research article high-level antimicrobial efficacy of...

Research ArticleHigh-Level Antimicrobial Efficacy ofRepresentative Mediterranean Natural PlantExtracts against Oral Microorganisms

Lamprini Karygianni1 Manuel Cecere1

Alexios Leandros Skaltsounis2 Aikaterini Argyropoulou2 Elmar Hellwig1

Nektarios Aligiannis2 Annette Wittmer3 and Ali Al-Ahmad1

1 Department of Operative Dentistry and Periodontology Center for Dental Medicine University of Freiburg Hugstetter Straszlige 5579106 Freiburg Germany

2Department of Pharmacognosy and Natural Product Chemistry Faculty of PharmacyNational and Kapodistrian University of Athens 15771 Athens Greece

3 Department of Hygiene and Microbiology Albert-Ludwigs University 79104 Freiburg Germany

Correspondence should be addressed to Ali Al-Ahmad alial-ahmaduniklinik-freiburgde

Received 24 April 2014 Revised 6 June 2014 Accepted 7 June 2014 Published 26 June 2014

Academic Editor Nikos Chorianopoulos

Copyright copy 2014 Lamprini Karygianni 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

Nature is an unexplored reservoir of novel phytopharmaceuticals Since biofilm-related oral diseases often correlate with antibioticresistance plant-derived antimicrobial agents could enhance existing treatment options Therefore the rationale of the presentreport was to examine the antimicrobial impact of Mediterranean natural extracts on oral microorganisms Five different extractsfromOlea europaea mastic gum and Inula viscosawere tested against ten bacteria and one Candida albicans strainThe extractionprotocols were conducted according to established experimental procedures Two antimicrobial assaysmdashthe minimum inhibitoryconcentration (MIC) assay and the minimum bactericidal concentration (MBC) assaymdashwere applied The screened extracts werefound to be active against each of the tested microorganisms O europaea presented MIC and MBC ranges of 007ndash1000mgmLminus1and 060ndash1000mgmLminus1 respectively The mean MBC values for mastic gum and I viscosa were 007ndash1000mgmLminus1 and 015ndash1000mgmLminus1 respectively Extracts were less effective against C albicans and exerted bactericidal effects at a concentration rangeof 007ndash500mgmLminus1 on strict anaerobic bacteria (Porphyromonas gingivalis Prevotella intermedia Fusobacterium nucleatum andParvimonas micra) Ethyl acetate I viscosa extract and total mastic extract showed considerable antimicrobial activity against oralmicroorganisms and could therefore be considered as alternative natural anti-infectious agents

1 Introduction

The Hippocratic statement ldquoNature without instruction orknowledge does what is necessaryrdquo wisely acknowledgesthe healing contribution of the vegetable kingdom to thetreatment of various diseasesThepotential of a small fractionof natural plant extracts to cure serious infections of thehuman body has beenwell established to date [1ndash4] In partic-ular various Mediterranean natural herb products have beenscreened profitably for their antioxidant and antimicrobialeffects [5ndash8] Origanum dictamnus Olea europaea Chios

mastic gum Inula viscosa Petroselinum crispum and Sideritisspp are some of the most well-known examples [7 9 10]Nevertheless the biological behavior of over 300000 existingplant species needs to be further studied [11]

The remarkable immunostimulatory activity of severalplant species is also responsible for the low occurrenceof infectious processes in their wild counterparts Themost known broad-spectrum defense mechanisms of plant-originated antimicrobial agents are related to the presenceof ldquophytoalexinsrdquo The latter are small-molecule antibiotics(molecular weight MW lt 500) and according to their

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 839019 8 pageshttpdxdoiorg1011552014839019

2 BioMed Research International

classification include polyphenols flavonoids terpenoidsand glycosteroids [12] In order to conquer the pathogenicattackers the aforementionedmild antimicrobials usually actsynergistically Moreover the production of callose a sugarpolymer with (1ndash3)-120573-D-glucan subunits at the microbialinvasion sites on the plant cell wall constitutes a typicalpathogen-specific response of vegetable organisms [13] Thesecretion of resistance (119877) proteins triggered by avirulence(Avr) genes in the presence of attacking microorganisms isalso another microbe-specific defense of plants [14]

Bacteria viruses and fungi are the cause of numerousinfectious diseases They are official residents of the humanbody and are capable of forming biofilms dynamic microbialnetworks on human substrata [15] In the oral cavity inparticular more than 700 microbial species have been recov-ered either being in a planktonic form or being embeddedin a polysaccharide-affluent extracellular layer [16] Despitethe unfriendly conditions on tooth surfaces and gingivaltissues oral microorganisms are skilled at surviving throughcomplicated physicochemical intercommunication patternswith the oral substrata [17] The harmful microbial impacton these surfaces results in the genesis of caries gingivitisor periodontitis

Recent years have seen a focus in oral microbiologyresearch on the elucidation and elimination of biofilm-relateddental diseases [18 19] Microbial biofilm communities havebeen found to be up to 1000 timesmore resistant to antibioticsagainst their equivalent planktonic cultures whilemultidrug-resistant oral microorganisms have been identified [19] Thepresence of vancomycin-resistant enterococci (VRE) andmethicillin-resistant Staphylococcus aureus (MRSA) under-lines the limits of antibiotics in facing this primary healthhazard with regard to the oral cavity [20] Furthermorean endocarditis-associated Enterococcus faecalis virulencegene named efaA has been recovered from therapy-resistantstrains in root canals [21]

The need to discover new efficient treatment strategiesagainst oral microbial species has raised interest in natu-ral vegetable sources Novel antimicrobial agents of plantorigin aim to reintroduce traditional treatment paradigmsto modern medicine Indeed plants exhibit a remarkablepharmaceutical range due to their secondary metabolism-mediated chemical responses to various microorganisms andthe synergistic paradigms they develop The latter preventmicrobes from resisting antibiotic monotherapy and there-fore becoming untreatable

