Composition and Antibacterial Activity of the Essential Oils of Orthosiphon stamineus Benth and

18 Pages·2017·28.04 MB·English

molecules
Article
Composition and Antibacterial Activity of the
Essential Oils of Orthosiphon stamineus Benth and
Ficus deltoidea Jack against Pathogenic Oral Bacteria
NuramirahAzizan1,2,ShahidaMohdSaid3 ID,ZamirahZainalAbidin2andIbrahimJantan1,*
1 DrugandHerbalResearchCentre,FacultyofPharmacy,UniversitiKebangsaanMalaysia,
KualaLumpur50300,Malaysia;[email protected]
2 DepartmentofClinicaloralBiology,FacultyofDentistry,UniversitiKebangsaanMalaysia,
KualaLumpur50300,Malaysia;[email protected]
3 DepartmentofPeriodontology,FacultyofDentistry,UniversitiKebangsaanMalaysia,
KualaLumpur50300,Malaysia;[email protected]
* Correspondence:profi[email protected];Tel.:[phone]
Received:2November2017;Accepted:30November2017;Published:5December2017
Abstract: Inthisstudy,theessentialoilsofOrthosiphonstamineusBenthandFicusdeltoideaJackwere
evaluatedfortheirantibacterialactivityagainstinvasiveoralpathogens,namelyEnterococcusfaecalis,
Streptococcusmutans,Streptococcusmitis,Streptococcussalivarius,Aggregatibacteractinomycetemcomitans,
Porphyromonas gingivalis and Fusobacterium nucleatum. Chemical composition of the oils was
analyzedusinggaschromatography(GC)andgaschromatography-massspectrometry(GC-MS).
The antibacterial activity of the oils and their major constituents were investigated using the
broth microdilution method (minimal inhibitory concentration (MIC) and minimal bactericidal
concentration (MBC)). Susceptibility test, anti-adhesion, anti-biofilm, checkerboard and time-kill
assayswerealsocarriedout.Physiologicalchangesofthebacterialcellsafterexposuretotheoilswere
observedunderthefieldemissionscanningelectronmicroscope(FESEM).O.stamineusandF.deltoidea
oils mainly consisted of sesquiterpenoids (44.6% and 60.9%, respectively), and β-caryophyllene
wasthemostabundantcompoundinbothoils(26.3%and36.3%,respectively). Othercompounds
presentinO.stamineuswereα-humulene(5.1%)andeugenol(8.1%),whileα-humulene(5.5%)and
germacrene D (7.7%) were dominant in F. deltoidea. The oils of both plants showed moderate to
strong inhibition against all tested bacteria with MIC and MBC values ranging 0.63–2.5 mg/mL.
However,noneshowedanyinhibitiononmonospeciesbiofilms. Thetime-killassayshowedthat
combinationofbothoilswithamoxicillinatconcentrationsof1×and2×MICvaluesdemonstrated
additiveantibacterialeffect. TheFESEMstudyshowedthatbothoilsproducedsignificantalterations
onthecellsofGram-negativebacteriaastheybecamepleomorphicandlysed.Inconclusion,thestudy
indicated that the oils of O. stamineus and F. deltoidea possessed moderate to strong antibacterial
propertiesagainstthesevenstrainspathogenicoralbacteriaandmayhavecauseddisturbancesof
membranestructureorcellwallofthebacteria.
Keywords: Orthosiphonstamineus;Ficusdeltoidea;essentialoils;antibacterial;oralpathogens
1. Introduction
Oralhealthproblems,particularlyperiodontaldiseases,dentalcariesandendodonticinfections,
arethemostsignificantdestructiveprocessesintheoralcavityandareacostlyburdentothepublic
globally. Periodontaldiseasesarepathologicconditionsofbacterialinfectionofthestructuresaround
the teeth (including the gums, the cementum that covers the root, the periodontal ligament and
the alveolar bone) that can lead to tooth loss affecting more than half of all adults. Dental caries
Molecules2017,22,2135;doi:10.3390/molecules22122135 www.mdpi.com/journal/molecules
Molecules2017,22,2135 2of18
(toothdecayorcavities)arethemostcommonandwidespreadchronicoraldiseasesthataffectchildren
andadults.Theyareirreversibleinfectiousdiseasesoftheteethleadingtocavitiesintheteethstructure,
thuscompromisingthestructureandfunctionoftheteeth[1,2]. Meanwhile,endodonticinfectionsare
polymicrobialinfectionsthataffectthetoothcanalsystemwhichallowtheteethtogrow,matureand
respond to stress. Oral diseases are initiated by bacterial infection in the oral cavity and trigger
inflammatoryresponsesthatwillcontinueuntilthesourceofinfectionisremovedthroughdental
treatment of choice [3,4]. The combined ill effects of these major oral diseases weaken bodily
defense and may also serve as portal of entry to other more chronic and opportunistic infections.
Indeed,oraldiseasescreateasilentepidemic,placingaheavyburdenonworldwidepopulations[5].
Previousstudieshaveindicatedthatclinicianswerenotinterveningearlyenoughwithdiagnosis
ortreatment. Despitesomeimprovements,ahighprevalenceofdentalcaries,periodontaldiseasesand
endodonticinfectionsarestillrecordedworldwideduetothecomplicationscausedbyemergenceof
antibioticsresistance,treatmentfailureandpoororalhygiene. Inaddition,inrecentyears,oraldisease
hasalsobeenlinkedtootherhealthproblemssuchascardiovasculardiseasesanddiabeticcomplication,
and associates the mouth as a potential source for bacteria to spread to the rest of the body [6,7].
