Chapter I

Introduction
Background and Rationale of the Study
Angiogenesis is a biological process that results in the formation of new blood vessels
which is controlled by chemical signals in the body such as vascular endothelial growth factor
(VEGF) and other endothelial growth factors. If these signals bind to their receptors on
endothelial cells, it strengthens the survival and initiates the growth of blood vessels.
Angiogenesis’ effect is dual in nature. It has both its positive and negative effects from aiding
in wound healing accelerating tumor growth (Ribatti, ​et al., 2​ 000; National Cancer Institute).
Chemotherapy and radiotherapy, two of the most conventional cancer therapies has
been administered through the years. While successful in suppressing cancer regression,
chemotherapy and radiotherapy are incapable of cancerous and normal cells differentiation
and often imposes a greater threat with multidrug resistance (MDR) a vital concern (Abdalla
et al., ​2018).
There are chemical signals that hinder the formation of blood vessels called
angiogenesis inhibitors or anti-angiogenic agents especially for angiogenesis of solid tumors.
Unlike chemotherapy and radiotherapy, these work by targeting angiogenesis signals like
VEGF antibody to block new vessel formation so that tumors can no longer receive nutrition
for growth (Yang, ​et al., ​2017).
The discovery of anti-angiogenesis resulted to the idea of providing anti-angiogenesis
therapies in treating cancer patients. Anticancer researchers continue to research natural
products as anti-angiogenic agents such as immunomodulatory drugs, agents that stimulate or
suppress the immune system that contains anti-angiogenic properties. One example of an
immunomodulatory drug is flavonoids which are commonly found in fruits and vegetables
(Quach, ​et al., ​2014; Gacche, ​et al., ​2011).
In the experimental study of Gacche ​et al., (​ 2011), a series of flavonoids obtained
from Sigma-Aldrich Co. (St. Louis MO, USA) were evaluated to determine its potential of
becoming an anti-angiogenic agent using the chorioallantoic membrane (CAM) model. The
chick embryo chorioallantoic membrane (CAM) allows the study of metastasis, angiogenic
and anti-angiogenic agents at a low-cost. The CAM model is quick and simple and does not
require the animal experimentation permit from the ethics committee because it may receive
transplantations due to its natural immunodeficiency (Ribatti, 2016). The result of Gacche ​et
al. (​ 2011) study shows that genistein, kaempferol and quercetin are effective inhibitors of
angiogenesis.
Sampa-sampalukan​, ​also known as ― ​Phyllanthus niruri,​ can be found anywhere as it
is a common roadside and garden weed throughout the Philippines. The whole plant is used as
medicine since it contains several medicinal properties (e.g. anti-bacterial, analgesic, anti-
hepatotoxic, etc.). Rusmana ​et al. (2​ 017), studied Sampa-Sampalukan (​Phyllantus niruri) a​ s a
source of flavonoids rutin and quercetin. Rusmana ​et al. ​(2017) also found out that rutin is a
weak flavonoid while quercetin, on the other hand, is an effective anti-angiogenic agent.
Objectives of the Study
The results and impacts of immunomodulatory flavonoid of Sampa-sampalukan
(​Phyllanthus niruri) f​ or anti-angiogenesis lead the production of possible reasons to follow up
after the project has been completed. Therefore, this study sought to attain the following
objectives:
1. To analyze the effects of isolated flavonoid using different concentrations.
2. To determine the exact dose in which the isolated flavonoid (quercetin) will be
most effective as an antiangiogenic substance.
Hypotheses of the Study
I. Null Hypotheses:
The isolated flavonoid from Sampa-sampalukan (​Phyllantus niruri) ​has no significant
anti-angiogenic property.
II. Alternative Hypotheses:
The isolated flavonoid from Sampa-sampalukan (​Phyllantus niruri) ​has significant
anti-angiogenic property.
Conceptual Framework
Significance of the Study
The goal of this study is to work out an anti-angiogenic substance that can prevent the
formation of blood vessels in the tumor. Thus, this study will be helpful for the following
people: doctors, Department of Health, students, teachers and future researchers. This study
will first highly benefit the doctors because it can be a breakthrough that they can study and
observe and can be a possible medication for patients. In addition, the study will be beneficial
to this sector of the government such as Department of Health since their focus is on the
health of the people. It can also help on their researches in discovering medications. The
students are also involved to be benefited in the study because the study can be a new source
of learning and it can be something that can really help them. This study will also be helpful
for the teachers, for it can be something that they can teach their students. Thus, the study will
help the future researchers to have more information regarding this topic, and they can have
ideas concerning the said problem.
Scope and Delimitations
The prime sample which is the ​Phyllantus niruri ​were gathered in Nueve Ecija only.
The study is limited only on determining if ​Phyllantus niruri ​possesses an anti-angiogenic
activity. The study did not also determine antioxidant, anti-inflammatory, and antimicrobial
activity of ​Phyllantus niruri.​ Phytochemical Screening conducted in the bulk there are
already several studies conducted that include such screening such as ​Phytochemical
Screening of ​Phyllanthus niruri collected from Kerala Region and its Antioxidant and
Antimicrobial Potentials (2017); Phytochemical analysis of Phyllanthus niruri L.
(Phyllanthaceae) extracts collected in four geographical areas in the Democratic Republic of
the Congo (2013); and Phytochemical Screening of ​Phyllanthus niruri collected from Kerala
Region and its Antioxidant and Antimicrobial Potentials (2017). The experiments were made
only in the Adamson University Technology Research and Development Foundation
Incorporated and University of the East--College of Science and Arts Laboratory.
Definition of Terms
To further understand the content of this research paper, the important terms used in
the study are defined.
Anti-angiogenesis​ is the inhibition of the formation of new blood vessels..
Flavonoids​ are class of plant cell that has been used in developing anti-angiogenic substance.
Immunomodulator ​refers as agents that stimulate or suppress the immune system.
Sampa-Sampalukan (​Phyllanthus niruri​) is a plant that has been used by the researchers in
developing an anti-angiogenic substance.
Chapter II
Review of Related Literature and Studies

