Rev. bras. farmacogn. vol.27 número5

RevistaBrasileiradeFarmacognosia27(2017)599–602

w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Anti-adhesion

potential

of

non-polar

compounds

and

extracts

from

Ficus

natalensis

Gbonjubola

V.

Awolola

_,

_Hafizah

_Chenia

_,

_Himansu

_Baijnath

_,

_Neil

_A.

_Koorbanally

a_{SchoolofChemistryandPhysics,UniversityofKwaZulu-Natal,Durban,SouthAfrica}

b_{SchoolofLifeSciences,UniversityofKwaZulu-Natal,Durban,SouthAfrica}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14April2017

Accepted5July2017

Availableonline1September2017

Keywords:

Ficusnatalensissubsp.natalensis

Fig

Bacterialadhesion

Biofilmreduction

a

b

s

t

r

a

c

t

Fourtriterpenoids,ergosta-4,6,8(14),22-tetraene-3-one1,stigma-4-ene-3-one2,3␤-hydroxy-21␤-H -hop-22(29)-ene3,sitosterolandaquinone,tectoquinone4,wereisolatedfromtheleaf,stembarkand fruitextractsofFicusnatalensissubsp.natalensis,Moraceae,amedicinalfigfoundinAfrica.Thepure compounds1–4andcrudeextractsweretestedfortheirantibacterialactivityagainstfiveGram-negative andsevenGram-positivestrainsandfortheirpotentialanti-biofilmactivity.Antimicrobial susceptibil-itywasobservedwithallpurecompoundstestedat250␮gagainstthemajorityofGram-negativeand Gram-positivestrains.Thedichloromethane-solublefruitextractwasactiveagainstsensitiveand resis-tantStaphylococcusaureusstrains,EnterococcusfaecalisandStaphylococcusxylosus.Compounds2,3and

4demonstratedbroad-spectrumantibioticeffectsagainsteightofthetwelvebacterialstrainstested. Intheanti-biofilmassay,exposuretoethylacetate,methanolandaqueousmethanolleaf,stembark andfruitextractsdecreasedadhesionwithabiofilmreductionof≥100%forallthreetestedorganisms: Escherichiacoli,PseudomonasaeruginosaandS.aureus.Themethanolleafextractdemonstratedthemost potentanti-adhesionpotentialagainstE.coli(218%biofilmreduction).Thegreatestabilitytodecrease adhesionwasobservedwithcompounds2,3and5againstP.aeruginosaatthelowestconcentration tested(100␮gml−1_).

©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Adhesionofmicroorganismstodiversesurfacesandtoother microbesisessentialforbiofilmdevelopmentandestablishmentof infections.Whenbacteriacoloniseandadheretosurfaces,theyare associatedwithsevere,recurrentinfectionsandbecome10–1000 timesmoreresistanttoantimicrobialagents(Borgesetal.,2015). Pharmaceuticalsorpharmaceuticalextractscapableofpreventing bacterialadhesiontosurfacesarethereforedesirableantibacterial agents.Onesourceofthesearemedicinalplantsusedintraditional medicine,since manyplant-basedremedies areknowntohave therapeuticantimicrobialactivityagainstclinicalpathogens and anincreasingnumberhave evenbeenidentifiedashaving anti-adhesionproperties(Borgesetal.,2016).

FicusnatalensisHochst.subsp. natalensis,Moraceae,is arock splittertreewhichgrowsuptoalmost20minheight.Itis geo-graphicallysituatedinSouthAfrica,Zimbabwe,Zambia,Malawi, Mozambique and Kenya. The different parts of the tree have

∗ _{Correspondingauthor.}

E-mail:koorbanally@ukzn.ac.za(N.A.Koorbanally).

variousapplicationsvaryingfromcultural,decorative,and com-mercialapplicationstofolkmedicine,isa majorsourceof bark clothandapotentialnaturaldye(BurrowsandBurrows,2003).The root,bark,leavesandlatexareusedinseveralAfricancountries totreatavarietyofmedicalailmentsincludingmalaria,influenza, whoopingcough,dysenteryandguinea worm(Hutchingsetal., 1996;BurrowsandBurrows,2003).

Inpreviousstudiesontheplant,antibacterialactivityhasbeen reportedforthemethanolextractoftheroot(RabeandvanStaden, 1997).Apartfromthis,Olaokunetal.(2013)alsoreportedthe anti-diabeticpotentialoftheacetoneextractoftheleaves.

Materialsandmethods

Thefruits,leavesandstembarkfromFicusnatalensisHochst. subsp.natalensis,Moraceae,werecollectedinDurban, KwaZulu-Natal,SouthAfricainDecember2012.Theplantwasidentifiedat theWardherbariumoftheUniversityofKwaZulu-Natal,wherea voucherspecimenwasdepositedunderthecollectornumberG.V. Awolola&H.Baijnath3.Thegroundleaves,stembarkandfruits wereextractedtwicesequentiallywithorganicsolventsof increas-ingpolarity,hexane(Hex),dichloromethane(DCM),ethylacetate

http://dx.doi.org/10.1016/j.bjp.2017.07.004

0102-695X/©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

600 G.V.Awololaetal./RevistaBrasileiradeFarmacognosia27(2017)599–602

(EtOAc)andmethanol(MeOH).Detailsoftheextractionaregiven inthesupplementarydata.