Theseconcernsarebeingusedtofindotherpossiblealternativestoefficientlymanagedestructiveoral
diseasesandultimatelyprovidebetterevidence-basedmanagementoforaldisease.
Herbal essential oils such as cinnamon and tea tree oils have been traditionally used for oral
care purposes. The benefits also come from their variety of pharmacological properties including
antimicrobial,anti-diabetic,antioxidants,treatmentforcancerandcardiovasculardiseases. Oilsfrom
theseherbsarerapidlygrowinginpopularitybecausetheyactasnaturalmedicineandhavebeen
proventohavebeneficialhealingpowerwithlittlesideeffects[8–10]. OrthosiphonstamineusBenth,
locally known as misai kucing or kumis kucing in Malaysia, is a medicinal herb belonging to the
LabiataeorLamiaceaefamily. Thenameisgivenduetothelongfilamentsthatlooklikecatwhiskers,
whichisoneoftheuniquemorphologicalfeaturesoftheflowers.Thisvaluableherbaceouswoodyplant
canbefoundinregionswithtropicalclimate[11]. Ithasbeenproventhatitsextracteffectivelytreated
various ailments as it is believed to have various pharmacological properties such as anti-allergic,
antihypertensive,diuretic,antioxidant,antidiabetic,anti-inflammatory,mildantiseptic,antifungal
and even exhibited considerable antibacterial and antiviral activities [12]. Ficus deltoidea Jack or
mistletoefog,whichisalsoknownasmascotekamongMalaysians,isaplantofthefamilyMoraceae.
Ethnobotanicalapproacheshaveclaimeditsmedicinalpotentialsuchastotreatdiabetes,rheumatism,
cold,sores,toothache,headacheandotherailments. Additionally,itisalsobeingusedtraditionally
forgeneralhealthbenefitssuchasasatonicforreproductionsystem. Scientificstudieshavefound
thattheseplantshaveawiderangeofbeneficialphytochemicalsandexhibitvariouspharmacological
activities such as anti-inflammatory, antidiabetic, anticancer, antifungal and antimicrobial [13–15].
However,thereisstilllimitedinformationregardingtheeffectsoftheessentialoilsofbothherbson
thehealthandwell-beingoftheoralcavity.
This study focused on assessing the antibacterial properties as well as profiling the bioactive
compounds of O. stamineus and F. deltoidea essential oils, with a high expectation to create and
developahighlyeffectiveinterventionagainstinvasiveoralinfectionscausedbydifferenttypesof
oralpathogens. Besides, answeringthesequestionswillalsoallowustogainbetterinsightofthe
significantcontributionofO.stamineusandF.deltoideatowardscombatingselectedoralpathogens.
Thislattermaypavethewaytopharmaceuticalapplicationsinthenearfutureandconsequentlyresult
inthedevelopmentofanovelpreventivestrategytopreventtheinfectionscausedbythesepathogens.
2. Results
2.1. AnalysisoftheComponentsoftheEssentialOils
Hydro-distillationofthewholepartsoftheplantsyielded0.33%ofO.stamineusoiland0.37%
of F. deltoidea oil, based on a dry weight. The GC analysis of the oils resulted in identification
Molecules2017,22,2135 3of18
of 30 and 40 compounds for O. stamineus and F. deltoidea, respectively. The quantitative and
qualitative data of the volatile components of both oils are shown in Table 1, in order of elution
onaDB-5typecolumn.OurstudyshowedthatO.stamineusoilcontainedmainlyβ-caryophyllene
(26.31%),bicyclogermacrene(7.7%),methyleugenol(7.4%),α-humulene(5.06%),α-copaene(2.26%)
andnaphthalene(1.13),whereasthemajorconstituentsofF.deltoideaoilwereβ-caryophyllene(36.3%),
eugenol(8.1%),germacreneD(7.7%),α-humulene(5.5%)andα-copaene(3.3%).
Table1.PercentagecompositionofO.stamineusandF.deltoideaessentialoils.
O.stamineus F.deltoidea Methodsof
No. Compound KovatIndexValue
PercentageComposition PercentageComposition Identification
1 α-Pinene 0.1 - 939 a,b
2 Camphene 0.05 - 954 a,b
3 1-Octen-3-ol 0.35 - 979 a,b
4 1,3-Benzenediamine 0.1 - 1169 a,b
5 Naphthalene 3.8 - 1181 a,b
6 trans-Geraniol 0.3 - 1253 a,b
7 Anethole 1.1 - 1253 a,b
8 Cinnamaldehyde 0.4 - 1270 a,b
9 Bornylacetate 0.16 - 1292 a,b
10 1-Methylnaphthalene 0.55 - 1289 a,b
11 2-Methylnaphthalene 0.5 - 1312 a,b
12 α-Cubebene 0.98 1.55 1351 a,b
13 Eugenol 8.1 - 1359 a,b
14 α-Copaene 2.26 3.3 1377 a,b
15 β-Bourbonene 0.57 0.09 1388 a,b
16 Methyleugenol 7.4 - 1392 a,b
17 7-Tetradecane 0.34 - 1400 a,b
18 β-Caryophyllene 26.31 36.3 1409 a,b
19 E-β-Damascenone 0.6 - 1414 a,b
20 γ-Elemene 0.25 - 1437 a,b
21 Aromadendrene 0.9 - 1441 a,b
22 α-Humulene 5.06 5.5 1455 a,b
23 γ-Muurolene 0.2 0.39 1480 a,b
