Evaluation of the Antibacterial Potential of Liquid and Vapor Phase Phenolic Essential Oil

17 Pages·2016·5.31 MB·English

RESEARCHARTICLE
Evaluation of the Antibacterial Potential of
Liquid and Vapor Phase Phenolic Essential Oil
Compounds against Oral Microorganisms
Tong-HongWang1,2,3☯,Shih-MinHsia4☯,Chi-HaoWu4,Shun-YaoKo5,6,Michael
YuanchienChen7,8,Yin-HuaShih9,Tzong-MingShieh10*,Li-ChuanChuang11,12,Ching-
YiWu13
1 TissueBank,ChangGungMemorialHospital,Tao-Yuan,Taiwan,2 ResearchCenterforIndustryof
HumanEcology,ChangGungUniversityofScienceandTechnology,Tao-Yuan,Taiwan,3 Graduate
InstituteofHealthIndustryTechnology,ChangGungUniversityofScienceandTechnology,Tao-Yuan,
a11111
Taiwan,4 SchoolofNutritionandHealthSciences,TaipeiMedicalUniversity,Taipei,Taiwan,5 Graduate
InstituteofMedicalScience,CollegeofHealthScience,ChangJungChristianUniversity,Tainan,Taiwan,
6 InnovateResearchCenterofMedicine,ChangJungChristianUniversity,Tainan,Taiwan,7 Department
ofOral&MaxillofacialSurgery,ChinaMedicalUniversityHospital,Taichung,Taiwan,8 SchoolofDentistry,
CollegeofMedicine,ChinaMedicalUniversity,Taichung,Taiwan,9 Mind-BodyInterfaceLab,ChinaMedical
UniversityHospital,Taichung,Taiwan,10 DepartmentofDentalHygiene,CollegeofHealthCare,China
MedicalUniversity,Taichung,Taiwan,11 DepartmentofPediatricDentistry,ChangGungMemorialHospital
atLinkou,Taoyuan,Taiwan,12 GraduateInstituteofCraniofacialandDentalScience,CollegeofMedicine,
OPENACCESS ChangGungUniversity,Taoyuan,Taiwan,13 InstituteofOralBiology,NationalYang-MingUniversity,
Taipei,Taiwan
Citation:WangT-H,HsiaS-M,WuC-H,KoS-Y,
ChenMY,ShihY-H,etal.(2016)Evaluationofthe ☯Theseauthorscontributedequallytothiswork.
AntibacterialPotentialofLiquidandVaporPhase *[email protected]
PhenolicEssentialOilCompoundsagainstOral
Microorganisms.PLoSONE11(9):e0163147.
doi:10.1371/journal.pone.0163147 Abstract
Editor:ImtaiyazHassan,JamiaMilliaIslamia,
INDIA Theaimofthepresentstudywastodeterminetheantibacterialactivitiesofthephenolic
Received:February22,2016 essentialoil(EO)compoundshinokitiol,carvacrol,thymol,andmentholagainstoralpatho-
gens.Aggregatibacteractinomycetemcomitans,Streptococcusmutans,Methicillin-resis-
Accepted:September2,2016
tantStaphylococcusaureus(MRSA),andEscherichia.coliwereusedinthisstudy.The
Published:September28,2016
minimuminhibitoryconcentrations(MICs),minimumbactericidalconcentrations(MBCs),
Copyright:©2016Wangetal.Thisisanopen
bacterialgrowthcurves,temperatureandpHstabilities,andsynergisticeffectsoftheliquid
accessarticledistributedunderthetermsofthe
andvaporEOcompoundsweretested.TheMIC/MBCoftheEOcompounds,rangingfrom
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and thestrongesttoweakest,werehinokitiol(40–60μg/mL/40-100μg/mL),thymol(100–
reproductioninanymedium,providedtheoriginal 200μg/mL/200-400μg/mL),carvacrol(200–400μg/mL/200-600μg/mL),andmenthol
authorandsourcearecredited.