The aim of the present report was to examine theantimicrobial activity of natural plant and fruit extracts ofMediterranean origin against variousmicrobial speciesMorespecifically five different extracts from olive leaves tableolives mastic gum and Inula viscosa (SynDittrichia viscosa)were screened against a panel of nine relevant pathogenicmicroorganisms which constitute typical residents of the oralmicroflora including one strain of Candida albicans Addi-tionally Staphylococcus aureus and Escherichia coli normallya part of skin and intestinal flora served as reference bacterialstrains The null hypothesis of this report was that theaforementioned extracts have no antimicrobial impact on thetested microorganisms For this purpose two antimicrobial

assaysmdashthe minimum inhibitory concentration (MIC) assayand the minimum bactericidal concentration (MBC) assaymdashwere utilized

2 Materials and Methods

21 Extraction Process

211 Olive Leaves Olea europaea leaves were dried in a well-ventilated shady place and subsequently stored in a darkroom Before their extraction the leaves were ground usingan Allen West type SCIS grinder with a sieve of 3mm Theleaves (35 Kg) were extracted by mechanical stirring for12 h with acetone (2 times 25 L) The extract was evaporatedcompletely and washed with a mixture of CH

2

Cl2

MeOH98 2 (3 L) The insoluble material was separated and driedunder reduced pressure producing a yellow powder (360 g)containing 60 oleuropein [22]

212 Table Olive Processing Wastewater Olive fruits werecured for a period of 4 months using an aqueous solutionof 8 NaCl (wv) The water extract (400mL) was appliedto Amberlite XAD-4 resin and the column eluted with 96ethanol After desorption ethanol was evaporated by dryingat 40∘C and the phenolic fraction (134 g) was recovered[22 23]

213 Conventional Extraction of Mastic Gum A quantity ofmastic gum (500 g) was diluted in ethyl acetate (500mL)1500mL of methanol was then added After a period of2 days a layer of poly-120573-myrcene (150 g) was decantedFiltration was applied in order to obtain a clear supernatantsolution and the solvent mixture was evaporated in a rotaryevaporator at 45∘C with an 80 kPa vacuum (extraction A)The resulting semisolid residue was dried in a desiccator at70∘C and 1000-mbar vacuum and resulted in a white powder(350 g) [24]

214 Inula viscosa Inula viscosa (Asteraceae) was extractedwith pressurized liquid extraction For that purpose aDionexaccelerated solvent extraction (ASE) 300 System (DionexSunnyvale CA) with 100mL stainless steel vessels was usedSpecifically 20 g of ground 119868 viscosa aerial parts was placedinto tubular extraction cells These were then placed intothe carousel and the samples were extracted under thespecified conditions temperature 70∘C pressure 120 bar(preset by the instrument) preheat time 1min heat time5min 2 extraction cycles of 5min static time each flushvolume 100 and purge 120 sec Separate preparations ofethyl acetate extracts and methanol extracts were madeAnalytical grade ethyl acetate and methanol were used andwere evaporated to dryness under reduced pressure usinga rotary evaporator (Buchi Rotavapor R-200) at 40∘C Theobtained yields were 208 gr for the ethyl acetate extractionand 351 gr for the methanol

22 Chemical Analysis of Extracts The qualitative and quan-titative determination of the tested extracts (olive leaves

BioMed Research International 3

table olive processing wastewater and Inula viscosa) wasperformed in a HPLC-DAD systemThermo FinniganHPLCsystem (Thermo Finnigan San Jose CA) coupled with aSpectral System UV6000LP PDA detector A two-solventgradient method was used (A) H

2

O and (B) acetonitrileThe flow rate was set at 1mLmin and the following elutionprogram was applied 0ndash60min linear gradient from 0A to50 B 60ndash65min linear gradient to 100 B 65ndash70min 100B isocratic 70ndash75min linear gradient to 0 B 75ndash85min0 B isocratic The analysis was performed at 25∘C andthe injection volume was 20120583L The detection was doneat 280 nm and the column used was Supelco AnalyticalDiscovery HS C18 (25 cm times 46mm id 50 120583m) Standardsolutions of oleuropein and hydroxytyrosol were preparedand run under the same conditions in the case of olive leavesand table olive processing wastewater extracts [22 23]

Concerning mastic gum the tested extract was dividedinto two fractions an acidic and a neutral oneThe acidic frac-tion after several chromatographic separations afforded themajor triterpenic acids oleanonic acid moronic acid 24Z-masticadienonic acid 24Z-isomasticadienonic acid 24Z-masticadienolic acid and 24Z-isomasticadienolic acid Theneutral fraction afforded five neutral triterpenic com-pounds tirucallol dammaradienone 28-norolean-12-en-3-one oleanonic aldehyde and oleanolic aldehyde All theabove constituents were identified by NMR (1H 13C COSYHMQC and NOESY) and MS and by comparison withdata in the literature Nuclear magnetic resonance (NMR)spectra were recorded on Bruker DRX 400 and BrukerAC 200 (503MHz for 13C NMR) instruments at 295KGas chromatography mass spectroscopy (MS) analysis wasperformed on a Finnigan GCQ Plus mass spectrometer [22]

23 Bacterial and Fungal Strains A total of ten bacterialstrains from eight different species and one Candida albicansstrain were investigated Eight of the tested bacterial strainsandC albicans occur in the oral flora whereas Staphylococcusaureus and Escherichia coli are mainly recovered from theskin and intestinal flora respectivelyThe latter twowere usedas reference microorganisms to specifically compare the oralinhibitory effect of natural extracts to their general antimi-crobial activity In particular Streptococcus mutans DSM20523 Streptococcus sobrinusDSM 20381 Streptococcus oralisATCC 35037 Enterococcus faecalisATCC 29212 and S aureusATCC 25923 are facultative anaerobic Gram-positive specieswhereas E coli ATCC 25922 is also facultative anaerobicbut has a Gram-negative cell wall Porphyromonas gingivalisW381 Prevotella intermedia ATCC 25611 Fusobacteriumnucleatum ATCC 25586 and Parvimonas micra ATCC 23195are obligate anaerobes All bacterial and fungal strains werekindly supplied by the Division of Infectious Diseases andthe Institute of Medical Microbiology and Hygiene of theAlbert-Ludwigs University Freiburg The microorganismswere deposited at minus80∘C in basic growth medium containing15 (vv) glycerol until their use

