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ABSTRACT
Category

Title: Anti-angiogenic and Antibacterial Property of Bangus (Chanos chanos) Bile Proponents: Jared Carlo O. nrile! Joegin Christian "i#! "loyd Joshua $o Ad%iser: &ogelio Ponte'o (chool: $eneral (antos )ope Christian (chool
Pick and shade only one box !iochemistry

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 !otany 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 Chemistry *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$e moisture. After drying" the proponents took the extract of the bangus bile through methanol soaking. The proponents bought Computer ,- Duck eggs from a professional breeder. .uickly thereafter" the proponents made three treatments" !angus bile extract" positi$e control *Sim$astatin+ and a negati$e control *%ater+. Science Then" filter papers %ere soaked on each treatment and placed on the CA) layer of the egg. The eggs %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 4ngineering 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 $ariance. 3or anti(bacterial screening" the proponents used three treatments( !angus bile extract" 4n$ironmental negati$e control *%ater+ and positi$e control *Amoxicillin+. 4ach treatment has three replications. 5esults sho%ed 6ones of inhibition in both Staphylococcus aureus and Escherichia Science 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 )edicine and to commercial medicine. 8ealth

I/We hereby certify that the above is the result of work done in my/our research . __________ Signature of Team Leader _________ Date

)icrobiology

Physics

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 *CE.1 in+ in length. They ha$e no teeth and generally feed on algae and in$ertebrates. They occur in the ndian <cean and across the Pacific <cean" tending to school around coasts and islands %ith reefs. The young fry li$e at sea for t%o to three %eeks and then migrate to mangro$e s%amps" estuaries" and sometimes lakes and return to sea to mature sexually and reproduce. !lood $essels are any tubular canals in %hich the blood circulatesH an artery" $ein" or capillary. The blood $essels are part of the circulatory system and function to transport blood throughout the body Through blood $essel count" %e can determine the anti(angiogenic property of a certain ob2ect. f more blood $essels appear in the medium" this means that the ob2ect has an angiogenic property %hile lesser blood $essels mean ha$ing an anti(angiogenic property. 3ertile eggs are eggs that %ill de$elop into a li$ing organism %hen incubated. 4ggs that are sold in the market for consumption are usually non(fertile. n order for an egg to be fertile" it must undergo a process in %hich %e call fertili6ation. This means there must be a male present in order for the egg to be fertile. A duck egg is a fertili6ed egg %ith a nearly(de$eloped embryo inside. The main significance is on the anti( angiogenic property of the bile" as %ell as the anti(bacterial. 1.! State"ent of the #ro$%e" This study aims to determine the antibacterial property" e$aluate the anti(angiogenic property and identify the phytochemical constituents of !angus bile. Specifically" this study %as conducted to ans%er the follo%ing /uestionsH 0. Does !angus !ile ha$e any anti(bacterial effectI ,. Does !angus !ile contain anti(angiogenic componentsI

1.&. 'enera% o$(ect)*es

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 $essel formation of the medium. The anti(bacterial effect %ill be tested by using Escherichia coli, Staphylococcus aureus and Pseudomonas aureginosa as the specimens in :irby(!auer Testing. There %ere three treatments used in this study J Positi$e Control *Amoxicillin+" ;egati$e Control *%ater+ and the bangus bile extract. The said experiments %ere conducted from Auly to September in the Science Laboratory of 9eneral Santos 8ope Christian School. 1.- Re*)e. of re%ated L)terature This chapter presents the re$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)$acter)a% Antibacterial is the destroying or suppressing the gro%th or reproduction *)iller(:eane 4ncyclopedia and Dictionary of )edicine" ;ursing" and Allied 8ealth+ Chor)oa%%anto)c "e"$rane of bacteria.

Also called the chorioallantois or abbre$iated to CA) L 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+

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 1D)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.

