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Majalah Farmasi Indonesia, 22(3), 174 – 181, 2011

Majalah Farmasi Indonesia, 22(3), 2011 174

Effect of Indonesian medicinal plants essential

oils on Streptococcus mutans biofilmPengaruh oil minyak atsiri dari beberapa tanaman obat

Indonesia terhadap biofilm Streptococcus mutans

Triana Hertiani*) , Sylvia Utami Tanjung Pratiwi, Irami Duma Kencana Irianto, Dian

Adityaningrum and Budi PranotoCentre for Natural Antiinfective Research, Faculty of Pharmacy, Gadjah Mada University, Seki utara, !o"jakarta, ##$%&

Abstract

Essential oil’s component such as menthol and eucalyptol were alreadyused as dental plaque inhibitors. In searching of potential dental plaque inhibitorfrom natural products, a study to explore the potency of essential oils extractedfrom several Indonesian medicinal plants against planktonic growth and biofilmadherence of S. mutans was performed. A total of ! essential oils from someselected Indonesian medicinal plants were extracted by steam"hydro distillation.Antibacterial assay was performed against S. mutans by micro dilution techniqueon nutrient broth media. #iofilm formation inhibition assay was conducted on aflexible $"bottom %&"wells '() micro plate by using #*I enriched with sucrose+ at -&.& ) for /"+! h. After staining with crystal violet, the opticaldensity was read at 0%0 nm. A mouthwash commercial product containingessential oil component was used as a positive control. 1esult showed that theessential oils of C. sintoc exhibited the highest biofilm formation inhibition 2I)03 4 3.3305, and Z. officinale showed the highest biofilm degradation with E)03

value of 3.3-. #oth were active against bacterial planktonic growth but C.sintoc showed lower 6I)%3 value 23.&5 in comparison to Z. officinale 23.3&5.6eantime, C. citratus was showed promising antibacterial and antibiofilmactivities with 6I)%3 value of 3.3&, 6#) 3.&, I)03 3.33/ and E)03 3.3+&.It is concluded that the essential oils of C. citratus, Z. officinale and C. sintoc arepotential to be developed as dental plaque inhibitors.Key words: essential oils, antibacterial, biofilm, Streptococcus mutans

Abstrak

7omponen minyak atsiri seperti mentol dan eukaliptol telah dilaporkanmampu menghambat pembentukan plak gigi. 'enelitian untuk mengetahuipotensi minyak atsiri dari beberapa tanaman obat Indonesia dalam

penghambatan pertumbuhan planktonik dan biofilm S.mutans telah dilakukan.8ebanyak ! minyak atsiri dipilih dari beberapa tanaman obat Indonesia yangdiekstraksi menggunakan metode destilasi air " uap air. $9i antibakteri dilakukanterhadap bakteri S.mutans menggunakan metode mikro dilusi dengan medianutrient broth. $9i penghambatan biofilm dilakukan pada flexible U-bottom

96-wells PVC micro plate menggunakan #*I yang diperkaya dengan sukrosa +pada suhu -&,& ) selama /"+! 9am. 8etelah diberi pewarnaan kristal violet, optical density dibaca pada pan9ang gelombang 0%0 nm. :bat kumur yangmengandung komponen minyak atsiri dan telah beredar di pasaran digunakansebagai kontrol positif. 'engu9ian dilakukan sebanyak tiga kali. *asil penelitianmenun9ukkan bahwa minyak atsiri dari C. sintoc memiliki penghambatan biofilmpaling tinggi 2I)03 4 3,3305, dan Z. officinale memiliki kemampuan degradasiyang paling tinggi dengan nilai E)03 sebesar 3.3-. 7eduanya mampumenghambat pertumbuhan bakteri planktonik tetapi C. sintoc memperlihatkan

nilai 6I)%3 yang rendah 23,&5 dibanding Z. officinale 23,3&5. 8ementara itu,



Triana Hertiani


C. citratus menun9ukkan aktivitas antibakteri dan antibiofilm yang potensialdengan nilai 6I)%3 3,3&, 6#) 3,&, I)03 3,33/ dan E)03 3,3+&. 6inyakatsiri dari C. citratus Z. officinale dan C. sintoc berpotensi untuk dikembangkan

sebagai inhibitor plak gigi.Kata Kunci: minyak atsiri, antibakteri, biofilm, Streptococcus mutans

