anti-angiogenic and antibacterial property of bangus bile research paper.doc
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ABSTRACT
I/We hereby certify that the above is the result of work done in my/our research.
__________ _________
Signature of Team Leader Date
2
Category
Pick and shade only one box
The milkfish is an important seafood in Southeast Asia and some Pacific slands.!ecause milkfish is notorious for being much bonier than other food fish" deboned milkfish"
called #boneless bangus# in the Philippines" has become popular in stores and markets" yet
bangus bile is not gi$en importance and is being thro%n a%ay. The proponents further
in$estigate the uses of this &left o$er' to pro$ide usage for this specimen" one of those is anti(
carcinogenic %hich %as conducted by the researchers by using Chorioallantoic )embrane*CA)+ Assay.
The proponents gathered !angus bile from the public market to be used for the
experiment. The !angus bile %ere then air dried to remo$e moisture. After drying" the
proponents took the extract of the bangus bile through methanol soaking. The proponents bought
,- Duck eggs from a professional breeder. uickly thereafter" the proponents made threetreatments" !angus bile extract" positi$e control *Sim$astatin+ and a negati$e control *%ater+.
Then" filter papers %ere soaked on each treatment and placed on the CA) layer of the egg. Theeggs %ere then /uickly sealed and incubated for another 011 hours. After the incubation process"
the blood $essels of the treated eggs %ere obser$ed under a microscope and compared to the
control unit. The anti(angiogenic screening of the bangus bile extract sho%ed a significant result
in decreasing the number of blood $essels in the embryo of the duck egg based on the re2ection
of the null hypothesis in the analysis of $ariance.
3or anti(bacterial screening" the proponents used three treatments( !angus bile extract"
negati$e control *%ater+ and positi$e control *Amoxicillin+. 4ach treatment has three
replications. 5esults sho%ed 6ones of inhibition in both Staphylococcus aureusandEscherichia
coli %hich can be compared to the positi$e control.
Thus" the proponents concluded that the methanol extract of !angus bile" has an
antibacterial and anti(angiogenic property7 therefore can be used as a much cheaper alternati$e
to commercial medicine.
!iochemistry
!otany
Chemistry
Computer
Science
4ngineering
4n$ironmental
Science
)edicine and
8ealth
)icrobiology
Physics
Title: Anti-angiogenic and Antibacterial Property of Bangus(Chanos chanos) Bile
Proponents: Jared Carlo O. nrile! Joegin Christian "i#! "loydJoshua $oAd%iser: &ogelio Ponte'o
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TABLE OF CONTENTS
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INTRODUCTION
This part includes the background of the study" statement of the problem" hypothesis" significance of the
study" operational definition of terms" and the scope and limitations that %ould lead the readers about the research.
1.1 Background of the stud
Angiogenesis simply means the formation of ne% blood $essels. n a more specific definition it is a
physiological process in$ol$ing the gro%th of ne% blood $essels from pre(existing $essels" especially blood $essels
that supply oxygen and nutrients to cancerous tissues. t is a normal process in gro%th and de$elopment of the
embryo and fetus" as %ell as in %ound healing. 8o%e$er" this is also a fundamental step in the transition of tumors
from a dormant state to a malignant state. =e can simply find out if an ob2ect can cure or lessen the effects of
certain cancers" by the means of blood $essel count. This is done due to the fact that %ithout the blood $essels" the
cancer tissues %hich are present in our body cannot and %ill not exist" these blood $essels supply the needed
nutrients for the cancer tissues. 3urthermore" angiogenesis cannot be done %ith non(fertile eggs because %ith these
kinds of eggs" there is no sperm cell and egg cell formation.
)ilkfish ha$e a generally symmetrical and streamlined appearance" %ith a si6able forked caudal fin. They
can gro% to but are most often about 0 meter *E.1 in+ in length. They ha$e no teeth and generally feed on algae and
in$ertebrates. They occur in the ndian
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The ob2ecti$e of this study is to e$aluate the anti(angiogenic property and anti(bacterial property of bangus
bile extract.
1.+ S)gn)f)cance of the stud
The significance of this study is to see the anti(bacterial and anti(angiogenic characteristics of !angus bile
and be able to find out if it could be an alternati$e and ne% source of antibiotic. Also" %ith this study" the researchers%ill be able to help and contribute additional information to the medical field of science.
