Rice Field Muck in Microbial Fuel Cells With: A Science Investigatory Project of

Rice Field Muck In Microbial Fuel Cells with Gelidium Corneum
(nutrient agar) Salt-Gel Solution Proton Exchange
Membrane for Power Generation
A Science Investigatory Project of:
Nerfa A. Minong
Kimberly Jeph C. Tagle
Chymee Grace C. Gagarra
Researcher
Florenda H. Quinte Jeffrey D.C. Barrera
Practical Research II Adviser Research Capstone Adviser
Regional Science High School for Region IX – Zamboanga Peninsula
Malasiga, San Roque, Zamboanga City

Rice Field Muck In Microbial Fuel Cells with Gelidium Corneum (nutrient agar) Salt- 1
Gel Solution Proton Exchange Membrane for Power Generation
February 208, 2018
Aloe Vera (Aloe barbadensis) Extract as Angiogenesis Inhibitor Using

Duck Chorioallantoic Membrane (CAM) Assay
By: Earl Humprey M. Bantug, Mariam Lujain J. Anwar Bahraq, Jamih Lynn J. Macario
Regional Science High School For Region IX – Zamboanga Peninsula
TABLE OF CONTENTS
INTRODUCTION………………………………………………………………………….. A
MATERIALS AND METHODS ………………………………………………………….. B
RESULTS ………………………………………………………………………………….. C
DISCUSSIONS ……………………………………………………………………………... D
CONCLUSION ………………………………………………………………………….. E
ACKNOWLEDGEMENT ………………………………………………………………… F
APPENDICES ……………………………………………………………………………... G
REFERENCES …………………………………………………………………………….. H
INTRODUCTION
Due to the technology’s impact on medicine, multiple diseases, viruses, and disorders
were discovered. For instance, cancer is one of the leading major causes of death worldwide
(World Health Organization [WHO], 2017). Each year, tens of millions of people are
diagnosed with cancer around the world, and more than half of the patients eventually die
from it. In many countries, cancer ranks the second most common cause of death following
cardiovascular diseases (Yu, 2014). With significant improvement in treatment and
prevention of cardiovascular diseases, cancer has or will soon become the number one killer
in many parts of the world. Unfortunately, there are only a few ways to combat cancer and
one if it is through chemotherapy. Chemo as a treatment for cancer has always been
controversial, with patients as well as medical professionals doubting its efficacy and safety
as a cancer treatment (Schmidt, 2016). Because of the chemicals used in chemotherapy, it
could also possibly kill some of the living cells of the body thus, many herbal, aquatic, and
organic plant extracts are used as an alternative (Cure Your Own Cancer [CYOC], 2011).
One example of this is the ornamental and abundant plant Aloe Vera which is famous for its
first-aid purpose (National Institute of Environmental Health Sciences [NIEHS], 2017).
Because of its significance in alternative medicine, the Aloe Vera plant will be used in the
study and will be tested by its effectiveness to inhibit angiogenesis.
Moreover, the study aimed to utilize varying concentrations of Aloe Vera
extracts as angiogenesis inhibitor using Duck Chorioallantoic Membrane (CAM) Assay. It
specifically sought to answer the following questions: a.) Is there a significant difference in
the mean number of branching points of the blood vessels from the CAM Assays setups
when subjected with the following extracts: a. Setup A: 100 mg/ml Aloe Vera Extract, b.
Setup B: 200 mg/ml Aloe Vera Extract, c. Setup C: 300 mg/ml Aloe Vera Extract and d.
Setup D: Distilled Water and b.) Is there a significant difference in the percentage of the
CAM Vascularity of the blood vessels when subjected with the following extracts: a. Setup
A: 100 mg/ml Aloe Vera Extract, b. Setup B: 200 mg/ml Aloe Vera Extract, c. Setup C: 300
mg/ml Aloe Vera Extract and d. Setup D: Distilled Water.
The hypotheseis of the questions above are as the followingfollows: a.) There is no
significant difference in the mean number of branching points of the blood vessels from the
CAM Assays setups when subjected in the 4 setups and b.) There is no significant difference
in the percentage of the CAM Vascularity of the blood vessels when subjected in the 4
setups.
Furthermore, this study will greatly contribute in the advancement and additional
knowledge about the different herbal, organic, and aquatic plants that could combat cancer.
Up-regulation of the activity of angiogenic factors is in itself not enough to initiate blood
vessel growth, and the functions of negative regulators or inhibitors of vessel growth may
need to be down-regulated. With the idea that the molecules of herbal, organic, and aquatic
plants will prevent or slow the growth of cancer, this study will also serve as a baseline study
for other researchers trying to find ways to block tumor growth linked with angiogenesis
around the Philippines. Moreover, these plants serve as more options for cancer patients to be
spared from using harmful chemicals through chemotherapy if proven effective in further
testing such as using cancer cell lines. They will now be able to utilize these plants since they
don’t contain immunotoxin chemicals that could harm other living and healthy cells within
the body.
Despite this, the study is only limited on to the usage of the Aloe Vera extracts of