24 Bicyclogermacrene 7.68 - 1500 a,b
25 β-Bisabolene 0.4 - 1506 a,b
26 cis-Z-α-Bisaboleneepoxide 0.4 - 1507 a,b
27 trans-Calamenene 0.5 - 1529 a,b
28 α-Calacorene 0.6 - 1546 a,b
29 Dimethyl-ionone 0.67 - 1567 a,b
30 Caryophylleneoxide 1.2 8.7 1583 a,b
31 (Z)-β-Ocimene - 0.1 1037 a,b
32 Linalool - 0.4 1097 a,b
33 α-Ylangene - 0.14 1375 a,b
34 β-Elemene - 0.1 1391 a,b
35 α-Santalene - 0.1 1418 a,b
36 β-Copaene - 0.3 1432 a,b
37 α-trans-Bergamotene - 0.4 1435 a,b
38 β-Farnesene - 0.5 1457 a,b
39 Alloaromadandrene - 1.5 1460 a,b
40 GermacreneD - 7.68 1485 a,b
41 β-Selinene - 1.1 1490 a,b
42 α-Selinene - 1.03 1498 a,b
43 α-Muurolene - 0.5 1500 a,b
44 α-Farnesene - 1.1 1506 a,b
45 GermacreneA - 1.0 1509 a,b
46 Myristicin - 1.7 1519 a,b
47 δ-Cadinene - 1.3 1523 a,b
48 α-Cadinene - 1.6 1539 a,b
49 GermacreneB - 1.5 1561 a,b
50 Dendrolasin - 0.7 1572 a,b
51 Himachaleneepoxide - 0.1 1580 a,b
52 cis-β-Elemonene - 0.2 1590 a,b
53 Humuleneepoxide - 0.1 1608 a,b
54 β-Himachaleneoxide - 1.4 1616 a,b
55 α-Muurolol - 0.3 1646 a,b
56 α-Cadinol - 0.2 1654 a,b
57 z-α-Santalol - 0.1 1675 a,b
58 α-Bisabolol - 0.2 1686 a,b
59 epi-β-Bisabolol - 0.3 1672 a,b
60 z-epi-β-Santalol - 0.1 1703 a,b
61 α-Vetivone - 0.1 1823 a,b
62 n-Hexadecanol - 0.1 1876 a,b
a = Kovat indices on a DB 5 column (1 µm thickness, 30.0 m length, 0.25 mm diameter);
b=Massfragmentationpattern.
Molecules2017,22,2135 4of18
2.2. AntibacterialActivityoftheEssentialOilsandTheirMajorCompounds
The antibacterial activity of the oils and their major compounds against the seven species
of pathogenic oral bacteria are shown in Table 2. The results indicated that there was no
different in the antibacterial activity of O. stamineus and F. deltoidea oils against all Gram-positive
and Gram-negative bacteria. In addition, the antibacterial activity of the essential oil standards
(trans-caryophyllene,α-humulene,eugenolandgermacreneD)wasgreateragainstGram-negative
bacteria as compared to Gram-positive bacteria. On the other hand, all of the monospecies
biofilms of the oral bacteria tested were found resistant against both oils and the essential oil
standardsatthetestedrangeof0.04–5.0mg/mL.Ourstudyalsoshowedthattheaerobicbacteria,
namely Enterococcus faecalis, Streptococcus mutans, S. mitis and S. salivarius, were moderately
susceptible to all the oils and the essential oil standards. However, the anaerobic Gram-negative
bacteria,Aggregatibacteractinomycetemcomitans,PorphyromonasgingivalisandFusobacteriumnucleatum,
wereslightlysusceptibletotheessentialoilstandards. Nevertheless,theantibacterialactivityofboth
oilsandthestandardswasstillattheintermediatelevelascomparedtotheactivityofamoxicillin
(positivecontrol),i.e.,MIC≤8µg/mLisconsideredsusceptibleandMIC≥16µg/mLisresistantfor
Gram-positivebacteria,whereasMIC≤0.5µg/mLisconsideredsusceptibleandMIC≥2µg/mLis
resistantforGram-negativebacteria[16].
Molecules2017,22,2135 5of18
Table2.TheantibacterialactivityofO.stamineusandF.deltoideaoilsandtheiractivecompoundsagainsttheoralpathogenicbacteria.
Samples O.stamineusEssentialOila F.deltoideaEssentialOila trans-Caryophyllenea α-Humulenea Eugenola GermacreneDa Amoxicillinb
L) L) L) L) L) L) L) L)
m m m m m m m m
L) L) mg/ L) L) mg/ L) L) mg/ L) L) mg/ L) L) mg/ L) L) mg/ mg/ mg/
m m ( m m ( m m ( m m ( m m ( m m ( ( (
Bacteria MIC(mg/ MBC(mg/ Adhesion MIC(mg/ MBC(mg/ Adhesion MIC(mg/ MBC(mg/ Adhesion MIC(mg/ MBC(mg/ Adhesion MIC(mg/ MBC(mg/ Adhesion MIC(mg/ MBC(mg/ Adhesion AndMBC Adhesion
Anti- Anti- Anti- Anti- Anti- Anti- MIC Anti-
E.faecalis 1.25 2.5 2.5 1.25 2.5 2.5 1.25 2.5 2.5 1.25 2.5 2.5 1.25 2.5 2.5 1.25 2.5 2.5 0.05 0.5
S.mutans 1.25 2.5 2.5 1.25 2.5 2.5 1.25 2.5 2.5 1.25 2.5 2.5 1.25 2.5 2.5 1.25 2.5 2.5 0.05 0.5
S.mitis 1.25 2.5 2.5 1.25 2.5 2.5 1.25 2.5 2.5 1.25 2.5 2.5 1.25 2.5 2.5 1.25 2.5 2.5 0.05 0.5
S.salivarius 1.25 2.5 2.5 1.25 2.5 2.5 0.63 1.25 1.25 0.63 1.25 1.25 1.25 2.5 2.5 1.25 2.5 2.5 0.05 0.5
A.actinomy. 1.25 2.5 2.5 0.63 1.25 1.25 0.63 1.25 1.25 0.63 1.25 1.25 1.25 2.5 2.5 1.25 2.5 2.5 0.05 0.5
P.gingivalis 1.25 2.5 2.5 0.63 1.25 1.25 0.63 1.25 1.25 0.63 1.25 1.25 1.25 2.5 2.5 1.25 2.5 2.5 0.05 0.5
F.nucleatum 1.25 2.5 2.5 0.63 1.25 1.25 0.63 1.25 1.25 0.63 1.25 1.25 1.25 2.5 2.5 1.25 2.5 2.5 0.05 0.5
aconcentrationsranging0.04–5.0mg/mL.bconcentrationat0.05mg/mL(positivecontrol).