(500-morethan2500μg/mL/1000-morethan2500μg/mL).Theantibacterialactivitiesof
DataAvailabilityStatement:Allrelevantdataare thefourEOphenoliccompoundbasedontheagardiffusiontestandbacterialgrowth
withinthepaperanditsSupportingInformation
curvesshowedthatthefourEOphenoliccompoundswerestableunderdifferenttempera-
files.
turesfor24h,butthethymolactivitydecreasedwhenthetemperaturewashigherthan
Funding:Thisstudywassupportedbygrantsfrom
80˚C.Thecombinationofliquidcarvacrolwiththymoldidnotshowanysynergisticeffects.
ChinaMedicalUniversity(CMU103-S-38),Taipei
Theactivitiesofthevaporouscarvacrolandthymolwereinhibitedbythepresenceofwater.
MedicalUniversity(03C0720007A,104-6202-015-
111),andtheNationalScienceCouncil,Taiwan Continualviolentshakingduringcultureenhancedtheactivityofmenthol.Bothliquidand
(MOST104-2320-B-182A-009-andNSC102- vaporoushinokitiolwerestableatdifferenttemperaturesandpHconditions.Thecombina-
2314-B-039-015-MY3).Thefundershadnorolein
tionofvaporoushinokitiolwithzincoxidedidnotshowsynergisticeffects.Theseresults
studydesign,datacollectionandanalysis,decision
topublish,orpreparationofthemanuscript. showedthattheliquidandvaporphasesofhinokitiolhavestronganti-oralbacteriaabilities.
PLOSONE|DOI:10.1371/journal.pone.0163147 September28,2016 1/17
AntibacterialPotentialofLiquidandVaporPhasePhenolicEssentialOilCompounds
CompetingInterests:Theauthorshavedeclared Hinokitiolhasthepotentialtobeappliedinoralhealthcareproducts,dentalmaterials,and
thatnocompetinginterestsexist. infectioncontrolstoexertantimicrobialactivity.
Introduction
Essentialoils(EOs)arevolatileoilyliquidsobtainedfromdifferentpartsofplants.EOsare
widelyusedinfoodpreservationandhealthcareproductsbecauseoftheirpotentantibacterial
activity[1–3],reductionofoxidativestress[4],andanti-inflammatoryactivities[5].ManyEOs
aregenerallyrecognizedassafebytheFoodandDrugAdministration(FDA)oftheUnited
Statesandhavebeenusedasartificialflavoringsandpreservatives.EOsareoftendilutedinsol-
ventsforspraysandrinsesorareheatedtovolatilizethemtoprohibitbacterialgrowthand
eliminateunpleasantodors.ManyEOscontainterpenoids,whichincludephenols,aldehydes,
ketones,alcohols,ethers,andhydrocarbons.Generally,phenolicEOshavestrongerantibacte-
rialactivitythanotherconstituents.Theantibacterialactivitiesoftheterpenoidsareaffectedby
theirfunctionalgroups,hydrophobicity, andenvironmentalconditions.
TheantibacterialactivityoftheconstituentsinEOsagainstcariogenicbacteriahasbeenexten-
sivelydiscussed[6,7].HinokitiolisanaturalcomponentisolatedfromChamacyparistaiwanensis.
Ithasalreadybeendemonstratedthatanoralcaregel(therapeuticdentifrice)containinghinoki-
tiolimprovedthequalityoflifefororallichenplanuspatients[8]andeffectivelyforreducedoral
malodor[9].TheEOofLippiagracilisSchauerleaveshassignificantsynergismwithseveralantibi-
otics[10].ThebioactivefractionsofLippiasidoidesdisrupttheintegrityandweakenthestructure
ofbiofilms[11].UsingL.sidoides-basedessentialoilmouthrinseforoneweekwasefficaciousin
reducingbacterialplaquesandgingivalinflammationinpatients[12],anditreducedthesalivary
Streptococcusmutanslevelsinchildrenwithcariesafterfivedaysoftreatment[13].
ThemajorconstituentsofL.gracilisandL.sidoidesarecarvacrolandthymol[10,14].Carva-
crolandthymolhavebeenusedasfoodadditivesbecauseoftheirantimicrobialandantioxi-
dantactivities[15,16].Thymolcanalsobeusedinvarnishtopreventcaries[17],andcarvacrol
haswell-knownanti-Candidapotentialandcanpreventdenturestomatitis[14].Mentholis
eithermadesyntheticallyorobtainedfrommint.Mentholisusedinconfections,chewinggum,
andoral-careproducts,suchastoothpasteandmouthrinse,toreducebacterialgrowth[18]
andoralmalodor[19].ThesefourphenolicEOcompoundsarevaluableforapplicationasfood
additivesororalhealthcareproducts.
