures (4)
Data and Analysis.
Part One, Purpose of the Study and Research Design
describes the objectives and the "blue print" of the study.
It provides the framework that has been created to seek
answers to the research questions.

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Part Two, Methods states the list of materials,
chemicals and apparatus used in conducting the study.
Part Three, Procedures presents the major methods for
measuring variables and collecting data to test the
hypotheses.
Part Four, Data Analysis gives vivid information about
the results of the study with the goal of discovering useful
information, suggesting conclusions, and supporting
decision-making.
The Research Design
The aim of this research study was to determine the
level of angiogenic inhibitory activity of Kappaphycus
alvarezii ethanolic extract on the chorioallantoic membrane
(CAM) of duck embryos.
An experimental method in which the researchers
manipulate one or more variables, and controls and measures
any change in other variables, was used to assess the effect

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of the different Kappaphycus alvarezii treatments on the
blood vessel formation of duck embryos.
The duck embryos were subjected to the screening of
angiogenesis to check the level of angiogenesis inhibitory
activity of Kapppahpycus alvarezii ethanolic extract. Lower
mean count of blood vessel formation indicates that the
extract has a potent antiangiogenic activity.
Methods
Materials. A rotary evaporator from West Visayas State
University’s central laboratory was used in separating
excess ethanol from the extracts. An incubator was used for
the incubation of duck eggs. Materials used in the study
were sterilized to ensure proper sanitation of materials
before using it on the duck eggs. The procedures followed an
aseptic technique in order to secure that it is free from

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pathogenic microorganisms that may affect the results of the
research study.
Test Organism. Duck eggs were incubated at the same
time until day ten, which was the day of inducing the
different treatments on the duck eggs. Eggs were divided
into three groups which correspond to the number of
treatments tested.
Procedure
Preliminary Activities
Collection of Algae. Algal samples were collected at
Brgy. Gogo, Estancia sealed tightly in a sterile plastic
bag. Agal sample (K. alvarezii) was identified by Dr. Gerard
Penecilla, a plant systematics professor at West Visayas
State University.
Extraction of Algae. The algal extract was obtained
using a blender. Supernatant was soaked in a 400 ml ethanol
for 72 hours and was filtered using a Whatman no. 1 filter
paper. A rotary evaporator was used to separate the excess
ethanol and was left to be air dried in a fume hood.

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Collection of Duck Eggs. About 72 hours old eggs of
duck (Anas luzonica) with an average weight of 50 g were
purchased in Trapiche Oton, Iloilo. About 9 eggs were used
per dose. All the eggs were disinfected with 70% ethanol
using a single swipe of cotton. Fresh, fertile duck eggs
were used in this test. Before incubation, the eggs were
stored in egg trays with blunt ends upward.
Incubation of eggs. The 72 hours old duck eggs were
incubated for nine days in an incubator at 37-400C
temperature under a relative humidity of 45-70%. Eggs were
rotated twice a day.
Screening of Eggs through Candling. All eggs were
incubated for 9 days. Their blunt ends were then illuminated
with a candling lamp on day 10. Eggs that have not been
fertilized or have not undergone embryogenesis were
rejected. Only eggs on which a distinct fine vascular system
can be recognized on the CAM were used for testing. Eggs
that do not meet this critical criterion were disposed in
the proper manner.
Determination of LD50. The LD50 study was assessed by
injecting the eggs with the arbitrary concentrations using
the method of Lorke (1983) and Carvalho et al. (1978). This

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phase involves the determination of the toxic range. The
eggs were grouped into 3 (n = 3) and the extract (100, 500,
and 1000 Mg/mL) suspended in distilled water were injected.
The treated eggs were observed for the next 24hrs. Then the
number of deaths in each group were recorded. The LD50 was
calculated using the formula: √2aXb where ‘a’ is the lowest
dose that brought death ‘b’ is the highest dose that did not
bring death (Lorke, 1983 and Carvalho et al., 1978). After
24 hours of incubation, the eggs were assessed for any death
of the embryos. Based on the result of determining the LD50,
the final concentration was 0.2236 µg/mL of K. alvarezii.
Experiment Proper
All instruments were sterilized using 70% ethanol
before use. The fertilized duck eggs were cleaned using 70%
ethanol and were incubated in an egg incubator for days. On
10th day, a small window was made into the shell with the aid
of small dissecting scissors the window was opened under
sterile conditions within a laminar-flow hood about 50
microliter of the K. alvarezii solution was injected

