1

Chapter I
INTRODUCTION

A. Background of the Study

Pineapple (Ananas comosus) (family: Bromeliaceae) is a perennial crop grown for its fruits

and used as a fresh and processed product. It is widely cultivated throughout tropical and sub-tropical

countries of the world including Philippines. The fruit has numerous medicinal applications and used

as traditional treatment for various illnesses such as, arthritis, Rheumatism, Arteriosclerosis,

Bronchitis, emphysema, wounds and cancer. The pharmaceutical importance of the peeling extracts

containing bromelain.

(http://www.fao.org/nr/water/cropinfo_pineapple.html)(https://www.youtube.com/watch?v=c2A2K51

cKs8In this study)

According to Major Fruit Crops Quarterly Bulletin, the production of pineapple in January-

March 2016 reached 591.25 thousand mt. This was 1.4% more than the 2015 level of 583.14

thousand mt.

Pineapples are abundant in Ormoc City; hence, it is not surprising to see many pineapple

vendors within the city. But these vendors usually just throw away the pineapple peelings and will

decay later on which will eventually become a food waste.

According to Junemy Pantig in her online article entitled “The Effects of Food Waste on the

Environment”, food wastes potentially contributes to greenhouse gas emissions especially with the

production of methane, a greenhouse gas that is twenty-one times more potent than carbon dioxide

and one of the most dangerous to the environment. Food waste contributes to greenhouse gas

emissions by the decomposition of food in landfills and the life cycle, which begins at the stage of

production all the way to consumption.

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A pineapple is a fruit wherein you have to peel off the peeling to eat it, like any other fruits.

The researchers chose this as their project to prove that pineapple peelings have other uses.

Pineapples are a funny-looking fruit with a serious impact on health, and its health and

medicinal benefits include their ability to improve respiratory health, cure coughs and colds, improve

digestion, help you lose weight, strengthen bones, improve oral health, boost eye health, reduce

inflammation, prevent cancer, increase heart health, fight off infections and parasites, improve the

immune system, and increase circulation. Aside from the interesting story and appearance of this

fruit, it is also a storehouse of huge health benefits due to its wealth of nutrients, vitamins,

and minerals, includingpotassium, copper, manganese, calcium, magnesium,vitamin C, beta

carotene, thiamin, B6, and folate, as well as soluble and insoluble fiber, and bromelain.

(https://www.organicfacts.net/health-benefits/fruit/pineapples.html)

As years go by, cancer related patients gradually increases every year. This is due to unhealthy

lifestyle and unconscious food intake, people diagnosed with cancer is rapidly growing. As of 2015,

there were an estimated 1,658,370 new cancer cases diagnosed and 589,430 deaths.

According to Dr. Joseph Mercola in his journal entitled Surprising Cancer-Fighting Benefits

of Pineapple Enzyme, one of the reasons why current cancer treatment is such a dismal failure is

because, it relies on chemotherapy. Chemotherapy drugs, by their very nature, are extremely toxic

and typically work against your body's natural ability to fight cancer, e.g. destroying host immunity

instead of supporting it. The cure is often worse than the disease, pushing scientists to search

for natural cancer-fighting solutions.

Nowadays, there are strategies one can use to reduce one’s risk of cancer; one of these is

through eating anti-cancer foods. Also, researchers have discovered many fruits and vegetables that

are anti-inflammatory and most importantly those that help fight cancer.
 3

As reported earlier, about 74% of the known anti-cancer medicines are derived from various

plant species. Indeed, there are many household dietary products exhibiting anti-cancer potential with

minimal side effect that are currently under clinical trials for cancer treatment. Among these is the

possibility of pineapple to have an anti-cancer potential because of the Bromelain, Vitamin C and

Dietary Fiber it contains.(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4545797/)

In this study the researchers focused on the anti-cancer potential of pineapple (Ananas

comosus) peeling extract. To determine the effectiveness of the extract, the researchers analyzed the

effect of the inoculum in the physical appearance of the embryo inside the eggs. The reason behind

selecting the inoculation process, its easy access to availability and is cost-effective.

This study aimed to determine the anti-cancer potential of pineapple (Ananas comusos)

peeling. In addition, if the blood vessels observed decreases and the appearance of the duck embryos

changes (after 3 days of injecting 0.3 mL and 0.5 mL of the extract into the two group of test

samples., it means that the extract is an antiangiogenic or an anticancer agent, however, if there are

new blood vessels observed then the extract is proangiogenic or it doesn’t have an anticancer

property. The novelty of this research is that the researchers will determine the anti-cancer potential

of waste pineapple peelings extract by injecting it into the eggs, which has not been studied in the

past.

B. Statement of the Problem

This study focused on determining if pineapple peelings extract has anti-cancer potential.

Specifically, this study sought to:

1. determine if pineapple peelings extract has anti-cancer potential in terms of:

a. amount of extract

b. status of the duck embryo

c. observed physical appearance of the yolk

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d. observed blood vessels

2. determine the significant difference in the status of the duck embryos, observed physical

appearance of the yolks, observed blood vessels, and the anti-cancer potential of pineapple

peelings extract as a whole between the first and second treated/experimental set-ups.

C. Hypothesis

HO – There was no significant difference in the status of the duck embryos, observed physical

appearance of the yolks, observed blood vessels, and the anti-cancer potential of pineapple peelings

extract as a whole between the first and second treated/experimental set-ups.

HA – There was a significant difference in the status of the duck embryos, observed physical

appearance of the yolks, observed blood vessels, and the anti-cancer potential of pineapple peelings

extract as a whole between the first and second treated/experimental set-ups.

D. Significance of the Study

This study is significant…

To the community

Pineapple is abundant in the community especially in a tropical country like Philippines.

Many people in the community sell pineapples as their livelihood. Usually, most of the

vendors just dispose or throw away the pineapple peelings or either uses it as a fertilizer.

Instead of throwing them away, the researchers find other uses of pineapple peelings. This

study’s significance to the community is, aside from the nutrients we can get from eating this

fruit, this study can also help to minimize solid wastes in the community and turn it into

something useful.

To the economy

This study is significant to the economy because this study can make or produce new,

innovative medical products. This product is low-cost since pineapple peelings can be found
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everywhere. Also, this product is better than other medical products because it is organic since

it is made out of pineapple peelings only. Lastly, it is helpful because of its medical purposes.

The products that will be produced by this study will surely catch people’s attention.

To the environment

Pineapple is abundant to the community and many people consume and sell pineapples.

