Antimicrobial Activity of the Essential Oil of Plectranthus neochilus

Research ArticleAntimicrobial Activity of the Essential Oil ofPlectranthus neochilus against Cariogenic Bacteria

Eduardo Joseacute Crevelin1 Soraya Carolina Caixeta2 Herbert Juacutenior Dias1

Milton Groppo3 Wilson Roberto Cunha2 Carlos Henrique Gomes Martins2

and Antocircnio Eduardo Miller Crotti12

1Departamento de Quımica Faculdade de Filosofia Ciencias e Letras de Ribeirao Preto Universidade de Sao Paulo14040-901 Ribeirao Preto SP Brazil2Nucleo de Pesquisas em Ciencias Exatas e Tecnologicas Universidade de Franca 14404-600 Franca SP Brazil3Departamento de Biologia Faculdade de Filosofia Ciencias e Letras de Ribeirao Preto Universidade de Sao Paulo14040-901 Ribeirao Preto SP Brazil

Correspondence should be addressed to Antonio Eduardo Miller Crotti millercrottiffclrpuspbr

Received 30 April 2015 Accepted 7 June 2015

Academic Editor Filippo Maggi

Copyright copy 2015 Eduardo Jose Crevelin et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

This work used the brothmicrodilutionmethod to investigate the antimicrobial activity of the essential oil obtained from the leavesof Plectranthus neochilus (PN-EO) against a representative panel of oral pathogens We assessed the antimicrobial activity of thisoil in terms of the minimum inhibitory concentration (MIC) PN-EO displayed moderate activity against Enterococcus faecalis(MIC = 250 120583gmL) and Streptococcus salivarus (MIC = 250 120583gmL) significant activity against Streptococcus sobrinus (MIC =625 120583gmL) Streptococcus sanguinis (MIC = 625 120583gmL) Streptococcus mitis (MIC = 3125 120583gmL) and Lactobacillus casei (MIC =3125 120583gmL) and interesting activity against Streptococcus mutans (MIC = 39 120583gmL) GC-FID and GC-MS helped to identifythirty-one compounds in PN-EO 120572-pinene (1 141) 120573-pinene (2 71) trans-caryophyllene (3 298) and caryophyllene oxide(4 128) were the major chemical constituents of this essential oil When tested alone compounds 1 2 3 and 4 were inactive(MIC gt 4000 120583gmL) against all the microorganisms These results suggested that the essential oil extracted from the leaves ofPlectranthus neochilus displays promising activity against most of the evaluated cariogenic bacteria especially S mutans

1 Introduction

Dental caries is associated with acidogenic and aciduricbacteria that adhere to the tooth surface as an oral biofilm(dental plaque) [1] Because this pathology can destroy dentalhard tissues [2ndash4] it has become a major public healthconcern worldwide The most efficient way to prevent cariesand periodontal diseases is to reduce and eliminate bacterialaccumulation on the top of and between teeth by brushing theteeth on a daily basis and conducting periodic dental cleaningor prophylaxis Unfortunately most people fail to maintain asufficient level of oral hygiene [5] which has called for theuse of oral products containing antimicrobial ingredients asa complementary measure to diminish biofilm formation onthe tooth surface [6]

Chlorhexidine has been the most effective antiplaqueagent tested to date but some reversible local side effects haveled dentists to recommend its use for short periods only [7]Several other antimicrobial agents including fluorides phe-nol derivatives ampicillin erythromycin penicillin tetra-cycline and vancomycin can inhibit bacterial growth [8]Nevertheless excessive use of these chemicals can disturb theoral and intestinal flora and cause microorganism suscepti-bility vomiting diarrhea and tooth staining [8] To find analternative to the substances currently employed to preventcaries and to control plaques researchers have investigatedthe antimicrobial activities of natural products especiallyessential oils [1 3 7 9ndash11]

The herbaceous and aromatic plant Plectranthus neochilusis popularly known as ldquoboldo-rasteirordquo in Brazil [12] In folk

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 102317 6 pageshttpdxdoiorg1011552015102317