IntroductionOne major problem in oral hygiene is

dental plaque. Accumulation of this slimy layer(biofilm) on teeth is not only give unpleasantlook, but also can lead to gingivitisand carries and even further damage suchas dental loss (Marsh, !!"). #evelopeddental biofilm plaque can consist of many

different microbes including those pathogenicin systemic. $hese microbes can harbor inmouth cavity and hide from immunologicalreaction (%asrolahei, et al ., !!&' i, et al ., !!!'%akano, et al ., !!).

Streptococcus mutans is the most frequentbacteria found in biofilm dental plaque(oesche, *++"). t is also kno-n as initiator ofdental biofilm formation and has cariogenicproperty by demineraliing enamels (slam, etal ., !!). $herefore to overcome dental carriesit is important to control mouth flora especially

S. mutans and at the same time control theircapabilities to build biofilm.

Mechanical treatments -ith dental flossand brush may reduce the plaque but addingchemicals can significantly reduce bacterialcounts (Addy, et al ., *+&' Ale/ander, *+*).Moreover, considering that electrostatic andhydrophobic interactions govern theattachment of pathogenic bacteria onto thesaliva0coated tooth surface suggests chemicalagents application in dental plaque controlroutine (Ouhayoun, !!1). At recent time,

available chemicals for dental paste andmouth-ash such as chlorhe/idine have beencorrelated -ith side effects on restorations(2asooli, et al ., !!&), imbalance mouthflora and dental staining (3idd, et al ., *++'4iancio, !!!). On the other hand,those un-anted effects are not commonlyseen -ith essential oils mouth-ashes (2asooli,et al ., !!&).

$his study aimed at finding naturalproducts to prevent the formation of dentalbiofilm -hich can be used in an oral hygiene

product. $he main focus -as on the effect of

essential oils e/tracted from selectedndonesian traditional plants against planktonicgro-th, biofilm formation and adherence of S.mutans . $hose selected plants and parts -ere5rhiomes of Curcuma xanthorrhiza rhizome(Temulawak), Zingiber aromaticum (Lempuyangwangi), and Z. officinale var rubrum . (ahe merah)'fruits of !momum car"amomum (#apulaga) and $.

cubeba ' leaves of $iper betle (Sirih), $. crocatum% $.retrofractum% Syzygium aromaticum , Citrus aurantifolia and Cymbopogon citratus ' barks of Cinnamommumzeylanicum , C. sintoc , and Litsea cubeba .

MethodologyPlant materials

6resh rhiomes of Curcuma xanthorrhiza 2o/b., Zingiber aromaticum 7ahl, Zingiber officinale var rubrum ., and !momum car"amomum 8all fruits(9ingiberaceae)' fresh leaves of $iper betle ., $ipercrocatum (2ui : ;av.), fruits of $iper retrofractum 7ahland $iper cubeba . (;iperaceae)' barks of

Cinnamommum zeylanicum <lume, C. sintoc <l., andLitsea cubeba (auraceae)' fresh leaves of Syzygiumaromaticum (.) Merrill : ;erry, Citrus aurantifolia (amiaceae)' and Cymbopogon citratus (;oaceae) -erecollected on 6ebruary, !!+ in =ogyakarta,ndonesia. $he species ta/onomy identification -asdone by >oko ?antoso, M.?c. (;harmacognosyaboratory, 6aculty of ;harmacy, @adjah Madaniversity, ndonesia).

Isolation of the essential oils

About B kg of each fresh sample -asseparated, triturated and steam0hydro distilled for "

hours. $he appearances of essential oils yielded -ereobserved. $hose oils -ere sealed and kept in darkglass vials for further analyses.

Bacteria and Culture condition

S. mutans A$44 *B (aboratory of 6oodand %utrition, nter niversity 4entre, @adjahMada niversity, ndonesia) -ere gro-n in %utrient<roth (O/oid) for C h at 1"."D4. A Mc6arlandstandard -as used to adjust inoculum density in%a4l solution for M4 assays. <acteria for biofilmassays -ere gro-n in <E enriched -ith sucrose Fand inoculums density -as adjusted to Mc6arlandstandard B.