Thecrude extracts weresubjected tovacuumcolumn chro-matography (CC) on silica gel using a Hex-DCM-EtOAc-MeOH gradient elution, to afford five major fractions (A–E) by TLC. Compound 3 (250.6mg), identified as 3␤-hydroxy-21␤-H -hop-22(29)-enecrystallisedoutoffractionAoftheDCMandHexcrude extractoftheleaves.FractionBofthesamecrudeextracts con-tained1ergosta-4,6,8(14),22-tetraene-3-one(140.5mg).Sitosterol (80.6mg)wasisolated fromfractionBof theEtOAc fractionof theleaves.Compound4,tectoquinone(70.8mg)wasobtainedby purificationoffractionBoftheHexandDCMcrudeextractsofthe stembark.Stigma-4-ene-3-one(2)(70.4mg)wasobtainedfrom fractionCofthesameextracts.Allcompoundswereisolatedand purifiedbysilicagelCCusingvariousratiosofHex:EtOAcand iden-tifiedbytheir1_Hand13_{CNMRdataandverifiedbydatainthe} literature,1(Fangkrathoketal.,2013),2(Liuetal.,2014),3(Sousa etal.,2012),sitosterol(Rasoanaivoetal.,2014)and4(Chengetal., 2008).

Antibacterial susceptibility testing (AST) was carried out in duplicateon12bacterialindicatorstrains(givenin the supple-mentarymaterial)usingthediscdiffusionmethodaccordingto standardprocedures(CLSI,2007).Bacterialadhesiononthree bac-terialstrains:EscherichiacoliATCC35218,Staphylococcusaureus ATCC43300andPseudomonasaeruginosaATCC27853basedonthe resultsfromtheASTaccordingtothecrystalvioletmicrotitreplate assaywerecarriedoutintriplicate(Bassonetal.,2008).Detailsof theassaysaregiveninthesupplementarydata.

Resultsanddiscussion

Thefractionationand purificationoftheleaf,stem barkand fruitextractsledtotheisolation andidentificationof five non-polarcompounds.Tothebestofourknowledge,this isthefirst reportofsecondarymetabolitesfromF.natalensissubsp.natalensis.

ThecrudeextractsofthedifferentorgansofF.natalensiswere testedalongwithcompounds1–4againstthebacterialstrainsto determinewhethertheyhadanyantibacterialactivityto substanti-atethereporteduseoftheplantasanantibacterialagent(Rabeand vanStaden,1997).Therehasbeenextensiveantibacterialstudies ontheubiquitoussitosterolandthereforeitsantibacterial proper-tieswasnottestedagain.

TheDCMextractofthefruitwasthemostactiveofallextracts demonstratingactivityagainstseveraloftheGram-positivestrains. Theantibacterialactivityofallextractsarereportedinthe sup-plementarymaterial.RabeandvanStaden(1997)havepreviously observedMICof4mgml−1 _{forS.aureusandStaphylococcus}

epi-dermidisand8mgml−1_{forBacillussubtilis,usingmethanolicroot} extractsofF.natalensis.

Varyingantibacterialactivitywasdemonstratedbytheisolated compounds,which rangedfromresistanttosusceptible against bothGram-negativeandGram-positiveorganisms(Table1).The antibacterialactivitywasdose-dependent,beingmore activeat 250␮g than at 100␮g. Compounds1–4 showed good activity against␤-lactamresistantE.coliATCC35218and theextended spectrum ␤-lactamase producing Klebsiella pneumoniae ATCC 700603 but was only moderately active against P. aeruginosa. AmongsttheGram-positive bacteria,thebestactivitywasseen bythemethicillin-resistantS.aureus(MRSA)strainATCC43300 byallfourcompoundsfollowing250␮gexposure(Table1).B. sub-tilisandEnterococcusfaecalisstrainsdemonstratedvaryinglevelsof susceptibilitytoallfourcompounds.Allcompounds,1–4,showed broad-spectrumantibioticeffects withas muchas10 ofthe12 indicatorbacterialstrainsbeingsusceptibletothem.Tectoquinone

4showedhighzonesofinhibitionof25mmand23mmagainst StaphylococcussciuriandK.pneumoniae,respectivelyand stigma-4-ene-3-one2at250␮gdemonstratedahighzoneofinhibition againstStaphylococcusxylosusof23mm.

Biofilmsinbodytissuescanbeformedbybacteriasuchas Acine-tobacterbaumannii,E.coli,P.aeruginosa,andS.aureus(Borgesetal., 2016).DecreasedadhesionwasobservedforallEtOAc,MeOHand aq/MeOHleaf,stembarkandfruitextractsagainstthreeresistant bacterialstrains,E.coliATCC35218,P.aeruginosaATCC27853and S.aureusATCC43300(seesupplementarydata).