2 Evidence-Based Complementary and Alternative Medicine

medicine this plant has helped to treat disturbed digestionskin infection respiratory ailments [13] hepatic insufficiencyand dyspepsia [14] The essential oil of P neochilus displaysantischistosomal [12] and insecticidal [15] activities Recentlyresearchers have applied diffusion in agar disc to assess theantimicrobial activity of the essential oil of a specimen of Pneochilus collected in Portugal against Bacillus cereus Bacil-lus subtilis Staphylococcus aureus Listeria monocytogenesEscherichia coli Pseudomonas aeruginosaHelicobacter pyloriand Saccharomyces cerevisiae [16] The authors reported thatthe activity of this essential oil against the selected microor-ganisms was between low and moderate

As part of our ongoing research on the antimicrobialactivities of essential oils [1 17ndash19] in this work we used thebroth microdilution method to evaluate the in vitro antimi-crobial activity of the essential oil obtained from the leavesof Plectranthus neochilus (Lamiaceae) against a representativepanel of cariogenic bacteria

2 Materials and Methods

21 Plant Material Adult P neochilus Schltr (Lamiaceae)leaves were collected at ldquoMay 13th Farmrdquo (20∘261015840S 47∘271015840W977m) in May 2011 The collection site was located near thecity of Franca state of Sao Paulo Brazil This species wasidentified by Professor Dr Milton Groppo one voucherspecimen (SPFR 12323) was deposited at the Herbarium ofthe Department of Biology (Herbarium SPFR) University ofSao Paulo Brazil

22 Essential Oil Extraction Fresh leaves of P neochilus weresubmitted to hydrodistillation in a Clevenger-type apparatusfor 3 h To this end 1200 g of the plant material was dividedinto three samples of 400 g each and 500mL of distilledwater was added to each sample Condensation of the steamfollowed by accumulation of the essential oilwater systemin the graduated receiver of the apparatus separated theessential oil from thewater which allowed for furthermanualcollection of the organic phase Anhydrous sodium sulfatewas used to remove traces of water Samples were stored in anamber bottle and kept in the refrigerator at 4∘C until analysisYields were calculated from the weight of the fresh leaves

23 Gas Chromatography (GC-FID) Analyses The essentialoil of P neochilus (PN-EO) was analyzed by gas chromatog-raphy (GC) on a Hewlett-Packard G1530A 6890 gas chro-matograph fitted with FID and a data-handling processorAn HP-5 (Hewlett-Packard Palo Alto CA USA) fused-silicacapillary column (length = 30m id = 025mm and filmthickness = 033 120583m)was employedThe column temperaturewas programmed to rise from 60 to 240∘C at 3∘Cmin andthen held at 240∘C for 5minThe carrier gas was H

2at a flow

rate of 10mLmin The equipment was set to the injectionmode the injection volume was 01120583L (split ratio of 1 10)The injector and detector temperatures were 240 and 280∘Crespectively The relative concentrations of the componentswere obtained by peak area normalization () The relativeareas were the average of triplicate GC-FID analyses

24 Gas Chromatography-Mass Spectrometry (GC-MS)Analy-ses GC-MS analyses were carried out on a ShimadzuQP2010Plus (Shimadzu Corporation Kyoto Japan) system equippedwith an AOC-20i autosampler The column consisted of Rtx-5MS (Restek Co Bellefonte PA USA) fused-silica capil-lary (length = 30m id = 025mm and film thickness =025 120583m)The electron ionization (EI-MS) mode at 70 eV wasemployedHelium (99999) at a constant flowof 10mLminwas the carrier gas The injection volume was 01 120583L (splitratio of 1 10) The injector and the ion source temperatureswere set at 240 and 280∘C respectivelyThe oven temperatureprogram was the same as the one used for GC-FIDThemassspectrawere registeredwith a scan interval of 05 s in themassrange of 40 to 600Da

25 Identification of the PN-EO Constituents PN-EO com-ponents were identified on the basis of their retention indicesrelative to a homologous series of n-alkanes (C

8ndashC24) To this

end an Rtx-5MS capillary column was employed under thesame operating conditions as in the case of GCThe retentionindex (RI) of each PE-EO constituent was determined asdescribed previously [20] The chemical structures werecomputer-matched with theWiley 7 NIST 08 and FFNSC 12spectral libraries of the GC-MS data system their fragmenta-tion patterns were compared with the literature data [21]