1., Sco#e and L)")tat)on
This study is only limited in the experimentation on the anti(angiogenic property of the !angus bile extract"
and the anti(bacterial property of the said extract. The anti(angiogenic property can be studied through the use of
sim$astatin as the positi$e control" %ater *negati$e control+" bangus bile extract" the combined property of the
bangus bile extract and sim$astatine and by solely depending on the blood $essel formation of the medium. The
anti(bacterial effect %ill be tested by usingEscherichia coli, Staphylococcus aureus and Pseudomonas aureginosa as
the specimens in :irby(!auer Testing. There %ere three treatments used in this study J Positi$e Control
*Amoxicillin+" ;egati$e Control *%ater+ and the bangus bile extract.
The said experiments %ere conducted from uly to September in the Science Laboratory of 9eneral Santos
8ope Christian School.
1.- Re*)e of re%ated L)terature
This chapter presents the re$ie% of related literature and the operational definition of terms used in this
research. Literatures %ere cited on the bases of their importance in the conduct of the study.
Ant)/Ang)ogen)c
a drug or substance used to stop the gro%th of tumors and progression of cancers by limiting the pathologic
formation of ne% blood $essels *angiogenesis+. *)osbyKs Dictionary of Complementary and Alternati$e )edicine+
Ant)$acter)a%
Antibacterial is the destroying or suppressing the gro%th or reproduction of bacteria.
*)iller(:eane 4ncyclopedia and Dictionary of )edicine" ;ursing" and Allied 8ealth+
Chor)oa%%anto)c "e"$rane
Also called the chorioallantois or abbre$iated to CA) is a $ascular membrane found in eggs of
someamniotes" such as birds and reptiles. t is formed by the fusion of the mesodermal layers of t%o de$elopmental
structuresH the allantois and the chorion. n mammals" this structure forms the placenta. *)osbyKs Dictionary of
Complementary and Alternati$e )edicine+
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Escher)ch)a co%)
Escherichia coli is found in large numbers as a normal inhabitant of the large intestine of %arm(blooded
animals. =hene$er they lea$e their usual habitat" these organisms can cause urinary tract infections" peritonitis"
endocarditis" and other diseases. Some strains cause se$ere gastroenteritis.
(9rolier 4ncyclopedia of :no%ledge)
0)r$/Bauer Ant)$)ot)c Test)ng D)sc D)ffus)on Test)ng2
:irby !auer Testing is a test %hich uses antibiotic(impregnated %afers to test %hether particular bacteria
are susceptible to specific antibiotics. :no%n /uantities of bacteria are gro%n on agar plates in the presence of thin
%afers containing rele$ant antibiotics. f the bacteria are susceptible to a particular antibiotic" an area of clearing
surrounds the %afer %here bacteria are not capable of gro%ing *called a 6one of inhibition+.
*en.%ikipedia.orgM%ikiM:irby(!auer_antibiotic_testing+
A"o3)c)%%)n
Amoxicillin is a moderate(spectrum" bacteriolytic" N(lactamantibiotic used to treat bacterialinfections
caused by susceptible microorganisms. t is usually the drug of choice %ithin the class because it is better absorbed"
follo%ing oral administration" than other N(lactam antibiotics. Amoxicillin is one of the most common antibiotics
prescribed for children.
*httpHMM%%%.medicinenet.comMnorfloxacin+
Sta#h%ococcus aureus
Staphylococcus is a genus of spherical bacteria" some species of %hich are normal inhabitants of the skin
and others of %hich causes disease. The most common pathogen" S.aureus" is fre/uently responsible for boils"
carbuncles" abscesses" osteomyelitis" and" sometimes" meningitis an pneumonia" and is the most common cause of
bacterial food poisoning and hospital infections.
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*9rolier 4ncyclopedia of :no%ledge+
Snerg
Synergy is the correlation or concurrence of action bet%een different organs in health or disease" or bet%een
different drugs. *=ebsterOs Dictionary of the 4nglish Language+
4ater)a%s and 4ethods
!.1 Research Des)gn
This study %ill follo% a simple research design. t attempts to sho% the importance and the medicinal effectof !angus *Chanos chanos)bile specifically the anti(angiogenic property of the said !angus bile.The study %ill
ha$e $arious tests conducted at the Science Laboratory of 9eneral Santos 8ope Christian School. The first part is%here the proponents extract the substance from the !angus bile *Chanos chanos).