Molecules2017,22,2135 6of18
2.3. MorphologicalChangesoftheBacterialCells
Anymodificationoralterationonthemorphologyofthebacterialcellsafterbeingtreatedwiththe
oilsandamoxicillinwereobservedunderthefieldemissionscanelectronmicroscope(FESEM).Theoral
bacteriaweretreatedwiththeoilsattheirrespectiveMICvaluesandresultsareshowninTables3
and4. Therewerenoteworthyalterationsonthebacterialcellswithregardstoitsshape, sizeand
arrangement.Thearrangementofthebacterialcellswasstillintactinclusters,whilethemorphologyof
thebacterialcellswasobservedslightlyalteredtobecomeroughandirregularwhencomparedtothe
controls(untreatedbacteria). Generally,treatmentwithO.stamineusandF.deltoideaoilsgavesimilar
effectonalltestedoralGram-positiveandGram-negativebacteria. Comparisonwithnormalbacterial
cellsshowedthatthecellsbecamepleomorphic,irregularinsizeandsomeruptured.Thebacterialcells
alsopresentedirregulararrangementastheymergedandattachedtoeachother. Fewcellsappeared
collapsedandlysed,causingpossiblyleakageoftheintracellularcontents. Theseobservationssuggest
thatthemechanismofantibacterialactivityofbothoilsmayberelatedtothedisturbanceofmembrane
structureorcellwallofthebacteriauponexposure[17,18]. Meanwhile,testedGram-negativebacteria
showeddrasticmorphologicalalterationsupontreatmentwithamoxicillinascomparedtothetested
oralGram-positivebacteria. Itispossiblethattheinhibitionandinterferencewithsynthesisofcell
wallmayhavecausedthetestedGram-negativebacterialcellstolosecontroloveritsshapeandsize
andfurtherdisruptioninhomeostasisleadstoshrinkageandeventuallydeathofthosecells.
2.4. HerbalOilsandAmoxicillinSynergisticEffect
The synergism activity of the essential oils combined with amoxicillin as determined by the
checkerboardmicrodilutiontechniqueispresentedinTable5. Thecombinationofbothoilsattheir
MICvalue(0.63to1.25mg/mL)andalsothecombinationofO.stamineusandF.deltoideaoilswith
amoxicillintestedattheirMICranges(0.63to1.25mg/mL)yieldedadditiveeffectasdefinedbythe
FIC index of 0.75. An additive effect is observed when there is two-fold decreasing in the MIC of
bothcomponentsandthustheeffectofthecombinationisequaltothesumoftheeffectofindividual
component[19,20]. Time-killexperimentwasperformedtoconfirmandvalidatetheactivityofthe
combinationofbothessentialoilswithamoxicillin.Allcombinationsofspecifiedconcentrationshowed
bacteriostatic and bactericidal effect in a time- and dose-dependent manner (Figure 1). The most
effectivecombinationwasthe2×MICasitconsistedofthehighestconcentrationofeachagentinthe
combination. Thebactericidalendpointsforalltheoralbacteriatestedwerereachedafter8and4hof
incubationatconcentrationsof1×and2×MICofthecombination,respectively.
Molecules2017,22,2135 7of18
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Molecules 2017, 22, 2135 7 of 19
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BaBcatceBtreiaarcilat cele rcileal lll cell
Bacterial cell
BtraBtecraaBtetecaaeBtrttcedriaeater dcewilaa tr tcewilieea rtcdhlililea t chwll el cli elt hll
treated with
OO.Bt rsa.Bett csaraaOBttetmtcearaea.Btemrti tdecsnriaeaatetir etdnacewilaueam tre tcdsewiiluee ta ir ocdsnhwliil elati e o clhwliul leit b lsch li eltb o h l il l b
O. stamineus oil b
BacterialcelltreatedwithO.stamOin.Ot esrO.utet arsasO.mtet tasorae.timet itdnasrlaetmeie btdnawuaemie tsndwiue tieo sndhwiu ite olshwiu itb olsh i itbo lh ib l b