Dentalcariesandperiodontitisrepresentthemajororalinfectiousdiseases.Bacterialpla-
quescomposedofnativeoralfloraaccumulateondentalsurfacesandaretheprimaryetiologi-
calagentsofperiodontaldiseaseanddentalcaries[20].Indentalplaques,S.mutansand
Aggregatibacteractinomycetemcomitans arerespectivelyconsideredtobehighlycariogenicand
periodontopathicmicroorganisms.Staphylococcalfoodpoisoningiscausedbyconsuming
foodscontaminatedwithenterotoxinsproducedbyStaphylococcusaureus[21].Methicillin-
resistantStaphylococcusaureus(MRSA)arefacultative-anaerobicStaphylococci,andtheyhave
beenreportedtocolonize77.8%oforalcancerpatientsfollowingsurgery[22].Escherichia.coli
cancauseseriousfoodpoisoninginhumans.Fecal-oraltransmissionisthemajorrouteby
whichE.coliistransmittedtoinduceentericdiseases.E.colihasbeenusedasanidealindicator
organismtotestenvironmentalsamplesforfecalcontamination.
Hinokitiol,carvacrol,thymol,andmentholhavesimilarstructuresandmolecularweights
(Fig1).Carvacrolandthymolarestructuralisomersbuthavedistinctphysicalcharacteristics.
Carvacrolisaliquidatroomtemperaturebecauseithasalowmeltingpoint,whiletheothers
arepowdersatroomtemperature.Mentholmeltsnearhumanbodytemperature,and
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AntibacterialPotentialofLiquidandVaporPhasePhenolicEssentialOilCompounds
Fig1.ThechemicalstructuresofthephenolicEOcompounds.(A)hinokitiol;(B)carvacrol;(C)thymol;(D)menthol.
doi:10.1371/journal.pone.0163147.g001
hinokitiolandthymolbothmeltat50°C.Thevaporpressureofhinokitiolislowerthanthatof
theothercompounds.Carvacrol,thymol,andmentholtendtoevaporateorvolatilizeeasilyat
moderatetemperatures,whilehinokitioldoesnot(Table 1).ThesephenolicEOcompounds
areusedincombinationwithothermaterialsatdifferentconcentrations,pH,andtemperatures
invarioushealthcareproducts.Theantibacterialactivityofhinokitiolissynergistically
increasedwhencombinedwithzincoxide,andthecombinationofcarvacrolwiththymolwas
alsoshowntohavesynergisticeffects[23,24].
FullyunderstandingtheantibacterialactivitiesofthesefourphenolicEOcompoundsindiffer-
entstatesandunderdifferentconditionswouldbehelpfulforchoosingsuitableadditivesforvari-
oushealthcareproducts.Inthisstudy,A.actinomycetemcomitans,S.mutans,MRSA,andE.coli
wereusedasdiseaseindicatorsforperiodontaldisease,caries,infection,andentericdiseases,
respectively,totesttheantibacterialpotentialofhinokitiol,carvacrol,thymol,andmentholinthe
liquidandvaporphasesundervarioustemperatureandpHconditionsandatdifferentmixratios.
Theresultsofthesestudiesprovideinformationthatcanhelptogenerateeffectivenewapplica-
tionsfornoveldentalformulations,foodadditives,oralhealthfoods,andinfectioncontrol.
MaterialsandMethods
Antimicrobialagentsandchemicals
Hinokitiol(469521),carvacrol(282197),thymol(T0501),menthol(M2772),zincoxide(ZnO,
721077),andchlorhexidine(CHX,282227)werepurchasedfromSigma-Aldrich(St.Louis,
Table1. Thephysicalcharacteristicsofhinokitiol,carvacrol,thymol,andmenthol.