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directly at the chorioallantoic membrane of the duck egg. It
was then sealed with transparent tape and was returned to
the incubator. This step was done in an enclosed area such
as a laminar-flow hood to minimize the risk of
contamination. The researchers used sterile gloves to
further minimize the risk of contamination. It was then
opened on the fourth day after the injection of the
different treatments to compare the vasculature formation in
the different treatment groups.
The following were the groups used in the different
treatment on duck embryos:
a. Positive Control. The 2.5% (w/v) concentration of
methotrexate (MTX) solution is based on the results of the
previous studies which have shown the submaximal efficacy of
the drugs, and the observations regarding its efficacy of
the concentration of the drug in the human body for
antiangiogenic studies. Low dose MTX inhibits endothelial
cell proliferation in vitro, and blocks endothelial cell
growth factor-induced neovascularization in the rabbit
cornea assay (Hirata et al., 1989). Cytotoxic agents such as
MTX (Steiner, 1992) markedly inhibited the growth of chick
embryo. It is therefore conventional cytotoxic agents may

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exert a tumor suppressive effect through an antiangiogenic
mechanism (Colleoni et al., 2002).
b. Experimental Group. A concentration of 0.2236 Mg/mL
at a volume of 50μL based on LD50 computation.
c. Negative Control. A concentration of 0.9 % NaCl was
used as a negative control. About 50 μL volume of 0.9 % NaCl
was injected to the chorioallantoic membrane of the eggs.
Isotonic saline solution was used in order not to disrupt
the normal fluid concentration needed by the egg to develop
its vascularization.
Data Collection
Angiogenesis Scoring. Eggs were incubated for 9 days
starting from the 20th of June to the 28th. Eggs were opened
and injected with the different treatments on 29th of June.
The vascular composition of the chorioallantoic membranes
was photographed and evaluated on the 12th day by using the
scoring system of Burgermeister et al. (2002) and Ribatti,
Nico, Vacca, & Presta (2006) as shown in Table 1.

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Table 1
Scoring System of Angiogenic Effect on the Chorioallantoic
Membrane of the Duck Embryo.
Score Evaluation
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Absence of any demonstrable
0 antiangiogenic effect (normal embryo and
no difference in surrounding capillaries)
A very weak (no capillary free area but
0.5 an area with reduced density of
capillaries, which is not larger than the
paper disc area)
A weak moderate (a small capillary-free
1 area or a small area with significantly
decreased density of capillaries
2 Strong antiangiogenic effect (a
capillary-free area around the paper
disc)
Data Analysis
Descriptive Data Analysis. After the scoring, the
equation developed by Krenn and Paper (2009) was used with
modification for the determination of the average mean score
for each drug concentration as indicated in the formula
below:
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The average mean score was then compared to the value
as shown in Table 2 for the level of antiangiogenicity in
the CAM of the duck embryo.
Table 2
Score Interpretation
<0.5 Very Good antiangiogenic effect
0.5-0.75 Good antiangiogenic effect
>0.75-1 Weak antiangiogenic effect
>1 No antiangiogenic effect
Descriptive Scales on the level of Angiogenic Inhibition
Inferential Data Analysis. One-Way Analysis of Variance
(ANOVA) was used to determine significant differences among
the treatments on the angiogenic inhibitory activity in the
CAM of embryos of duck eggs. Least Significant Difference
(LSD) was used for pairwise comparison between treatments.
The level of significance was set at 0.05 alpha level.

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Chapter 4
Results and Discussion
Chapter Four presents the data obtained from the
results of the experiment, the analysis and interpretation,
and the discussion involved.

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Part One, Results of the Study deals with the
presentation, analysis and interpretation of the angiogenic
inhibitory effect of K. alvarezii extract on duck embryo
using chorioallantoic membrane (CAM) assay. It also provides
the test result on the significant difference between the
inhibition of angiogenesis on the duck CAM.
Part Two, Discussion, reviews the significance of the
findings based on the results. Statistical findings are
highlighted and inferred for the significant difference of
the treatments. Justifications on the results are supported
by studies as to give light to the possible mechanism behind
the outcome of the results.
Descriptive Data Analysis
Table 3 shows the level of angiogenic inhibitory
activity among the treatments. Both the experimental groups
(M =0.667; SD=0.250) and positive control (M =0.722;
SD=0.263) were evaluated with good antiangiogenic effect.