26 Bacterial Strains and Antimicrobial Assays The in vitroantimicrobial activity of PN-EO and its major constituentswas assessed by minimum inhibitory concentration (MIC)values calculated by means of the broth microdilutionmethod accomplished in 96-well microplates The followingstandard ATCC strains were used Streptococcus salivarius(ATCC 25975) Streptococcus sanguinis (ATCC 10556) Strep-tococcus mitis (ATCC 49456) Streptococcus mutans (ATCC25175) Streptococcus sobrinus (ATCC 33478) Enterococcusfaecalis (ATCC 4082) and Lactobacillus casei (ATCC 11578)Individual 24-h colonies from blood agar (Difco LabsDetroit MI USA) were suspended in 100mL of trypticsoy broth (Difco) Standardization of each microorganismsuspension was carried out on a spectrophotometer (FemtoSao Paulo Brazil) operating at a wavelength (120582) of 625 nm tomatch the transmittance of 81 (equivalent to 05 McFarlandscale or 15 times 108 CFUmL) The microorganism suspensionwas diluted to a final concentration of 5 times 105 CFUmL PN-EOwas dissolved in DMSO (Merck Darmstadt Germany) at160mgmL and diluted in tryptic soy broth (Difco) to yieldconcentrations between 4000 and 39 120583gmL Compounds1 (120572-pinene) 2 (120573-pinene) 3 (trans-caryophyllene) and 4(caryophyllene oxide) were purchased from Sigma-Aldrich(St Louis MA) and evaluated by means of the samemethodology and at the same concentrations as PN-EO A1 120583M solution of each compound was tested individually Inthe case of the mixture 1 + 2 + 3 + 4 the constituents weremixed in the same proportion that they occurred in PN-EOAfter dilutions the DMSO concentrations were between 4and 00039 (vv)Three inoculated wells containing DMSOat concentrations ranging from 4 to 1 were used as neg-ative controls One inoculated well was included to control

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Minimum inhibitory concentration (MIC) values (120583gmL) obtained for the essential oil of P neochilus (PN-EO) compounds 1 2 3and 4 and the mixture 1 + 2 + 3 + 4 against selected cariogenic bacteria

Microorganisms PN-EO CHDa 1b 2b 3b 4b 1 + 2 + 3 + 4c

E faecalis 2500 148 gt4000 gt4000 gt4000 gt4000 gt4000S salivarius 2500 74 4000 4000 4000 4000 4000S mutans 39 18 gt4000 gt4000 gt4000 gt4000 1000S mitis 313 148 4000 4000 4000 4000 4000S sobrinus 625 18 gt4000 gt4000 gt4000 gt4000 4000S sanguinis 625 74 4000 gt4000 gt4000 gt4000 gt4000L casei 313 37 4000 4000 4000 4000 500aChlorhexidine dihydrochloridebFor compounds 1 2 3 and 4 the concentration of 4000 120583gmL corresponds to 294 294 196 and 181mM respectivelyc1 + 2 + 3 + 4 mixture of 120572-pinene 120573-pinene trans-caryophyllene and caryophyllene oxide

the adequacy of the broth for organismgrowthOnenoninoc-ulated well free of antimicrobial agent was also included toassess the medium sterility Twofold serial dilutions of chlo-rhexidine dihydrochloride (CHD) (Sigma-Aldrich St Louis)were performed in tryptic soy broth (Difco) to achieve con-centrations ranging from 59 to 0115 120583gmL These dilutionswere used as positive control The microplates (96-well) weresealed with parafilm and incubated at 37∘C for 24 h Afterthat 30mL of 002 resazurin (Sigma-Aldrich St LouisMOUSA) aqueous solutionwas poured into eachmicroplatereservoir to indicate microorganism viability [22] The MICvalue (ie the lowest concentration of a sample capable ofinhibiting microorganism growth) was determined as thelowest concentration of the essential oil and or major constit-uents capable of preventing a colour change of the resazurinsolution [23] Three replicates were conducted for eachmicroorganism

3 Results and Discussion

This work relied on minimum inhibitory concentration(MIC) values to evaluate the antimicrobial activity of theessential oil of P neochilus (PN-EO) against a panel of car-iogenic bacteria chlorhexidine dihydrochloride (CHD) wasthe positive control Samples with MIC values lower than100 120583gmL between 100 and 500120583gmL and between 500and 1000 120583gmL were considered to be promising moder-ately active and weak antimicrobials respectively Sampleswith MIC values greater than 1000 120583gmL were deemedinactive [11 24ndash26]