Hinokitiol Carvacrol Thymol Menthol
Molecularweight 164.2 150.22 150.22 156.27
Formula C H O C H O C H O C H O
10 12 2 10 14 10 14 10 20
Density,25˚C(g/cm3) 1.127 0,977 0,965 0,89
Vaporpressure,25˚C(mm/Hg) 8.9×10−5 2.96×10−2 3.76×10−2 3.20×10−2
Boilingpoint,1atm(˚C) 303.4 236~237 231~232 214~216
Meltingpoint,1atm(˚C) 48~53 3~4 49~51 34~36
doi:10.1371/journal.pone.0163147.t001
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AntibacterialPotentialofLiquidandVaporPhasePhenolicEssentialOilCompounds
MO,USA).TheEOsweredissolvedordilutedinDMSO,andZnOwasdissolvedin2.5NHCl.
Allofthecompoundsweremadeasstocksolutionsof100mg/mLandwerestoredat-20°C.
ThechemicalstructuresandphysicalcharacteristicsofthesefourphenolicEOcompoundsare
showninFig1andTable 1.
Microorganismsandmedia
A.actinomycetemcomitans (ATCCnumber:33384),S.mutans(ATCCnumber:25175),Methi-
cillin-resistantS.aureus(MRSA,ATCCnumber:33591),andE.coli(ATCCnumber:10798)
wereusedinthestudy.A.actinomycetemcomitanswasculturedinbrainheartinfusion(BHI)
broth,S.mutansandMRSAwereculturedintrypticsoybroth(TSB),andE.coliwascultured
inLysogenybroth(LB).Thebacteriawereinoculatedbylooptransferfromfrozentubesinto3
mLslantnutrientbroth,thenweresubjectedto200rpmshakingcultureat37°Cfor24h.Bac-
teriafromtheseculturesweretransferredontoanappropriatesolidmediumandincubated
overnight.Selectedcoloniesweretransferredtotheappropriateliquidmediumandwereincu-
batedfor4–6htoachievelogphasegrowth.Theopticaldensityofeachcultureat600nm
(OD600)wasadjustedto1.0usingfreshbrothtogiveastandardinoculumof106cfu/mL.
Stockculturesweremaintainedat-80°Cingrowthbrothcontaining25%sterileglycerol.
Directcontactagardiffusiontests
Fordirectcontactagardiffusiontests,5mLoffreshbrothagarwaspreparedin6-cmPetri
dishes,andbacteriawerespreadat5×105cfuonthebrothagarsurface.Aliquots(4–10μL)of
thedifferenttestcompounds(200μg-1000μg)wereplacedon6-mmdiameterfilterdiscs.
Usingthedirectcontactmethod,thediscswereplacedonthecenterofthesolidifiedagarsur-
face.Thecultureswereincubatedfor24–96hat37°C,andthediameteroftheinhibitionzone
wasthenrecorded.
Minimuminhibitoryconcentration(MIC)andminimumbactericidal
concentration(MBC)ofphenolicEOcompoundsdeterminedbythe
brothdilutionmethod
Cellsuspensionswerepreparedin2mLofbrothwithvariousconcentrationsofthephenolic
EOcompoundsin15mLculturetubesbyinoculationwith2μlof106cfu/mLfromeachglyc-
erolstock.Thecultureswereincubatedat37°Cat200rpmfor24h.Tubesshowingnovisible
turbiditywereconsideredtorepresenttheMICandweresubsequentlyinoculatedontosterile
6cmnutrientagarplateswithoutanyphenolicEOcompoundandincubatedfor24h.Thelow-
estconcentrationatwhichnogrowthwasobservedwasconsideredtobetheMBC[23].
Growthcurveassay
Thegrowthcurveassaywasconductedina96-wellformatthatwasadaptedfromapreviously
describedmethod[25].Bacterialsuspensionspreparedwithvariousconcentrationsofphenolic
EOcompoundsin1mLofliquidbrothin1.6mLmicrocentrifugetubeswereinoculatedwith
1μLof106cfu/mLfromtheglycerolstocks,200μLwerethentransferredto96-wellplatesfor
testing,and200μLofsterileliquidbrothwasusedasablank.The24-hgrowthcurveanalyses
wereperformedforthefouroralpathogensat37°C.Thekineticanalysisincludeda10-sshak-
ingstepbeforeeachofthetimepointmeasurementsoftheOD600,whichwererecordedat30
minintervals.ThedatawereanalyzedusingtheVersaMaxTMandSoftmax1Pro(version
5.4.1,California,US)softwareprograms.