varying concentration as the cultivar that is to be used as an angiogenesis inhibitor.
Furthermore, this study will only use the Duck Chorioallantoic Membrane (CAM)
Assay wherein fertilized duck eggs will be used. In this assay, the branching points of the
blood vessels and the CAM vascularity will be the only parameters for testing and
verification for angiogenic inhibition of the extracts.
The fertilized eggs will only be incubated for 2 days after the application of the
extracts at 10 days while the extracts will only be prepared by drying and soaking in ethanol.
The whole duration of the experimentation will only last for one month.
Due to the technology’s impact on medicine, multiple diseases, viruses, and disorders
were discovered. For instance, cancer is one of the leading major causes of death worldwide
(World Health Organization [WHO], 2017). Unfortunately, there are only a few ways to
combat cancer and one if it is through chemotherapy however, because of the chemicals used
in chemotherapy, it could also possibly kill some of the living cells of the body thus, many
herbal, aquatic, and organic plant extracts are used as an alternative (Cure Your Own Cancer
[CYOC], 2011). One example of this is the ornamental and abundant plant Aloe Vera which
is famous for its first-aid purpose (National Institute of Environmental Health Sciences
[NIEHS], 2017). Because of its significance in alternative medicine, the Aloe Vera plant will
be used in the study and will be tested by its effectiveness to inhibit angiogenesis.
The study aimed to utilize varying concentrations of Aloe Vera extracts as
angiogenesis inhibitor using Duck Chorioallantoic Membrane (CAM) Assay. The study
specifically seeked to answer the following questions: a.) Is there a significant difference in
the mean number of branching points of the blood vessels from the CAM Assays setups
when subjected with the following extracts: a. Setup A: 100 mg/ml Aloe Vera Extract, b.
Setup B: 200 mg/ml Aloe Vera Extract, c. Setup C: 300 mg/ml Aloe Vera Extract and d.
Setup D: Distilled Water and b.) Is there a difference in the percentage of the CAM
Vascularity of the blood vessels when subjected with the following extracts: a. Setup A: 100
mg/ml Aloe Vera Extract, b. Setup B: 200 mg/ml Aloe Vera Extract, c. Setup C: 300 mg/ml
Aloe Vera Extract and d. Setup D: Distilled Water.
The hypothesis of the questions above are the following: a.) There is no significant difference
in the mean number of branching points of the blood vessels from the CAM Assays
setupswhen subjected in the 4 setups and b.) There is no difference in the percentage of the
CAM Vascularity of the blood vessels when subjected in the 4 setups.
This study will greatly contribute in the advancement and additional knowledge about
the different herbal, organic, and aquatic plants that could combat cancer. Up-regulation of
the activity of angiogenic factors is in itself not enough to initiate blood vessel growth, and
the functions of negative regulators or inhibitors of vessel growth may need to be down-
regulated. With the idea that the molecules of herbal, organic, and aquatic plants will prevent
or slow the growth of cancer, this study will also serve as a baseline study for other
researchers trying to find ways to block tumor growth linked with angiogenesis around the
Philippines. Moreover, these plants serve as more options for cancer patients to be spared
from using harmful chemicals through chemotherapy if proven effective in further testing
such as using cancer cell lines. They will now be able to utilize these plants since they don’t
contain immunotoxin chemicals that could harm other living and healthy cells within the
body.
The study is only limited on to the usage of the Aloe Vera extracts of varying
concentration as the cultivar that is to be used as an angiogenesis inhibitor. Furthermore, this
study will only use the Duck Chorioallantoic Membrane (CAM) Assay wherein fertilized
duck eggs will be used. In this assay, the branching points of the blood vessels and the CAM
vascularity will be the only parameters for testing and verification for angiogenic inhibition
of the extracts.
The fertilized eggs will only be incubated for 2 days after the application of the extracts at 10
days while the extracts will only be prepared by drying and soaking in ethanol. The whole
duration of the experimentation will only last for one month.
Cancer and Tumor
Cancer is one of the leading causes of death around the world. It is described as the
group of diseases involving abnormal cell growth with the potential to invade or spread to
other parts of the body. Moreover, the spreading of infected cancer cells happen through the
bloodstream, once in the blood, they can go to any part of the body. Many of these cells die,
but some may settle in a new area, start to grow, and form new tumors. This spread of cancer
to a new part of the body is called metastasis. Therefore, one major platform of the spreading
of cancer around the body is the rapid multiplication of blood vessels (American Cancer
Society [ACS], 2015). This physiological process through which new blood vessels forms