The proponents then conduct the anti(angiogenic screening %herein Duck eggs %ere used as the medium.
The proponents conducted the :irby(!auer testing to see if the extract is effecti$e in inhibiting the gro%th of
bacteria. The bacteria used areEscherichia coli, seudomonas aeru!inosa,and Staphylococcus aureus.
Data gathered %as obser$ed after the treatment using the solution then analy6ed through blood $essel
count for each of the concentration.
!.! 4ater)a%s and E5u)#"ents
This sub(section presents the ra% materials and laboratory e/uipments to be used in the experimental
setup. These %ere categori6ed based on the antibacterial susceptibility test and anti(angiogenic testing.
4ater)a%s and Instru"ents used for ant)/ang)ogen)c screen)ng
The said study used $arious instruments and e/uipments to perform the anti(angiogenic screening.
Ta$%e !.1 4ater)a%s used for Ant)/Ang)ogen)c Screen)ng Ta$%e !.! 4ater)a%s used for 0)r$/Bauer Test)ng
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!.& 6rocedure F%o Chart
This part sho%s the procedures undertaken for the study7 a more detailed chart of the procedure is sho%nbelo%.
8
Primary)aterials
Laboratory4/uipments
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!.+ E3#er)"entat)on and 'enera% 6rocedure
6rocedure of e3tract)ng the su$stance fro" Bangus $)%e Chanos chanos2.
!angus !ile *Chanos chanos+ %ere dried to remo$e unpleasant odor. Dried !angus bile %as crushed using
mortal and pestle and a 0GmL $olume of distilled %ater added to the po%dered bile then use for experimentation.
0)r$/Bauer Ant)$)ot)c Test)ng7D)sc D)ffus)on Test)ng
After mixing the )ueller 8inton Agar %ith distilled %ater" it %as heated on a hot plate. ;ine Petri Dishes"
along %ith the inoculating loop and the )ueller 8inton Agar %ere sterili6ed in the autocla$e. The Petri dishes %ere
di$ided into three treatments ( %ater"bangus bile and Amoxicilin. Also" the Petri dishes %ere further labeled as
positi$e for Staphylococcus aureus and negati$e for 4scherichia coli. As the agar is still in its li/uid state" the
proponent /uickly added the E. coli"S. aureus and . aeru!inosabacteria on the petri dishes. 3ilter papers %ere
soaked in the !angus bile" %ater and dissol$ed amoxicilin. =hen the agar solidified" these treated paper discs %ere
then placed on each of the three replications. After ,1 hours" the 6one of inhibition of each treatment %as then
measured %ith the use of the Bernier Caliper.
Ant)/Ang)ogen)c Screen)ng
3irst and foremost" the proponents bought fresh Duck eggs from a professional handler. After purchasing
the duck eggs" they %ere /uickly incubated using the incubator of the school. T%el$e filter papers %ere then used"
three for each treatment. t %as dipped onto the said concentration. The soaked filter papers %ere then placed on the
CA) layer of the eggs and %ere sealed tightly to a$oid unnecessary entry of bacteria. After placing the filter paper
on the CA) of the eggs" the eggs %ere then incubated for 011 hours and %ere closely obser$ed. The Duck eggs
%ere then remo$ed after the said time period and the contents %ere closely obser$ed on a Petri Dish. Samples of the
blood $essels of the eggs %ere then taken and obser$ed under the microscope. The data gathered %as then analy6ed
and compared.
6RESENTATION8 ANAL9SIS AND INTER6RETATION OF DATA
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This part presents the data gathered through actual obser$ation and the changes that happened to the blood
$essels of the Duck eggs incubated for - days.
Resu%ts of the 0)r$/Bauer Test)ng
The results sho%ed that the extracts did ha$e anti(bacterial properties.Escherichia coliand Staphylococcus
aureusboth sho%ed positi$e results %hile Pseudomonas aureginosa did not.
The analysis of $ariance sho%ed the re2ection of the null hypothesis and accepted the alternati$e hypothesis
of ha$ing differences. 8o%e$er" Tukey test of difference in the mean of each group sho%ed that the !angus bile
mean has no significant difference %ith that of the mean of amoxicillin. t sho%ed that !angus bile has comparable
inhibitory effect %ith that of amoxicillin in inhibiting the gro%th of Staphylococcus aureus.