Table 1 summarizes the MIC values PN-EO displayedmoderate activity against S salivarius (MIC = 250 120583gmL)and S faecalis (MIC = 250 120583gmL) and significant antimi-crobial activity against Streptococcus sobrinus (MIC =625 120583gmL) Streptococcus sanguinis (MIC = 625 120583gmL)Streptococcus mitis (MIC = 3125 120583gmL) Lactobacillus casei(MIC = 3125 120583gmL) and Streptococcus mutans (MIC =39 120583gmL) The antimicrobial activity of PN-EO againstS mutans was an interesting result this microorganism isconsidered to be the main cariogenic agent [10 27] and veryfew natural compounds can inhibit it [26]

Hydrodistillation of P neochilus leaves afforded PN-EOin 003 plusmn 001 (ww) yield Gas chromatography revealedthe presence of 31 compounds in PN-EO namely fifteenmonoterpenes (360) fifteen sesquiterpenes (635) andaliphatic alcohol (02)Themajor PN-EO constituents were120572-pinene (1 141) 120573-pinene (2 71) trans-caryophyllene(3 298) and caryophyllene oxide (4 128) as shown inTable 2 The chemical composition of PN-EO differed signif-icantly from the chemical composition of P neochilus spec-imens collected in South Africa whose major constituentswere citronellol (290) citronellyl formate (110) linalool(98) and isomenthone (92) [28] but it resembled thechemical composition previously reported for P neochilusspecimens collected inBrazil [12 15]These different chemicalcompositions may be associated with environmental factorsor growing conditions which can greatly affect the chemicalcomposition of volatile oils [29 30]

The antimicrobial activity of essential oils has been asso-ciated with the lipophilicity of their chemical constituentsmainly monoterpenes and sesquiterpenes which are oftenthe main chemicals thereof [31] The hydrophobicity of ter-penoids would allow these compounds to diffuse across thecell membranes easily and to kill microorganisms by affectingthe metabolic pathways or organelles of the pathogen Inaddition synergistic interactions between essential oil com-ponents could enhance their activity [31] For this reason themajor chemical constituents of some essential oils deserveantimicrobial evaluation alone or as a mixture [32ndash34]This study evaluated the individual antimicrobial activityof 120572-pinene (1) 120573-pinene (2) trans-caryophyllene (3) andcaryophyllene oxide (4) Alone all of these compoundswere much less effective against the selected cariogenicbacteria than PN-EO their MIC values were higher than4000 120583gmL (Table 1)The antimicrobial activity of a mixturecontaining compounds 1 + 2 + 3 + 4 in the same relativeproportion compared to their relative areas in the CG-FIDchromatogram of PN-EO displayedmoderate activity againstL casei (MIC = 500 120583gmL) and weak activity against Smutans (MIC = 1000 120583gmL) but it was inactive againstthe other bacteria (MIC gt 4000 120583gmL) Although the MICvalues obtained for the mixture suggested a very discrete


Table 2 Chemical composition of the essential oil from the leaves of P neochilus as identified by GCMS