Sampa-Sampalukan (​Phyllantus niruri​)

 Sampa-Sampalukan (​Phyllantus niruri​) is commonly used as herbal medicine, and
therapeutic effects are proven by different studies (Filho, 2018). Sampa-Sampalukan
(​Phyllantus niruri)​ has been proven a remedy to diabetes, hypertension, jaundice,
hypercalcuiria, and urolithiasis. Any proof of toxics and side effects are not seen in the
previous studies (Bagalkotkal ​et al., 2006). In traditional medicine, it has been tested that
Sampa-Sampalukan (​Phyllantus niruri​) escalates the immune system (Nisar ​et al.,​ 2018).
Sampa-Sampalukan (​Phyllantus niruri​) is usually used as a remedy to ailments such
as disturbances of kidney and urinary bladder, intestinal infections, diabetes, hepatitis B virus,
pain disorders, dyspepsia, vaginitis, tumors, diarrhea, epilepsy, malaria, hypertension, fever,
inflammatory and dolorous processes. The therapeutic effect of this plant is proven by
different studies (Filho, 2018). Proofs of toxicity and side effects are not seen in the said plant
(Bagalkotkal ​et al.,​ 2006).
In traditional medicine, Sampa-Sampalukan (​Phyllantus niruri​) has been tested that it
escalates the immune system (Nisar ​et al., 2018) In the study of Putri ​et al. (2018), it is said
that the aqueous extract of Sampa-Sampalukan (​Phyllantus niruri​) have immunomodulatory
activity on persons with Tuberculosis (Tb). The activity done by Sampa-Sampalukan
(​Phyllantus niruri​) is dose dependent. Sharma ​et al. (2009), stated that Sampa-Sampalukan
(​Phyllantus niruri​) has anti-tumor activity which supports the traditional medicine practiced
in the past.
Phytochemicals on Sampa-Sampalukan (​Phyllantus niruri​)
The active phytochemicals – flavonoids, terpenes, coumarins, lignans, tannins,
saponins, and alkaloids were determined from various parts of Sampa-Sampalukan
(​Phyllantus niruri) through phytochemical screening. Rutin, quercetin, quercitrin, astragalin,
and catechin are the types of flavonoids present in Sampa-Sampalukan (​Phyllantus niruri)
(Bagalkotkar ​et al., 2006). It is said that rutin and quercetin exhibit anticancer and antioxidant
properties, quercitrin possess anti-inflammatory properties, astragalin has an immunological
regulatory property and catechin inhibits the growth of human colon and hepatic epithelial
cancer cells (Gao ​et al., 2003; Saija ​et al., ​2003; Muzitano ​et al., 2006; Gan, 1998; Uesato ​et
al., 2001, as cited in Bagalkotka ​et al., 2006). Ramandeep ​et al. (2017) evaluated phenol,
flavonoid, terpene, and saponin as antioxidant and antibacterial substance using
phosphormolybdenum method for determining the total antioxidant activity and agar well
diffusion assay for the assessment of ​Lactobacillus acidophilus​, ​Pseudomonas aeruginosa​,
Staphylococcus and Escherichia coli.​
Chick Chorioallantoic Membrane (CAM) Assay
When tumor reaches 1 to 2 mm or more diameter of size, there is a possibility that
tumors will be induced and supplied and could cause harm to individuals (Steiner, 1992).
Facing the situation, a report of Denekamp of 1982 suggested using anti-cancer agent such as
drugs would be a great idea to slow down the process of tumors induced. Chick
Chorioallantoic Membrane (CAM) Assay has been renowned for its significant contribution
in preventing angiogenesis and invading tumor cells. The study of Lokman ​et al. (2012)
further discussed that the presence of extracellular matrix proteins such as laminin, collagen
type I, fibronectin and more can be possibly found on CAM assay which was proven as
multilayer epithelium, an ectoderm at the air interface, mesoderm and endoderm at the
interface of the sac.
The number of studies to assess inhibition of anti-angiogenesis using CAM is limited.
As tested, Chick Chorioallontoic Membrane (CAM) assay rapidly grows as soon as it reaches
its appearance on the third or fourth day of incubation. In 1974, when Tanaka and his
colleagues successfully utilized CAM assay to capture tumor- induced angiogenesis. After the
experiment, tons of samples were proven that it could be screened by using CAM assay and
different researchers across the globe adopted the process (Tanaka ​et al., 1986). As Folkman
and Cotran (n.d. as cited by Tanaka ​et al., 1986) said that the density of newly induced
vessels by substances was assigned a grade of 0 to 5. Tanaka ​et al. (1986) also narrates that
positive angiogenesis was tested and proven histologically. Jakob and Voss (n.d. as cited by
Tanaka ​et al., 1986) used automatic image analyzer to calculate neovascular responses on
CAM while Harris-Hooker ​et al. (n.d. as cited by Tanaka ​et al., 1​ 986) took photographs on
the sample of CAM assay and counted the number of arranged vessels.
In addition, Richardson and Singh (2003) stated that Chick Chorioallantoic Membrane
(CAM) Assay is very cheap and very simple yet it is effective, and have high consistency.
The study of Marks ​et al. (2002) entitled Effects of Putative Hydroxylated
Thalidomide Metabolites on Blood Vessel Density in the Chorioallantoic Membrane (CAM)