OO. s.Ot asO.tm as.mti nastimenaumiesnu ieosnu ieolsu ibols ibol ibl b

MMoleMoclueMoclleeuoscll uee2scl0e u21sl0 7e21s,0 7221,20 72,1, 2 272,1, 2 232,1 5223 ,1 523 153 5 8 o8f o81 f 9o 81 f 9 o1 f9 1 9
BaBcaBtecaBtrceiatarecli artceilea rcllil eac llel clle ll
BacterialcelltreatedwithF.deltoidtreeatraotetriaeelttdracee tdwae dtwiet dhwit hwi t hit h
F.F d .Fe dl.t Fedol.ite ddolteieodalite dooaei idaol e icoal i col ic l c

aAllspeciesofbacterialcellsshoweda Asal ilAam sAlapli llAesla cplsrileep pscsepi aceoeistfec t sioebe fraos nc bfo taebfoc rabfticeaatmrlec icartoeiela rrlcllip saec llhse lcshlol eossll owhlssogh eswiodhcw eoasdweilm dcse iihdsmlai amsrinli ampiglraa iertlpast aeprdrt atnpetu atroeetnrtf e ntomro fno o tmforh pmfoe hrmopofroolhporrohgpmloiohclgaooaitllgc oiicaogchlani accl anhoclgah fcenachsgn raedgynseu sgsdete audst leoudl ietutzo hete eo t th sfotteoh r rtefuhmo efcrao tmftruoimoarrtnmeai otoai(noatf i nrooc rrfnoo y cfwso rtcfyar )sclyltsrsiayzutlaeslritl zlrasieotlzlr eiusuz tncesrttu durscutritrnecuu tgr(uceatr utre(hrr aoe(eraw r(rcoa)rew orslrwu)lo srws)r utos)hru ursaronurtudordrniunodinugsid nrntiugdhn piegtn h tgctehei ttleclhs seec slet lthcslrl easutlth l ctsah tttua htra et and
disrupt its structure and arrangement; b and c All species of bacterial cells showed similar pattern of morphological changes due to the presence of sticky and
arrangement;bandcAllspeciesofbacrcytdesitdrsaiirdlasulriilpzsucretpu e istplt tlsirtst ussis cttssrthtu urosrcutwetrcu u(teraucedrtr ureaors newaidm na) d nasi lurdaarr rarrarnoarpgrunaaengntmdetgmeieenrnmegntn ;et tohnb; fetba; m ncabed nola ldnsrc pdAtc hh Alcal o tlAsl ld polsliegp sscreipiucceeapisec ltsio eci ftosh s bf ao sabtfncr abtguceacetrectsiuarteidrla reclui aeacellne lscdtll eo ssal hltrsshorh asweohnweopgdweer mdese sidesmeni mnsitli.cam ilera irlpoa afprt astpettatirectnrtk enoyr fno a mfo nmfod rmopcrohprroyhplosohtlgooailglcoliaigczlai eccl ahcslah tcnrahugnaecgnste gusde rudse eud (etauo ret or ttoh otewh teph) repse ruspeerrsnreeocsneeucn eonc feod sfio tnsifct giksctytkihc yake nyacd nae dnl lds that
disruptitsstructureandarrangement.
crcyrscytrsaytlaslitlzlaielzl eiszt sretu rscuttrcuutrucetr ue(ar (erar (roarworrw)o sw)u s)ru rsrouruorrnuodnuidnnigdn igtnh tgeh tech ecelel csll etslh ltsah tta hdta idst irdsuripsurtpu ittp sitt ssit tsrstu rscuttrcuutrucetr uear naedn a dan rdarr aarnargrnaegnmegmeenmetn.e tn. t.
TaTbaTlbeaTl b4eal. be 4El .4e lEe. 4clEe.t lrcEeotclrnetor cnmotr nmioc nmrico mirgcorrigcoarrpgaorhpga hropa fhop t fhho t feho tnfeh otenhr onmer onmarolm aralman ald ana ldan al dtane ldatrele tardeel rtdmee rdmoe drmop rmohproohplroohplgoohilgcooailgcoli agcolai fco lt afho lt feho tGfeh terGha Germa rGm-anrm-aengm-enag-etnaigvetaiegvt aibevt aiebvc abteec atbrceiatarecli artceilae rcllil eacsll el ucsll epsulo lpusno pu ntopr netora ntetra mettrameetnamettn mewtn ewitnt hwitt hwaitm hait mohaxm oaixcmoiixlcoliiicxlnliii lcn(lipi ln(lopi ns(opi ts(oipivstoieivts iceivto iecvn ocetnro octnrolto)nr laot)rn lao)d nla )dn a dn d
thteh tOeh te.Oh sOe.t a s.Omt sa.tim ansmtienauimensu ieansun easdu nas dFn a .dF nd .dFe dl. t Fedol.ite dodlteieodalite dooaeii daolse io.al si Eol. sniE.ll snaE.rl naEglrneagmrleagmreegnmeetn:me t1n:e0 t1n:, 00t10:,0 001,00×000., 0×0.0× 0. ×.