24 Determination of the Minimum Inhibitory Concentration(MIC) First an overnight culture of each bacterial and

fungal strain was prepared according to the CLSI guidelines[25 26] Each dilution was plated on Columbia blood agar(CBA) plates or yeast-cysteine blood agar (HCB) platesFacultative anaerobic bacteria andC albicanswere incubatedon CBA agar plates at 37∘C and 5ndash10 CO

2

atmosphere for24 h Anaerobic bacteria were placed on HCB agar plates at37∘C for 48 h (anaerobic chamber Genbox BioMerieux SAMarcyEtoile France) For the microdilution assay all fac-ultative anaerobic strains were inoculated in Mueller-Hintonbroth (MHB) anaerobic bacteria in Wilkins-Chalgren broth(WCB) and C albicans in Sabouraud dextrose broth (SDB)each to be tested at 106 colony forming units (CFU) mLminus1Afterwards appropriate volumes of the MHBWCBSDBmicrobial cultures were transferred into a 96-well microtiter-plate using a multichannel pipette The prepared naturalextracts were then dissolved in dimethyl sulfoxide (DMSOSigma Steinheim Germany) and diluted in aqua destillataAll extract solutions in DMSO were screened in a concentra-tion series ranging from 10mgmLminus1 to 002mgmLminus1 at dilu-tion levels starting from 2-fold to 512-fold The experimentswere performed in duplicate A 051 A McFarland standardsuspension was prepared in normal saline for bacteria andfungi respectively Each well of the 96-well microtiter-plate had a total volume of 200120583L In order to excludepotential antimicrobial effects of the DMSO residuals adilution series of DMSO was examined in parallel Wellscontaining solely MHBWCBSDB or 02 chlorhexidine(CHX) served as positive and negative controls for bacterialgrowth respectively The possibility of contamination wasminimized by using sterile MHBWCBSDB Thereafter fac-ultative anaerobic bacteria and C albicans were incubated at37∘C and 5ndash10 CO

2

atmosphere for 24 h while anaerobicbacteria were kept at 37∘C for 48 h (anaerobic chamberGenbox BioMerieux SA MarcyEtoile France) All assaysfor each bacterial and fungal strain were performed at leastin duplicate The highest minimum inhibitory concentration(MIC) values were taken into consideration in case the MICvalues of a specific strain were not identical MICwas definedas the lowest concentration of each natural extract at whichvisible inhibition of bacterial growth was induced and wasthus expressed by the percentage of bacterial growth at thatparticular concentration The inhibitory impact of DMSOwas taken into consideration if bacterial growthwas observedin the cotested DMSO dilution series

25 Determination of the Minimum Bactericidal Concen-tration (MBC) The minimum bactericidal concentration(MBC) was also assessed according to the CLSI guidelines[25 26] After completion of the MIC assay the aforemen-tioned 96-well microtiter-plates were further incubated forMBC testing In brief 10120583L from each well containing thetested natural extract concentration series was plated onColumbia blood agar (CBA) or yeast-cysteine blood agar(HCB) plates In particular the facultative anaerobic bacteriaand C albicans were incubated on CBA plates at 37∘Cand 5ndash10 CO

2

atmosphere for 2 days Strictly anaerobicbacteria were cultivated on HCB plates at 37∘C for 5 days(anaerobic chamber Genbox BioMerieux SA MarcyEtoile


France) The colony forming units (CFU) were determinedvisuallyTheMBCwas defined as the concentration atwhich athree-log decrease in bacterial growth (=999) was detectedcompared to the positive control

3 Results

31 Olea europaea Two extracts produced from olive pro-cessing byproducts were tested Extraction of olive leaveswith acetone afforded a polar fraction which was defattedwith a mixture of CH

2

Cl2

MeOH 98 2The obtained extractcontained 60 oleuropein The extract coming from tableolive processing wastewater contained as itsmain compoundthe degradation product of oleuropein hydroxytyrosol in apercentage around 15

The mean MIC and MBC values for each of the Oeuropaea extracts as well as the tested bacterialfungal strainsare presented in Table 1

In general table olive extract was more active than oliveleaf extract It was effective against almost all of the testedmicroorganisms with a mean concentration range of 015mgmLminus1 (Porphyromonas gingivalis Parvimonas micra) to1000mgmLminus1 (Candida albicans) For the facultative anae-robic bacteria MIC values varied between 125mgmLminus1(Streptococcus mutans Streptococcus oralis) and 500mgmLminus1 (Enterococcus faecalis) The MBC values of thetable olive extract ranged from 060 to 1000mgmLminus1Obligate anaerobes (Prevotella intermedia P micra) weremore easily eradicated (060mgmLminus1) when compared to Efaecalis and C albicans (1000mgmLminus1)

Olive leaf extracts showed a milder inhibitory effectagainst oral pathogens The MIC values of the eradicatedmicrobial strains were between 007mgmLminus1 (S oralis) and1000mgmLminus1 (C albicansEscherichia coli)TheMBCvaluesdemonstrated the persistence of facultative anaerobes inthe presence of O europaea leaf extracts (1000mgmLminus1)However P gingivalis was inhibited by 060mgmLminus1 of theextract while 125mgmLminus1 killed 999 of Fusobacteriumnucleatum and P micra

32 Mastic Gum Table 2 summarizes the MIC and MBCvalues of the tested mastic gum extract for all screenedmicrobial strains A total mastic extract without polymer wasprepared after removal of the contained insoluble polymerThe extensive characterization of the extract by NMR andMS revealed that it contained triterpenic acids triterpenicalcohols and aldehydes

Total mastic extract was effective against all of themicroorganismswithMICvalues ranging from002mgmLminus1(P gingivalis) to 10mgmLminus1 The mean MBC values werebetween 007mgmLminus1 (P gingivalis P micra) and1000mgmLminus1 (S mutans S sobrinus E faecalis C albicansand E coli) Extract concentrations between 007 and250mgmLminus1 exerted bactericidal effect mainly on strictanaerobic Gram-negative bacteria (P gingivalis P inter-media and F nucleatum)