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The negative control (M=2.000; SD=0.000) has no
antiangiogenic effect.
Table 3
Level of Angiogenic Inhibitory Activity Among the
Treatments.
Group N Mean Score Std. Description
Deviation
Experimental 9 0.667 0.25 Good antiangiogenic
effect
Positive 9 0.722 0.26 Good antiangiogenic
Control effect
Negative 9 2.000 0.00 No antiangiogenic
Control effect
Inferential Data Analysis
Table 4 shows the inferential data analysis results
using One-way ANOVA on the difference in the angiogenesis
inhibition on the duck embryo among the treatments of K.
alvarezii extract (experimental), Methotrexate (positive

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control) and the negative control. There is a significant
difference in the inhibition of angiogenesis on the duck
embryo among the treatments, F (2,24) = 116.421, p=0.00) <
0.05. Thus, the null hypothesis stating no significant
difference on the angiogenesis inhibitory activity of the
different treatments of K. alvarezii using chorioallantoic
membrane (CAM) assay on duck embryo is rejected.
The eta squared (η² = 0.91) has a large effect size. It
implies that the angiogenic inhibitory activity is due to
the treatments. It also suggests that the treatment used
have potent capabilities of reducing angiogenesis on CAM of
duck embryos.
Table 4
One-Way ANOVA on the level of Angiogenic Inhibitory Activity
Among the Treatments.
Treatment Sum of df Mean F Sig. Eta

Squares Square Square
d
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Between 10.241 2 5.120 116.421 .000 .91

Groups *
Within 1.056 24 .044
Groups
Total 11.296 26
*p<0.05 is significant.
Furthermore, results of the LSD pairwise comparison
test revealed that angiogenesis inhibition on duck embryo
are statistically significant between the negative control
and experimental (p=0.00) and positive control (p=0.00)
treatments, <0.05. However, there is no significant
difference in the inhibition of vascularization on the duck
embryo between the experimental (p=0.579) and positive
control (p=0.579) > 0.05. This means that the experimental
and positive control groups have comparable angiogenic
inhibitory activity on the CAM of duck embryos.
Discussion
The statistical and descriptive data showed that K.
alvarezii has antiangiogenic activity which resulted to the
significant decrease in the vascularization of the CAM of
the duck embryos. Presence of avascular zone of inhibition

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or lower count of blood vessel formation on the duck embryo
chorioallantic membrane indicated that the algal crude
extract has an antiangiogenic activity.
The angiogenesis scoring value by Burgermeister et al.
(2002) and Ribatti, Nico, Vacca & Presta (2006) showed both
the K. alvarezii extract and the positive control
(Methotrexate) have good antiangiogenic effect. Thus, making
both groups to have comparable effects while the negative
control has no antiangiogenic effect.
The efficacy of the K. alvarezii extract is supported by
phytochemical studies of Rajasulochana, Dhamotharan and
Krishnamoorthy (2009) on Kappaphycus sp. revealing that the
algae contain substantial amounts of phytochemicals
especially carotenoids such as beta-carotene. Beta-carotene
is known to exhibit radical scavenging and antiangiogenic
activity.
In a study by Ponesakki, Matsubara, Sugawara and Hirata
(2013), they showed an anti-angiogenic potential of marine
algal carotenoids: beta-carotene, fucoxanthin and
siphonaxanthin. Three carotenoids exhibited molecular
mechanisms of anti-angiogenic activity using human umbilical
vein endothelial cells. They have concluded that all three
carotenoids suppressed the mRNA expression of fibroblast

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growth factor 2 (FGF-2) and its receptor (FGFR-1) as well as
their trans-activation factor, EGR-1. These growth factors
called Vascular endothelial growth factor (VEGF) are
endothelium-specific secreted protein which plays a vital
role in angiogenesis and progression of tumor and metastasis
(Kumaragu, 2001).
In addition, studies of Niebauer et al. (2005),
suggested that beta-carotene is a potential anti-cancer and
anti-aging phytochemical. Beta-carotene is a powerful
antioxidant, protecting the cells of the body from damage
caused by free radicals.
Furthermore, Guruvayoorappan and Kuttan (2007)
stressed that beta - carotene exerts its antiangiogenic
effect by altering the proinflammatory cytokine production
and inhibits the activation and nuclear translocation of
p65, p50, c-Rel subunits of nuclear factor-kappa B, and
other transcription factors such as c-fos, activated
transcription factor-2, and cyclic adenosine monophosphate
response element-binding protein in B16F-10 melanoma cells.
It could be drawn that beta-carotene acts as regulator and
inhibitors in the activation and nuclear translocation of
transcription factors which are significant to angiogenesis.