After eating, usually they just throw away its peelings. Sometimes pineapple peelings were

not properly disposed which eventually decays and will emit foul odor which people find

disgusting and disturbing. Because of this, the researchers find other uses of pineapple

peelings for at least to minimize the disposal of pineapple peelings. This study is significant to

the environment because it can lessen the pollution.

To the researchers

By studying the pineapple peelings, the researchers can gain new information or

knowledge. The researchers can use their knowledge in many ways. One way is by creating

new medicine for different diseases. They can also use it by creating new studies related to the

study. Moreover, they can also use the products or medicines that they made as their

livelihood.

To the school

This study will be of great help in providing more facilities and supplies needed in the

school. This is to continue conducting more innovative researches. The school cannot afford

any more to lose a study just because of the scarcity of laboratory equipment and supplies. If

done so, the school will be fully equipped with laboratory equipment. Also, this study can

help the teachers and students for new additional knowledge about pineapple peelings other

uses.
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E. Scope and Limitations of the Study

This study was limited to duck eggs (16 days old) and pineapple peelings extract as the main

components. This study focused on determining if pineapple peelings extract has an anti-cancer

potential. The researchers searched 10 incubated duck eggs (about 16 days). They also used 1 kilo

of ripe pineapple peelings that was collected from a pineapple vendor. The researchers used

laboratory equipments like: incubator, laminar hood, and stereoscope equipped with a digital

camera. There were no chemicals needed in conducting the study. This study lasted for 1 week.

Furthermore, this study was limited to the researchers and lab technicians who helped them

perform the investigations that concern the topic.

F. Time and Place of the Study

This study was conducted at the grounds of Ormoc City, City Agriculture Office and Visayas

State University from July-September, 2016.

G. Definition of Terms

Inoculum – Is the substance injected into the egg

Inoculation – Is the process of injecting the extract into the egg

Laminar flow hood – enclosed cabinet-like apparatus designed to prevent contamination of the

samples under investigation.

Proangiogenic – promotes the growth of new blood vessels formation.

Antiangiogenic – prevents the growth of new blood vessel formation and sometimes lead to

the disappearance of pre-existing vessel networks.

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Chapter II
REVIEW OF RELATED LITERATURE

Pineapple is delicious and juicy and it has many nutritional benefits and the thick skin of a

pineapple that is often discarded also has many nutritional benefits, such as strengthening immune

system, keeping teeth healthy, and reducing skin inflammation. In its core, pineapple is full of bromelain,

an enzyme which acts as a natural anti-inflammatory. Vitamin C, fiber, manganese, and copper can all be

found in the core as well. Pineapple skin is also nutritious, containing vitamin C and bromelain.

Bromelain is the pineapple enzyme that digests proteins and it exhibits this ability. You can also get small

quantities in juice, but for true cancer-fighting power, you need to consider a pineapple enzyme extract.

(http://www.seattleorganicrestaurants.com/vegan-whole-food/fruit-nutrition-seeds-

skin.php#sthash.R82WoSo9.dpuf)

Pineapple has been used as a medicinal plant in several native cultures and these medicinal

qualities of pineapple are attributed to bromelain, which is a crude extract from pineapple that contains,

among other compounds, various closely related proteinases, exhibiting various fibrinolytic,

antiedematous, antithrombotic, and anti-inflammatory activities in vitro and in vivo. (EC 3.4.22.32)

(Mondal S, Bhattacharya S, Pandey JN, Biswas M. Evaluation of acute anti-inflametry effect of Ananas

Comosus leaf extract in Rats. Pharmocologyonline. 2011;3:1312–1315.)

Scientists found that the anti-tumor effects of bromelain were superior to the effects of the

chemotherapy drug 5-fluorouracil. Yes, it worked better than chemo.

5-fluorouracil (5-FU) has been used as a cancer treatment method for about 40 years. It’s highly

toxic and its effectiveness is questionable at best. But like many cancer treatment methods, it’s used

regardless.
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The problem with 5-FU and other chemo drugs is they don’t discriminate. They kill healthy cells

not just the cancer cells. And, even when they are indiscriminately killing cells, they don’t kill all of the

cancer cells. On the other hand, bromelain’s toxicity is rated at 1.5 pounds, the same as 5-FU at 7.5

grams. In other words, bromelain isn’t only safer, but measures safer and more effective.

(http://naturalsociety.com/pineapples-been-used-cancer-treatment/)

Beyond cancer, bromelain’s extremely high anti-inflammatory properties make it a natural

treatment for arthritis and bowel disorders. Inflammation is believed to be a root cause of hundreds of

diseases. (http://undergroundhealthreporter.com/pineapple-enzyme-fights-cancer/)

It is well known that increased consumption of dietary fiber helps to maintain health and

wellness. Fibers have numerous health benefits, more and more are being discovered every day. They

can reduce constipation and promote the health of your colon; lower cholesterol, blood pressure, and the

risk of cardiovascular disease; and improve blood sugar control after a meal and aid in weight loss. They

can also help reduce diarrhea in some instances. (http://fiberfacts.org/for-health-professionals/benefits-of-

a-high-fiber-diet/)

Dietary fiber is made up of the non-digestible carbohydrates and lignin that naturally occur in

plants; fruits, vegetables, and grains. Dietary fiber has been shown to reduce the risk of developing many

diseases, including cancer. Current evidence from prospective meta-analyses suggests that, through

various suggested mechanisms, dietary fiber may reduce the risk of developing highly prevalent

gastrointestinal and hormonal cancers, most notably colorectal and breast cancer.

There are many mechanisms by which dietary fiber may protect against the development of

cancer. These mechanisms include insulin resistance, modulation of immunity including the inflammatory

response, and DNA damage-repair. While all of these mechanisms may be important for cancer

prevention, reducing insulin resistance is thought to be the one key mechanism. Among individuals who
 9

demonstrate insulin resistance, even a moderate intake of low-fiber and high-carbohydrate foods results in

a significant elevation in plasma insulin levels. Furthermore, high intake of dietary fiber may reduce the

risk of overweight or obesity, which is an established risk factor for some cancers.