Assay and on Tumor and Endothelial Cell Proliferation showed the presence of incubated
thalidomide in a human’s liver microorganisms resulting for an increase of anti-angiogenic
activity of the drug they used. Furthermore, Marks and his team jumped into a conclusion that
active anti-angiogenic substance exists. In addition, the idea of the study was built because of
the definition of Sheu and Brooks about CAM Assay. Sheu and Brooks (n.d. as cited by
Marks ​et al.,​ 2002) called CAM assay as a revised method.
Angiogenesis
Tumor growth requires a constant blood supply to support prior formation of blood
vessels called vasculogenesis and express new capillary blood vessel growth and maturation,
neovascularization or angiogenesis. Triggered by angiogenic factors such as different forms
of the Vascular Endothelial Growth Factors (VEGFs) and angiopoietin gene groups, tumor
angiogenesis buttresses progression of cancer and metastasis, especially when cancer cells
reach the separation stage from the primary site of the tumor (Saaristo ​et al., 2000).
Angiogenesis’ role in this status quo provides a vital focal point in the management of cell
proliferation and metastasis of cancer (Bielenberg & Zetter, 2015). With this considered,
studies to look for angiogenesis inhibitors has been a wide and extensive research (Folkman,
2000).
Anti-angiogenesis
Suppression of angiogenesis may be achieved through different ways. Some
angiogenesis inhibitors function by insinuating blockage of the VEGF and the VEGF
receptors therefore incapacitating the VEGF receptor from initiating angiogenesis.
Monoclonal antibodies are one type that particularly target VEGF and bind itself to the
growth factor. (Bagri ​et al., 2010; Chauhan ​et al., 2​ 012; Jain, 2005). One example is the
tissue inhibitor of metalloproteinases (TIMP3) that binds to the VEGF and thus prevents
binding to VEGF receptor (Qi ​et al., 2003). Another is the ability of tea catechins of in vitro
inhibition of angiogenesis by blocking VEGF receptor binding process (Kondo ​et al., 2002).
Some anti-angiogenic drugs also operate with immunomodulation. Such drugs suppress or
boost the immune system while manifesting angiogenesis inhibition. Sutinib and sorafenib are
examples of this having antiangiogenic properties, commonly administered orally for renal
cell carcinoma patients which blocks a number of tyrosine kinase species, a type of VEGF
receptor. Both drugs exhibit immunomodulatory effects on some investigation in mice and
men (Sheng ​et al., 2016; Heine ​et al., 2011; Álvarez-Aznar, Muhl, & Gaengel, 2017). In the
new milieu, about 17 existing anti-angiogenic drugs are present in the field with 14 of them
approved and is prescribed for cancer therapy while the latter in the preclinical stage.
Approved angiogenesis inhibitors include Axitinib (Inlyta), Bevacizumab (Avastin),
Cabozantinib (Cometriq), Everolimus (Afinitor), Lenalidomide (Revlimid), Lenvatinib
mesylate (Lenvima), Pazopanib (Votrient), Ramucirumab (Cyramza), Regorafenib (Stivarga),
Sorafenib (Nexavar), Sunitinib (Sutent), Thalidomide (Synovir, Thalomid), Vandetanib
(Caprelsa), Ziv-aflibercept (Zaltrap) (Abdalla ​et al., 2018). Previous studies showed that
anti-angiogenic therapy’s efficacy is enhanced if combined with other therapies. A recent
study depicted better use of the combination therapy of anti-angiogenetic drug and
chemotherapy for the management of tumor-induced angiogenesis and normalization of
tumors (Yonucu ​et al., 2017). A separate study by Tailor ​et al. ​(2010) also supports this that
probed the effect combined liposome and anti-angiogenic therapy using the inhibitor
pazopanib on tumor bearing animals.
Flavonoids
Flavonoids were defined and described as a set of plant metabolite which is known to
contain health benefits by its cell transmitting pathways and antioxidant effects. These were
also said to be found in a lot of fruits and vegetables, or in general, in plants. They were also
said to be prominent in plants, they are one of the substances in the plant that plays a big role.
Flavonoids are the one in charge of generating colors required to attract pollinating insects.
And in higher order plants, they are in charge of UV filtration, nitrogen fixation, cell cycle
inhibition, and as chemical messengers. Flavonoids are as well sometimes a plant protector
against plant diseases. It was all stated in the article of Robertson (2018)
It was also seen in the article of Robertson (2018), that due to the affluence of
flavonoids joint with its irrelevant toxicity level, it was said that humans and animals could
devour immense amount of it, considering its benefits. The article discussed that flavonoids
are in fact essential antioxidant and produce extensive health effects such as of it being
anti-viral, anticancer, anti- inflammatory, and anti-allergic. There is also this subgroup of
flavonoid named quercetin that was said to help ease eczema, sinusitis, asthma, and hay fever.
Aside from those said, flavonoids according to other studies contain more benefits
especially for humans.