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Chapter 5
Summary, Findings, Conclusions, Implications, and
Recommendations
Chapter Five subsumes four parts: (1) Summary, (2)
Conclusions, (3) Implications, and (4) Recommendations.

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Part One, Summary, presents the salient points and the
overall findings of the study.
Part Two, Conclusions, states the conclusions drawn
from the findings of the study
Part Three, Implications, gives the connotations that
can be obtained from the study.
Part Four, Recommendations, offers some possible
suggestions for additional practical applications in terms
of findings and conclusions.
Summary:
This study focused on the understanding and
determination of the anti-angiogenic effect of Kappaphycus
alvarezii ethanolic extract on the blood vessel formation
using chorioallantoic membrane (CAM) assay on duck embryo.

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The study sought primarily to answer the following
questions:
1. What is the level of effect of Kappaphycus
alvarezii ethanolic extract on the formation of blood vessel
on the duck embryo using chorioallantoic membrane (CAM)
assay?
2. Is there a significant difference on the level of
angiogenesis inhibitory activity on the duck embryo using
chorioallantoic membrane (CAM) assay using Kappaphycus
alvarezii ethanolic extract?
In this study, the experimental method was employed.
The K. alvarezii ethanolic extract was obtained through
blending the algae until a liquid paste is obtained.
Supernatant was soaked in a 400 ml ethanol for 72 hours and
was filtered using a Whatman no. 1 filter paper. A rotary
evaporator was used to separate the excess ethanol and was
left to be air dried in a fume hood. 50 microliter of the
K. alvarezii solution was injected directly at the
chorioallantoic membrane of the duck egg on its tenth day.
It was left incubated for four days before opening and
comparing the blood vessel development on the duck eggs.

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Findings:
Analysis of data showed the following findings:
1. Kappaphycus alvarezii ethanolic extract showed a
significant reduction on the formation of blood vessel on
the duck embryo. Therefore, Kappaphycus alvarezii has a
potential anti-angiogenic property.
2. There is a significant difference on the angiogenesis
inhibitory activity on the duck chorioallantoic membrane
(CAM) using Kappaphycus alvarezii ethanolic extract.
Conclusions:
Based on the findings, the following conclusions were
drawn:
1. The Kappaphycus alvarezii extract showed a good anti-
angiogenic effect on the CAM of duck embryos which is
comparable to the positive control, methotrexate.
2. There is a statistically significant difference in the
inhibition of angiogenesis on the different treatments at
0.05 level of significance among the treatments.
Implications:
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Based on the drawn conclusions, the following
implications were revealed.
1. The study reveals that inhibition in the blood vessel
formation in the chorioallantoic membrane (CAM) of duck
embryo after it was applied with K. alvarezii ethanolic
extract. It implies that K. alvarezii has a potent
antiangiogenic constituent which may have inhibited the
translocation of vascular endothelial growth factors (VEGF)
which is an essential signaling protein for the
vascularization of blood vessels. Therefore, K. alvarezii
has a promising antiangiogenic constituent and possible
source of chemotherapeutic agent against tumors.
2. Based on the statistical result angiogenesis
inhibition on duck embryo was statistically significant
between the positive and K. alvarezii extract vs. the
negative control. Treatment using K. alvarezii extract have
shown significant reduction in the vascularization on the
duck CAM in ovo and has been observed to significantly
inhibit the development of capillary networks in CAM. The
observation in this study implies that K. alvarezii extract
exhibits a strong antiangiogenic activity and therefore, can
be considered a potential angiogenic inhibitor which might be