from pre-existing vessels is termed as Angiogenesis. Even though cancer cells are abnormal,
they still require oxygen and nutrients. The development of blood vessels is an essential step
in the growth of a tumor. Without vessels tumors cannot grow to be larger than a small
fraction of an inch (Emory Winship Cancer Institute [EWCI], 2016). With this in mind,
scientists and doctors in this generation incorporate the process of angiogenesis and apply
several different or varying chemicals, substances, and extracts, which may help increase or
decrease its inhibition.
Cancer and its TreatmentsTreatments for Cancer
Chemotherapy is the only standard remedy for cancer treatment. Most of the
anticancer drugs currently used in chemotherapy are cytotoxic to normal cells and cause
immunotoxicity; this affects not only the tumor development, but also aggravates patient’s
recovery (Das, Pradhan, Sadique, & Nayak, n.d). Hence, so many researches were interested
to finding new drugs from terrestrial plants, marine organisms/microorganisms, and herbal
plants. Now a day, drug discovery has been developed greatly in finding a pure organic
compounds or crude extracts to provide new lead in preventing the spread of cancer. One of
the common extracts used in treating cancer is vincristine and vinblastine which are
substances extracted from the plant Periwinkle (The Living Rainforest [TLR], 2010).
Furthermore, another known plant, specifically a marine macroalga is the red algae or termed
as tambalang within the local market. This specie of marine macroalga is discovered to
contain the polysaccharide Fucoidan which is known to have different anticancer properties
(Duraikannu et al., 2014). Fucoidans may suppress tumor growth by inhibiting tumor-
induced angiogenesis (Koyanagi, Tanigawa, Nakagawa, Soeda, & Shimeno, 2003). And
lastly, tThe plant aloe vera is also researched to be an effective inhibitor of cancer because
they exert their chemo-preventive effect through modulating antioxidant and detoxification
enzyme activity levels, as they are one of the indicators of tumorigenesis (Liu, Chen, & Shi,
2013).
Angiogenesis
According to Folkman (2002) in his study titled Role of angiogenesis in tumor
growth and metastasis, it has been further established that angiogenesis is extremely needed
for the formation and growth of tumor. It also constitutes to the metastasis that in turn
contributes to the progression of the cancer itself. Thus, the inhibition of angiogenesis can
play a vital role in the stopping the spreading of cancer throughout the body. Although it can
be used as an alternative cancer therapy for invasive approach, there are still problems
especially in the application of the cultivar in the assays for testing and the nature of the
cultivars themselves especially their costing, parameters, availability and accessibility as
explained in the study of Auerbach, Akhtar, Lewis and Shinners (2005) titled Angiogenesis
Assays: Problems and Pitfalls.
As the time passes by, the demand for anti-cancer treatments are rising exponentially
thus more and more noninvasive therapies are greatly needed. Chemotherapy is already an
option but it was also stated in the study of Weeks et. al titled Patients' Expectations about
Effects of Chemotherapy for Advanced Cancer that what is perceived to be beneficial can
have at most harmful effects for the whole duration of the application. It is already given that
chemotherapy does not only target the cancer cells but also the healthy cells thus causing
certain side effects such as the formation of new type of cancer cells from the preexisting
one.
With this, as quoted from the study of Tran et. al titled Angiogenesis Inhibitors and the Need
for Anti-angiogenic Therapeutics, depriving a tumor of its vascular supply by means of anti-
angiogenic agents has been of great interest since its proposal in the 1970s.
Angiogenesis and Tumor Growth
Angiogenesis is a progressive, multistep physiological process by which new blood
vessels are generated from pre-existing vasculature. Adult vasculature is maintained mostly
in an angiostatic state that must be switched off to allow for new blood vessel formation.
This angiogenic switch is a part of normal physiologic responses, for example to tissue
injury, as well as a critical step in the pathology of tumor progression. It is commonly
accepted that specific mechanisms underlining the angiogenic switch involve a selective
remodeling of the extracellular matrix (ECM) by proteolytic enzymes and the induction,
generation or release of angiogenic growth factors, which induce endothelium sprouting,
followed by reorganization and formation of new blood vessels. During cancer progression,
the newly formed tumor-associated blood vessels serve first as feeding/nurturing tubes for a
growing tumor and next, as conduits for dissemination of tumor cells that escaped from an
established primary tumor. Therefore, control of tumor angiogenesis has become a central
issue in the fight against cancer progression since anticancer therapy could be ineffective
once tumor cells reach favored secondary organs and generate metastatic foci.
CAM Assay