Chemical compound RT [min]a RIexpb RIlit

c Content []d Identificatione

120572-Thujene 500 921 924 63 RL MS120572-Pinene (1) 519 929 932 141 RL MSThuja-24(10)-diene 543 939 941 02 RL MSCamphene 562 943 947 01 RL MSSabinene 621 966 971 19 RL MS120573-Pinene (2) 637 975 977 71 RL MS120573-Myrcene 665 985 988 03 RL MSOctan-3-ol 690 993 996 02 RL MS120572-Terpinene 753 1015 1016 05 RL MSo-Cymene 779 1022 1023 03 RL MSLimonene 794 1026 1027 02 RL MS(Z)-120573-Ocimene 814 1030 1033 04 RL MS(E)-120573-Ocimene 850 1040 1043 18 RL MS120574-Terpinene 894 1052 1055 14 RL MS120572-Terpinolene 994 1080 1084 02 RL MS4-Terpineol 1375 1177 1179 12 RL MS120572-Cubebene 2078 1341 1344 05 RL MS120572-Copaene 2197 1366 1372 12 RL MS120573-Bourbonene 2229 1380 1379 11 RL MS120573-Cubenene 2250 1378 1384 03 RL MStrans-Caryophyllene (3) 2380 1412 1415 298 RL MS120572-Humulene 2525 1448 1450 15 RL MSGermacrene D 2630 1470 1476 62 RL MSEremophilene 2751 1504 1505 39 RL MS120572-Amorphene 2761 1506 1508 04 RL MS120575-Cadinene 2783 1514 1513 19 RL MS(E)-Nerolidol 2963 1554 1559 03 RL MSCaryophyllene oxide (4) 3026 1571 1575 128 RL MSUnknown 3206 mdash 1622 03 mdashepi-120572-Cadinol 3262 1634 1637 15 RL MS120575-Cadinol 3270 1636 1639 08 RL MS120572-Cadinol 3312 1647 1650 13 RL MSMonoterpenes hydrocarbons 348Oxygenated monoterpenes 12Sesquiterpenes hydrocarbons 468Oxygenated sesquiterpenes 167Others 02Not identified 03aRT retention time determined on the Rtx-5MS capillary columnbRIexp retention index determined on the Rtx-5MS column relative to n-alkanes (C8ndashC20)cRIlit retention indexdCalculated from the peak area relative to the total peak areaeRL comparison of the retention index with the literature [21] MS comparison of the mass spectrum with the literature

synergism between compounds 1 2 3 and 4 the mixture1 + 2 + 3 + 4 was much less active than PN-EO Henceonly the presence of compounds 1 2 3 and 4 does notaccount for the antimicrobial activity of PN-EO In factthe antimicrobial activity of PN-EO may also be related tothe other minor chemical constituents identified in the oilwhich may underlie or even increase the activity of the majorchemical constituents of this essential oil

4 Conclusions

The essential oil of P neochilus (PN-EO) displays promis-ing antimicrobial activity against some cariogenic bacteriaincluding Streptococcus mutans which is one of the maincausative agents of dental caries Taken together our resultssuggest that this essential oil might be promising for thedevelopment of new oral care products Further studies to


identify the active chemical constituents of PN-EOare under-way

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Theauthors are grateful to the Brazilian Foundations FAPESP(Proc 200854241-8) for financial support and CNPq forfellowships

References

[1] G P Aguiar C E Carvalho H J Dias et al ldquoAntimicrobialactivity of selected essential oils against cariogenic bacteriardquoNatural Product Research vol 27 no 18 pp 1668ndash1672 2013

[2] B R da Silva V A A de Freitas V A Carneiro et al ldquoAnti-microbial activity of the synthetic peptide Lys-a1 against oralstreptococcirdquo Peptides vol 42 no 4 pp 78ndash83 2013

[3] B Kouidhi T Zmantar and A Bakhrouf ldquoAnticariogenic andcytotoxic activity of clove essential oil (Eugenia caryophyllata)against a large number of oral pathogensrdquo Annals of Microbiol-ogy vol 60 no 4 pp 599ndash604 2010

[4] W G Wade ldquoCharacterisation of the human oral microbiomerdquoJournal of Oral Biosciences vol 55 no 3 pp 143ndash148 2013

[5] J J Jardim L S Alves and M Maltz ldquoThe history and globalmarket of oral home-care productsrdquo Brazilian Oral Researchvol 23 supplement 1 pp 17ndash22 2009

[6] A Furiga A Lonvaud-Funel G Dorignac and C Badet ldquoInvitro anti-bacterial and anti-adherence effects of natural poly-phenolic compounds on oral bacteriardquo Journal of AppliedMicro-biology vol 105 no 5 pp 1470ndash1476 2008

[7] K W Albertsson A Persson and J W V van Dijken ldquoEffect ofessential oils containing and alcohol-free chlorhexidinemouth-rinses on cariogenic micro-organisms in human salivardquo ActaOdontologica Scandinavica vol 71 no 3-4 pp 883ndash891 2013

[8] S EMoon H Y Kim and J D Cha ldquoSynergistic effect betweenclove oil and its major compounds and antibiotics against oralbacteriardquo Archives of Oral Biology vol 56 no 9 pp 907ndash9162011

[9] TOyanagi J Tagami andKMatin ldquoPotentials ofmouthwashesin disinfecting cariogenic bacteria and biofilms leading toinhibition of cariesrdquo Open Dentistry Journal vol 6 no 1 pp23ndash30 2012