(http://www.esciencecentral.org/ebooks/food-as-source-of-anticancerigen/the-health-benefits-of-dietary-

fiber-evidence-of-its.pdf)

Recently, exciting research has been published that documents the ability of vitamins C, E and

selenium orally to prevent most cancers, and to decrease the mortality of pancreatic cancers, stomach

cancers, prostate cancers and other cancers. It sounds too good to be true. The Vitamin C Foundation is

interested in the how and why vitamin C prevents and treats cancers. Almost all vitamin C experts

agree, hydrogen peroxide produced by high levels of ascorbate (vitamin C) kill cancer cells. However,

vitamin C by itself is relatively weak at this job so don't try low dosages. Experts recommend continuous

high dosages of ascorbate (vitamin C) combined with specific adjuncts (e.g.Alpha Lipoic Acid, Vitamin

K3, and/or D'Fraction Maitake Mushroom.). Most experts recommend incorporating high-dose

intravenous vitamin C (IV/C) infusions to achieve the high blood concentrations necessary to kill the

most tumor cells. (http://vitamincfoundation.org/vitcancer.php)

Cancer is a dangerous disease in which certain cells in our body grow in an uncontrolled way. It

is one of the world’s most serious illnesses. Together with heart attacks, it kills more people than any

other disease in the world.

Cancer begins in the building blocks of your body which are the cells. To replace the dead

cells, your body forms new cells as you need them. Sometimes this process goes wrong. New cells grow

even when you don't need them, and old cells don't die when they should. These extra cells can form a

mass called a tumor. Tumors can be harmless or malignant. Benign tumors aren't cancerous while
 10

malignant ones are. Cells from malignant tumors can invade nearby tissues. They can also break away

and spread to other parts of the body. (https://medlineplus.gov/cancer.html)

Chemotherapy is the most common form of treatment for cancer in the United States. It has

been used for the past four decades despite the fact that it destroys the body, weakening the immune

system until it can no longer function properly.

According to Dr. Joseph Mercola in his journal entitled Surprising Cancer-Fighting Benefits of

Pineapple Enzyme, one of the reasons why current cancer treatment is such a dismal failure is because, it

relies on chemotherapy. Chemotherapy drugs, by their very nature, are extremely toxic and typically work

against your body's natural ability to fight cancer, e.g. destroying host immunity instead of supporting it.

One of the biggest disadvantages of chemotherapy is the fact that it kills healthy cells along

with cancer cells, a side effect that doesn’t lead to healing but to accelerated death instead. Another

devastating side effect of chemotherapy is the way it supports chemo resistant and malignant cells within

tumors (e.g cancer stem cells), killing harmless cells that slow down the tumor’s growth.

The cure is often worse than the disease, pushing scientists to search for natural cancer-fighting

solutions.

Nowadays, there are strategies one can use to reduce one’s risk of cancer; one of these is through

eating anti-cancer foods. Also, researchers have discovered many fruits and vegetables that are anti-

inflammatory and most importantly those that help fight cancer.

According to research, bromelain is a traditional anticancer medicine of Southeast Asia. It is a

water extract of pineapple. Protease is believed to be the reason for the anticancer activity of bromelain.

Bromelain is easily absorbed in the gastrointestinal tract. Antacids preserve bromelain’s proteolytic

activity. Doses up to 3 g/day are well tolerated. Doses of 1-2.4 g/day are effective.
 11

There are very few clinical trials of bromelain in cancer. Most studies of bromelain have been of

anti-inflammatory activity. In studies of chemically-induced skin tumors in mice, bromelain reduced the

formation of tumors, reduced tumor volumes and induced cellular apoptosis (natural cell death.)

Bromelain has been reported to reduce metastasis of cancers.

A wide range of therapeutic benefits have been claimed for bromelain, such as reversible

inhibition of platelet aggregation, sinusitis, surgical traumas , thrombophlebitis, pyelonephriti angina

pectoris, bronchitis , and enhanced absorption of drugs, particularly of antibiotics. ( Livio M, Gaetano

GDe, Donati MB. Effect of bromelain of fibrinogen level, protrombin complex and platelet aggregation in

the rat-a preliminary report. Drugs under Experimental and Clinical Research. 1978;1:49–53.)

(NeubaurR Ra.A plant protease for potentiation of and possible replacement of antibiotics.Exp Med Surg.

1961; 19():143-60.)(Varengo R. Absorption of tetracycline in presence of bromelain after oral

administration.Arzneimittelforschung. 1972 Feb; 22(2):410-2.)(Maurer HR. Bromelain: biochemistry,

pharmacology and medical use.Cell Mol Life Sci. 2001 Aug; 58(9):1234-45.)

Several studies have been carried out indicating that bromelain has useful phytomedical

application. However, these results are yet to be amalgamated and critically compared so as to make out

whether bromelain will gain wide acceptance as a phytomedical supplement. (Tochi BN, Wang Z, Xu SY,

Zhang W. Therapeutic application of pineapple protease (Bromelain): a review. Pakistan Journal of

Nutrition. 2008;7(4):513–520. )

Pineapple peelings are not yet proven to be a cure for cancer. Some people already made use of

the extract as an alternative cure for cancer but is still questioned for its accuracy specifically on its proper

dosage. Also, pineapple peeling extract is not yet clinically proven officially. Because of these, it is still

questioned as they may cause massive harm or damage to those vulnerable people who are desperately

looking for miracles.

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The mechanism by which bromelain works against cancer is not fully known but bromelain has

shown benefit against a number of cancers. (http://www.rainbow.coop/library/bromelain-as-an-

anticancer-agent/)

Bromelain is considered as a food supplement and is freely available to the general public in

health food stores and pharmacies in the USA and Europe. Existing evidence indicates that bromelain can

be a promising candidate for the development of future oral enzyme therapies for oncology patients. (Ley

CM, Tsiami A, Ni Q, Robinson N. Zhong Xi Yi Jie He Xue Bao. A review of the use of bromelain in

cardiovascular diseases. 2011 Jul; 9(7):702-10.)( Chobotova K, Vernallis AB, Majid FA. Bromelain's

activity and potential as an anti-cancer agent: Current evidence and perspectives. 2010 Apr 28;

290(2):148-56.)

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

generated from pre-existing vasculature. 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.

(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2699944/)

Arianwen Ledesma Rollan, a 17-year-old girl from Barangay Kalunasan, Cebu City and a Grade

11 student of the Cebu City National Science High School that was recently recognized by an

international award-giving body for her research on the anti-tumor properties of malunggay seeds,

explained that tumors often developed due to angiogenesis, or the development of new blood vessels in

pre-existing vessels. (http://www.sunstar.com.ph/cebu/local-news/2016/06/19/girl-17-studies-plant-vs-

tumor-475145)
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“When there is a tumor, there will be a rapid development of blood vessels. The more blood

vessels, there will be more nutrients supplied to the tumor. In this case, we need to inhibit the

development of these blood vessels in order to cut off its source of nutrients,” Arianwen Ledesma Rollan

told GMA News. News. (http://www.gmanetwork.com/news/story/567513/scitech/science/pinay-student-

bags-int-l-prize-for-malunggay-anti-tumor-research#sthash.yfxlm2qf.dpuf)

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).