To analyze the mechanisms underlying normal and pathological angiogenesis,
numerous in vivo angiogenic assays have been established employing different species of
laboratory animals, including mammals (mouse, rat, hamster, and rabbit), birds (chicken and
quail), and fish (mainly zebra fish). In this chapter, we will focus on major models of
angiogenesis in the chick embryo. The use of chick embryo models for angiogenic studies is
facilitated by the existence in avian species of a specialized respiratory tissue, named the
chorioallantoic membrane (CAM) that allows for gas exchange between the embryo and the
atmosphere surrounding the egg and in effect performs the function of a lung during
embryonic life (Romanoff, 1960). Several CAM angiogenic assays have been introduced
since almost a century ago when rat Jensen sarcoma cells, implanted into the CAM on the
day 6 of incubation, were demonstrated to develop large tumors showing signs of tumor-
induced angiogenesis (Murphy, 1913). All modifications of the original angiogenic assay in
the chick embryo involve grafting of test material onto developing CAM. The grafting is
often performed through a window cut in the egg shell over the CAM. The angiogenic
material is usually introduced in the form of small disks soaked in angiogenic factors or
small pieces of polymerized materials such as gelatin sponges or biologically inert synthetic
polymers, containing either purified angiogenic factors or impregnated with tumor cells.
Aloe Vera
Aloe is made up of more than seventy-five compounds. Complex carbohydrates,
steroids, organic acids, enzymes, antibiotic agents, amino acids, and minerals are all included
in a plant made of succulent green leaves. An enzyme in aloe has been found to be
responsible for the gel’s ability to heal burns. But it is the complex carbohydrates in aloe that
can strengthen the immune system cells, kick the abnormal cells’ behinds and prevent or cure
cancer. Complex carbohydrates are compounds that causes the cells to open their door to
nutrients coming in and also open their door to push out toxins. The complex carbohydrates
make up a long chain sugar that puts itself into all cells. The cells absorb nutrients and repel
toxins because this sugar chain makes the cells able to do this. Cells metabolize better and
energy production improves. The immune system is stronger and is better able to fight off
abnormal cells, or cancer. The body’s immune system is all over the body, not located in one
spot like the heart is, for example. Each organ or area in the body has its own team fighting
infection and they are all inter-related. When something foreign is taken into the body, such
as the chemicals that color, flavor or preserve our foods, the foreign things build up in the
intestines and colon. They can’t be removed because the body does not recognize them, so
they sit there. They irritate the linings of the body’s systems, causing swelling, ulcers and
evil. Aloe’s anti-viral, anti-bacterial, anti-inflammatory and other properties make aloe
essential to good health and prevention of disease. Its vitamins, minerals, amino acids and
other good properties make aloe necessary to replenishing the body’s defenses so it can fight
better for us. Its cancer preventing and cancer fighting properties make aloe a no-brainer for
anyone suffering from the disease. Aloe’s cancer-fighting goal: Kill the abnormal cells, make
the good cells better and improve the overall quality of life for the cancer victim.
Ethanolic Extraction
Medicinal Plants are currently in considerable significance view due to their special
attributes as a large source of therapeutic phytochemicals that may lead to the development
of novel drugs. Most of the phytochemicals from plant sources such as phenolics and
flavonoids have been reported to have positive impact on health and cancer prevention. The
study of medicinal plants starts with the pre-extraction and the extraction procedures, which
is an important step in the processing of the bioactive constituents from plant materials. One
of the extraction methods is the Maceration. Maceration is a technique use in wine making
and has been adopted and widely used in medicinal plants research. Maceration involved
soaking plant materials (coarse or powdered) in a stoppered container with a solvent and
allowed to stand at room temperature for a period of minimum 3 days with frequent agitation.
The processed intended to soften and break the plant’s cell wall to release the soluble
phytochemicals. After 3 days, the mixture is pressed or strained by filtration. In this
conventional method, heat is transferred through convection and conduction and the choice
of solvents will determine the type of compound extracted from the samples.
MATERIALS AND METHODS
Research Design: This study will useused the Complete Randomized Design (CRD)
wherein all of the other variables will be keptwere kept constant such as the type of egg,
temperature, and amount of extract applied through the filter disc for the duration of
the whole experimentation. . This study will also hadve 4 setups, 3 replicates and with
35 samples totaling with 3660 eggs for the entire experimentation.