[10] B Kim S Park M Kim et al ldquoInhibitory effects ofChrysanthe-mum boreale essential oil on ibofilm formation and virulencefactor expression of Streptococcus mutansrdquo Evidence-BasedComplementary and Alternative Medicine vol 2015 Article ID616309 11 pages 2015

[11] W S Alviano R R Mendonca-Filho D S Alviano et alldquoAntimicrobial activity of Croton cajucara Benth linalool-richessential oil on artificial biofilms and planktonic microorgan-ismsrdquoOralMicrobiology and Immunology vol 20 no 2 pp 101ndash105 2005

[12] S C Caixeta L G Magalhaes N I de Melo et al ldquoChemicalcomposition and in vitro schistosomicidal activity of the essen-tial oil of plectranthus neochilus grown in southeast BrazilrdquoChemistry amp Biodiversity vol 8 no 11 pp 2149ndash2157 2011

[13] C W Lukhoba M S J Simmonds and A J Paton ldquoPlectran-thus a review of ethnobotanical usesrdquo Journal of Ethnopharma-cology vol 103 no 1 pp 1ndash24 2006

[14] M F Duarte and J F Lopes ldquoStem and leaf anatomy of Plec-tranthus neochilus Schltr Lamiaceaerdquo Revista Brasileira deFarmacognosia vol 17 no 4 pp 549ndash556 2007

[15] E L L Baldin A E M Crotti K A L Wakabayashi et alldquoPlant-derived essential oils affecting settlement and oviposi-tion of Bemisia tabaci (Genn) biotype B on tomatordquo Journal ofPest Science vol 86 no 2 pp 301ndash308 2013

[16] L Mota A C Figueiredo L G Pedro et al ldquoVolatile-oils com-position and bioactivity of the essential oils of Plectranthus bar-batus P neochilus and P ornatus grown in PortugalrdquoChemistryamp Biodiversity vol 11 no 5 pp 719ndash732 2014

[17] J A Alves A L L Mantovani M H G Martins et al ldquoAnti-mycobacterial activity of some commercially available plant-derived essential oilsrdquoChemistry of Natural Compounds vol 51no 2 pp 353ndash355 2015

[18] M A Ferreira T C Carvalho I C C Turatti et al ldquoAntimicro-bial activity of Aegiphila sellowiana Cham Lamiaceae againstoral pathogensrdquoBrazilian Journal of Pharmacognosy vol 20 no2 pp 246ndash249 2010

[19] L C Keles F M Gianasi R C Souza et al ldquoAntibacterialactivity of 15-deoxygoyazensolide isolated from the stems ofMinasia alpestris (Asteraceae) against oral pathogensrdquo NaturalProduct Research vol 25 no 4 pp 326ndash331 2011

[20] H Vandendool and P D Kratz ldquoA generalization of the reten-tion index system including linear temperature programmedgas-liquid partition chromatographyrdquo Journal of chromatogra-phy vol 11 pp 463ndash471 1963

[21] R P Adams Identification of Essential Oils Components by GasChromatographyMass Spectrometry Allured Publishing CarolStream Ill USA 4th edition 2007

[22] J-C Palomino A Martin M Camacho H Guerra JSwings and F Portaels ldquoResazurin microtiter assay plate sim-ple and inexpensive method for detection of drug resis-tance inMycobacterium tuberculosisrdquo Antimicrobial Agents andChemotherapy vol 46 no 8 pp 2720ndash2722 2002

[23] S D Sarker L Nahar and Y Kumarasamy ldquoMicrotitre plate-based antibacterial assay incorporating resazurin as an indica-tor of cell growth and its application in the in vitro antibacterialscreening of phytochemicalsrdquo Methods vol 42 no 4 pp 321ndash324 2007

[24] J L Rıos and M C Recio ldquoMedicinal plants and antimicrobialactivityrdquo Journal of Ethnopharmacology vol 100 no 1-2 pp 80ndash84 2005

[25] M A Botelho R A dos Santos J G Martins et al ldquoCompara-tive effect of an essential oil mouthrinse on plaque gingivitisand salivary Streptococcus mutans levels a double blind ran-domized studyrdquo Phytotherapy Research vol 23 no 9 pp 1214ndash1219 2009