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AntibacterialPotentialofLiquidandVaporPhasePhenolicEssentialOilCompounds
Heatstabilitytest
ToevaluatethestabilitiesofthephenolicEOcompoundsatdifferenttemperatures,thetestcom-
poundswerepre-incubatedat4°C,25°C,50°C,80°C,and100°Cfor1hforaheatstabilitytest,
followedbydirectcontactdiffusiontests.Thediameteroftheinhibitionzonewasrecorded.
Vaporphaseagardiffusiontests
TheagardiffusiontestwasusedtoevaluatetheantibacterialactivitiesofthephenolicEOcom-
poundsinthevaporphase,anditwastechnicallysimilartothedirectcontactdiffusiontest,with
thesame6cmPetridishformat,bacterialculture,filterdiscsize,andEOcompoundloading[26].
However,thefilterdiscswereplacedinthecenterofthecoverofthePetridishinthisexperiment.
Thedisheswerethensealedusinglaboratoryparafilmtoavoidevaporationofthetestcompounds,
followedbyincubationat37°Cfor24–96h.Thediameteroftheinhibitionzonewasrecorded.
StabilityofthephenolicEOcompoundsundervariouspHconditions
ThepHofthewaterwasadjustedtopH3,pH5,pH7,pH9,andpH11byaddingHClor
NaOH,anditwasmeasuredbyapHmeterbeforeuse.Atotalof500μgofeachphenolicEO
compoundwasdissolvedin5μLDMSO,whichwasthenmixedwith5μLofwaterwithdiffer-
entpHvalues(pH3topH11).Then,thevaporphaseagardiffusiontestwasperformed.The
diameterofthediffusionzonewasrecorded.
Statistics
Alloftheassayswereperformedinduplicateortriplicate.Differencesbetweenspecificmeans
wereanalyzedbyaone-wayanalysisofvariance(ANOVA). Groupmeanswerecompared
usingaone-wayANOVA andTukey’s test.Thedataareshownasthemeans±standarddevia-
tion(SD).DifferencesbetweenthevariantswereconsideredsignificantwhenP<0.05.The
CompuSynsoftware(Version1.0,ComboSynInc.,USA)wasusedtoquantifysynergismand
antagonismforthedrugcombinations.AlltherawdatawasshowedinS1File.
Results
AntibacterialactivityofthefourphenolicEOcompounds
Allofthetestcompoundswereusedat500μginthedirectcontactdiffusiontests.Hinokitiol
showedthelargestinhibitionzone,andmentholshowedlittleinhibitioninthisstudy.
Althoughcarvacrolandthymolarestructuralisomers,theyshoweddifferentinhibitionzones
forallofthebacteriatested.Fig2Ashowstheresultsofthedirectcontactagardiffusiontestof
thefourphenolicEOcompoundsagainstMRSA.A.actinomycetemcomitans wasmoresensi-
tivetothephenolicEOcompoundsthantheotherbacteria.TheinhibitionzonesforA.actino-
mycetemcomitans,S.mutans,andMRSAwerethelargestforhinokitiol,followedbythymol,
carvacrol,thenmenthol.However,E.coliwasmoresensitivetocarvacrolthanthymol.The
diameteroftheinhibitionzoneformentholwas0.667±0.116cminA.actinomycetemcomitans
and0.667±0.058cminE.coli,buttherewasnoinhibitionzoneinthedisheswithS.mutans
andMRSA(Fig2B).Thediameteroftheinhibitionzoneinouranalysisisshownbythesolid
column/symbolandhollowcolumn/symbolrepresentingthedirectcontactandvaporphase
agardiffusionmethod,respectively.Thedottedlinerepresentsthe0.6cmdiameterofthefilter
discusedinthedirectcontactagardiffusionmethod,whilethiswasnotusedinthevapor
phaseagardiffusionmethod.Becausethediametersoftheinhibitionzonesweretotallyformed
bythegaseousphenoliccompoundsinthevaporphasestudies,wedidnotincludethefilter
disccoverageforthosesamples.