*9rolier 4ncyclopedia of :no%ledge+

S nerg

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 effect of !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 are Escherichia 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

Primary )aterials Laboratory Primary Laboratory 4/uipments )aterials 4/uipments 0. Duck 4gg 0. Petri Dish 0. 4scherichia coli 0. !eaker ,. !angus ,. 3ilter Paper bile extract ,. Staphylococcus ,. Alcohol C. !eaker aureus Lamp C. noculating C. !angus bile 1. Scalpel Loop extract @. 3orceps 1. ;utrient Agar 1. 3orceps @. %ater @.Autocla$e -. )icroscope

<ther <ther )aterials )aterials

0. )asking Tape 0. Laboratory go%n ,. Alcohol ,. Surgical C. Distilled 9lo$es =ater C.Surgical Safety 9lo$es 1. )ask @. 1.Laboratory Lighter 9o%n @.Pentel Distilled -. Pen =ater Slide P. Sticks -.Psedomaonas P. 9lass -. Bernier -.)atch )asking Slip D. Tissue auregenosa D. Co$er Caliper Tape P. 9raduated P. Pentel Pen E. Alcohol Lamp Cylinder 0G. 8ot Plate D. 8ot Plate 00. 3lask 0,. ncubator

!.& belo%.

6rocedure F%o. Chart This part sho%s the procedures undertaken for the study7 a more detailed chart of the procedure is sho%n

!.+

E3#er)"entat)on and 'enera% 6rocedure 6rocedure of e3tract)ng the su$stance fro" Bangus $)%e 1Chanos 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$e for 4scherichia coli. As the agar is still in its li/uid state" the proponent /uickly added the E. coli" S. aureus and . aeru!inosa bacteria on the petri dishes. 3ilter papers %ere soaked in the !angus bile" %ater and dissol$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

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$e anti(bacterial properties. Escherichia coli and Staphylococcus aureus both sho%ed positi$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.D@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 $essels of 1,.CCC !angus bile %ith ,,.,,, and sim$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.

5uptured $isible blood $essel formed sho%ing branched $essels under the naked eye treated %ith !angus !ile.

SU44AR9 OF FINDIN'S AND CONCLUSIONS

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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$e bacterium %ere cultures in the schoolOs science laboratory. 9lass %ares %ere then prepared for the antibacterial testing. The testing %as then follo%ed using aseptic techni/ue. The Petri dishes used in the testing %ere then put in the incubator in ,1 hours. <bser$ations of the 6one of inhibition %ere measured on the next day and the results %ere recorded. The results sho%ed that the extracts of the bangus bile ha$e antibacterial properties. After the antibacterial screening" the anti(angiogenic testing %as done. The researchers prepared the materials as %ell as the bangus bile extract. The researchers bought fresh duck eggs and they incubated it for six days. Then" the prepared bile solution %as put on each concentration and placed on the CA) layer of the egg. The eggs %ere then /uickly sealed and incubated for another 1D hours. After the incubation process" the blood $essels of the treated eggs %ere obser$ed under a microscope and compared to the control unit. 5esults sho%ed that both the !angus !ile and sim$astatin exhibit anti(angiogenic properties. C<;CL>S <;S !ased on the experimentation" results and data gathered" the researchers %ere able to formulate the follo%ing conclusionsH 0. ,. The extract of the bangus bile has an anti(bacterial property that inhibit the gro%th of bacteria such as E. coli and S. aureus and non for . aeru!inosa The extract of the bangus bile has anti(angiogenic properties.

RECO4ENDATIONS 3rom the findings of the study" it can be obser$ed that there %ere research gaps. The follo%ing %ould ser$e as research agenda and methodological suggestions in future studies. 0. ,. C. 1. 3urther studies to be conducted %ith more bacterial cultures to pro$e the $alidity of the inhibitory effect. Testing %ith other a$ailable antibiotics aside from the ones used in this study. Testing %ith anti(cancer medicines than that of using cholesterol(lo%ering medicines. Conducting the anti(angiogenesis screening after pro$iding more days for the incubation of the duck eggs.

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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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6)ctor)a%s

Bangus 6etr)

Soak)ng of B)%e )n A%coho% 6re#ar)ng the 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 ANAL9SIS OF ;ARIANCES

Incu$at)ng the Treated Egg

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