Benefits of Flavonoids
In the study of Kozlowska and Szostak-Wegierek (2014), it was said that flavonoids
are really abundant in health benefits, especially when it is taken regularly and religiously. It
was said that if it is being ingested routinely, it will then be linked to reduced risk of number
of chronic diseases such as cancer, cardiovascular disease, and neurodegenerative disorders.
Flavanones, flavones, flavonols, flavanols, anthocyanins and isoflavones are the subgroups in
which flavonoids are segmented and classified to. Burgeoning group of scientific evidence
states that flavonoids do a favorable move in preventing variety of diseases, nevertheless, it
still needs further clinical and epidemiological trials to be proven beneficial for humans. As
stated in the first paragraph, flavonoid sources can be found in fruits, vegetables, and in
addition, nuts, seeds, and spices. This study also proven that consumption of this substance
along with your diet is actually harm-free. This study suggests that a diet abundant in
flavonoids has favorable effects and its advocacy is valid.
However, in the study of Williams ​et al. (2004), it was claimed that there were indeed
a lot of studies showing the neuroprotective, cardioprotective, and chemopreventive acts of
dietary flavonoids. In those studies, the focus is only at the flavonoids being antioxidant or
the antioxidant properties of flavonoids. However, as he studied, he had witnessed that
flavonoids and their metabolites have potentials that do not act as antioxidant but rather may
exert modulatory actions in cells by actions at protein and lipid kinase signaling pathways. It
was also stated that in recent studies, flavonoids seen acted at some kinase signaling pathways
they should not acted at because Inhibitory or stimulatory actions at those pathways are
probably to damage cellular function extremely. But as he studied it furtherly, the results
showed that flavonoids are truthfully beneficial. But flavonoids interactions with intercellular
signaling pathways could have unforeseeable results, it will depend on the cell type, the
disease studied, and the stimulus applied. Nevertheless, it was proven that flavonoids are
evident and potent bioactive molecules that could help in chronic diseases.
The study of Procházková, Boušová, and Wilhelmová (2011) is somehow similar with
the study of Williams, ​et al. (2004). This study focused to determine and review both the
antioxidant and pro-oxidant effects of flavonoids. This was caused by the rapidly growing
research about the health benefits of flavonoids relating to their antioxidant activities but
there were only few studies regarding their pro-oxidant properties. It was said that because of
the pro-oxidant properties of flavonoids, they were able to create an oxidative damage by
reacting with different biomolecules such as lipids, proteins, and DNA. By the end of the
study, Procházková and his team (2011) were able to analyze the anti and pro-oxidant
activities of flavonoids. It was said that the properties mostly depend on their concentration.
They were also able to state that the perception on the pro- oxidant effect of flavonoids as
negative or toxic is misleading since in fact, pro-oxidant activities could also be beneficial
since, by imposing a mild degree of oxidative stress, the levels of antioxidant defenses and
biotransformation enzymes might be raised, leading to overall cytoprotection.
And according to their conclusion, Procházková ​et al. (2011) have concluded that
flavonoids cannot be only considered as antioxidant because it also imposes the
characteristics of pro-oxidant activities.
While in the study of Cushnie and Lamb (2005), the flavonoids anti-microbial activity
was highlighted. Flavonoids, for years, have been an active element to use in treating human
diseases. It was also said in their study that, progressively, flavonoids are becoming the center
of medical research since there were countless report saying flavonoids possess numerous
beneficial properties such as anti-inflammatory activity, oestrogenic activity, enzyme
inhibition, anti-allergic activity, antioxidant activity, vascular activity, cytotoxic antitumor
activity, and antimicrobial activity. It was not just these years wherein flavonoids have been
used as antimicrobial treatment, in fact it was used for centuries (Cushnie & Lamb, 2005). In
