TheinvestigationofantibacterialactivitiesofCloveessentialoilon

Escherichiacoli
.

Introduction

AccordingtoThaiNationalListofEssentialMedicine,cloveiscommonlyknowasherbal
medicineinwhichusuallyusedfordentalcareandindigestion .[5] Insomeof
ThaiHerbal
Pharmacopoeia,suchasThaiBunjob,hasclaimedthatclovecanalsousedfordiarrheathat
doesntcausedbyinfection .[6] However,manyThaipeopleunderstandthatclovecancures
diarrheawithoutknowingthedetail.Themisunderstandingofdrugusagecanleadtothe
distressoftheuser,asclovehasachemicalcompound,eugenol,thatmightbeunsafeforsome
groupofpeoplesuchasinchildrenand
patientswhohavecoagulopathy .[9]

Syzygiumaromaticumo
rclovehasaneugenolasamajorcompound,especiallyinclove
oilwhichcouldbemadeupof80to90percentofeugenol .[2] Thismakesclovesometimes
uses
[9]
[6]
tothreatenbloodimpurities becauseitcaninhibittheplateletactivitypowerfully .Moreover,
eugenolisalsoanantisepticandantiinflammatory , [2][8] whichoftenusedfortoothachesorpain
andswellingofmouthandthroat .[7][9] Nevertheless,itislikelytobeunsafewhenusingina
largeproportionorrepeated,anddangeroustotakenbychildrenorpeoplewhohas
coagulopathy.Cloveoilcanbeirritatedanddamagetotissueswhenpeoplefrequentlyapply
cloveoilontheskinorgum.Especiallyinchildren,itcancausesseveresideeffectssuchas
seizures,liverdamage,andfluidimbalances,becausechildrenhasamoredelicateskin.
Furthermore,eugenolinclovealsocausedslowbloodclotting,whichisdangeroustousewith
peoplewhohasbleedingdisorder .[2][9]

However,inrecentyears,manyresearchescameoutfortheantimicrobialactivityfrom
clove.Forinstance,accordingtoXies
research(2015),theresultcameoutthatcloveoilwith
the90.6percentofeugenolhaskilledalltermitesandresiststothetreefungi.Hence,itcould
beconcludethattheyhaveastrongantitermicandantifungalactivities .[10] Moreover,insomeof
foodpackagingindustryhasusedclovebudpowdertocoatedonfilmsorpackages.These
powderwillprotectfoodfromcontaminationofpathogens.Thesupportthisidea,studyfrom
Abdali(2015)showsthesolutionthattheclovebudpowderinhibitsthegrowthoftested
microorganismsincluding
E.coli,L.innocua,a
nd
S.aureus .[1] Therefore,itispossibleforclove
oiltohaveanantibacterialactivityandthreatendiarrheathatcausedbybacteria.

I
nthisexperiment,wesetupwith5differentconcentrationofcloveoilemulsion0.05
mL/mL,0.1mL/mL,0.25mL/mL,0.50mL/mL,and1mL/mL.Byassumingthatthediarrheais
causedbygramnegative
E.coli,
wetesteachofcloveoilconcentrationonthem.Thepetridish
willbedividedin4sections,inwhicheachconcentrationwillhaveaholefordroppingcloveoil
emulsion.Lastly,wewillobserveandcalculateitsclearzonetoseewhetherthecloveoilcan
eliminateorinhibittheantibacterialactivitiesornot.


Materials

Reagents
XL1bluecells(
Escherichiacoli)
Cloveoil
Tryptone
YeastExtraction
BacteriologicalAgar
NaCl

Equipments
LaboratoryAutoclave
Incubator
Labvortex
ImageJprogramme

Procedure

GeneralNotes
E.coli
isanaerobicbacteria,itrequiredapproximately5timesofairinthecentrifugal
tube.
Store
E.coli
inanincubatorat37degreecelsius.
Warmthemediabeforestartingbacteriaculture.
Thisexperimentsetupwith16petridishes.
Cloveoilwillbemixedwithbrothtomakeanemulsionofcloveoil.

PartI:PreparationofMedia.
Thepreparationof750mLofbrothconsistof
7.5gramsofTryptone
3.75gramsofYeastextraction
7.5gramsofYeastextraction
5gramsofNaCl
750mLofroomtemperaturewater
750mLofbrothisseparatedinto250mLofBroth,and500mLofmediabyadding7.5
gramsofBacteriologicalAgar.
Bothof250mLofbrothand500mLofmediasterilizebyLaboratoryAutoclaveat121
degreecelsiusfor20minutes.

PartII:InoculationofE.Coli
Sterilizetheworkingareawith70percentofethanol.
Using15mLofcentrifugaltubeandapproximately2mLofBrothtoinoculatewith
XL1bluecells.

Storethemintheincubatorfor18hours.

PartIII:OvernightcultureofE.Coli
Prepare5mLofdifferentconcentrationsofcloveoilemulsion.
0percentofCloveoil(pureBroth)
5percentofCloveoil
10percentofCloveoil
25percentofCloveoil
50percentofCloveoil
100percentofCloveoil
Dividedaplateinto4differentconcentrationswhicheverydisheshastohave1of
control.
UsingLabvortextoprecipitate
E.coli.