33 Inula viscosa Table 3 summarizes the range of MICand MBC values of the two tested I viscosa extracts againstthe selected oral bacterialfungal strains Aerial parts of Iviscosa were extracted with two solvents of different polarityfollowing a separate extraction Ethyl acetate extract affordedmedium polarity compounds mainly flavonoids such as3-O-acetylpadmatin 7-O-methylaromadendrin hispidulinapigenin luteolin sesquiterpenic compounds and triter-penoids While the methanol extract contained some com-mon compoundswith the ethyl acetate extract phenolic acidsand flavonoids were also detected

Among the two I viscosa extracts ethyl acetate extractpresented a more robust antimicrobial effect against thescreened microorganisms compared to the methanol extractUnder its influence a mean inhibitory concentration rangeof 007mgmLminus1 (P gingivalis) to 250mgmLminus1 (S sobrinusE faecalis and E coli) was observed The MBC values ofthe ethyl acetate extract varied from 015 to 500mgmLminus1Obligate anaerobes such as P gingivalis (015mgmLminus1) wereefficiently eliminated by reduced extract concentrations ascompared to the more persistent E faecalis and E coli strains(500mgmLminus1)

The I viscosa methanol extract also had a considerableinhibitory impact on oral bacteria and fungi The MICvalues of the eradicated microorganisms ranged between015mgmLminus1 (P gingivalis) and 1000mgmLminus1 (E coli) TheMBC values ranged from 030 to 1000mgmLminus1 Hence Pgingivalis was eliminated by 030mgmLminus1 of the extractwhile 060mgmLminus1 induced a three-log reduction in bacte-rial growth for P intermedia and S oralis

4 Discussion

Taking the antibiotic resistance of oral biofilms into consid-eration the present study aimed to introduce novel plant-derived antimicrobial agents in the treatment of therapy-persistent dental diseases Hence the antimicrobial impact offive different Mediterranean natural plant and fruit extractswas investigated on representative oral bacterial strains andC albicans To the best of our knowledge this is the firstreport on the antimicrobial efficacy of Olea europaea masticgum and Inula viscosa against microorganisms which belongto the commensal flora of the oral cavity

In this study the tested O europaea extracts were foundto be especially effective against Gram-negative anaerobicperiodontal pathogens such as Porphyromonas gingivalisPrevotella intermedia and Fusobacterium nucleatum Indeedthere are several reports on the excellent antibacterial activityof O europaea extracts and pure compounds using diversemicrobial species [7 27ndash29] Sudjana et al substantiated thenarrow-spectrum antibacterial action of commercial oliveleaf extract against Campylobacter jejuni Helicobacter pyloriand Staphylococcus aureus as well as against methicillin-resistant S aureus (MRSA) [30] This highlights the regula-tory interference of O europaea extracts with gastric Gram-negative microorganisms such as C jejuni and H pylori Inanother report olive leaf extracts succeeded in eliminatingthe Gram-negative Escherichia coli Pseudomonas aeruginosa


Table 1 Antimicrobial activity in mgmLminus1 of olive leaf and table olives extracts (O europaea)

119874119897119890119886 119890119906119903119900119901119886119890119886

Sample Olive leaf extract Table olives extract DMSO ()cmgmLminus1 MIClowast MBClowastlowast MIC MBC MIC MBCStreptococcus mutans DSM 20523 125 NA 125 500 500 4000Streptococcus sobrinus DSM 20381 250 NA 250 500 2000 2000Streptococcus oralis ATCC 35037 007 NA 125 250 1000 2000Enterococcus faecalis ATCC 29212 060 NA 500 NA 2000 8000Candida albicans DSM 1386 NA NA NA NA 1000 1000Escherichia coli ATCC 25922 NA NA 500 NA 2000 4000Staphylococcus aureus ATCC 25923 250 500 250 250 2000 4000Porphyromonas gingivalisW381 030 060 015 125 500 2000Prevotella intermedia ATCC 25611 250 500 030 060 250 500Fusobacterium nucleatum ATCC 25586 060 125 060 250 1000 1000Parvimonas micra ATCC 23195 030 125 015 060 1000 2000NA no activity observed MIC or MBC was measured at 1000mgmLminus1lowastMIC extract concentration at which the optical density (OD) measurement revealed minimal bacterial growthlowastlowastMBC extract concentration at which a three-log reduction (999) of the bacterial growth was induced

Table 2 Antimicrobial activity in mgmLminus1 of four mastic gum extracts

Mastic gumSample Total mastic extract DMSO ()cmgmLminus1 MIClowast MBClowastlowast MIC MBCStreptococcus mutans DSM 20523 NA NA 500 4000Streptococcus sobrinus DSM 20381 NA NA 1000 2000Streptococcus oralis ATCC 35037 250 500 1000 2000Enterococcus faecalis ATCC 29212 NA NA 1000 8000Candida albicans DSM 1386 NA NA 1000 1000Escherichia coli ATCC 25922 NA NA 1000 1000Staphylococcus aureus ATCC 25923 250 500 2000 8000Porphyromonas gingivalisW381 002 007 1000 2000Prevotella intermedia ATCC 25611 007 030 250 500Fusobacterium nucleatum ATCC 25586 250 250 1000 2000Parvimonas micra ATCC 23195 060 060 250 1000NA no activity observed MIC or MBC was measured at 1000mgmLminus1lowastMIC extract concentration at which a three-log reduction (999) of the bacterial growth was inducedlowastlowastMBC extract concentration at which a three-log reduction (9999) of the bacterial growth was induced

and Klebsiella pneumoniae [31] However Gram-negativemicroorganisms are often resistant to conventional antimi-crobial drugs because of their expression of active effluxpumps [32] Their tenacious nature is also assisted by therelease of degrading enzymes and molecular metamorphosisof antibiotic targets [33] Moreover the overdelicate struc-ture of Gram-negative bacteria which involves structuraldiscrepancies between the cell wall and exterior membraneinfluences their susceptibility to various antimicrobial agents[34]