their study that in Argentine folk, a certain plant named ​Tagetes minuta containing quercetin
has been used to cure infectious disease. Flavonoids were also used as early as Ancient Greek
for the treatment of sores an ulcer (Cushnie & Lamb, 2005). It was also studied in their
research the different uses of flavonoid, mainly as antimicrobial and it was concluded that
even in existence of antimicrobial drugs and medicines, flavonoids still represent a peculiar
set of leads. The study suggested that existing flavonoid structures could be altered to produce
another generation of antimicrobial treatments other than those that were made already.
Quercetin
In the article, ​Overviews of Biological Importance of Quercetin: A Bioactive
Flavonoid ​of David et al. (2016), a kind of flavonoid named, quercetin has been the main
spotlight. Quercetin according to his study is recognized for its anti-inflammatory,
antihypertensive, vasodilator effects, anti-obesity, anti-hypercholesterolemic, and
anti-atherosclerotic activities. Quercetin in his study was described as a plant pigment, a
dominant antioxidant flavonoid, certainly, a flavonol which is located mostly in wines,
berries, citrus fruits, and a lot more plants. It was said that it is an all-around antioxidant
known to contain protective characteristics against tissue damages generated by different drug
toxicities. It was concluded that quercetin is indeed a potent antioxidant element. It has
numerous benefits that people could get, especially for their health. In this study, there were
two subjects, who David ​et al., (2016) asked to take a high dosage flavonoid, and after several
tests, they were able to conclude that the flavonoid, quercetin is a great medicine to reduce the
risk of cardiovascular disease. Then, they confirmed that flavonols are the most dominant
flavonoids in fruits and vegetables, while quercetin is the most usually taken in the human
diet.
Cancer
Siegel, Miller and Jemal (2017) stated that cancer is a major public health problem
and is the second leading cause of death in the United States. In the recent research about
cancer, there was an estimated 1,735,350 new cases of cancer in the United States alone and
about 609,640 people will die from the disease. (National Cancer Institute, 2018). Cancer is
also one of the four epidemic non-curable diseases (NCDs) or lifestyle related diseases
(LCDs). This include cardiovascular diseases, chronic respiratory diseases and ​diabetes
melliticus. Dans (2014, as cited in Department of Health, 2018) mentioned that NCDs are
considered a “silent disaster” that is affecting the Filipinos. Oxales (2018, as cited in
manilastandard.net, 2019) said that 110,000 new cases of cancer are recorded every year and
about 66,000 Filipinos die from the disease. He also mentioned that since the year 2004,
cancer has been the third leading cause of death in the country. Hassanpour and Dehghani
(2017) mentioned that in the broader sense, cancer refers to more than 227 different types of
disease. Scientists indicated that in cancer pathogenesis, several gene mutations are involved
after they have identified different stages of cancer. DNA exists in the individual genes of
every cell. It has instructions that instructs cells what function to perform, how to grow and
how to divide. Mutations occur frequently but most often, cells are able to correct these
mistakes. However, a cell can become cancerous once a mistake is not corrected.
Healthline (2016) defined mutations as the one responsible for the survival of cells
that are the product of mistakes and the cells that should be replaced. It also causes new cells
to form even when the formation of new ones is not needed. These extra cells can divide
uncontrollably causing growths also called tumors to form. Not all tumors are cancerous and
are divided into benign and malignant forms. Benign tumors resemble normal tissue and have
a limited capacity when it comes to growing freely. They exhibit little tendency to invade
locally. Meanwhile, malignant tumors or sarcomas are aggressive and have the capability of
destructive growth, the chances of occurring again and distant metastasis. They also
mentioned that radical surgery is needed to ensure the removal of malignant tumors.
(Goldblum, Folpe & Weiss, 2013).