[26] M Saleem M Nazir M S Ali et al ldquoAntimicrobial naturalproducts an update on future antibiotic drug candidatesrdquo Nat-ural Product Reports vol 27 no 2 pp 238ndash254 2010

[27] D Kalemba D Kusewicz and K Swiader ldquoAntimicrobialproperties of the essential oil ofArtemisia asiaticaNakairdquoPhyto-therapy Research vol 16 no 3 pp 288ndash291 2002

[28] O A Lawal A H Hutchings and O Oyedeji ldquoChemicalcomposition of the leaf oil of Plectranthus neochilus SchltrrdquoJournal of Essential Oil Research vol 22 no 6 pp 546ndash547 2010


[29] C M O Simoes and V Spitzer ldquoOleos volateisrdquo in Farma-cognosia Da Planta ao Medicamento C M O Simoes E PSchenkel G Gosmman J C P Mello L A Mentz and P RPetrovick Eds pp 467ndash495 Editora da UFRGS Porto AlegreBrazil 2003

[30] K B G Torsell Natural Product Chemistry A MechanisticBiosynthetic and Ecological Approach Swedish PharmaceuticalPress Stockholm Sweden 2nd edition 1997

[31] A E Edris ldquoPharmaceutical and therapeutic potentials ofessential oils and their individual volatile constituents a reviewrdquoPhytotherapy Research vol 21 no 4 pp 308ndash323 2007

[32] M A Botelho N A P Nogueira G M Bastos et al ldquoAntimi-crobial activity of the essential oil fromLippia sidoides carvacroland thymol against oral pathogensrdquoBrazilian Journal ofMedicaland Biological Research vol 40 no 3 pp 349ndash356 2007

[33] H J D Dorman and S G Deans ldquoAntimicrobial agents fromplants antibacterial activity of plant volatile oilsrdquo Journal ofApplied Microbiology vol 88 no 2 pp 308ndash316 2000

[34] G Iscan N Kirimer M Kurkcuoglu K H C Baser and FDemirci ldquoAntimicrobial screening ofMentha piperita essentialoilsrdquo Journal of Agricultural and Food Chemistry vol 50 no 14pp 3943ndash3946 2002

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


medicine this plant has helped to treat disturbed digestionskin infection respiratory ailments [13] hepatic insufficiencyand dyspepsia [14] The essential oil of P neochilus displaysantischistosomal [12] and insecticidal [15] activities Recentlyresearchers have applied diffusion in agar disc to assess theantimicrobial activity of the essential oil of a specimen of Pneochilus collected in Portugal against Bacillus cereus Bacil-lus subtilis Staphylococcus aureus Listeria monocytogenesEscherichia coli Pseudomonas aeruginosaHelicobacter pyloriand Saccharomyces cerevisiae [16] The authors reported thatthe activity of this essential oil against the selected microor-ganisms was between low and moderate

As part of our ongoing research on the antimicrobialactivities of essential oils [1 17ndash19] in this work we used thebroth microdilution method to evaluate the in vitro antimi-crobial activity of the essential oil obtained from the leavesof Plectranthus neochilus (Lamiaceae) against a representativepanel of cariogenic bacteria

2 Materials and Methods

21 Plant Material Adult P neochilus Schltr (Lamiaceae)leaves were collected at ldquoMay 13th Farmrdquo (20∘261015840S 47∘271015840W977m) in May 2011 The collection site was located near thecity of Franca state of Sao Paulo Brazil This species wasidentified by Professor Dr Milton Groppo one voucherspecimen (SPFR 12323) was deposited at the Herbarium ofthe Department of Biology (Herbarium SPFR) University ofSao Paulo Brazil

22 Essential Oil Extraction Fresh leaves of P neochilus weresubmitted to hydrodistillation in a Clevenger-type apparatusfor 3 h To this end 1200 g of the plant material was dividedinto three samples of 400 g each and 500mL of distilledwater was added to each sample Condensation of the steamfollowed by accumulation of the essential oilwater systemin the graduated receiver of the apparatus separated theessential oil from thewater which allowed for furthermanualcollection of the organic phase Anhydrous sodium sulfatewas used to remove traces of water Samples were stored in anamber bottle and kept in the refrigerator at 4∘C until analysisYields were calculated from the weight of the fresh leaves