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helpful in treating angiogenesis related diseases such as
cancer and the like.
Recommendations:
In view of the foregoing conclusions, following
recommendations are proposed for future studies of similar
nature:
1. Make a further study the biological component of
Kappaphycus alvarezii that will further validify its
potential anti-angiogenic factor.
2. It is advised to further study the anti-angiogenic
effect of Kappaphycus alvarezii to other experimental
animals to further prove its effectivity.
3. It is also advised to use other solvents aside
from ethanol to extract the biological active components in
the algae.
4. It would be also a benefit to further study the
different components of algae in order to further enrich our
knowledge about its uses.
5. Additional study on the antiangiogenic mechanism of
K. alvarezii.
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10.1007/s11010-013-1651-5. Retrieved November 2016,
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Rajasulochana, P., Dhamotharan, R., and Krishnamoorthy, P.
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Ranganayaki, P., Susmitha, S., and Vijayaraghavan, R.
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alvarezii and its in-vitro. International Journal of
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Appendix A
Multiple Comparisons
Dependent Variable: Score
LSD
(I) (J) Mean Std. Sig. 95%
Treatment_ Treatment Differe Error Confidence
Type _ nce (I- Interval
Type J) Upper
Lower Bound
Bound
Experimental Positive -.05556 .0988 .579 -.2596 .1485
(Methotre 6
xate)
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- .0988 .000 - -
Negative 1.33333 6 1.5374 1.1293
*
Experiment .05556 .0988 .579 -.1485 .2596

al 6
Positive
- .0988 .000 - -
Control Negative
1.27778 6 1.4818 1.0737
Control *
Experiment 1.33333 .0988 .000 1.1293 1.5374

Negative al *
6
Control 1.27778 .0988 .000 1.0737 1.4818
Positive *
6
*. The mean difference is significant at the 0.05 level.
Appendix B
Photos of the different vascularization on the CAM Assay of

the duck embryos using different treatments.
Representative Representative Representative Photo:

Photo: CAM Assay Photo: CAM Assay CAM Assay result for
result for the result for the the Negative Control
Experimental Positive Control
Photo taken by the
Photo taken by the Photo taken by researchers
researchers the researchers
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Appendix C
Specimen Identification Certification

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Appendix D
Letter to the Central Laboratory Supervisor

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February 09, 2016
Mr. Audie Suladay

Head, Central Science Laboratory
This University
Dear Mr. Suladay,
We are Third Year BS Biology - Premed students currently conducting our research
entitled “Angiogenesis Inhibitory Activity of Kappaphycusalvarezii(Tambalang) Crude
Extract on Duck Embryo using Chorioallantoic Membrane (CAM) Assay” in partial
fulfilment of the requirements for the course, BIO225B: Thesis Writing for Biological
Sciences.
In lieu of this, we would like to request the following chemicals and laboratory apparatus
during the duration of the conduct of the experiment from the period of April – June
2016.
Attached herewith is the list of request needed for the said activity.
Your favourable response on this matter will be highly appreciated.
Very truly yours,
Angelie Kate Canto

Researcher
John Stephen Clavel III

Researcher
Noted:
Dr. Rey Tantiado

Thesis Adviser
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Dr. Nancy Surmieda

Dean
College of Arts and Sciences
Approved:
Mr. Audie Suladay

Head, Central Science Laboratory
List of Apparatus, Chemicals and Equipment
Apparatus
 Two (2) Mortar and pestle

 One (1) Funnel
 Three (3) 250 ml Erlenmeyer flask
Equipment
 Rotatory Evaporator
 Oven
 Refrigerator
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Appendix E
Letter to the Panel

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November 29, 2016
Dr. Jeannemar Genevieve Yap-Figueras

Faculty
Biological Science Department
Dear Dr. Yap-Figueras,
We would like to invite you as panelist for the panel defense of our undergraduate
thesis entitled, “Angiogenesis Inhibitory Activity of K. alvarezii extract using
Chorioallantoic Membrane (CAM) Assay on Duck Embryo” on December 1, 2016, 3:00
pm – 5:00 pm.
Attached herewith is a manuscript of our study.
Your positive response on this matter is highly appreciated.
Very truly yours,
Angelie Kate Canto

BS Biology Premed 4-A

Noted:
Dr. Rey G. Tantiado
Thesis Adviser
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November 29, 2016
Prof. Prency Yerro

Faculty
Biological Science Department
Dear Prof. Yerro,
We would like to invite you as panelist for the panel defense of our undergraduate
thesis entitled, “Angiogenesis Inhibitory Activity of K. alvarezii extract using
Chorioallantoic Membrane (CAM) Assay on Duck Embryo” on December 1, 2016, 3:00
pm – 5:00 pm.
Attached herewith is a manuscript of our study.
Your positive response on this matter is highly appreciated.
Very truly yours,
Angelie Kate Canto