TrTeTraertTamertameetamnettmnetnetnt AA. aA. ca.At cai.tn caiotncimontimoynmcoyemcyteecytmeectmecetomcemomcmoictmoiatmniatsnai tsna sn s P.P gP. ig.nP iggn. iiggnviiganvilgaviislavi lsaislis F.F n.F un.F cunl. ceunlaceutlaceutlameutamu tmum
UUnUtnrUtenratenrtaeetrtdaeet daebt dabec dabtce abtrceaitarceiltra eilar ila l
ceclelc l(elcn l(elon l(rlon m(ronmaromla)r mla)l a) l)

PoPsoPitsoPiivstoiievts iiecvto iecvn ocetno rcotnorlton rlot rlo l
(tr(te(ratert(aeetrtdaeet daewt dwei tdwhi t wiht hit h
AAmAmoAmxoimxociixoclliixiclnliiicl)nli ial)n l ia)n a) a
MMoollMMeeccuuoolllleeeeccsMsM uu 22lloo00eellss11ee cc7722uu,,00 ll2211ee22ss77 ,,,,22 22220011221133,,77 5522,, 1122332255,, 22113355 88 ooff88 11 oo99 ff 118899 oo ff 1199
BBaaccBBtteeaarrcciittaaBBeellrr aacciiccaaeettllllee llcc rreeiiaallllll cceellll
ttrreeaattttrreeeeddaa tt ttwweerrddeeiitata wwhhttee iidd tthh ww iitthh
FF.. ddFFeell..t tddooeeiidFdFllttee..oo aaddii ddeoeoeelliittaalloo cciooi ddiieell aacc ooiill cc
Molecules2017,22,2135 8of18

aa AAaalll l AAssppllllaaee ssccAAppiieelleellsscc ssiiooeeppffssee bbccooiiaaffeecc ssbbtt eeaaoorrccffiitt aaeebbllrr aaciicccaaeettllllee llccrrssieei aassllllllhhss ococ sseewwhhlllleeoossdd wwss hheessiioddommww ssiieiiellmmaaddrr ii ssllppaaiimamarr tttpptiielelaaaarrttrrnntt ee ppoorraannfftt ttmmooeerffroo nnrmmr ppoooohhffrr ooppmmllohhoooggoorriillppccooaahhggllooii ccccllaaoohhllggaa iccinncchhggaaaaelel nn ssccgg hhddeeaauussnn eeddgg etuuetoossee ttddtthhoouuee ee tt f hhfototeeoorr mmffttoohhaarreettmm iiofofaaoonnttrr ii mmoooonnffaa ttccooiirrooffyy nnsscc ttrrooaayyffllssl l iittcczzaarreellyyll iisssszzttttaeearr lluullssiiccttzzrrtteeuuuu rrccsseettttuu rr((uurraaeeccrr ttrr((uuooaarrwwrreerr ))oo(( wwaassurur))rrrr oorssrwwoouuuurr))rr n noossdduuuuiirrnnnnrrddggoo ii uunnttnhnhggdede ttiichhcnneeeeggll l lccsstteehh ttlleehhllss aacc ttttee hh llllaasstt tthhaatt
ddiissrrdduuiipssprrttuu ddiipptitissssttr r ssuiiuttttppssrr uuttss ccttiirrttttususuu rrccsseettttuu rraauurrnneeccd dttaauu nnarareeddrrr r aaaaaannnnrrrrddggaae e nnaammrggrrreeeeaannmmnnttg;g;ee nneebb mmttaa;; nneebbnn ddaatt nn;;cc ddbbAA aallccl lnn AAsdsdppll llccee ssccAAppiieelleellsscc ssiiooeeppffssee bbccooiiaaffeecc ssbbtt eeaaoorrccffiitt aaeebbllrr aaciicccaaeettllllee llccrrssieei aassllllllhhss ococ sseewwhhlllleeoossdd wwss hheessiiddoommww ssiiiielelmmaaddrr ii sllsppaaiimamarr tttpptiileleaaaarrttrrnntt ee ppoorraannfftt ttmmooeerffroo nnrmmr ppoooohhffrr ooppmmllohhoooggoorriillppccooaahhggllooii ccccllaaoohhllggaa iccinncchhggaaaalele nn ssccgg hhddeeaauussnn eeddgg etuuetoossee ttddtthhoouuee ee tt p hhpttreerooee ppssttheherrnneeeess c cppeeeenn r rooeeccsfsfee ee ssoonnttffiicccc eesskk ttyyooiicc f fkk a assnnyytt didiccaa kknnyydd aanndd
Table4.Electronmicrographoftheccrryynssccottrraayyrllssllmiittcczzaarreellyyall iisssszzlttttaeearrauu llssllccniittzztrrtuueuude rrccsseettttauu rr((uulrraateeccrre rtrt((uuooaarwrwrrreeerr)d) oo (( swwsaauurrm))rrrr oorssroouuowwuurrr))rrnn poossdduuuuhiirrnnnnrroddggoo ii luuttnnohhnnggeegdd ttiiccihhnneeceegglla ll ccssttlee hhttllhheeollss aacc fttttee hhddtlllaalhiissttss tertrddhhuuiiaapGsspttrrtt uu ddriippttiiasssstt rrm ssiiuuttttrssrpp-uu tsstnc c ttiirrtttteuuuuss grrccsseettttauu rraauurrtnneeiccdd vttaauu nnaaerrrrddeerrb aaaaaannnanrrrrggddcaaee tnnaammerggrrreereeaannmminnattg.g.ee lnneemmctt.. eeelnnltts.. upontreatmentwithamoxicillin(positivecontrol)andthe
O.stamineusandF.deltoideaoils.EnTTaalbabTTllraaeegbb 44lleTT..ee EmEaa 44bblle..e lleccEEeettn llrr44eeoot..cc nn:ttEErr llmmoo1eenncc0ii ctctmmr,rrr0ooooiinng0gcc rrrr0mmooaaggp×piiccrrhhrraa . ooppooggffhh rrt taaoohhppffee hh ttnnhh ooooeeffrr nnmmtthhooaaerrellmm nnaaaaoonnllrrdd mmaa nnaaaallddltlt ee aaaarrnnlleettddddee rr aammeellddttooee rmmrrrppeeoodhdhrro o ppmmllohhoooggoorriillppccooaahhggllooii ccoollaaooffll ggt toohhiiccffeeaa ttllGGhh ooeerrffa a GGtmtmhhrr-e-eaann mmGGeegg--rrnnaaaaeemmttiiggvv--aanenett iieebbvvggaaeeaacc ttbbttiieevaavrrcceeiittaa eebbllrr aacciiccaaeettllllee llccssrree iiuuaallllllppss cc oouueennlppll l tstsoorr nnuuee aappttttrroommeennaaee ttttnnmmrrttee eeaawwnnttmmittitt wwhhee nniiaattttmmhh ww ooaaiixmmxttihihccoo iiaaxxllllmmiiiiccnniiooll (ll(xxiippiinnccoo ii((ssllpplliiiittoonniivvss ((iieettpp iiccovvooosseennii ttccttiioorrvvoonneelltt ))ccrr ooaoaonnllnn))ddtt aarr oonnlldd)) aanndd
tthhee ttOOhhee.. ssOOtttthaha..m meess ttiiOOaannmmee.. uussiinnttssaa eeaammuunnssiiddnn aa e ennFFuu.dd.ss dd aaFFeennll..t tddddooeeii dFdFllttee..oo aaddii ddoeoeeelliittaallooss ooii..dd iiEEeellssaann.. llooEEaaiirrnnllsgsgll..aaee EEmrrmggnneeeellnanammrrttg:g:ee 11nnee00mmtt::,, 00ee1100nn0000,,tt00××:: 00..11 0000××,,00.. 0000××..