Chapter III
Methodology
Research Design
 This study pursues an experimental research design that attempts to establish the cause
and effect relationship between the dependent variable – anti-angiogenic activity and the
independent variable – dosage of flavonoid. In this kind of design, the independent variable
must be manipulated, while the dependent variable will be measured and the other variable
will be controlled. There are three steps in this design: (1) Manipulation, manipulate the
independent variable then (2) Measurement, measure the effects or the results of the
manipulation, followed by (3) Comparison, compare the results of each tests done. The
remaining variables will be controlled and could not be changed.
Materials
×​ 15 Pateros duck (​Anas platyrhynchus)​ eggs
​

×​ Isolated Flavonoid (Quercetin) of Sampa-sampalukan (​Phyllantus niruri)​

​

×​ Incubator
​

×​ Filter paper
​

×​ Test compounds with volume to volume concentrations of 25%, 50%, 75% and 100%.
​

Methods

Collection and Authentication of ​P. niruri ​samples

Samples of ​P. niruri were collected from a specific rural area in Nueva Ecija and was
subjected to washing process with tap water and settled in shade for air drying. The plant
sample were taxonomically identified by Dr. Edwino S. Fernando, Curator, Jose Vera Santos
Memorial Herbarium (PUH), Institute of Biology, University of the Philippines.

Extraction and Isolation of Plant Sample

Air-dried samples were shredded until a coarsely powdered form was achieved. Pure
ethanol (98.7%) was used for 24-hour maceration in preparation for the extraction. Soaked
samples were rotary-evaporator extracted under 40◦C in Adamson University Technology
Research Development Center (AUTRDC). The plant extract in solid form was stored in a
amber glass for future use.
 Column chromatography was then utilized, conducted in AUTRDC as well. The
column was prepared for the separation of compounds found in the extract specifically the
flavonoids (quercetin). The stationary phase for the column was silica gel together with a
mobile-phase-gradient of water and ethanol. After passing the plant extract diluted in
deionized water through the column, four fractions were then retrieved from the column and
labeled as W1, W2, W3 and W4 respectively.

Thin Layer Chromatography

To verify what fraction from the column contains flavonoids, Thin Layer Chromatography
(TLC) was executed. Using a solvent system of ethanol:hexane 5-30% (used to move very
polar compounds) for
the TLC, the fractions
W1, W2 and W3 were
spotted in the TLC
plate until it became
palpable enough for
observation. The
flavonoid Quercetin
was used as the
standard in the
process. After doing
the TLC in triplicates,
the plates were then examined under a TLC viewer in AUTRDC. Rf values were then
computed upon measuring the distance travelled by both the solvent and the solutes used.
Table 1 shows that the fraction W3 contains flavonoids as its rf value (0.33) is almost the
same as the standard (0.32). This can be attributed to the study of Bagalkotkar ​et al. ​(2006)
where they subjected the ​P. niruri ​to phytochemical screening and found that flavonoids
specifically flavonols (quercetin) have a huge portion in the plant extract. David ​et al. (​ 2016)
also had a similar conclusions of flavonols being the most dominant flavonoid in plants.

Preparation of Test Compounds

The fraction W3 in liquid form was diluted in distilled water. Volume to volume
concentrations of 25%, 50%, 75% and 100% was done with the W3 fraction with flavonoids.
Small filter papers with dimensions 1X1 cm were immersed in each concentration that will be
induced.

CAM Assay

The researchers performed the CAM assay of ​Phyllantus niruri to determine the
anti-angiogenic activity of Flavonoid (quercetin) as described in the study of Indap et al.
(2006). Fertile Pateros duck (​Anas platyrhynchus​) eggs purchased from Mandaluyong were
incubated without any test compounds within the incubator set in 37 ± .05°C for five days.

Application of the test compound through filter paper with dosages 25%, 50%, 75% and
100% was done directly on the CAM after a 1x1 cm window was created into the shell upon
five days of incubation. Covering process of the window was accomplished through the use
of surgical tape. To prevent the rupture of the blood vessels and causing injury to the embryo,
extraction of 3-4 ml of albumen was initially performed. Incubation in succession of the
induced eggs for three days has then situated. The inhibition of angiogenesis in the CAM was
observed after the application of the test compounds. After, all of the eggs’ surgical tape
cover was retracted and photo capturing was carried out around the induced test compound on
the CAM.

Quantification of Blood Vessels

The researchers utilized a MATLAB-based software in quantifying angiogenesis called

Angioquant v1.33. The software presents the mean number of tubules and mean number of
joints of each egg.