The gro%th inhibition of Pseudomonas aureginosa %ith !angus bile sho%ed a greater difference of 0.@1
mm. The analysis of $ariance sho%ed re2ection of the null hypothesis and accepted the alternati$e hypothesis of
ha$ing differences. 8o%e$er" Tukey test for difference in the mean of each group sho%ed that the each treatment is
significantly different from each other" %hich sho%ed that !angus bile is not effecti$e in inhibiting the gro%th of
Pseudomonas aurigonosa.
=hile the gro%th inhibition of 4. coli %ith !angus !ile under study. t is clear that the gro%th inhibitin of
!angus !ile and amoxicillin sho%ed a slight difference of G.-@ mm. The analysis of $ariance sho%ed the re2ection
of the null hypothesis and accepted the alternati$e hypothesis of ha$ing differences. 8o%e$er" Tukey test for
difference in the mean of each group sho%ed that the each !angus !ile and amoxicillin has no signicantly different
from each other" %hich sho%ed that !angus bile is effecti$e in inhibiting the gro%th of 4. coli
AN'IO'ENIC SCREENIN'
The test sho%ed the number of blood $essel formed on the choreoallantoic membrane assay of the fertile
duck egg. =ater %ith the greatest number of blood $essels of 1,. !angus bile %ith ,,.,,, and sim$astatin %ith
,0.GGG.
The analysis of $ariance sho%ed the re2ection of the null hypothesis and accepted the alternati$e hypothesis
of ha$ing differences. 8o%e$er" Tukey test for difference in the mean of each group sho%ed that the !angus bile
mean has no significant difference %ith that of the mean of sim$astatin. This only sho%ed that !angus bile has
comparable anti(angiogenic effect %ith that of sim$astatin in inhibiting the gro%th and formation of abnormal blood
$essels.
SU44AR9 OF FINDIN'S AND CONCLUSIONS
10
5uptured $isible blood $essel formed
sho%ing branched $essels under the
naked eye treated %ith !angus !ile.
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This portion presents the summary of findings and conclusions made from the study and recommendations
gi$en by the researchers.
F)nd)ngs
The researchers started %ith the collection of the bangus bile . The collected samples %ere air dried for @
days. After drying" they soaked %ith )ethanol in 4rlenmeyer flasks in the science laboratory of the said school.
There %ere t%o screenings used. After each screening" results %ere again recorded despite the positi$e or negati$e
outcomes. The results on some of the screening %ere positi$e and some are negati$e.
Antibacterial screening %as conducted using three treatments. A pure culture of a gram positi$e and a gram
negati$e bacterium %ere cultures in the schoolOs science laboratory. 9lass %ares %ere then prepared for the
antibacterial testing. The testing %as then follo%ed using aseptic techni/ue. The Petri dishes used in the testing %ere
then put in the incubator in ,1 hours.
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B)$%)ogra#h
Internet
httpHMMen.%ikipedia.orgM%ikiMChorioallantoic_membrane
en.%ikipedia.orgM%ikiM:irby(!auer_antibiotic_testing
httpHMM%%%.medicinenet.comMnorfloxacin
en.%ikipedia.orgM%ikiMScreening
%%%.scientificcommons.orgMstatinManti(angiogenic
!ooks
)osbyKs Dictionary of Complementary and Alternati$e )edicine
The American 8eritage Dictionary of the 4nglish Language
)iller(:eane 4ncyclopedia and Dictionary of )edicine" ;ursing" and Allied 8ealth
=ebsterOs Dictionary of the 4nglish Language
9rolier 4ncyclopedia of :no%ledge
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http://www.scientificcommons.org/statin/anti-angiogenichttp://www.scientificcommons.org/statin/anti-angiogenic -
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6)ctor)a%s
Soak)ng of
Bangus B)%e )n A%coho%
6re#ar)ng the
6etr) D)shes
Ster)%):at)on of 6etr) D)shes
6etr) D)shes )th Agar
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4ak)ng a ho%e )n the $%unt end of the egg Incu$at)ng the Treated Egg
ANAL9SIS OF ;ARIANCES
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