Ee!t o Indonesian medi!inal "lants####


etermination of the minimum inhibitoryconcentration !MIC"

M4s -ere determined by the micro titer

broth method (Amsterdam, *++") in sterile flatbottom +"0-ell polystyrene plates. ?erial dilution ofessential oils in methanol -ere used to determinethe M4s of samples after *&0C h gro-th at 1"."D4. %egative controls (cells G media), positive control(cells G media G mouth-ash product), vehiclecontrols (cells G media G methanol), and mediacontrols -ere included. <lank samples -ereprepared -ith same treatment as for samples, only

-ithout cells added. All tests -ere performed at leastin triplicate. Optical density readings -ere takenusing micro plate reader (<iorad <enchmark, >apan)at B+B nm for *&0C h post0inoculation. $o account

for the effect of the essential oils color, a formulafor calculating percent inhibition -as used. $hemean F inhibition of replicate tests -as used todetermine the final M4 values -ith formulamodified from Huave and collaborators (!!&) asfollo-s5

.........(*) -here O#sample5 Optical #ensity of samples Gbacterial suspension' O#sample0blank 5 Optical #ensityof samples G saline' O# vehicle5 Optical #ensity ofcontrol G bacterial suspension' O# vehicle0blank 5Optical #ensity of vehicle control G saline. M4

value -as determined as +!F of inhibition or more.?amples mi/tures -ere re0inoculated on

%utrient Agar plates. M<4 value -as determined asthe lo-est concentration -ith no visible gro-th.

etermination of the biofilm formationinhibition

4B! -as determined by the adherence assaymethod modified from OI$oole and 3olter, (*++&).?erial dilutions of sample solutions -ere put onfle/ible 0bottom +"0-ell polystyrene plates(<ecton #ickinson, ?A) and -ere incubated for*&0C h gro-th at 1"."D4. 4ontrols -ere prepared

as in the M4 assay. <lank samples -ere obtained byperforming the same treatment as for samples, only

-ithout cells added. All tests -ere performed at leastin triplicate. After *&0C h incubation, the contentsof the -ells -ere aspired, rinsed 1 times -ithdistilled -ater, and -ere fi/ed for *! min. $hen, *BJ of *F crystal violet stain -as added to the -ellsand left for *B min. $he e/cess stain -as rinsed off

-ith tap -ater. A !! J methanol -as added toeach -ells, left for *B min and then *B!J -astransferred to a flat bottom +"0-ell plates. Opticaldensity readings -ere taken using micro plate readerat B+B nm. $o account for the effect of the essential

oils color, a formula for calculating percent

inhibition -as used. $he mean F inhibition ofreplicate tests used -as the same as for M4calculation. 4B! -as determined as a concentrationof sample -hich inhibited B!F of biofilm formationin comparison to vehicle controls and -as calculatedby using probit analysis -ith ?;?? (?tatistical;ackage for the ?ocial ?ciences) version *B.!.

etermination of the biofilm degradationacti#ity

K4B! -as determined by the adherence assaymethod modified from OI$oole and 3olter, (*++&).?erial dilutions of sample solutions -ere put onfle/ible 0bottom +"0-ell polystyrene plates(<ecton #ickinson, ?A). 4ontrols -ere preparedas in the M4 assay. <lank samples -ere obtained by

performing the same treatment as for samples, only -ithout cells added. All tests -ere performed at leastin triplicate. After C h incubation at 1"."D 4, thecontents of the -ells -ere aspired. ?erial dilution ofessential oils in media -ere added to the -ells andincubated for another C h in 1"."D 4 incubator.6urther treatment -as as done for biofilmformation inhibition determination. K4B! -asdetermined as a concentration of sample -hichdegrade B!F of C h0old0biofilm formed incomparison to vehicle controls and -as calculatedby using probit analysis -ith ?;?? (?tatistical;ackage for the ?ocial ?ciences) version *B.!.