(Romanoff AL.1960. The Avian Embryo. New York: Macmillan, New York)

Inoculation is the act or process of introducing something to where it will grow or reproduce. It is

most commonly used in respect of the introduction of a serum, vaccine, or antigenic substance into

the body of a human or animal, especially to produce or boost immunity to a particular disease. It may

also refer to the communication of a disease to a living organism by transferring its causative agent into

the organism, to implant microorganisms or infectious material into a culture medium.

(http://www.biology-online.org/dictionary/Inoculation)

According to microbiologists, to inoculate means simply to introduce -- or insert -- one substance

into another. For example, you could inoculate a bacteria culture with a certain chemical compound -- in

other words, you introduce the chemical compound into the bacteria culture .

(http://classroom.synonym.com/inoculate-mean-microbiology-12655.html)

To many people, the word "inoculate" relates specifically to vaccination, in that to inoculate

someone means to administer a vaccine to that person. While that definition of the word is true, the word

"inoculate" also has a more general meaning in the field of microbiology, one that still applies to the

process of vaccination, but isn't related at all to the goal of bolstering a person's immune system.

(http://classroom.synonym.com/inoculate-mean-microbiology-12655.html)
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Prior to the advent of cell culture, animal viruses could be propagated only on whole animals or

embryonated chicken eggs. Good pasture in 193 first used the embryonated hen’s egg for the cultivation

of virus. The process of cultivation of viruses in embryonated eggs depends on the type of egg which is

used. The egg used for cultivation must be sterile and the shell should be intact and healthy. A hole is

drilled in the shell of the embryonated egg, and a viral suspension or suspected virus- containing tissue is

injected into the fluid of the egg. Viral growth and multiplication in the egg embryo is indicated by the

death of the embryo, by embryo cell damage, or by the formation of typical pocks or lesions on the egg

membranes. An embryonated egg offers various sites for the cultivation of viruses (Fig 3). The different

sites of viral inoculation in embryonated eggs are: chorioallantoic membrane(CAM), amniotic cavity,

allantoic cavity and yolk sac. (http://vlab.amrita.edu/?sub=3&brch=76&sim=1223&cnt=1)

Among various animal model systems designed to study the mechanisms underlying

angiogenesis, chick embryo models have been useful tools in analyzing the angiogenic potential of

purified factors and intact cells. The chorioallantoic membrane (CAM), a specialized, highly vascularized

tissue of the avian embryo, serves as an ideal indicator of the anti- or pro-angiogenic properties of test

compounds. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2699944/#R23)

Until day 11 or 12 of chick embryo development, the blood vessel system of the CAM is highly

angiogenic, that is, undergoing maturation through a constant generation of new blood vessels as well as

establishment of new blood vessel anastomoses. Therefore, between day 8 and day 10, the developing

CAM vasculature is ready to sprout in response to additional proangiogenic stimuli and, in turn, is very

responsive to antiangiogenic factors. This feature renders the chick embryo CAM models well suited for

experimental validation of pro- and anti-angiogenic compounds.

(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2699944/#R23)
 15

The available CAM area under the window is only enough for the application of one disk per

embryo, thus dictating the use of large groups of animals to get reliable, quantitative readouts. In this

assay, quantitation is performed usually 3 days after implantation and involves counting the number of

CAM vessels in the area of filter disk. In response to proangiogenic stimuli, the newly formed blood

vessels appear converging toward the disk in a wheel-spoke pattern. Inhibition of angiogenesis by

antiangiogenic compounds results in the lack of new blood vessel formation and sometimes in

disappearance of pre-existing vessel networks. Angiogenesis levels can be also determined by counting

branch points in the vessels adjacent to the disks. Time course of new blood vessel formation in the CAM

can be readily performed by analyzing the images taken at different time points after grafting of the disk

using a stereoscope equipped with a digital camera.

(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2699944/#R23)

The amniotic cavity is the closed sac between the embryo and the amnion, containing the

amniotic fluid. The amniotic cavity is formed by the fusion of the parts of the amniotic fold, which first

makes its appearance at the cephalic extremity, and subsequently at the caudal end and sides of

the embryo. As the amniotic fold rises and fuses over the dorsal aspect of the embryo, the amniotic cavity

is formed. (https://en.wikipedia.org/wiki/Amniotic_sac)

Allantois is a sac-like structure that forms part of a developing amniote's conceptus(which

consists of all embryonic and extra-embryonic tissues). It helps the embryo exchange gases and handle

liquid waste. This sac-like structure is primarily involved in nutrition and excretion, and is webbed

with blood vessels. The function of the allantois is to collect liquid waste from the embryo, as well as to

exchange gases used by the embryo. (https://en.wikipedia.org/wiki/Allantois)

The earliest stages of development take place not just inside the egg, but inside the mother duck,

or hen. There, the egg develops from the inside out, starting with the yolk -- this serves as a food source

for the duck throughout his development, and around which the first cells form. These cells are called the
 16

gastrula, and over the course of the duck's incubation, they will develop to form the different parts of his

body, like his lungs, muscles, nerves and skin. The last part of the duck's egg to develop before it is laid is

the shell, which protects the duck inside and also provides the calcium that will form his bones. Once this

layer is complete, the hen lays the egg. (http://animals.mom.me/stages-duck-development-egg-7868.html)

A hen only lays one egg per day, and she lays them all in the same place -- a nest of her own

making. The hen prepares the nest over the course of several days as she lays her eggs one by one, and

during this period, development is slow. This is because the hen wants her eggs to hatch at approximately

the same time, so her offspring will be easier to manage. If she starts incubating them too early, the first-

laid eggs will hatch before the later-laid eggs. The developing ducks are not particularly sensitive to the

cold at this stage in their development, so until she is ready to incubate them all herself, she simply leaves

them in the nest and shelters them from predators and the elements with besting material like grass and

her own feathers, which she plucks out. (http://animals.mom.me/stages-duck-development-egg-

7868.html)

The incubation period is when the duck develops inside his egg. His mother lays on his egg and

the rest of the eggs at the same time, rotating them periodically so that they all benefit from the same

amount of body heat. This heat causes the duck embryo's cells to divide, an approximately 21- to 28-day

process during which the gastrula develops into a living duck. During this period, the duck gets oxygen

through the pores of his shell and obtains all of the nutrients he needs from the yolk of the egg.