1. Research Test Samples:
A. Aloe Vera and Duck Eggs Identification
The aloe vera identification of the plants will be donewas done at the Bureau of
Plant Industry, Department of Agriculture and the duck eggs certification of
identification will be donewas done in the Bureau of Animal Industry, Zamboanga City.
The formulation of the extracts and the Duck Chorioallantoic Membrane (CAM) Assay will
be done at the Regional Science High School Chemistry Laboratory Zamboanga City.
B. Egg Collection
Thirty six (36) pieces of ten-day old fertilized eggs of Anasplatyrhynchos were
obtained from Sobtained from a reputable poultry farm at Kambal Poultry House, San
Roque poultry farmTugbungan, Zamboanga City. The eggs are to be were to be randomly
grouped and labeled according to the treatment. The eggs will be were placed in the
incubator at a constant temperature and at a constant humidity which are all controlled
by the incubator.
2. Data Gathering Procedure
A. Preparation of Aloe Vera
Fresh aloe vera plants were collected at Anwar Bahraq’s residence. The plants were
sun dried for one week and were, later on, powdered using a blender for 2 minutes. The
blended plant were then strained to obtain a fine flour. It was then weighed using a weighing
scale which yielded 255 grams.
B. Ethanolic Extraction and Extract Preparation

The powdered leaves were placed in a 500 ml Erlenmeyer Flash and were subjected
to extraction using maceration. It was soaked with in with 300 ml of ninety – five percent
(95%) ethanol for 72 hours at room temperature. After maceration, the filtrate was filtered
using a filter paper. The filtrate was stored in a flask and the solid residue was treated again
with 95% ethanol and was placed at room temperature for another 72 hours. After three days,
the mixture was again filtered separating the residue and the filtrate. The solid residue is
discarded while the filtrate was combined with existing filtrate from the first extraction.
C. Preparation of test solution
Distilled water was used as a vehicle for the preparation of the Aloe Vera extracts.
The concentrated extract was diluted with 100 ml of distilled water; usd as a solvent in the
preparation of the test extracts namely 100mg/ml, 200mg/ml and 300mg/ml. These were
stored in labeled test tubes in a test tube rack. Concentration for the control set up is only
pure distilled water.
D. Filter Disk Preparation
A filter paper was cut into a paper disk with approximately 4 cm in diameter size.
Only 20 ul of test solutions and test control was placed and absorbed by the filter paper.
E. Duck Chorioallantoic Membrane (CAM) Assay
Twenty-four (24) pieces of 10-day old fertilized eggs of Anasplatyrhynchos were
utilized in this study. The eggs were placed inside the incubator with constant temperature
and humidity and were positioned horizontally.
The eggs were disinfected by wiping 70% ethanol using a cotton. Candling was done
to inspect the egg’s viability, position of embryo and airspace. At the site of the airspace, the
egg was marked with a marker. The marked area was opened, exposing the CAM for the
experimental manipulation. Using an 18-gauge size tuberculin needle, the syringe carefully
entered the blunt end (air space) and extracted about two milliliters of the albumen fluid. This
allows separation of the vascularized CAM from the vitelline membrane and the shell
The filter paper disks were introduced with 20 uL of each concentration of Aloe Vera
extract, control (Distilled Water) until fully absorbed. Then, the treated filter paper disks
were placed directly onto the CAM. The treated and untreated eggs were sealed with
micropore tape and were incubated for 48 hours. While incubating, the temperature was
maintained at 36.
After 48 hours of incubation, the micropore tape was removed. The site where the filter paper
disk was placed was photographed for examination. After observing, the paper disk was
placed again in the site where it was originally located. The micropore tape was used again
for sealing the eggs and was placed back into the incubator.
D. Visual Assessment of the CAM
On the 12th day of incubation, reactions of the CAM will be observed. CAM will be
harvested carefully by removing the hard shell leaving the soft membrane intact. The shell
less embryo will be transferred to a petri dish. The number of branching points of blood
vessels will be manually counted.
DE. Photomicrography technique and Quantification
On the 12th day of incubation, Tthe egg embryos were removed from the
incubation and the CAM was harvested carefully by removing the hard shell leaving the soft
membrane intact. The shell less embryo was transferred to a petri dish. and aA
photomicrography was taken below of each disk to count the total number of branch
points, the width and length of each blood vessel. The blood vessel found below the disk
was recorded using an OPPO F1S 13 megapixel. It was then imported to Angiotool
software. The researchers made use of this software in order to decrease the chance of
human error. The total number of branch points in eggs after the application of
treatments was then recorded.
3. Statistical Analysis
Data will be subjected to Analysis of Variance (ANOVA) of the Complete
Randomized Design (CRD). The branching point of each extract will be individually
compared to the negative control which is the Setup D with the Distilled water. Moreover,
the results were interpreted clinically wherein more than 50% CAM vascularity indicates an
anti – angiogenic property.
4. Ethical Considerations and Waste Disposal
Fertilized Anas pPlatyrynchos Embryos were used as test samples to be screened
with different concentrations of the test extract and test controls. Covered in the
Institutional Animal Care and Use Committee (IACUC) guidelines, any experiment
that uses animals must require to obtain ethical clearance. With this consideration, this
study was not required to obtain ethical clearance for the reason that the seventy-
twothirty six (7236) fertilized duck eggs used in this study was not covered in the IACUC
guidelines.
Eggs were disposed in accordance to the guidelines wherein the duck embryos were
autoclaved at a temperature of 212oC for 1 hours to avoid contamination. The embryos were
sealed properly and buried in a compost pit.
RESULTS
Table 1. Mean Number of Branching Points after the Application of Varying