Furthermore table olive extract presented milder anti-staphylococcal activity suggesting an additional impact onGram-positive bacteria A previous report described theantimicrobial efficacy of O europaea leaves towards theGram-positive Bacillus cereus [27] The exact infiltration anddestruction mechanism of the Gram-positive cell wall by O

europaea extracts probably implies the presence of antiquo-rum sensing (QS) compounds [35] Since the pathogenicityof Gram-positive microorganisms such as S aureus is basedon small peptides named ldquoquormonesrdquo triggered by theagr operon or analogous QS communication patterns Oeuropaea could assumingly interfere with their action [36]Contrary to the results of an earlier study Candida albicanswas not found susceptible to table olive extract [27] Theconflicting outcomes can be attributed to the differing extrac-tion methods which were used including boiling Howeverwhich of the numerous phenolic or other compounds wasresponsible for this favorable effect remains unknown Inthe present study the two main compounds of the extractswere oleuropein in olive leaves and hydroxytyrosol in tableolive processing wastewater A lot of studies claim the strongantimicrobial activity of the two compounds [22 37]


Table 3 Antimicrobial activity in mgmLminus1 of I viscosa extracts

119868119899119906119897119886 V119894119904119888119900119904119886Sample Ethyl acetate extract Methanol extract DMSO ()cmgmLminus1 MIClowast MBClowastlowast MIC MBC MIC MBCStreptococcus mutans DSM 20523 015 125 125 500 500 4000Streptococcus sobrinus DSM 20381 250 250 250 500 2000 2000Streptococcus oralis ATCC 35037 030 030 060 060 1000 2000Enterococcus faecalis ATCC 29212 250 500 500 500 2000 8000Candida albicans DSM 1386 125 250 500 500 1000 1000Escherichia coli ATCC 25922 250 500 NA NA 2000 4000Staphylococcus aureus ATCC 25923 060 125 125 250 2000 4000Porphyromonas gingivalisW381 007 015 015 030 500 2000Prevotella intermedia ATCC 25611 015 030 030 060 250 500Fusobacterium nucleatum ATCC 25586 030 060 060 125 1000 1000Parvimonas micra ATCC 23195 015 060 060 125 1000 2000NA no activity observed MIC or MBC was measured at 1000mgmLminus1lowastMIC extract concentration at which the optical density (OD) measurement revealed minimal bacterial growthlowastlowastMBC extract concentration at which a three-log reduction (999) of the bacterial growth was induced

Mastic gum constitutes a stem-derived resinous exudateof the Mediterranean tree Pistacia lentiscus var chia Pgingivalis P intermedia F nucleatum and P micra demon-strated high susceptibility toall of the mastic gum extractsstudiedThe numerous reports on the favorable antimicrobialproperties of mastic gum also underline its effectivenessagainst various pathogens [6 24 38ndash40] More specificallya commercial product containing mastic in the liquid form(2) showed a narrow antibacterial spectrum against Gram-negative P gingivalis and Prevotella melaninogenica [40]Gram-positive bacteria (Streptococcus mutans S aureus) aswell as fungi (C albicans) were not influenced Takahashiet al confirmed a decrease in the total number of bacterialsalivary colonies after 4 hours of chewing mastic gum [41]Since side effects such as tooth discoloration and enhancedcell toxicity accompany the application of conventional oralantibacterial chemicals such as chlorhexidine (CHX) theintroduction of mastic-derived mouth rinses could promoteantiplaque activity with minimal drawbacks

In general mastic gum is composed of various volatileingredients the most important of which are 120572-pinene 120573-myrcene 120573-caryophyllene 120573-pinene and limonene [6] Theherein tested extract though contained mostly triterpenicacids which could be the active principles It was previouslyfound that total mastic extract without polymer containingmostly major triterpenic acids can efficiently eliminate otherpersistentmicroorganisms such asH pylori Reduced activitywas observed for the triterpenic alcohols and aldehydes[24] The additionally contained triterpenic alcohols andaldehydes have not showed a respective antimicrobial actionKoutsoudaki et al detectedmoderate to high susceptibility ofB subtilis and S aureus to 120573-myrcene while E coli exhibitedno sensitivity to the substance [39] Furthermore 120573-pineneproved to be ineffective against E coli and S aureus whereasB subtilis showed only a mild response These varyingoutcomes suggest that a synergistic activity of the differentcomponents of mastic gum extracts may be responsible for

its antimicrobial actionThus the results of the present studyindicate that the interdependent antimicrobial activity of theparticular mastic ingredients focuses on eradicating therapy-resistant Gram-negative oral microorganisms

In this study I viscosa was highly effective against notonly Gram-negative anaerobic pathogens but also strepto-cocci (S mutans S oralis) Although its advantageous effectsin the oral cavity were demonstrated here for the first timethere are many reports appraising its general antibacterialantifungal and antiviral properties [42ndash45] The differencebetween the activities of the two extracts could be attributedto the different chemical profile Generally the two extractscontain a lot of common metabolites however the mostpolar ones are found at the methanol extract In a previousreport methanol extracts of the plant demonstrated effectiveantimicrobial behavior against Gram-positive bacteria suchas S aureus andE faecalisC albicans andCandida tropicaliswere also found to be sensitive to I viscosa whereas Gram-negative bacteria were less susceptible [45]The antimicrobialimpact of I viscosa was also observed on Bacillus cereus andSalmonella typhimurium In addition it was revealed thata compound of the plant named 331015840-di-O-methylquercetincan deteriorate the cytoplasmic membranes after penetratingvarious bacterial cell walls [44] Moreover Wang et alreported that I viscosa presented fungicidal propertiesagainst pathogens of the families Oomycetes Ascomycetesand Basidiomycetes [46] This is in agreement with thefindings of this study in which I viscosa exerted bacteri-cidal effects at a concentration range of 250ndash500mgmLminus1towards C albicans

Medicinal plants have proven to be a bountiful reservoirof numerous biologically active components with advancedantibacterial properties It is well known that syntheticchemicals which are commonly used in dental productssuch as toothpastes and oral mouthwashes can induce toothdiscoloration cell toxicity regurgitation or diarrhea [47 48]Thus plant extracts are considered to be potent alternative


compounds for the new generation of oral pharmaceu-ticals In fact novel plant-derived constituents of dentalchemotherapeutics (mouth rinses irrigation solutions andintracanal medicaments) are crucial future sources of thedental industry specializing in plaque-control strategiessince they allow for efficient treatment regarding antibiotic-resistant pathogens immunocompromised individuals andfinancially weak developing countries