Bioautography

<ioautography assay -as performedaccording to @ibbons and @ray (*++&) -ithmodification. ?everal $4 systems -ere evaluatedto find out the best separation of the samplecomponents (stationary phase used -as precoatedsilica gel "! 6 BC, Merck, @ermany' toluene 5 ethylacetate (+15) v L v as mobile phase). After drying thesolvents, one replication of the eluted samples -ereput on to the %utrient Agar containing bacteria in;etri dish for * h, -hile others -ere sprayed -ithanisaldehyde E?OC and 6e4l1 separately. Afterincubation in 1"."D4 for *&0C h, the diameters ofinhibition ones of each spot -ere measured in mm.

$esults and iscussion $his research has revealed that all

essential oils tested -ere active againstthe planktonic gro-th and biofilm of S. mutans .%evertheless, the essential oils of $. crocatum ,$. retrofractum , and !. car"amomum hadM4+! value higher than !." F (ma/imumconcentration tested). All of the essential oilssho-ed lo-er planktonic gro-th inhib0ition incomparison to the biofilm inhibition ($able ).



Triana Hertiani


?ome of the essential oils e/hibited higherdegradation activity than biofilm formationinhibition, -hile others sho-ed the reverse.

<ioautography assays results -ere asdescribed in table . $he spot of C. zeylanicum

essential oil e/hibited the highest activity,

follo-ed by S. aromaticum. C. sintoc e/hibited thelo-est 4B! value of biofilm formationinhibition (!.!!BF). t sho-ed moderatebiofilm degradation activity, -hile the essentialoil from Z. officinale e/hibited the highest

activity (K4B! !.!*1F).

$able . nhibition data of the essential oils against the planktonic gro-th and biofilm ofStreptococcus mutans

Samples MIC90 MBC IC50 EC50 Z. officinale !.!"F !."F !.!**F !.!*1FC. xanthorriza !.!"F !."F !.!*!F !.!&1F A. cardamomum !."F !."F !.!*BF !.!1*FZ. aromaticum !.!"F !."F !.!*CF !.!1CFiper !etle !.!"F !."F !.!"*F !.!1F. crocatum !."F !."F !.!*F !.!*F. retrofractum !."F !."F !.!BBF !.!*BF. cu!e!a !.!"F !."F !.!"F !.!1&FCinnamomum ze"lanicum !.!"F !."F !.!**F !.!C"F#itsea cu!e!a !.!"F !."F !.!*!F !.!1FC. sintoc !."F !."F !.!!BF !.!&1FC"m!opo$on citratus !.!"F !."F !.!!&F !.!"FS"z"$ium aromaticum !.!"F !."F !.!1!F !.!1FCitru aurantifolia !.!"F !."F !.!F !.!"F

$able . 2esult of <ioautografi Assays (stationary phase5 silica gel 6 BC, mobile phase5 toluene0ethyl acetateN+15 vLv, loading one5 J of essential oils *F in toluene)

Color chan$eSample h%f

&' (5)nm Anisaldeh"de

*(S+) ,eCl-

Inhi!itionzone .φφφφ

mm/

Actiecompoundprediction

Z. officinale CC 1 <ro-n 0 ".! $erpenoidC. xanthorriza B+ 1 =ello- <lue C.! ;henolic A. cardamomum C" 1 0 0 1.! unidentifiedZ. aromaticum C* 1 =ello- 0 " $erpenoidiper !etle C& 1 0 <ro-n C.C ;henolic. crocatum 0 2 0 0 0 0. cu!e!a 2 ;urple 0 C.! $erpenoid. retrofractum 1* 2 0 ngu C.B ;henolicCinnamomumze"lanicum

1! 2 0 ;urple *& ;henolic

#itsea cu!e!a CC 2 <ro-n ;urple ".B ;henolicC. sintoc CB 1 <lack 0 &.B $erpenoid

C"m!opo$oncitratus

CC 1 <lack ;urple ".B ;henolic

S"z"$iumaromaticum

C" 1 0 <ro-n &.& Kugenol

Citrus aurantium " 2 <ro-n ;urple B.1B ;henolic





<iofilm plaque in mouth cavity is actuallyconsisted of diverse microbes, but mostlyinitiated by S. mutans . Moreover, ability of this@ram positive bacterium to convert sugars cangenerate acidic environment and causecariogenic effect (Marsh, !!"). $herefore,finding of agents active against the planktonikas -ell as biofilm gro-th of this bacterium ise/pected to be valuable agents in oral hygieneproducts.