Pour30mLofwarmmediatothesterilizeddish.
Afterthemediaissetup,gentlypipette0.2mLofcellsupandtransferintothedishwith
amedia.
Spreadthemwiththespreaderuntilitisnotsoaked.
Creating4holesonthemediawithpasteurpipette.
Fillineachholewithapproximately0.1mLofemulsion.
Storeplatesintheincubatorforatleast18hours
Seetheresultandcollectthedata.
AnalyzetheresultbyusingImageJprogramme.

Results

Figure1Exampleofthepetridishwith5percentofcloveoil(topright),
10percentofcloveoil(bottomleft),and25percentofcloveoil(bottomright).


Figure2Exampleofthepetridishwith0percentofcloveoil(topright),
25percentofcloveoil(bottomleft),and50percentofcloveoil(bottomright).

Figure3Exampleofthepetridishwith0percentofcloveoil(topright),
25percentofcloveoil(bottomleft),and100percentofcloveoil(bottomright).


Figure4Totalclearzoneareaindifferentconcentrationsofcloveoilemulsion.

Discussion

Accordingtotheresult(seefigure13),wecanseethatcloveoilwipeoutthecoloniesof
XL1bluecells,whichisagramnegativebacteriathatresistancetothetetracycline.Moreover,
Theclearzonewillbelargerasthecloveoilismoreconcentrated(seefigure4),butusingthe
dilutedcloveoilisstilleffectiveasat5percentconcentrationcanstilleliminatethesurrounded
E.coli
.Thepredictionofthisoutcomeisthecloveoilcoulddamagethebacterialcellwalland
outermembrane,whichcausethecellularcomponents,suchasnucleicacidandATP,leaksout
fromthecellandmakethemdie .[3][4] Therefore,cloveoilhasanantibacterialactivitywhich
doesntcontainanycomponentsofdrugthatisatetracycline.Also,itispossibletousecloveoil
totreatdiarrheathatcausedbygramnegative
E.coli.

However,thiscannotbeconcludethatcloveoilcankillallthebacteria.Accordingto
research
Thespecificantibacterialactivityofliposomeencapsulated
Clove
oilandits
applicationintofu(2015),cloveoilhasnoeffectonsometypeofbacteriasuchas
E.Coli
that
[3]
doesntsecretePFTs . Theusageofcloveoilstillhastobecarefulasitssideeffectscanbe
severe,soitshouldbedilutedbeforeingestionassmallproportioncanstillkillbacteria.Also,its
antibacterialactivitycanalsokillessentialbacteriainhumanbodysuchasE.coliinintestine
thatassistindigestion.Additionally,thedeviationmightbeoccursinthisexperimentasbroth
consistsofnutrientsforcelltogrow.Thismightreducetheefficiencyofantibacterialactivityof
cloveoil,andcausethedatatobeinaccurate.Thus,furtherresearchesandexperimentsare
neededtoconcludethehypothesis.

Conclusion

Cloveoilisanantibacterialagentthatdoesntcontaintetracyclineinitscomponents.It
hasapossibilitythatcloveoilcanthreatendiarrheathatcausedbybacteria.Nevertheless,
cloveoilcannotkillallthebacteria.Furtherresearchesarestillneededtoconcludethe
hypothesis.

References.

1 Abdali,H.,&Ajji,A.(2015).
Developmentofantibacterialstructuresandfilmsusingclovebud
powder.

IndustrialCropsandProducts,
72,
214219.

2 Broadhurst,C.L.,&Duke,J.A.(1997).OilofCloves:TheBenefitsofEugenol.
MotherEarth
Living
.Retrievedfrom
http://www.motherearthliving.com/plantprofile/eugenol.aspx
[9/3/2016]

3 Cui,H.,Zhao,C.,&Lin,L.(2015).Thespecificantibacterialactivityofliposomeencapsulated
Clove
oilanditsapplicationintofu.
FoodControl,56,1
28134.

4 Cui,H.,Zhao,C.,&Lin,L.(2016).Synergeticantibacterialefficacyofcoldnitrogenplasma
andcloveoilagainst
Escherichiacoli
O157:H7biofilmsonlettuce.
FoodControl,66,8
16.

5 DepartmentofMedicalSciences,MinistryofPublicHealth.(2012).
NationalListofEssential
Medicines(volumeII)
.Retrievedfrom
http://kpo.moph.go.th/webkpo/tool/Thaimed2555.pdf
[2/3/2016]

6 FacultyofPharmaceuticalSciences,UbonRatchathaniUniversity.(2010).
.ThaiCrude
Drug
.Retrievedfrom
http://www.thaicrudedrug.com/main.php?action=viewpage&pid=18
[2/3/2016]

7 LisBalchin,M.(2006).Cloveoil.
AromatherapyScience:Aguideforhealthcareprofessionals
(pp.170172).London,UK:PharmaceuticalPress.

8 NationalCenterforBiotechnologyInformation.Eugenol.
PubChemCompoundDatabase.

Retrievedfrom
https://pubchem.ncbi.nlm.nih.gov/compound/eugenol#section=MechanismofAction
[16/3/2016]


9 U.S.NationalLibraryofMedicine.(2015).Clove.
MedlinePlus.
Retrievedfrom
https://www.nlm.nih.gov/medlineplus/druginfo/natural/251.html
[2/3/2016]

10 Xie,Y.,Yang,Z.,Cao,D.,Rong,F.,Ding,H.,&Zhang,D.(2015).Antitermiticandantifungal
activitiesofeugenolanditscongenersfromtheflowerbudsof
Syzygiumaromaticum(
clove).
IndustrialCropsandProducts,77,
780786.