5 Conclusion

In conclusion the results of this report promote interest in thediscovery of alternative natural compounds with antimicro-bial activity against therapy-resistant oral microorganismsOverall extracts fromO europaea mastic gum and I viscosawere active against the tested oral pathogens especiallyGram-negative anaerobic bacteria and could therefore beconsidered as alternative natural anti-infectious agents whichcould be used against periodontitis

Conflict of Interests

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

References

[1] A Sofrata E M Santangelo M Azeem A Borg-Karlson AGustafsson and K Putsep ldquoBenzyl isothiocyanate a majorcomponent from the roots of Salvadora persica is highly activeagainst Gram-Negative bacteriardquo PLoS ONE vol 6 no 8Article ID e23045 2011

[2] C J Paddon P J Westfall D J Pitera et al ldquoHigh-level semi-synthetic production of the potent antimalarial artemisininrdquoNature vol 496 no 7446 pp 528ndash532 2013

[3] Y Takebe C J Saucedo G Lund et al ldquoAntiviral lectins fromred and blue-green algae show potent in vitro and in vivoactivity against hepatitis C virusrdquoPLoSONE vol 8 no 5 ArticleID e64449 2013

[4] D S Trentin D B Silva M W Amaral et al ldquoTanninspossessing bacteriostatic effect impair Pseudomonas aeruginosaadhesion and biofilm formationrdquo PLoS ONE vol 8 no 6Article ID e66257 2013

[5] C Spanou D Stagos L Tousias et al ldquoAssessment of antioxi-dant activity of extracts from unique Greek varieties of Legumi-nosae plants using in vitro assaysrdquo Anticancer Research vol 27no 5A pp 3403ndash3410 2007

[6] S Paraschos S Mitakou and A-L Skaltsounis ldquoChios gummastic a review of its biological activitiesrdquo Current MedicinalChemistry vol 19 no 14 pp 2292ndash2302 2012

[7] EM Odiatou A L Skaltsounis and A I Constantinou ldquoIden-tification of the factors responsible for the in vitro pro-oxidantand cytotoxic activities of the olive polyphenols oleuropein andhydroxytyrosolrdquoCancer Letters vol 330 no 1 pp 113ndash121 2013

[8] N Silva S Alves A Goncalves J S Amaral and P PoetaldquoAntimicrobial activity of essential oils from Mediterraneanaromatic plants against several foodborne and spoilage bacte-riardquo Food Science and Technology International vol 19 no 6pp 503ndash10 2013

[9] C C Liolios K Graikou E Skaltsa and I Chinou ldquoDittany ofCrete a botanical and ethnopharmacological reviewrdquo Journal ofEthnopharmacology vol 131 no 2 pp 229ndash241 2010

[10] E Gonzalez-Burgos M E Carretero and M P Gomez-Serranillos ldquoSideritis spp uses chemical composition andpharmacological activitiesmdasha reviewrdquo Journal of Ethnopharma-cology vol 135 no 2 pp 209ndash225 2011

[11] L T Ngo J I Okogun and W R Folk ldquo21st Century nat-ural product research and drug development and traditionalmedicinesrdquoNatural Product Reports vol 30 no 4 pp 584ndash5922013

[12] S Hemaiswarya A K Kruthiventi and M Doble ldquoSynergismbetween natural products and antibiotics against infectiousdiseasesrdquo Phytomedicine vol 15 no 8 pp 639ndash652 2008

[13] RMaor and K Shirasu ldquoThe arms race continues Battle strate-gies between plants and fungal pathogensrdquo Current Opinion inMicrobiology vol 8 no 4 pp 399ndash404 2005

[14] J L Dangl and J D G Jones ldquoPlant pathogens and integrateddefence responses to infectionrdquo Nature vol 411 no 6839 pp826ndash833 2001

[15] T J Battin W T Sloan S Kjelleberg et al ldquoMicrobiallandscapes New paths to biofilm researchrdquo Nature ReviewsMicrobiology vol 5 no 1 pp 76ndash81 2007

[16] B J Keijser E Zaura S M Huse et al ldquoPyrosequencinqanalysis of the oral microflora of healthy adultsrdquo Journal ofDental Research vol 87 no 11 pp 1016ndash1020 2008

[17] S S Socransky and A D Haffajee ldquoDental biofilms difficulttherapeutic targetsrdquo Periodontology 2000 vol 28 no 1 pp 12ndash55 2002

[18] A Al-Ahmad A Wunder T M Auschill et al ldquoThe invivo dynamics of Streptococcus spp Actinomyces naeslundiiFusobacterium nucleatum and Veillonella spp in dental plaquebiofilm as analysed by five-colour multiplex fluorescence in situhybridizationrdquo Journal of Medical Microbiology vol 56 no 5pp 681ndash687 2007

[19] L Karygianni M Follo E Hellwig et al ldquoMicroscope-basedimaging platform for large-scale analysis of oral biofilmsrdquoApplied and Environmental Microbiology vol 78 no 24 pp8703ndash8711 2012

[20] K Smith D P Robertson D F Lappin and G RamageldquoCommercial mouthwashes are ineffective against oral MRSAbiofilmsrdquo Oral Surgery Oral Medicine Oral Pathology and OralRadiology vol 115 no 5 pp 624ndash629 2013

[21] A Al-Ahmad H Ameen K Pelz et al ldquoAntibiotic resistanceand capacity for biofilm formation of different bacteria isolatedfrom endodontic infections associated with root-filled teethrdquoJournal of Endodontics vol 40 no 2 pp 223ndash230 2014

[22] J D Kyriazis N Aligiannis P Polychronopoulos A Skaltsou-nis and E Dotsika ldquoLeishmanicidal activity assessment of olivetree extractsrdquo Phytomedicine vol 20 no 3-4 pp 275ndash281 2013