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Fig2.TheantibacterialactivitiesofthephenolicEOcompounds.(A)MRSAtreatedwith500μgphenolicEOcompounds,asassessedusing
directcontactagardiffusiontests.(B)ThephenolicEOcompoundswerealltestedat500μg.ThemicroorganismsexaminedwereA.
actinomycetemcomitans(Aa),S.mutans(Sm),MRSA,andE.coli.Dottedline,the0.6cmdiameterofthefilterdisc.*P<0.05,**P<0.01,***
P<0.001comparedwithA.a.ineachcompoundgroup;a,b,c,anddwereP<0.05,comparewithA.a.,S.m.,MRSA,andE.coliinthehinokitiol
group,respectively;e,P<0.01basedonacomparisonofthecarvacrolandthymolgroups.
doi:10.1371/journal.pone.0163147.g002
TheMICandMBCofthefourphenolicEOcompounds
DifferentconcentrationrangesofthefourphenolicEOcompoundsweretestedbythebroth
dilutionmethodtodeterminetheMICandMBC.Hinokitiolwastestedfrom20to120μg/mL,
carvacrolandthymolfrom50to1000μg/mL,andmentholfrom250to2500μg/mL.Chlorhex-
idine(CHX)iscommonlyusedasanactiveingredientinmouthrinsetoreducedentalplaques
andoralbacteria.Hence,CHXwasusedasapositivecontrolandwastestedatconcentrations
rangingfrom0.5to4μg/mL.TheMICandMBCofthetestphenolicEOcompoundsagainst
thefouroralpathogensarelistedinTable 2.Theresultsoftheinhibitionzone(Fig2B)and
MIC/MBC(Table2)experimentswereconsistent.Hinokitiolwasastrongantiseptic,carvacrol
andthymolwererelativelymoderateantiseptics,andmentholwasaweakantiseptic.
Microorganismgrowthisdelayedinaconcentration-dependentmanner
bythefourphenolicEOcompounds
Thekineticmicroplatemethodwasusedtoanalyzethebacterialgrowthinhibitionfor24h.A
logphasedelayoradelayinthestationaryphaseofthegrowthcurveaftera24-hincubation
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Table2. TheMICandMBCofthefourphenolicEOcompoundsagainstfourmicroorganisms(μg/mL).
Aa Sm MRSA E.coli
MIC MBC MIC MBC MIC MBC MIC MBC
Hinokitiol 40 40 40 100 60 60 40 100
Carvacrol 200 200 400 600 400 600 400 400
Thymol 100 200 200 400 200 200 200 400
Menthol 500 1000 1000 1000 1000 1000 >2500 >2500
CHX 1 1 1 1 1 2 1 1
MICandMBCdataforphenolicEOcompoundsandchlorhexidine(CHX;positivecontrol)inA.actinomycetemcomitans(Aa),S.mutans(Sm),MRSA,and
E.coliasdeterminedinthreeindependentexperimentsusingthebrothdilutionmethod.
doi:10.1371/journal.pone.0163147.t002
impliesthatbacterialgrowthwasinhibitedorthatthephenolicEOcompoundskilledthebac-
teria,respectively.Interestingly,thecultureswithadelayinthestationaryphase(basedonthe
OD600ofbacterialcultures)weremorecommoninthesamplestreatedwithlowconcentra-
tionsofphenolicEOcompoundsthaninthecontrolsamples,whichmighthavebeencaused
bybacterialaggregationintheculture,suchasinMRSAculturestreatedwith100μg/mLcarva-
crol.TheresultsforthehinokitiolgroupwereconsistentwiththeMICofeachmicroorganism
examinedinthestudy.Inthecarvacrolandthymolgroups,theconcentrationsthataffectedthe
microorganisms’growthcurves(lessthan100–200μg/mL)werelowerthantheMIC(100–
400μg/mL),butthisfindingwasinvertedinthementholgroup(Fig3).Overall,thelogphases
ofthemicroorganisms’growthcurvesweredose-dependentlydelayed,exceptfortheS.mutans
groupstreatedwith10and20μg/mLhinokitiol.