Experiment was executed in a triplicate manner in order to assure the precision of the result.
Each experiment consisted of five fertile eggs: one egg for each dosage and each trial
(triplicates) and one for the untreated control group. To know the effectiveness of flavonoid at
a certain dosage, the anti-angiogenic activity of each test subject will be determined in terms
of percentage through the equation as described in the study of Gacche ​et al.​ (2011):

angiogenic activity (%) = 1 - CT ×100

where,
T = no. of blood vessels within the area of the filter containing flavonoid
C = no. of blood vessels of the control group

Statistical Analysis
The data gathered are also treated with one-way ANOVA data analysis to determine the
statistical significance between the experimental and control group (Lokman ​et al., 2012)
using Microsoft Excel (2016). P values<0.05 were considered significant.
 Chapter IV
Results and Discussion
This chapter covers the presentation, analysis and discussion of the results gathered
from experiments done by the researchers with accordance to the objectives of the study.
Presentation, Interpretation and Analysis of Data
Third and fourth day of incubation mark the rapid vasculogenesis of duck embryo that can
also be attributed in the study of Tanaka​ et al. (​ 1986). Visible blood vessels formed after five
days of incubation upon the creation of a window.
Digitized Illustration of Chorioallantoic Membranes in Day 8 of Incubation

Figure 4.1 |Flavonoid extract in different concentration: (a) 25%, (b) 50%, (c) 75%, (d)
100%, treated in CAM for 3 days upon grafting and determined its effectiveness by
comparing to the (e) untreated CAM as control group.
Figure 4.1 shows the digitized illustration of the CAM after eight days of incubation captured
around the filter paper area.

Presentation of Data

Number of Blood Vessels

Concentration Trial 1 Trial 2 Trial 3 Mean

25% 67 65 63 65.00

50% 48 48 50 48.67

75% 46 38 38 40.67

100% 21 15 10 15.33

CONTROL 69 113 71 84.33

Figure 4.2 ​|Table of values showing the number of blood vessels in day 8 of incubation in
three trials. In 25% concentration, – blood vessels were formed. In 50% concentration, –
blood vessels were formed. In 75% concentration, – blood vessels were formed. In 100%
concentration, – blood vessels were formed. The number of blood vessels were calculated by
getting the mean values of the three trials retrieved from Angioquant.

Anti-angiogenic Activity (%) of Quercetin

Figure 4.3 ​|The anti-angiogenic activity of quercetin in different dosage were calculated
using the given formula:
T
Anti-angiogenic activity (%) = 1 - C
x 100
In 25% concentration, the anti-angiogenic activity is 22.92%, 42.29% in 50% concentration,
51.77% in 75% concentration, and 81.82% in 100% concentration. This concludes that the
most effective dosage for anti-angiogenesis is at 100% concentration of quercetin wherein it
has an anti-angiogenic activity of 81.82%. Figure 4.3 also underlines the anti-angiogenic
activity of isolated Flavonoid from ​P. niruri i​ n a dose-dependent manner. This buttresses the
statement of Sharman ​et al. (​ 2009) in their study that the plant extract of ​P. niruri ​exhibits an
antitumor activity in a dose dependent manner. It is proven further using statistical analysis in
Figure 4.4.
Statistical Analysis
ANOVA: Single Factor

Source of SS df MS F P-value F crit

Variation

Between Groups 8073.733 4 2018.433 14.96614 0.000317 3.47805

Within Groups 1348.667 10 134.8667

Total 9422.4 14

Figure 4.4 ​|The researchers used one-way ANOVA to analyze the results of the experiments.