Treatment TTrreeTTaarrtteemmaaTTeettrrmmnneettaaeettnnmmtteenntt AAA.. aa.AAacc..tct aaiitnnAiAccnotto..oii mmaannmccooyyyttmmccciieenenyytttooeeccemmmmeemttcyyceecoccommomeemmccttieeootiiammttmmaanccsniinoottssaamm nniisstt aannss PP.. ggPPPii..nn. gggggiiPPiiinnnvv..ggg aaggiiillviivviinnassaaggl lliiiiisvvss aalliiss FF.. nnFFuu.. ccnnlluueFeFFaacc... llttnnneeuuuuuaammcccttllluu eeemmaaattt uuummm
Untreatedbacterialcell(noUUrnmnUUttarrlnnee)aattUrrUtteeeennaaddtttt rreebbeeddaaaa ccbbtttteeeeaaddrrccii ttbabaeellaarr iiccaattllee rriiaall
cceellllcc ee((nnlllloo cc((rrnneemmllooll a arr((mmllnn)) ooaarrll))mm aall))

PPoossPPiittooiissvviiPPeettii oocvvcssooeeiinn ttcciittvvoorreennoo llttcc rroooonnll ttrrooll
Positivecontrol(treatedwithAmox((ittcrrieel((laattirrntteeee)aadd((atttt rreewweeddaaii ttwwtthhee diidtt hhww iitthh
AAmmAAoommxxiiooAAcciixxmlmliillcciinonoiillxx))ll iiiaiannc c ii))ll aall iinn)) aa
MMoollMeeMccuuoolllleeeescMsMc uu22olol00eell1s1ese c c7722uu,,00 ll221e1e22ss77 ,,,,22 22220011221133,,77 5522,, 1122332255,, 22113355 99 oo ff 9 911 9o9o ff 119999 oo ff 1199

BBaaccBBtteeaarrcciittBBaaeellaarr icccciaateteelelll llrrcc iiteteaarrlllleell acacttreretteeeelllldada tttt rreeeeddaa tteedd
BacterialcelltreatedwithO.stamwwiniittewwhhus iiOOttohhww..i l ssOOiibttttaa.h.h mmss OtOtiiaann.m.m eessuuiittnnaasse m emu u iissnn ee uuss
ooiill oobb iill bboo iill bb

BBaaccBBtteeaarrcciittBBaaeellaarr icccciaateteelelll llrrcc iiteteaarrlllleell acacttreretteeeelllldada tttt rreeeeddaa tteedd
BacterialcelltreatedwithF.dewwltoiiittwwdhhe iaiFFtthoh..ww id d lFFiieectt..ll hh ttddoo eeFFiilddl.t.t e eooddaaiieed doolleettiiaoaoll i i oocdcd ieiellaa c c oo iill cc

aAllspeciesofbacterialcellswerepleoaa mAAllaaoll AAssrppplllleeaa hs cs cAAppiiieeelclessllcc sso(oiieppeaffss eerbb cocoraaiifofcece btbtsswee aaoorrcc)iifftata ebbedllr raacciuicceaeattlellleel l cscsrrte eiiwwoaallllllsees rcc rrwwueeeell eepllppssrrll teeweweu ooppeermmllrreeeeeoooo apprrmmppnlleeoohhdoorriipmcmcp lhh((yooaaiirrcrcrsppr r i((oohhsaawwr;iriccrrb)) o o(( ddawawCrruu))rre ee oodd lwtwtuuloosee)) rr ddtutouoouuppf rerettAuuuu ttpporor.eet trrau uaauucrrnnppeetdd it taanuu llnnryryodedessm i iaallssyynny;; ssbdbdcii sCeCs ll;;tyy eebbessll mCllCiisssse e ;;coo llbbolffl ss C CmAA ooee..fifl l aa ltl AAsscca tt.oo.nii annaffs ccooAAttmmaii..nn nyyaaooccccdmmetetiittyynneeFccmmoo.eemmtctcneeooyymmmumcceeccciittototleeaammemmnnaiissccttt aooaaaunmmnnnmssidid ttaaa a FFnnwnn..ddss nn e aaFFuurnn..cce ddnnlleeu u paaFFcctt..lulul eenenmmaaouutt cucuwmwllmmeeeeaao rrttwweeuur emmpepprrll h eeewew ooippeecmmllrreeee(ooooa pprrmmrppllreeoohhooorriipcmcmpw hh((ooaai)irrcrcrppr dr ((oohhaauwwririccrre)) o o(( ddwaawtorruu))rre e ooddr wwuuuee))p ddtuuueer eandlysis
whilecellsofP.gingivaliscollapsedandttoob rrettuuoocpp arrttuuuumttpooprree tterru uauaurrtnnppeerdd t taaauu nnllnrryyededsss p iiaallssyynna wwssddriis e shhll yyniiwwllsseethiih sscc(ii leelawweellr ll hhcscsre eiiooolllleellffwss ccPP ooee).. fl lfggl ld sPsPii nnu..oo ggggffeii iivvPPnntaa.g.go lliggiiivsvsiil annaccyllggooiisssiillvv illccaaasaooplpl;iillssssllc a aeeccpdpdAooss llaaellelaanlnddppdd s asas peenbbnddeeeddcc c aabaabinnmmeeeddccseeaa bb momtteerrfeacacea an nbttmmrssraapapeecnnaa tststrrerrpepeaarnnaannitrtras s