(http://animals.mom.me/stages-duck-development-egg-7868.html)

Embryonated hen’s eggs will support the growth of some viruses. Not all viruses will grow in the

tissues of embryonated eggs initially but many can be adapted to growth in eggs without much difficulty.

Eggs provide a suitable means for the primary isolation and identification of viruses, the maintenance of

stock cultures and the production of vaccines. The viruses grow in the cells of the embryo and membranes
 17

and can be detected in several ways. These include mortality, deformity or haemorrhages in the embryo,

lesions on the membrane in the form of pocks, oedema of the developing membranes, inclusion bodies in

sections prepared from embryo tissues or the presence of viral antigens in the egg fluids. An embryonated

hen’s egg contains cells (the embryo and its membranes) that will support the growth of some viruses.

They grow either in the cells of the embryo or its membranes, or in both and when these cells die they are

liberated into the egg fluids. Collection of the virus-infected egg fluid is relatively simple, if somewhat

messy. Eggs are inexpensive and easy to maintain. Eggs come in a usually sterile package surrounded by

a porous shell. As they should arrive clean in the laboratory, they should not be washed or immersed in

water as this may allow bacteria to enter the egg. Use a quickly evaporating agent, such as alcohol,

tincture of iodine or ether, to sterilize the eggshell at the site re inoculation. Eggs are freely available,

especially hens’ eggs but ducks’ eggs have also been used. The immune system of the embryo has not

matured; therefore antibodies are not produced against the inoculated virus. However, maternal antibodies

are transferred from the hen to the egg which implies that eggs should be obtained from non-vaccinated

(especially against Newcastle disease virus), mycoplasma-free flocks.

(http://www.afrivip.org/sites/default/files/01_virology_eggs_complete.pdf)

Advantages of eggs over animal host systems are: eggs are readily available, cheap and easy to

maintain, preliminary incubation of the eggs is carried out at 38 – 39°C and 60 – 70% humidity. The eggs

need to be turned at least twice a day or rolled continually in a specially designed egg incubator, once

inoculated, the eggs are incubated at temperatures suitable for the growth of the virus, but still

maintaining a high degree of humidity, eggs are easily manipulated under sterile conditions, eggs come in

a sterile package surrounded by a porous shell (to sterilize the site of inoculation, use a quickly

evaporating agent, such as alcohol, tincture of iodine or ether), eggs are sheltered from the natural

diseases often observed in laboratory animals, and are relatively free from bacterial and many latent viral

infections, eggs are generally free from natural factors of defence, specific or non-specific, that

sometimes intervenes and prevents passage in adult animals, the immune system of the embryo has not
 18

matured therefore antibodies are not made against the inoculated virus. Also the embryo is sensitive of

some viruses that are harmless to the adult bird and eggs are easily identified and labelled with details of

date, virus inoculated and experimental procedure.

(http://www.afrivip.org/sites/default/files/01_virology_eggs_complete.pdf)

Many viruses such as fowl plague and Newcastle disease virus, grow readily in the endoderm of

developing chicken eggs. Other viruses such as influenza, may require repeated amniotic passages before

becoming adapted to the egg and grown in the allantoic cavity. Allantoic inoculation is a quick and easy

method that yields large amounts [8 – 15 ml] of virus-infected egg fluids.

(http://www.afrivip.org/sites/default/files/01_virology_eggs_complete.pdf)

The analysis of the experiments described above indicates that tumors of the white rat or white

mouse inoculated into parenchymatous organs acquire a different biological character from those

inoculated subcutaneously. The latter are a great deal more benign in their behavior than human cancer or

spontaneous tumors in the same species of animals. Tumors inoculated into organs, on the other hand, are

quite identical in their biological behavior with the malignant tumors of animal and man. A conclusion

must then be drawn, even a priori, that the method of inoculation into organs is a very important aid in

the experimental investigation of cancer. It is true that the method is a great deal more complicated and

time-consuming than the ordinary subcutaneous inoculation. (http://jem.rupress.org/content/16/2/155)

The subcutaneous method is satisfactory for a number of cancer problems. One of these is the

study of general susceptibility and resistance of the organism of the host to the inoculation of the tumors,

and this is a subject of paramount importance in cancer research. On the other hand, the investigations of

the writer (10) have shown that an animal may be susceptible to a subcutaneous inoculation of a certain

tumor and resist the inoculation of the same tumor into the testicle. Undoubtedly this method of

inoculation will reveal the existence of a number of other phenomena.

(http://jem.rupress.org/content/16/2/155)
 19

Chapter III
METHODOLOGY

Research Design

This study was an experimental research, since the researchers tested the anti-cancer potential of

pineapple (Ananas comosos) peelings extract using fertilized duck eggs (about 16 days of incubation).

The independent variables were: pineapple peeling and the fertilized duck eggs; and the dependent

variable were the blood vessels observed and the appearance of the duck embryos after 3 days of injecting

0.3 mL and 0.5 mL of the extract into the two group of test samples.

Materials/Equipments

For the Pineapple Peelings Extract:

Waste pineapple peelings (1 kilo)

Juicer

Knife

Chopping board

For the Chick Embryo Chorioallantoic Membrane Assay:

10 fertilized duck eggs

Incubator

Egg candler/flashlight

Laminar flow hood

2 bottles of 500 mL Ethyl Alcohol (70% solution)

1 bottle of 350 mL Mineral Water

2 liters of Mineral Water

Sterilized stirring rod

 20

Marker

Sterile laboratory scissors

Cotton and/or cotton balls

Sterilized forceps

Clean/sterilized tray

Tissue

Alcohol lamp

Sterilized petri dish with cover

Clean/sterilized incubator trays

Sterilized beaker

3 new syringes

Stereomicroscope equipped with a digital camera

Treatment of the Study/General Procedure

For the Pineapple Peelings Extract:

1. Collect 1 kilo of pineapple peelings/skin from a pineapple vendor.

2. Rinse/wash the peelings with mineral water.

3. Slice the pineapple peelings using a knife into small pieces for the juicer to easily extract its

juice. Also, be sure to use a chopping board.

4. Put the pineapple peelings on the juicer. Use the juice collector cup of the juicer as the

container.