ThephenolicEOcompoundsareheatstable
After500μgofhinokitiol,carvacrol,andthymolwerepre-incubatedatdifferenttemperatures
(4to100°C)for1h,theinhibitionzoneswerenotsignificantlydifferentforthefouroralpatho-
gensbasedonthedirectcontactagardiffusiontest(Fig4).Theantibacterialactivitiesofthe
heatedphenolicEOcompoundsfromstrongesttoweakestwereconsistentwithpreviousfind-
ingsforthecompounds(Fig2B,Table 2).However,whentwotothreeEO-loadeddiscswere
placedina10-cmdishtoperformdirectcontactdiffusiontests,thebacterialcolonynumber
andsizeweredecreased,andtheinhibitionzonesincreased.Thephenomenonwasnot
observedintheCHXgroup(datanotshown).TheseresultssuggestedthattheEOphenolic
compoundsmightevaporatetointerferewithbacterialgrowth,andthemoleculardiffusion
couldbeexcludedasafactoraffectingthefindings.Theinhibitionzonesof500μgmenthol
wereexcludedduetoitsweakantibacterialactivity.
ThevaporphenolicEOcompoundsdisplayantibacterialactivity
To verifytheantibacterialactivityofthephenolicEOcompoundsduetoevaporationat37°C,
thevaporphaseagardiffusiontestwasperformed(Fig5A).Vaporous hinokitiolalsoshowed
thebestantibacterialactivityoutofthefourcompoundstestedinthestudy.Vaporous carva-
crolandthymolshowedsmallandclearinhibitionzonesinGram-negativebacteria(A.actino-
mycetemcomitans andE.coli)butweakactivityagainstGram-positivebacteria(S.mutansand
MRSA).TheS.mutansandMRSAcoloniesweresmallandthin,meaningthattherewasweak
inhibitionbyvolatilecarvacrolandthymol.Thevaporousmentholdidnotshowanyinhibition
zone(Fig5B).However,theindistinctmarginofinhibitionzonemeasurementsmayhaveled
tosomeerrorindeterminingthesizesoftheinhibitionzones(Fig5C).
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Fig3.PhenolicEOcompoundsdelaythemicroorganismgrowthcurvesinaconcentration-dependentmanner.Variousconcentrationsofthe
phenolicEOcompoundswereusedtotesttheirimpactonthebacterialgrowthcurves.Thebacterialgrowthcurvesinthepresenceofvariousphenolic
EOconcentrations(hollowdiamond,triangle,square,andcircle)werecomparedtoeachcontrol(solidcircle).Broth-onlytreatmentservedasa
negativecontrol(solidsquare).Yaxis,OD600;Xaxis,time(sec).
doi:10.1371/journal.pone.0163147.g003
We usedA.actinomycetemcomitans tocomparetheantibacterialactivitiesofliquidand
vaporphenolicEOcompoundspre-incubatedatvarioustemperaturesbydirectcontactand
evaporationconditions,respectively.Theinhibitionzonesofhinokitiolwerenotsignificantly
differentbetweenthedirectcontactandvaporphasesafter24h.Theinhibitionzonesofboth
directcontactandthevaporphaseforhinokitiolwerereducedafter96h,andtheinhibition
zonesofthevaporphaseweresmallerthanthoseinthedirectcontactgroup.Theliquidand
vaporformsofhinokitiolwerestablewhensubjectedtofreezing,refrigeration,roomtempera-
ture,andhightemperature,andtheantibacterialactivityofthisEOwasnotsignificantlydiffer-
entforthedifferentformsorafterstorageatdifferenttemperatures(Fig5D).Theinhibition
zonesofvaporcarvacrolandthymolweresmallerthanthoseobtainedbythedirectcontact
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Fig4.ThephenolicEOcompoundswereheatstable.ThephenolicEOcompounds(500μg)werepre-incubatedat4–100˚Cfor1hbeforethedirect
contactagardiffusiontest.(A)Hinokitiol;(B)carvacrol;(C)thymol.Dottedline,the0.6cmdiameterofthefilterdisc.
doi:10.1371/journal.pone.0163147.g004
methodatboth24and96h.Although,theantibacterialactivityofthymolwasstrongerthan
thatofcarvacrol(Figs2and4andTable 2),carvacrolshowedamoreprolongedeffectthan
thymol(Fig5Eand5F).Theinhibitionzoneproducedbyvaporouscarvacrolwasapproxi-
mately0.6cm,butthezoneforvaporousthymolhaddisappearedby96h.Theinhibitionzone
ofdirectcontactthymoldecreasedat96hinatemperature-dependentmanner(Fig5F).Carva-
crolwasmorestablethanthymolwhenthetemperaturewashigherthan80°C.