[23] A Agalias P Magiatis A L Skaltsounis et al ldquoA new processfor the management of olive oil mill waste water and recoveryof natural antioxidantsrdquo Journal of Agricultural and Food Chem-istry vol 55 no 7 pp 2671ndash2676 2007

[24] S Paraschos P Magiatis S Mitakou et al ldquoIn vitro and in vivoactivities of chios mastic gum extracts and constituents againstHelicobacter pylorirdquo Antimicrobial Agents and Chemotherapyvol 51 no 2 pp 551ndash559 2007

[25] NCCLS National Committee for Clinical Laboratory StandardsNCCLS Wayne Pa USA 1999


[26] NCCLS National Committee for Clinical Laboratory StandardsNCCLS Villanova Pa USA 6th edition 2003

[27] A P Pereira I C F R Ferreira F Marcelino et al ldquoPhenoliccompounds and antimicrobial activity of olive (Olea europaeaL Cv Cobrancosa) leavesrdquo Molecules vol 12 no 5 pp 1153ndash1162 2007

[28] O-H Lee and B-Y Lee ldquoAntioxidant and antimicrobial activi-ties of individual and combined phenolics inOlea europaea leafextractrdquo Bioresource Technology vol 101 no 10 pp 3751ndash37542010

[29] E Zoidou E Melliou E Gikas A Tsarbopoulos P Magiatisand A Skaltsounis ldquoIdentification of throuba thassos a tradi-tional Greek table olive variety as a nutritional rich source ofoleuropeinrdquo Journal of Agricultural and Food Chemistry vol 58no 1 pp 46ndash50 2010

[30] A N Sudjana C DrsquoOrazio V Ryan et al ldquoAntimicrobialactivity of commercial Olea europaea (olive) leaf extractrdquoInternational Journal of Antimicrobial Agents vol 33 no 5 pp461ndash463 2009

[31] D Markın L Duek and I Berdıcevsky ldquoIn vitro antimicrobialactivity of olive leavesrdquo Mycoses vol 46 no 3-4 pp 132ndash1362003

[32] J Pages and L Amaral ldquoMechanisms of drug efflux andstrategies to combat them challenging the efflux pump ofGram-negative bacteriardquo Biochimica et Biophysica Acta vol1794 no 5 pp 826ndash833 2009

[33] L J V Piddock ldquoClinically relevant chromosomally encodedmultidrug resistance efflux pumps in bacteriardquo Clinical Micro-biology Reviews vol 19 no 2 pp 382ndash402 2006

[34] M Friedman P R Henika and R E Mandrell ldquoBactericidalactivities of plant essential oils and some of their isolatedconstituents against Campylobacter jejuni Escherichia coli Lis-teria monocytogenes and Salmonella entericardquo Journal of FoodProtection vol 65 no 10 pp 1545ndash1560 2002

[35] B Gray P Hall and H Gresham ldquoTargeting agr- and agr-likequorum sensing systems for development of common ther-apeutics to treat multiple gram-positive bacterial infectionsrdquoSensors vol 13 no 4 pp 5130ndash5166 2013

[36] J S Wright III K E Traber R Corrigan S A Benson J MMusser and R P Novick ldquoThe agr radiation an early event inthe evolution of staphylococcirdquo Journal of Bacteriology vol 187no 16 pp 5585ndash5594 2005

[37] H K Obied P D Prenzler S H Omar et al ldquoPharmacology ofolive biophenolsrdquo Advances in Molecular Toxicology vol 6 pp195ndash242 2012

[38] P Magiatis E Melliou A Skaltsounis I B Chinou and SMitaku ldquoChemical composition and antimicrobial activity ofthe essential oils of Pistacia lentiscus var chiardquo Planta Medicavol 65 no 8 pp 749ndash752 1999

[39] C Koutsoudaki M Krsek and A Rodger ldquoChemical compo-sition and antibacterial activity of the essential oil and the gumof Pistacia lentiscus Var chiardquo Journal of Agricultural and FoodChemistry vol 53 no 20 pp 7681ndash7685 2005

[40] H Sakagami K KishinoMKobayashi et al ldquoSelective antibac-terial and apoptosis-modulating activities of masticrdquo In Vivovol 23 no 2 pp 215ndash224 2009

[41] K Takahashi M Fukazawa H Motohira K Ochiai HNishikawa and T Miyata ldquoA pilot study on antiplaque effectsof mastic chewing gum in the oral cavityrdquo Journal of Periodon-tology vol 74 no 4 pp 501ndash505 2003

[42] M Maoz and I Neeman ldquoEffect of Inula viscosa extracton chitin synthesis in dermatophytes and Candida albicansrdquoJournal of Ethnopharmacology vol 71 no 3 pp 479ndash482 2000

[43] A B Sassi F Harzallah-Skhiri N Bourgougnon andMAounildquoAntiviral activity of some Tunisian medicinal plants againstHerpes simplex virus type 1rdquo Natural Product Research vol 22no 1 pp 53ndash65 2008

[44] W H Talib M H Abu Zarga and A M Mahasneh ldquoAntipro-liferative antimicrobial and apoptosis inducing effects of com-pounds isolated from Inula viscosardquoMolecules vol 17 no 3 pp3291ndash3303 2012

[45] A Gokbulut O Ozhan B Satilmis K Batcioglu S Gunal andE Sarer ldquoAntioxidant and antimicrobial activities and phenoliccompounds of selected inula species from Turkeyrdquo NaturalProduct Communications vol 8 no 4 pp 475ndash478 2013

[46] W Wang B H Ben-Daniel and Y Cohen ldquoControl of plantdiseases by extracts of Inula viscosardquo Phytopathology vol 94no 10 pp 1042ndash1047 2004

[47] J Y Chung J H Choo M H Lee and J K Hwang ldquoAnticar-iogenic activity of macelignan isolated from Myristica fragrans(nutmeg) against StreptococcusmutansrdquoPhytomedicine vol 13no 4 pp 261ndash266 2006