5. Wait for it to finish the extraction process.

For the Duck Embryo Amniotic/Allantoic Inoculation:

1. Find 10 organic and fertilized eggs of the same age (1 day old). 3 of the eggs will be the first

group of test samples, another 3 will be the second group of test samples, another 3 will be
 21

the controlled samples and the last egg will serve as the basis in determining the average

density of the blood vessels of the eggs of the same age.

2. Label the first group of test samples as: T1.A, T1.B and T1.C; label the second group of test

samples as: T2.A, T2.B and T2.C; label the controlled samples as: C1, C2 and C3. Don’t do

anything to the last egg.

3. Place tissue papers on the tray. Put all of the sterilized equipments (e.g sterilized stirring rod,

sterilized forceps, sterilized beaker, sterilized petri dish with cover, sterilized laboratory

scissors, sterilized iron and 3 new syringes) in it.

4. Turn on the laminar flow hood and put the tray with sterilized equipments in the laminar flow

hood.

5. Get the incubator tray and place it on the laminar flow hood.

6. Candle only the eggs of the first and second group of test samples with the use of an egg

candler. Locate where the air sac is and mark it with a small ‘x’. Use a marker.

7. Place the candled and marked eggs in the incubator tray placed on the laminar flow hood.

8. On the sterilized beaker, pour 100 mL of ethyl alcohol (70% solution) and a 150 mL of

mineral water. Stir well with a stirring rod.

9. Soak cotton on the solution made with the use of a sterilized forceps (WARNING: Don’t over

soak the cotton). Make sure that the eggs are disinfected, especially around the area you want

to bore into (air sac area).

10. Get the extract of the pineapple peelings by following the steps above.

11. Take the desired concentration of your prepared inoculum and inject it in the eggs, use 1

syringe in the samples with the same concentration.

12. For the first group of test samples, inject 0.3 mL of the pineapple peelings extract to the

vessels of the eggs with the use of a syringe. For the second group of test samples, inject 0.5

mL of the pineapple peelings extract to the vessels of the eggs with the use of a syringe.

13. Seal the holes with candle wax

 22

14. After sealing the bores, incubate the eggs (treated samples) for 72 hours/3 days with the

controlled samples.

15. Get the last egg.

16. After 3 days, compare the physical appearance of the eggs with respect to concentration. The

untreated sample is expected to be hatched. Analyze whether the pineapple peelings extract is

proangiogenic or antiangiogenic.
 23

Table 1. Amount of Pineapple Peelings Extract Inoculated to the Experimental Set-up No.1
TREATMENT SAMPLE A SAMPLE B SAMPLE C
(T1.A) (T1.B) (T1.C)

Pineapple Peelings Extract 0.3 mL 0.3 mL 0.3 mL

Table 2. Amount of Pineapple Peelings Extract Inoculated to the Experimental Set-up

No.2 TREATMENT SAMPLE A SAMPLE B SAMPLE C
(T2.A) (T2.B) (T2.C)
Pineapple Peelings 0.5 mL 0.5 mL 0.5 mL
Extract

Table 3. Amount of Pineapple Peelings Extract Inoculated to the Controlled Set-up

TREATMENT SAMPLE A SAMPE B SAMPLE C
(C1) (C2) (C3)
none none none none
 24

Gathering of Inoculation of the

Sealing of the eggs
Materials extract

Extraction of
Purchase of duck Incubation of the
pineapple peelings
eggs eggs for 3 days
extract

Collection of
Candling of eggs Gathering of data
Pineapple Peelings

Dividing of eggs
(Set-up 1, Set-up 2, Labeling of eggs
Controlled Set-up according to set-up
and basis)

Figure 1. Methodology Flowchart

 25

Table 4. Conceptual Framework of the Study

Pineapple Peelings 1. Collect 1 kilo of pineapple peelings/skin

from a pineapple vendor.

2. Rinse/wash the peelings with mineral

water.

3. Slice the pineapple peelings using a knife

into small pieces for the juicer to easily

extract its juice. Also, be sure to use a

chopping board.

4. Put the pineapple peelings on the juicer.

Use the juice collector cup of the juicer as

the container.

5. Wait for it to finish the extraction process.

Duck Eggs 1. Collection of organic fertilized eggs (one

day old)
2. The eggs were divided into groups: 3 eggs
were the first group, 3 eggs were the
controlled samples and the remaining one
served as a basis in determining the
average density of the blood vessels of the
same age.
3. The eggs were labelled-first group of
treated samples as: T1.A, T1.B, T1.c;
second group of treated sample as: T2.A,
T2.B, T2.C and the controlled samples as:
C.1, C.2, and C.3.
4. All of the sterilized equipments were put in
the tray where tissue papers were placed on
it.
 26

5. Put the tray, where sterilized equipments

are placed, on the laminar flow hood.
6. The incubator tray was placed on the
laminar flow hood.
7. Egg candler was used to locate the air sac
of the egg and using a marker, air sac was
marked with a small ‘x’.
8. In making the disinfectant solution, pour
100 mL of ethyl alcohol and 150 mL of
mineral water on a sterilized beaker and
stirred with a stirring rod.
9. To make sure that the eggs are disinfected,
soak cotton on the solution with the use of
forceps and rub on the area especially
around the area to bore to.
10. Take the desired inoculum and inject it in
the eggs, use one syringe in the samples
with the same concentration.
11. 0.3 mL of the pineapple peelings extract
was injected to the first group, 0.5 mL to
the second group.
12. Candle wax was used to seal the holes.
13. After sealing the holes, eggs were
incubated for 72 hours/3days with the
controlled samples.
14. Get the last egg.
15. After 3 days, compare the physical
appearance of the eggs with respect to
concentration. The untreated samples were
expected to hatch. Analyze whether the
pineapple extract is
proangiogenic/antiangiogenic.
 27

Chapter IV
RESULTS AND DISCUSSION

Presentation of Results

At this point, the extract has been inoculated into the different treated samples and was ready

to be tested. Moreover, the researchers prepared 3 set-ups (2 treated set-ups, controlled set-up).

The 2 treated/experimental set-ups were inoculated with 0.3 mL and 0.5 mL of pineapple

peelings extract respectively. After injecting/inoculating the extract, the researchers incubated the eggs for

3 days. After the incubation, they break eggs and put its contents inside a petri dish, gathered the data and

analyzed the significant differences by comparing the treated/experimental set-ups from the controlled

set-up. The tables below shows the data obtained from the experiment conducted by the researchers.