HinokitiolisstableunderdifferentpHconditions
MostbiochemicalreactionsoccuratneutralpH.EnvironmentalpHisamajorfactorthatsup-
pressesmicrobialcolonization[27],butsomeentericbacteriaproduceacidandhavehighpH
resistance[28].Thevaporphasemethodwasusedtotestthestabilitiesofhinokitiol,carvacrol,
andthymolundervariouspHconditionstodeterminewhetheracidityoralkalinityinthe
brothagarwouldinterferewithbacterialgrowth.Inthehinokitiolgroup,theinhibitionzones
forallmicroorganismsweresimilarunderthevariouspHconditions(Fig6A).Theinhibition
zonemarginsofS.mutans,MRSA,andE.coliwereallcloudy.Inthecarvacrolandthymol
groups,therewasnovisibleinhibitionzoneundervariouspHconditions(Fig6Band6C),
evenwhenthenumberofinoculatedbacteriawasincreasedfrom106to108cfu.Theseresults
showedthattheantibacterialactivityofvaporhinokitiolwasnotaffectedbypHorthepresence
ofwater.Theeffectsofvaporouscarvacrolandthymolantibacterialactivitywereinhibitedby
water,andtheimpactofpHontheactivityofthesecompoundscouldthereforenotbeverified.
ThephenolicEOcompoundsexhibitsynergisticantibacterialeffects
CombinationtreatmentwithhinokitiolandZnOresultedinstrongsynergisticantibacterial
activityandcytotoxicity[29–31].A.actinomycetemcomitans wasusedtostudythepotential
synergisticantibacterialeffectsofdifferentcombinations.Thesizeoftheinhibitionzonesin
thedirectcontactmethod(fromlargesttosmallest)was250μghinokitiol,followedby250μg
hinokitiolcombinedwith500μgZnO,then500μgZnO.Therewasnoinhibitionzoneinthe
samplestreatedwith500μgZnO,orinthesamplestreatedwith500μgZnOcombinedwith
250μgvaporoushinokitiolasdeterminedbyvaporphasemethoddetection(Fig7A).Ithas
previouslybeenreportedthatEOscontainingcarvacrolandthymolcanhavesynergisticeffects
incombinationwithantibiotics[32].Thecombinationof50%thymoland50%carvacrolwas
foundtohavethehighestsynergisticantimicrobialactivityinanotherstudy[33].However,
twodifferentcombinations(200μgcarvacrol+200μgthymol,and500μgcarvacrol+500μg
PLOSONE|DOI:10.1371/journal.pone.0163147 September28,2016 9/17
AntibacterialPotentialofLiquidandVaporPhasePhenolicEssentialOilCompounds
Fig5.ThevaporousphenolicEOcompoundsdisplayantibacterialactivity.(A)Thevaporphaseagardiffusionexperimentaldevice.(B)MRSAtreated
with500μgphenolicEOcompoundswasexaminedbyvaporphaseagardiffusiontests.(C)Thevaporsfrom500μgphenolicEOcompoundsweretested
byvaporphaseagardiffusion.Theliquidandvaporphasesof(D)hinokitiol,(E)carvacrol,and(F)thymolshoweddifferentantibacterialactivitiesafter
incubationatdifferenttemperatures.*P<0.05,comparedwith4˚Cineachcurve.Dottedline,the0.6cmdiameterofthefilterdisc.
doi:10.1371/journal.pone.0163147.g005
thymol)showednosynergisticeffectsinthedirectcontactmethodinthepresentstudy
(Fig7B).
Discussion
ThestrengthsoftheantibacterialactivitiesfortheEOswerehinokitiol>thymol>
carvacrol>menthol.Theantibacterialworkingdoseandphenotypeofcarvacrolandthymol
weresimilar,consistentwiththefindingsofXuetal.[34].BasedontheMICrange,whichcan
beusedasaparametertodeterminetheactivityofessentialoils[18,35],hinokitiol(MIC=40–
60μg/mL)hadverystrongactivity,carvacrolandthymol(MIC=100–400μg/mL)hadstrong
activities,andmenthol(MIC=500–1000μg/mL)hadrelativelymoderateactivityinthisstudy
PLOSONE|DOI:10.1371/journal.pone.0163147 September28,2016 10/17