23 Gas Chromatography (GC-FID) Analyses The essentialoil of P neochilus (PN-EO) was analyzed by gas chromatog-raphy (GC) on a Hewlett-Packard G1530A 6890 gas chro-matograph fitted with FID and a data-handling processorAn HP-5 (Hewlett-Packard Palo Alto CA USA) fused-silicacapillary column (length = 30m id = 025mm and filmthickness = 033 120583m)was employedThe column temperaturewas programmed to rise from 60 to 240∘C at 3∘Cmin andthen held at 240∘C for 5minThe carrier gas was H

2at a flow

rate of 10mLmin The equipment was set to the injectionmode the injection volume was 01120583L (split ratio of 1 10)The injector and detector temperatures were 240 and 280∘Crespectively The relative concentrations of the componentswere obtained by peak area normalization () The relativeareas were the average of triplicate GC-FID analyses

24 Gas Chromatography-Mass Spectrometry (GC-MS)Analy-ses GC-MS analyses were carried out on a ShimadzuQP2010Plus (Shimadzu Corporation Kyoto Japan) system equippedwith an AOC-20i autosampler The column consisted of Rtx-5MS (Restek Co Bellefonte PA USA) fused-silica capil-lary (length = 30m id = 025mm and film thickness =025 120583m)The electron ionization (EI-MS) mode at 70 eV wasemployedHelium (99999) at a constant flowof 10mLminwas the carrier gas The injection volume was 01 120583L (splitratio of 1 10) The injector and the ion source temperatureswere set at 240 and 280∘C respectivelyThe oven temperatureprogram was the same as the one used for GC-FIDThemassspectrawere registeredwith a scan interval of 05 s in themassrange of 40 to 600Da

25 Identification of the PN-EO Constituents PN-EO com-ponents were identified on the basis of their retention indicesrelative to a homologous series of n-alkanes (C

8ndashC24) To this

end an Rtx-5MS capillary column was employed under thesame operating conditions as in the case of GCThe retentionindex (RI) of each PE-EO constituent was determined asdescribed previously [20] The chemical structures werecomputer-matched with theWiley 7 NIST 08 and FFNSC 12spectral libraries of the GC-MS data system their fragmenta-tion patterns were compared with the literature data [21]

26 Bacterial Strains and Antimicrobial Assays The in vitroantimicrobial activity of PN-EO and its major constituentswas assessed by minimum inhibitory concentration (MIC)values calculated by means of the broth microdilutionmethod accomplished in 96-well microplates The followingstandard ATCC strains were used Streptococcus salivarius(ATCC 25975) Streptococcus sanguinis (ATCC 10556) Strep-tococcus mitis (ATCC 49456) Streptococcus mutans (ATCC25175) Streptococcus sobrinus (ATCC 33478) Enterococcusfaecalis (ATCC 4082) and Lactobacillus casei (ATCC 11578)Individual 24-h colonies from blood agar (Difco LabsDetroit MI USA) were suspended in 100mL of trypticsoy broth (Difco) Standardization of each microorganismsuspension was carried out on a spectrophotometer (FemtoSao Paulo Brazil) operating at a wavelength (120582) of 625 nm tomatch the transmittance of 81 (equivalent to 05 McFarlandscale or 15 times 108 CFUmL) The microorganism suspensionwas diluted to a final concentration of 5 times 105 CFUmL PN-EOwas dissolved in DMSO (Merck Darmstadt Germany) at160mgmL and diluted in tryptic soy broth (Difco) to yieldconcentrations between 4000 and 39 120583gmL Compounds1 (120572-pinene) 2 (120573-pinene) 3 (trans-caryophyllene) and 4(caryophyllene oxide) were purchased from Sigma-Aldrich(St Louis MA) and evaluated by means of the samemethodology and at the same concentrations as PN-EO A1 120583M solution of each compound was tested individually Inthe case of the mixture 1 + 2 + 3 + 4 the constituents weremixed in the same proportion that they occurred in PN-EOAfter dilutions the DMSO concentrations were between 4and 00039 (vv)Three inoculated wells containing DMSOat concentrations ranging from 4 to 1 were used as neg-ative controls One inoculated well was included to control

















4 Conclusions






Acknowledgments


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4 Conclusions






Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















4 Conclusions






Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgments


References









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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Antimicrobial Activity of the Essential Oil of Plectranthus neochilus