Concentration of Extractsa
Setups Replicate 1 Replicate 2 Replicate 3 Mean

A 250.00 202.00 221.00 224.33
B 299.00 185.67 141.33 208.67
C 140.67 256.67 238.67 212.00
D 176.00 834.33 567.33 525.89

a
Replicate values are the means of the samples per replicate (see appendix D for the
raw data)
Table 1 above shows the mean number of branching points per Setup after the
application of the extracts in respective samples. It is shown here that Setup D, the setup with
distilled water only, have the largest number of branching points with 525.89 while Setup B,
the setup with 200 micrograms/milliliter, has the smallest number of branching points
compared to the other 3 setups.

Figure 1. Comparison of Branching Points after the Application of Varying

Concentration of Extracts
600
500
400 Setup A
Setup B
300 Setup C
Setup D
200
100
0
Mean Number of Branching Points
Similarly to table 1, Figure 1 shows the relationship of the setups and their
comparison in respect to other setups. Still, the Setup D as the setup with the highest mean
branching points while Setup C has the smallest number of branching points but are highly
close to the other two setups.
Table 2. ANOVA Results on the Mean Number of Branching Points after the
Application of Varying Concentration of Extracts
sum of degrees of mean square F
source p-value
squares SS freedom νν MS statistic
treatmen 217,875.444
653,626.3333 3 4.0321 0.0154
t 4
1,729,142.888
error 32 54,035.7153
9
2,382,769.222
total 35
2
The p-value corresponing to the F-statistic of one-way ANOVA is lower than 0.05,
suggesting that the one or more treatments are significantly different.