Table 5. Results for the Experimental Set-up No. 1

I. AMOUNT STATUS
COLOR OF
SAMPLE OF OF THE ODOR YOLK BLOOD VESSELS
THE YOLK
EXTRACT EMBRYO
T1.A 0.3 mL lifeless foul intact pale yellow had many blood
vessels but not as
many as that of the
blood vessels of the
controlled set-up.
T1.B 0.3 mL lifeless foul damaged pale red no blood vessels
and observed.
scattered
inside the
petri dish
T1.C 0.3 mL lifeless foul damaged dark yellow there were few blood
and vessels observed
scattered
inside the
petri dish
 28

Findings:

Table 5 shows that for sample T1.A, the duck embryo was lifeless and had foul odor. Its yolk

was still intact and was pale yellow in color. It had blood vessels but not as many as that of the blood

vessels of the controlled set-up (refer to appendix, figure 12). For sample T1.B, the duck embryo was

lifeless and had foul odor. Its yolk was damaged and was scattered inside the petri dish. The yolk was

pale red in color. There were no blood vessels observed up (refer to appendix, figure 13). For sample

T1.C, the duck embryo was lifeless and had foul odor. Its yolk was also damaged and was scattered inside

the petri dish. The yolk was dark yellow in color. There were few blood vessels observed. up (refer to

appendix, figure 14).

Analysis of Data:

The data from the table indicated that the pineapple peelings extract had an effect to the

growth and over-all development of the duck embryos inoculated with 0.3 mL of the extract though the

effects to the 3 samples were not the same in some aspects. As stated earlier, to inoculate means simply to

introduce -- or insert -- one substance into another. It is also a known fact that chorioallantoic membrane

(CAM) in embryonated eggs is one of the different sites of viral inoculation. This can be related to

chorioallantoic membrane (CAM) assay, wherein in this assay, quantitation is performed usually 3 days

after implantation and involves counting the number of CAM vessels in the area of filter disk. In

connection to this, angiogenesis is a progressive, multistep physiological process by which new blood

vessels are generated from pre-existing vasculature. 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. In CAM assay, in

response to proangiogenic stimuli, the newly formed blood vessels appear converging toward the disk in a

wheel-spoke pattern. Inhibition of angiogenesis by antiangiogenic compounds results in the lack of new

blood vessel formation and sometimes in disappearance of pre-existing vessel networks. Angiogenesis

levels can be also determined by counting branch points in the vessels adjacent to the disks. Since there
 29

were lack and disappearance of blood vessels, these data could be interpreted to mean that pineapple

peelings extract is antiangiogenic and thereby has anti-cancer potential.

Table 6. Results for the Experimental Set-up No. 2

SAMPLE AMOUNT STATUS ODOR YOLK BLOOD VESSELS
COLOR OF
OF OF THE
THE YOLK
EXTRACT EMBRYO
T2.A 0.5 mL lifeless foul covered pale yellow no blood vessels
the whole observed.
embryo
T2.B 0.5 mL lifeless foul damaged yellow there were few blood
and vessels observed
scattered
inside the
petri dish
T2.C 0.5 mL lifeless foul damaged yellow orange there were few blood
and vessels observed
scattered
inside the
petri dish

Findings:

Table 6 shows that for sample T2.A, the duck embryo was lifeless and had foul odor. Its yolk

covered the whole embryo and was pale yellow in color. There were no blood vessels observed up (refer

to appendix, figure 16). For sample T2.B, the duck embryo was lifeless and had foul odor. Its yolk was

damaged and was scattered inside the petri dish. The yolk was yellow in color. There were few blood

vessels observed (refer to appendix, figure 17). For sample T2.C, the duck embryo was lifeless and had

foul odor. Its yolk was also damaged and was scattered inside the petri dish. The yolk was yellow orange

in color. There were few blood vessels observed (refer to appendix, figure 18).
 30

Analysis of Data:

The data from the table indicated that the pineapple peelings extract had an effect to the

growth and over-all development of the duck embryos inoculated with 0.5 mL of the extract though the

effects to the 3 samples were not the same in some aspects. As stated earlier, to inoculate means simply to

introduce -- or insert -- one substance into another. It is also a known fact that chorioallantoic membrane

(CAM) in embryonated eggs is one of the different sites of viral inoculation. This can be related to

chorioallantoic membrane (CAM) assay, wherein in this assay, quantitation is performed usually 3 days

after implantation and involves counting the number of CAM vessels in the area of filter disk. In

connection to this, angiogenesis is a progressive, multistep physiological process by which new blood

vessels are generated from pre-existing vasculature. 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. In CAM assay, in

response to proangiogenic stimuli, the newly formed blood vessels appear converging toward the disk in a

wheel-spoke pattern. Inhibition of angiogenesis by antiangiogenic compounds results in the lack of new

blood vessel formation and sometimes in disappearance of pre-existing vessel networks. Angiogenesis

levels can be also determined by counting branch points in the vessels adjacent to the disks. Since there

were lack and disappearance of blood vessels, these data could be interpreted to mean that pineapple

peelings extract again, is antiangiogenic and thereby has anti-cancer potential.

 31

Table 7. Results for the Controlled Set-up

SAMPLE AMOUNT STATUS ODOR YOLK BLOOD VESSELS
COLOR OF
OF OF THE
THE YOLK
EXTRACT EMBRYO
C1 0 mL alive and none intact and yellow there were a lot of
moving healthy blood vessels
observed
C2 0 mL alive and none intact and yellow there were a lot of
moving healthy blood vessels
observed
C3 0 mL alive and none intact and yellow there were a lot of
moving healthy blood vessels
observed

Findings:

Table 7 shows that for sample C1, the duck embryo was alive and moving. It doesn’t have

odor. Its yolk was intact and healthy and was yellow in color. There were a lot of blood vessels observed

(refer to appendix, figure 20). For sample C2, the duck embryo was alive and moving. It doesn’t have

odor. Its yolk was intact and healthy. The yolk was yellow in color. There were a lot of blood vessels

observed (refer to appendix, figure 21). For sample C3, the duck embryo was also alive and moving. It

doesn’t have any odor. Its yolk was intact and healthy and was yellow in color. There were a lot of blood

vessels observed (refer to appendix, figure 22).

Analysis of Data:

The data from the table indicated that the duck embryos were alive and moving because they

weren’t inoculated with any amount of pineapple peelings extract.

 32

II.