[48] R A Neumegen A R Fernandez-Alba and Y Chisti ldquoToxici-ties of triclosan phenol and copper sulfate in activated sludgerdquoEnvironmental Toxicology vol 20 no 2 pp 160ndash164 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


classification include polyphenols flavonoids terpenoidsand glycosteroids [12] In order to conquer the pathogenicattackers the aforementionedmild antimicrobials usually actsynergistically Moreover the production of callose a sugarpolymer with (1ndash3)-120573-D-glucan subunits at the microbialinvasion sites on the plant cell wall constitutes a typicalpathogen-specific response of vegetable organisms [13] Thesecretion of resistance (119877) proteins triggered by avirulence(Avr) genes in the presence of attacking microorganisms isalso another microbe-specific defense of plants [14]

Bacteria viruses and fungi are the cause of numerousinfectious diseases They are official residents of the humanbody and are capable of forming biofilms dynamic microbialnetworks on human substrata [15] In the oral cavity inparticular more than 700 microbial species have been recov-ered either being in a planktonic form or being embeddedin a polysaccharide-affluent extracellular layer [16] Despitethe unfriendly conditions on tooth surfaces and gingivaltissues oral microorganisms are skilled at surviving throughcomplicated physicochemical intercommunication patternswith the oral substrata [17] The harmful microbial impacton these surfaces results in the genesis of caries gingivitisor periodontitis

Recent years have seen a focus in oral microbiologyresearch on the elucidation and elimination of biofilm-relateddental diseases [18 19] Microbial biofilm communities havebeen found to be up to 1000 timesmore resistant to antibioticsagainst their equivalent planktonic cultures whilemultidrug-resistant oral microorganisms have been identified [19] Thepresence of vancomycin-resistant enterococci (VRE) andmethicillin-resistant Staphylococcus aureus (MRSA) under-lines the limits of antibiotics in facing this primary healthhazard with regard to the oral cavity [20] Furthermorean endocarditis-associated Enterococcus faecalis virulencegene named efaA has been recovered from therapy-resistantstrains in root canals [21]

The need to discover new efficient treatment strategiesagainst oral microbial species has raised interest in natu-ral vegetable sources Novel antimicrobial agents of plantorigin aim to reintroduce traditional treatment paradigmsto modern medicine Indeed plants exhibit a remarkablepharmaceutical range due to their secondary metabolism-mediated chemical responses to various microorganisms andthe synergistic paradigms they develop The latter preventmicrobes from resisting antibiotic monotherapy and there-fore becoming untreatable

The aim of the present report was to examine theantimicrobial activity of natural plant and fruit extracts ofMediterranean origin against variousmicrobial speciesMorespecifically five different extracts from olive leaves tableolives mastic gum and Inula viscosa (SynDittrichia viscosa)were screened against a panel of nine relevant pathogenicmicroorganisms which constitute typical residents of the oralmicroflora including one strain of Candida albicans Addi-tionally Staphylococcus aureus and Escherichia coli normallya part of skin and intestinal flora served as reference bacterialstrains The null hypothesis of this report was that theaforementioned extracts have no antimicrobial impact on thetested microorganisms For this purpose two antimicrobial

assaysmdashthe minimum inhibitory concentration (MIC) assayand the minimum bactericidal concentration (MBC) assaymdashwere utilized

2 Materials and Methods

21 Extraction Process

211 Olive Leaves Olea europaea leaves were dried in a well-ventilated shady place and subsequently stored in a darkroom Before their extraction the leaves were ground usingan Allen West type SCIS grinder with a sieve of 3mm Theleaves (35 Kg) were extracted by mechanical stirring for12 h with acetone (2 times 25 L) The extract was evaporatedcompletely and washed with a mixture of CH

2

Cl2

MeOH98 2 (3 L) The insoluble material was separated and driedunder reduced pressure producing a yellow powder (360 g)containing 60 oleuropein [22]

212 Table Olive Processing Wastewater Olive fruits werecured for a period of 4 months using an aqueous solutionof 8 NaCl (wv) The water extract (400mL) was appliedto Amberlite XAD-4 resin and the column eluted with 96ethanol After desorption ethanol was evaporated by dryingat 40∘C and the phenolic fraction (134 g) was recovered[22 23]

213 Conventional Extraction of Mastic Gum A quantity ofmastic gum (500 g) was diluted in ethyl acetate (500mL)1500mL of methanol was then added After a period of2 days a layer of poly-120573-myrcene (150 g) was decantedFiltration was applied in order to obtain a clear supernatantsolution and the solvent mixture was evaporated in a rotaryevaporator at 45∘C with an 80 kPa vacuum (extraction A)The resulting semisolid residue was dried in a desiccator at70∘C and 1000-mbar vacuum and resulted in a white powder(350 g) [24]

214 Inula viscosa Inula viscosa (Asteraceae) was extractedwith pressurized liquid extraction For that purpose aDionexaccelerated solvent extraction (ASE) 300 System (DionexSunnyvale CA) with 100mL stainless steel vessels was usedSpecifically 20 g of ground 119868 viscosa aerial parts was placedinto tubular extraction cells These were then placed intothe carousel and the samples were extracted under thespecified conditions temperature 70∘C pressure 120 bar(preset by the instrument) preheat time 1min heat time5min 2 extraction cycles of 5min static time each flushvolume 100 and purge 120 sec Separate preparations ofethyl acetate extracts and methanol extracts were madeAnalytical grade ethyl acetate and methanol were used andwere evaporated to dryness under reduced pressure usinga rotary evaporator (Buchi Rotavapor R-200) at 40∘C Theobtained yields were 208 gr for the ethyl acetate extractionand 351 gr for the methanol

22 Chemical Analysis of Extracts The qualitative and quan-titative determination of the tested extracts (olive leaves



2






2


2



2




3 Results


2

Cl2










4 Discussion





119874119897119890119886 119890119906119903119900119901119886119890119886

















5 Conclusion




References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



2






2


2



2




3 Results


2

Cl2










4 Discussion





119874119897119890119886 119890119906119903119900119901119886119890119886

















5 Conclusion




References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



3 Results


2

Cl2










4 Discussion





119874119897119890119886 119890119906119903119900119901119886119890119886

















5 Conclusion




References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



119874119897119890119886 119890119906119903119900119901119886119890119886

















5 Conclusion




References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











5 Conclusion




References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article high-level antimicrobial efficacy of...

Documents