Noted:
Dr. Rey G. Tantiado
Thesis Adviser
Iloilo City
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RESEARCH CERTIFICATION
This is to certify that ANGELIE KATE CANTO and JOHN
STEPHEN CLAVEL III have submitted to us their manuscript
entitled “Angiogenic Inhibitory Activity of Tambalang
(Kappaphycus alvarezii) Extract Using Chorioallantoic
Membrane (CAM) Assay On Duck Embryo”.
I have examined the same and found it in order.
REY TANTIADO, Ph.D. ____________

Adviser (Name & Signature) Date
JEANNEMAR GENEVIVE YAP – FIGUERAS, Ph.D. ____________

Department Editor (Name & Signature) Date
JEANNEMAR GENEVIVE YAP – FIGUERAS, Ph.D. ____________

Student Research Coordinator (Name & Signature) Date
GERARD L. PENECILLA Sr., Ed.D. ____________

Department Chair (Name & Signature) Date
LEAH MAE CABALFIN, Ph.D. ____________

Dean (Name & Signature) Date
Iloilo City
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Note:
►This form should be accomplished by CAS students who are

enrolled in thesis writing.
►One copy of this certification should be submitted to the

Dean’s office together with the bound copy and a photocopy
of the abstract.

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West Visayas State University

COLLEGE OF ARTS AND SCIENCES

Introduction to the Study

Chapter One consists of five parts: (1) Background and

Conceptual Framework of the Study, (2) Statement of the

Problem and Hypothesis, (3) Significance of the Study, (4)

Definition of Terms; and (5) Scope and Delimitation.

Part One, Background and Conceptual Framework of the

Study, justifies the need for conducting the study, and

provides the rationale for conducting the research.

Part Two, Statement of the Problem and Hypothesis,

states the specific questions which the study seeks to

answer. It also states what the researchers expect to find

Part Three, Significance of Study, discusses the

importance of the study to the community.

Part Four, Definition of Terms, defines both conceptual

and operational definition of some terms mentioned in the

Part Five, Scope and Delimitation of the Study,

explains what information or subjects is being analyzed and

defines the limits and boundaries of the study.

Background of the Study

Seas and oceans represent a big store for beneficial

algae. It is a real fact that the importance of marine

organisms as a source of new substances is growing. With

marine species comprising approximately half of the total

global biodiversity, the sea offers an enormous resource for

novel compounds and it is classified as the largest

remaining reservoir of natural molecules to be evaluated for

drug activity (Rajasulochana, 2009).

According to Lalopua et al. (2012), seaweeds are one of

the most extensively used functional foods and medicinal

herbs with a long history in Asian countries. They are known

as functional food because of their richness in lipids,

minerals and certain vitamins, and also several bioactive

substances like polysaccharides, proteins, and polyphenols,

with potential medicinal uses against cancer, oxidative

stress, inflammation, allergy, diabetes, thrombosis,

obesity, lipidemia, hypertensive, and other degenerative

Hayashi et al. (2012) describes Kappaphycus alvarezii

synonymously known as red algae as one of the millions of

the diverse plants in the ocean. It is an important source

of a variety of commercial applications as gelling,

thickening, and stabilizing agents, especially food products

such as frozen desserts, chocolate milk, instant products,

yogurt, jellies, and in sauce preparation (Ranganayaki,

Susmitha and Vijayaraghavan, 2014).

Furthermore, it has been reported through the study of

Ferraces-Casais et al. (2011) and Senthil et al. (2011) that

it is a possible rich source of bioactive compounds such as

the presence of alkaloids, saponin, phenols, steroids,

protein, phytosterols, amino acids, sugars, reducing sugars,

flavonoids, tannins, high fiber and mineral content, with

significant amounts of protein, vitamins, and trace elements

and a wide range of secondary metabolites not found in other

organisms. Because of this additional uses as a natural

source of functional ingredients, this resource is

particularly attractive in the field of herbal medicine.

In addition, a recent study conducted by Lau, Vittal

and Chew (2014) established a name for anti-inflammatory,

anti-bacterial, antioxidative diuretic, choleretic and

hemostatic properties and has been approved for food use in

South Eastern Asia in Japan, Korea, and Taiwan.

A phytochemical screening conducted by Rajasulochana,

Dhamotharan and Krishnamoorthy (2009) analyzed Kappaphycus