Table 3. Turkey Test on the Mean Number of Branching Points after the Application of
Varying Concentration of Extracts
treatments Tukey HSD Tukey HSD Tukey HSD
pair Q statistic p-value inferfence
A vs B 0.2022 0.8999947 insignificant
A vs C 0.1592 0.8999947 insignificant
A vs D 3.8918 0.0454171 * p<0.05
B vs C 0.0430 0.8999947 insignificant
B vs D 4.0940 0.0326308 * p<0.05
C vs D 4.0509 0.0350456 * p<0.05

(insert discussion here)
Figure 2. Comparison of CAM Vascularity of Samples after the Application of Varying

Concentration of Extractsb
60.5
60
59.5
59
Setup A
58.5
Setup B
58 Setup C
57.5
57
56.5
56
55.5
CAM Vascularity %
b
The percentage values are in respect to the formula given below.
(insert discussion here)
Table 4 and table 5 Anova and Turkey
DISCUSSIONS
O Decrease number of branching points -> reason kung bakit may decreased number
of branching points
O Significance ng Angiogenesis sa Cancer Metastasis -> Ways of mitigating
O VEGF Vascular endothelial growth factor
O Decreased CAM Vascularity and link of tumor growth
CONCLUSIONS
ACKNOWLEDGMENTS
The researchers would like to give their heartfelt gratitude and warmest thanks to
those who helped them in the completion of their research study.
First, to the parents of the researchers who helped and supported the researchers
reach their goals and complete the study;
To the researchers’ classmates who gave their support and encouragement to carry on
their research especially Jessie B. Locsin.
To the researchers’ attending veterinarian, Luzvanessa Grace N. Jimenea, for giving
her knowledge to complete the study and her supportive help during the
experimentation.
To the school’s principal, faculty and staff for their support and consideration.
And lastly, to our Almighty God for physically and spiritually giving the researchers
the strength they needed every day, the wisdom which helped them in the completion
of the research study and giving them hope to complete the study.
APPENDICES
A. Certification of Botanical and Agricultural Verification

B. Certification of Attending Veterinarian

C. Process Flow Chart
Cutting of Aloe Vera Leaves Washing of Aloe Vera Leaves

Leaving the Mixture for 72 hours

Mixing the Ethanol and the Dried Aloe Vera
Diluted extracts for the different set-ups

(100mg/ml, 200mg/ml, 300mg/ml, and
400mg/ml)
Transferring the diluted extracts to their

designated test tubes
Weighing of extracts for different set-ups
Filtering the Mixture using Filter Paper
The diluted extracts for the different set-

The extracts with different weights (1g, 2g, 3g) for ups (SA, SB,
Diluting the extracts in aSC, and distilled
100ml SD) and water
distilled
different set-ups water
Purchasing of Eggs
Incubating the eggs before the experiment proper
Labeling of Eggs
Candling of Eggs
Opening of Eggs Extracting the Albumen

Applying the Aloe Vera Extract on the

Closing the open area using Micropore Tape
Filter Paper
Checking of the results

Incubating the eggs after the application
of extract
D. Raw Data
Autoclaving of eggs for waste disposal

Table 3 Data for the Number of Branching Points in the Samples for Set Up A
Samples Branching Points Mean
SAR1S1 242.00
SAR1S2 278.00
SAR1S3 230.00 250.00
SAR2S1 366.00
SAR2S2 109.00
SAR2S3 131.00 202.00
SAR3S1 236.00
SAR3S2 190.00
SAR3S3 237.00 221.00
224.33
Table 3 shows the raw data of the number of branching points per sample of each
replicate for the set up A. The mean per replicate is also given above and the mean number of
branching points for set up A with 100 micrograms/milliliter is 224.33.
Table 4 Data for the Number of Branching Points in the Samples for Set Up B
SBR1S1 122.00
SBR1S2 638.00
SBR1S3 137.00 299.00
SBR2S1 101.00
SBR2S2 350.00
SBR2S3 106.00 185.67
SBR3S1 48.00
SBR3S2 182.00
SBR3S3 194.00 141.33
208.67
replicate for the set up B. The mean per replicate is also given above and the mean number of
branching points for set up B with 200 micrograms/milliliter is 208.67.
Table 5 Data for the Number of Branching Points in the Samples for Set Up C