The number of blood vessels formed in each concentration and the control group were
processed through one-way ANOVA and yielded a p-value of 0.000317. Therefore, reject the
null hypothesis, there is a significant difference between the experimental group and control
group, and thus, proves that the isolated Flavonoid from the sample shows significant
anti-angiogenic activity as compared to the control group.
This further emphasizes the results of study of Gacche ​et al. (​ 2011) where they concluded that
genistein, kaempferol and quercetin are effective inhibitors of neovascularization using CAM
assay. Rusmana ​et al.’s (​ 2017) results can also be attributed to this study as they found out
that quercetin is an effective anti-angiogenic agent.
 Chapter V
Summary, Conclusions and Recommendations
This chapter presents the summary of the research study. It is composed of the
significant findings, derived conclusions, and proposed recommendations.
Summary
Blood vessel formation commonly known as angiogenesis is essential in wound healing
however, uncontrolled production of blood vessels can also cause the metastasis of tumors.
Chemotherapy and radiotherapy are the current methods for stopping the proliferation of
cancerous cells. Although it is effective, it could also kill normal cells. Recent studies found
out that there are certain chemical signals that could inhibit the proliferation of blood vessels
called anti-angiogenesis. This led to the idea on researching natural products that possess
anti-angiogenic substances like immunomodulatory drugs, one that stimulate or suppress
immune system such as flavonoids which are commonly found in fruits and vegetables.
Anti-cancer researchers such as Gacche and Rusmana ​et al. ​studied the anti-angiogenic
response of flavonols using CAM assay which had positive results. Thus, the researchers of
the University of the East, Manila studied the anti-angiogenic activity of isolated flavonoid of
Sampa-sampalukan (​Phyllanthus niruri) u​ sing ​in ovo CAM assay. Samples were gathered
from the local farm and poultry. The leaves of Sampa-sampalukan were extracted through
rotary evaporation and went through column chromatography to isolate the flavonoid. The
isolated phytochemicals labeled as W1, W2, W3, and W4 undergo Thin Layer
Chromatography (TLC) to determine the flavonoid and divided it into 25%, 50%, 75%, and
100% volume-to-volume concentration. The eggs were pre-incubated for 5 days and grafted
the filter paper soaked in flavonoid concentrations and studied its results after 3 days of
incubation. Based on the results, the researchers concluded that the most effective flavonoid
concentration to inhibit angiogenesis is 100% which has 81.82% anti-angiogenic activity.
With a p-value of 0.000317, there is a significant difference between the experimental group
and control group, therefore, it rejects the null hypothesis.
Conclusions
Sampa-sampalukan (​Phyllanthus niruri), a​ common roadside and garden weed, is a good
source of flavonoids such as rutin and quercetin. The isolated flavonoid from the samples
gathered has significant anti-angiogenic property as seen in the result of the Chorioallontoic
Membrane Assay.
Recommendations

1. The researchers highly suggest the use of dose beads in CAM Assay because it
reduces the chances of injuring the chick embryo.
2. For wider and more specific data, future researchers should provide a positive and a
negative control in dealing with their control group.
3. Oncology Institute of the Philippines should make greater use of the advance
technology that we now have to make and provide better treatment for the growing
number of cancer patients in the country.
4. Other institutional leaders must actively advocate and support programs that will
benefit cancer patients.
5. Government officials, in collaboration with local barangay officers, should
incorporate fostering a safe and considerable environment for cancer patients.

Chapter VI
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 Chapter VII
Appendices
Gantt Chart

Month

Description November December January February March

Days

Research 20, 21, 22,

Orientation 23

Chapters 1 and 2 26, 27, 28,

29
Writing of Gantt 26
Chart and Budget

Chapter 3 5, 6, 7

Statistics 13
Seminar

Resumption of 7
Classes

Third Quarter 16, 17, 18

Examination

Experimentation 7-18
and Gathering
Data

Writing Chapters 19-26

4 to 6

Creating Poster 21

for Scientific
Conferences
Seminar

Final 6, 7, 8
Examination

Final Defense 11, 12, 13,

Proposal 14, 15, 18, 19

Finalization of 11, 12, 13,

Manuscript 14, 15, 18, 19
 Balagdiwa
Research TBA
Conference

Competition
(Oral and TBA
Defense)

Budgeting
Contribution: 100 pesos per person per week

Month Collection Total collection

November, 2018 (3 weeks) P 1800.00 P 1800.00

December, 2018 (3 weeks) P 1800.00 P 3600.00

January, 2019 (4 weeks) P 2400.00 P 6000.00

February, 2019 (4weeks) P 2400.00 P 8400.00

March, 2019 (2 weeks) P 1200.00 P 9600.00

Date Materials Quantity Prices Total

November 2018 Printing 44 P5.00 per P220.00

Piece

December 2018 Printing 115 P2.00 per P230.00

Piece

January 2019 Printing 103 P2.00 per P206.00

Piece

Transportation 1 - P180.00

February 2019 Laboratory Fee - - P2579.00

 Plant Identification 1 P150 per P150.00
Species

Transportation - - P50.00

March 2019 Eggs 30 P12 per Piece P360.00

Transportation - - P120.00

Chicken wire - - P70.00

Masking Tape 1 P 20.00 per P20.00

Roll

Vials 4 P 10.00 per P40.00

piece

Thin Layer - - P1000

Chromatography

Column - - P2000
Chromatography

Laboratory Fee - - P1000

Total P8225.00

Documentation
A. Laboratory Transactions
B. Laboratory Test Results
C. Group Planning and Distribution of Labors

D. Thin-layer Chromatography at Adamson University Technology Research and

Development Center (Documentation)
E. Column Chromatography (Documentation)

F. Rotary Evaporation (Documentation)

G. Incubation of Eggs (Documentation)

H. Collection of Sampa-sampalukan (​Phyllantus niruri) (​ Documentation)

I. Egg Candling (Documentation)