ee((ppnlnaaaarrttc r rrr((eoeoeaanlnwwrrlrttrs) ) oo(( dwadawwrruu))rre e eoodd rwwttuueooe))e lpl ddyyttooluusse iieellssoyy ;;tt ssmoocci i s A sAll;;oyy llccllssr AAiisspssppl;;lh lleecc scsicAApcpiieelleeslslacc ssniooieppeffdss eebb ccooaabiiffeecc esbstbtee caaoorracciffitata mebbellr raacciieccaeaettlllleie ll rcscsrre ir eiwwaalelllllssge e cc rrwwueeeell lelleppssarr lleerwwee looppeyemmllrreeeeaoooo prprrmmpprlleeoaohhoorrniipmcmcpg hhaaooeininrrccdddpp aa h hnbbn(iiaedecdccc r aabaabrnnmmeeoddccweeaa bb mmee)ecceda a mmueee torupture
andlysis. iirrrreeiiggrruurreellagagiirrurrullrrlyyleeaa ggraralluurryyrrll aaaaaanrnrrrllrggryyaaee nanaddrrgg rr((eeaaaaddnrnr rr gg((ooaaeewwrrddrr)) oo(( ddwaawrruu))rre e oodd wwttuuooe)e) r r ddttuuoouupp ererttuu u uttpoporree t trru uaauurrnnppeedd t taauu lnlnrryydeedss iiaallssyynn.. ssddiis s ll..yy ssiiss..
Molecules2017,22,2135 9of18
Table5.Synergisticactivityoftheessentialoilsandamoxicillin.
MIC(mg/mL)
Bacteria EssentialOils Alone(EssentialOil)a Alone(Amoxicillin)b Combination(EssentialOil)c Combination(EO+Amoxicillin)d FICIndexe Activity
O.stamineus 1.25 0.05 0.625 0.0125 0.75 additive
E.faecalis
F.deltoidea 1.25 0.05 0.625 0.0125 0.75 additive
O.stamineus 1.25 0.05 0.625 0.0125 0.75 additive
S.mutans
F.deltoidea 1.25 0.05 0.625 0.0125 0.75 additive
O.stamineus 1.25 0.05 0.625 0.0125 0.75 additive
S.mitis
F.deltoidea 1.25 0.05 0.625 0.0125 0.75 additive
O.stamineus 1.25 0.05 0.625 0.0125 0.75 additive
S.salivarius
F.deltoidea 0.63 0.05 0.625 0.0125 0.75 additive
O.stamineus 1.25 0.05 0.625 0.0125 0.75 additive
A.actinomycetemcomitans
F.deltoidea 0.63 0.05 0.625 0.0125 0.75 additive
O.stamineus 1.25 0.05 0.625 0.0125 0.75 additive
P.gingivalis
F.deltoidea 1.25 0.05 0.625 0.0125 0.75 additive
O.stamineus 1.25 0.05 0.625 0.0125 0.75 additive
F.nucleatum
F.deltoidea 0.63 0.05 0.625 0.0125 0.75 additive
aMICvalueofessentialoil.bMICvalueofAmoxicillin.cMICvalueofthecombinationofbothessentialoils.dMICvalueofthecombinationofeachessentialoilwithAmoxicillin.
efractionalinhibitoryconcentrationindex.
Molecules2017,22,2135 10of18
Molecules 2017, 22, 2135 12 of 19
15 15 15
mL10 mL10 mL10
log10 cfu/ 5 log10 cfu/ 5 log10 cfu/ 5
0
0 0
0 2 4 6 8 12 24 48
0 2 4 6 8 12 24 48 0 2 4 6 8 12 24 48
Time (h)
Time (h) Time (h)
E. faecalis S. mutans S. mitis E. faecalis S. mutans S. mitis E. faecalis S. mutans S. mitis
S. salivarius A.a P.g S. salivarius A.a P.g S. salivarius A.a P.g
F.n F.n F.n
(a) i. (b) i. (c) i.
15 20 20
mL mL
log10 cfu/150 log10 cfu/10 10 cfu/mL10
g
o
l
0 0 0
0 2 4 6 8 12 24 48 0 2 4 6 8 12 24 48 0 2 4 6 8 12 24 48
Time (h) Time (h)
Time (h)
E. faecalis S. mutans S. mitis E. faecalis S. mutans S. mitis E. faecalis S. mutans S. mitis
S. salivarius A.a P.g S. salivarius A.a P.g S. salivarius A.a P.g
F.n F.n F.n
(a) ii. (b) ii. (c) ii.
Figure 1. Time-kill curves of the antibacterial activity of the combination of both oils and amoxicillin (i: O. stamineus; and ii: F. deltoidea) at: (a) 0.5 MIC concentration
Figure1.Time-killcurvesoftheantibacterialactivityofthecombinationofbothoilsandamoxicillin(i:O.stamineus;andii:F.deltoidea)at:(a)0.5MICconcentrationof
coofm cobminbatinioanti;o(nb;) (1b×) 1M× IMCICco cnocnecnetnratrtiaotinonof ocfo cmombibniantaiotino;na; nadnd(c ()c2) ×2×M MIICCc coonncceennttrraattiioonn ooff ccoommbbiinnaattiioonn..