Kssential oils -ere reported to kill

microorganisms by disrupting cell -allsand inhibiting enymatic activities, thereforecan prevent bacterial aggregation, slo- multipli0cation and e/tract endoto/ins (Ouhayoun,!!1). ?everal essential oils component suchas menthol, eucalyptol (Ouhayoun, !!1),cynamaldehyde (%iu and @ilbert, !!C) and/anthorrhiol (E-ang and 2ukayadi, !!")have been reported to be potential antibacterialand antibiofilm.

Kssential oils assayed in this research -ere selected based on the antibacterial activity

reported in the literatures (%iu and @ilbert,!!!C' Martins, et al ., !!*' %alina and2ahim, !!", !!' >uliantina, et al ., !!+5 8angand iu, !*!' 3han and ?iddiqui, !!'@upta, et al ., !!+' 4handarana, et al ., !!B'nouye, et al ., !!*' Aneja and >oshi, !!+'E-ang and 2ukayadi, !!"' ?u-ondo, !!' Akin0Osanaiye, et al ., !!). $heir abundantavailability as -ell as their -ide usage astraditional medicine in ndonesia -ere also theconsideration of selection.

2egarding the prominent antibacterial

activity of the C. zeylanicum essential oil, can be

e/plained by the presence of cynamaldehydecompound -hich -as supported by thebioautography result. %iu and @ilbert (!!C)reported prominent planktonic and biofilminhibition activity of cynamaldehyde, a majorconstituent of C. zeylanicum (cinnamon) against &scherichia coli and selected $seu"omonas species.4love oil sho-ed also prominent activity inbioautography result. Kugenol as the majorconstituent is a responsible component -hichcontributes to the antibacterial activity.

Moreover, 3han and collaborators (!!)reported anti quorum sensing activity ofcinnamon and clove oils. Anti quorum sensingactivity can inhibit communication bet-eenbacteria -hich further can inhibit bacterialbiofilm formation (8illiams, et al ., !!).

t is interesting to note that not allof active spots -ere phenolic compounds,even though all of those essential oilscontain phenolics. ?ome factors such asdiffusion capacity of the essential oilcomponents through media may influence the

bioautography results (2amre0Mares, et al .,!*!). ?ynergistic effects of several compoundsin the essential oils to e/pose the antibacterialactivity may also occur. $4 method prior tothe assay separated the active components -hich -ere then too dilute to e/hibit theactivity.

Curcuma xanthorrhiza ethyl acetate e/tract -as reported by 3im and collaborators (!!&)to be a potential anti plaque agent based on itsantibacterial activity against planktonic andbiofilm of mouth cavity microbes. $he

constituent responsible for the activity -as

(a) (b) (c)

6igure *. 4hemical structure of eucalyptol (a), cynamaldehyde (b), and /anthorrhiol (c).



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reported to be the /anthorrhiol. $hiscompound is phenolic and should be e/tracted

as essential oils components.

Conclusions $he essential oils of C. citratus , Z.

officinale% and C. sintoc -ere proven to bepotential dental plaque inhibitors. 2esultsho-ed that the essential oils of C. sintoce/hibited the highest biofilm formationinhibition (4B! N !.!!BF), and Z. officinale sho-ed the highest biofilm degradation -ithK4B! value of !.!*1F. <oth -ere active against

bacterial planktonic gro-th but C. sintoc sho-edM4+! value at !."F, -hile Z. officinale (!.!"F).

C. citratus -as sho-ed promising antibacterialand antibiofilm activities -ith M4+! value of!.!"F, M<4 !."F, 4B! !.!!&F and K4B! !.!"F.

Acknowledgements $his research -as funded by Eibah

<erkualitas ;rima !!& %o. @ML6AL"!"aLML!BL!* from 6aculty of ;harmacy, @adjahMada niversity, ndonesia.

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