SCR1S1 152.00
SCR1S2 110.00
SCR1S3 160.00 140.67
SCR2S1 206.00
SCR2S2 470.00
SCR2S3 94.00 256.67
SCR3S1 112.00
SCR3S2 164.00
SCR3S3 440.00 238.67
212.00
replicate for the set up C. The mean per replicate is also given above and the mean number of
branching points for set up C with 300 micrograms/milliliter is 212.00.
Table 6 Data for the Number of Branching Points in the Samples for Set Up D
SCR1S1 216.00
SCR1S2 172.00
SCR1S3 140.00 176.00
SCR2S1 220.00
SCR2S2 1219.00
SCR2S3 1064.00 834.33
SCR3S1 643.00
SCR3S2 591.00
SCR3S3 468.00 567.33
525.89
replicate for the set up D. The mean per replicate is also given above and the mean number of
branching points for set up D with distilled water is 525.89.
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Rice Field Muck In Microbial Fuel Cells with Gelidium Corneum

(nutrient agar) Salt-Gel Solution Proton Exchange

Membrane for Power Generation

A Science Investigatory Project of:

Kimberly Jeph C. Tagle

Chymee Grace C. Gagarra

Florenda H. Quinte Jeffrey D.C. Barrera

Practical Research II Adviser Research Capstone Adviser

Regional Science High School for Region IX – Zamboanga Peninsula

Malasiga, San Roque, Zamboanga City

February 208, 2018

Aloe Vera (Aloe barbadensis) Extract as Angiogenesis Inhibitor Using

cardiovascular diseases (Yu, 2014). With significant improvement in treatment and

as a cancer treatment (Schmidt, 2016). Because of the chemicals used in chemotherapy, it

first-aid purpose (National Institute of Environmental Health Sciences [NIEHS], 2017).

study and will be tested by its effectiveness to inhibit angiogenesis.

Moreover, the study aimed to utilize varying concentrations of Aloe Vera

extracts as angiogenesis inhibitor using Duck Chorioallantoic Membrane (CAM) Assay. It

mg/ml Aloe Vera Extract and d. Setup D: Distilled Water.

varying concentration as the cultivar that is to be used as an angiogenesis inhibitor.

verification for angiogenic inhibition of the extracts.

The study aimed to utilize varying concentrations of Aloe Vera extracts as

Aloe Vera Extract and d. Setup D: Distilled Water.

CAM Vascularity of the blood vessels when subjected in the 4 setups.

concentration as the cultivar that is to be used as an angiogenesis inhibitor. Furthermore, this

duration of the experimentation will only last for one month.

Cancer and Tumor

decrease its inhibition.

Cancer and its TreatmentsTreatments for Cancer

According to Folkman (2002) in his study titled Role of angiogenesis in tumor

cultivars themselves especially their costing, parameters, availability and accessibility as

Assays: Problems and Pitfalls.

Angiogenesis and Tumor Growth

Angiogenesis is a progressive, multistep physiological process by which new blood

injury, as well as a critical step in the pathology of tumor progression. It is commonly

generation or release of angiogenic growth factors, which induce endothelium sprouting,

To analyze the mechanisms underlying normal and pathological angiogenesis,

Aloe is made up of more than seventy-five compounds. Complex carbohydrates,

phytochemicals. After 3 days, the mixture is pressed or strained by filtration. In this

MATERIALS AND METHODS

35 samples totaling with 3660 eggs for the entire experimentation.

1. Research Test Samples:

A. Aloe Vera and Duck Eggs Identification

Plant Industry, Department of Agriculture and the duck eggs certification of

2. Data Gathering Procedure

A. Preparation of Aloe Vera

scale which yielded 255 grams.

B. Ethanolic Extraction and Extract Preparation

C. Preparation of test solution

pure distilled water.

D. Filter Disk Preparation

E. Duck Chorioallantoic Membrane (CAM) Assay

Twenty-four (24) pieces of 10-day old fertilized eggs of Anasplatyrhynchos were

and humidity and were positioned horizontally.

D. Visual Assessment of the CAM

vessels will be manually counted.

DE. Photomicrography technique and Quantification

treatments was then recorded.

Data will be subjected to Analysis of Variance (ANOVA) of the Complete