Tables 5 and 6 shows that there was a significant difference, but not in all aspects, in the

status of the duck embryos, observed physical appearance of the yolks, observed blood vessels, and the

anti-cancer potential of pineapple peelings extract as a whole between the first and second

treated/experimental set-ups. In terms of the physical appearance of the yolks, the second

treated/experimental set-up had the worse results than that of the first (refer ro appendix, figures 14&18).

In terms of the status of the duck embryos, both the embryos of the first and second treated/experimental

set-ups were lifeless. In terms of the observed blood vessels, the first treated/experimental set-up had a

better result than the second for it had much more blood vessels (refer to appendix, figure 14&18). Based

from the results, the bigger the amount of the extract to be inoculated, the higher its antiangiogenic effect.

On the other hand, the smaller the amount of the extract to be inoculated, the lower its antiangiogenic

effect. The extract’s antiangiogenic effect depicts that it has anti-cancer potential.
 33

Chapter V
SUMMARY AND CONCLUSION

Summary

This study was performed to determine if pineapple peelings extract has anti-cancer potential

and specifically in terms of: amount of extract, status of the duck embryo, observed physical appearance

of the yolk and observed blood vessels.

Extract from the pineapple peelings was prepared. 10 duck eggs (16 days old) were also

prepared and divided into 3 different set-ups. Each of the these set-ups has 3 replicates. 3 samples for the

first treated/experimental set-up which were inoculated with 0.3 mL of the extract, 3 samples for the

second treated/experimental set-up which were inoculated with 0.5 mL of the extract and 3 samples for

the controlled set-up which weren’t inoculated with the extract. After inoculation, all of the eggs were

incubated for 3 days.

Conclusion

At the end of the experiment, the researchers were able to conclude that pineapple peelings

extract has an anti-cancer potential since there were lack and disappearance of blood vessels. Based from

the results, the bigger the amount of the extract to be inoculated, the higher its antiangiogenic effect. On

the other hand, the smaller the amount of the extract to be inoculated, the lower its antiangiogenic effect.

The extract’s antiangiogenic effect depicts that it has anti-cancer potential.

Recommendations

The researchers would like to recommend to use other inoculation process or assays in a way

that the next researcher will be able to count the blood vessels of the embryos to have a statistical data and

will make the study/the results of the experiment more accurate. Also, they would like to recommend to

use other fruit like: mangosteene, kerson fruit, passion fruit, etc.
 34

BIBLIOGRAPHY

Websites:

http://www.fao.org/nr/water/cropinfo_pineapple.html

https://www.youtube.com/watch?v=c2A2K51cKs8In this study

https://www.organicfacts.net/health-benefits/fruit/pineapples.html

http://undergroundhealthreporter.com/pineapple-enzyme-fights-cancer/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4545797/

http://www.seattleorganicrestaurants.com/vegan-whole-food/fruit-nutrition-seeds-
skin.php#sthash.R82WoSo9.dpuf

http://naturalsociety.com/pineapples-been-used-cancer-treatment/

http://fiberfacts.org/for-health-professionals/benefits-of-a-high-fiber-diet/

http://www.esciencecentral.org/ebooks/food-as-source-of-anticancerigen/the-health-benefits-of-dietary-
fiber-evidence-of-its.pdf

http://vitamincfoundation.org/vitcancer.php

https://medlineplus.gov/cancer.html

http://www.rainbow.coop/library/bromelain-as-an-anticancer-agent/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2699944/

http://www.jove.com/video/52411/the-ovo-chick-chorioallantoic-membrane-cam-assay-as-an-efficient

http://www.gmanetwork.com/news/story/567513/scitech/science/pinay-student-bags-int-l-prize-for-
malunggay-anti-tumor-research#sthash.yfxlm2qf.dpuf

http://animals.mom.me/stages-duck-development-egg-7868.html

http://classroom.synonym.com/inoculate-mean-microbiology-12655.html

http://www.afrivip.org/sites/default/files/01_virology_eggs_complete.pdf

http://jem.rupress.org/content/16/2/155
 35

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2699944/#R23

http://www.biology-online.org/dictionary/Inoculation

http://classroom.synonym.com/inoculate-mean-microbiology-12655.html

https://en.wikipedia.org/wiki/Amniotic_sac

https://en.wikipedia.org/wiki/Allantois

Books:

Mondal S, Bhattacharya S, Pandey JN, Biswas M. 2011. Evaluation of acute anti-inflammatory effect of

Ananas Comosus leaf extract in Rats. Pharmocologyonline.3:1312–1315.

Livio M, Gaetano GDe, Donati MB. 1978. Effect of bromelain of fibrinogen level, prothrombin complex

and platelet aggregation in the rat-a preliminary report. Drugs under Experimental and Clinical Research.

1:49–53

NeubaurR Ra.1961. A plant protease for potentiation of and possible replacement of antibiotics. Exp Med

Surg. 19:143-60.

Varengo R. 1972 Feb. Absorption of tetracycline in presence of bromelain after oral

administration.Arzneimittelforschung. 22(2):410-2.

Maurer HR. 2001 Aug. Bromelain: biochemistry, pharmacology and medical use.Cell Mol Life

Sci.58(9):1234-45.

Tochi BN, Wang Z, Xu SY, Zhang W. 2008.Therapeutic application of pineapple protease (Bromelain): a

review. Pakistan Journal of Nutrition. 7(4):513–520.

(Ley CM, Tsiami A, Ni Q, Robinson N. Zhong Xi Yi Jie He Xue Bao. 2011 Jul. A review of the use of

bromelain in cardiovascular diseases.9(7):702-10

 36

APPENDICES
 37

Figure 2. Purchasing of duck eggs

Figure 3.Collection of pineapple peelings

 38

Figure 4. Preparation of the materials to be used

Figure 5. Unpacking of the gloves

 39

Figure 6. Candling of the eggs

Figure 7. Marking of the air sac

 40

Figure 8. Inoculation of the extract

Figure 9. Sealing of the eggs

 41

Figure 10. Preparation of the eggs for incubation

Figure 11. Breaking of the eggs for the gathering of data

 42

Figure 12. Sample T1.A

Figure 13. Sample T1.B

 43

Figure 14. Sample T1.C

Figure 15. Experimental Set-up No. 1

 44

Figure 16. Sample T2.A

Figure 17. Sample T2.B

 45

Figure 18. Sample T2.C

Figure 19. Experimental Set-up No. 2

 46

Figure 20. Sample C1

Figure 21. Sample C2

 47

Figure 22. Sample C3

Figure 23. Controlled Set-up

 48

Figure 24. The samples and set-ups of the experiment