The MeOH leaf extract displayed the greatest anti-adhesion potentialagainstE.coli,whilemajorityoftheextracts,withthe exceptionoftheDCMandHexextract(leavesandfruit)showed reduced biofilm formation by P. aeruginosa. For S. aureus, the aq/MeOH leaf,EtOAcstembarkand fruitandMeOH stem bark extractsreducedbiofilmformation.Allthepolarextractsdecreased adhesionsuggestinginterferencebytheextractsontheabilityof thesemicroorganismstoadheretopolystyrenesurfaces.Thereare varyingreportsonanti-adhesioneffectsofpolarextractsagainst differentbacterialstrainsintheliterature,whichcorroborateour findings. The anti-adhesion effects of polar extracts have been reportedforE.coliandBacilluscereus(Bräunlichetal.,2013),oral bacteriaStreptococcusmutans andStreptococcussobrinus (Rahim andKhan,2006),bothS.aureusandMRSA(Chusrietal.,2012)and P.aeruginosa(Sarkaretal.,2014).

Theactivityoftheisolatedcompoundswithregardto bacte-rialadhesionwasvariable.Allfourcompoundsincreasedadhesion ofE.coliATCC35218atallconcentrations(Table2). Stigmast-4-en-one(2)decreasedadhesionofP.aeruginosaATCC27853atall concentrations,with100␮gml−1_{beingthemosteffective.} Ergost-4,6,8(14),22-tetraen-3-one(1)increasedadhesionat100␮gml−1_, but decreased adhesionat inhibitory and supra-inhibitory con-centrations.For 3and4,decreasedadhesionwasonlyobserved at sub-inhibitory (100␮gml−1_{)concentrations, while increased} adhesionwasobservedatinhibitoryandsupra-inhibitory concen-trations.IncreasedadhesionofS.aureusATCC43300wasobserved withallfourcompoundsatallconcentrationstested(Table2).

The variableactivity observed withtheisolated compounds wasinaccordancewiththeeffectsofoleanolicacidand␤-amyrin acetate from Vernonia auriculifera against biofilm formation by seven different bacterial strains (Kiplimo et al., 2011). Strain-specificeffects havebeen observedwiththetriterpenes ursolic andbetulinicacids,fromtheliverwortLepidoziachorduliferawhich inhibitedbiofilmformationandelastolyticactivityofP. aerugin-osaATCC27853,butincreasedadhesionofS.aureusATCC6538 (Gilabertetal.,2015).Theincreasedadhesionbeingobservedwith some of the isolated compounds may be due to promotion of microbialadhesionbyproviding asuitableconditioningfilmfor enhancementofcellattachment(Selimetal.,2014).

G.V.Awololaetal./RevistaBrasileiradeFarmacognosia27(2017)599–602 601

Table1

Antibacterialactivityofisolatedcompounds(zonesofinhibitioninmm)fromFicusnatalensissubsp.natalensis.

Compound Zonesofinhibition(mm)

Gram-negativebacteria

E.coliATCC25922 E.coliATCC35218 P.aeruginosa ATCC27853

P.aeruginosa ATCC35032

K.pneumoniae ATCC700603

100␮g 250␮g 100␮g 250␮g 100␮g 250␮g 100␮g 250␮g 100␮g 250␮g

1 10R ₂₀S ₉R ₁₅S ₇R ₁₅S ₉R ₁₅S ₉R ₁₉S

2 10R ₁₈S ₉R ₁₆S ₀R ₁₅S ₁₀R ₁₄I ₉R ₁₇S

3 0R ₁₆S ₀R ₁₅S ₀R ₁₄1 ₀R ₁₈S ₇R ₁₆S

5 9R ₁₈S ₁₀R ₂₀S ₈R ₁₃I ₁₀R ₁₅S ₀R ₂₃S

AMP10 22S ₀R ₀R ₀R ₀R

TE20 28S ₂₃S ₁₅S ₁₄R ₁₂R

Compound Zonesofinhibition(mm)

Gram-positivebacteria

B.subtilisATCC 6633

E.faecalisATCC 29212

E.faecalisATCC 51299

S.aureusATCC 29213

S.aureusATCC 43300

S.sciuriATCC 29062

S.xylosusATCC 35033

100␮g 250␮g 100␮g 250␮g 100␮g 250␮g 100␮g 250␮g 100␮g 250␮g 100␮g 250␮g 100␮g 250␮g

1 0R ₂₀S ₀R ₁₃I ₀R ₁₃I ₉R ₁₆S ₉R ₁₅S ₉R ₁₅S ₁₁I ₁₆S

2 15S ₁₈S ₀R ₁₄I ₀R ₁₆S ₁₀R ₁₈S ₈R ₂₂S ₁₀R ₁₉S ₀R ₂₃S

3 0R ₀R ₀R ₁₇S ₀R ₁₆S ₇R ₂₀S ₇R ₁₉S ₀R ₁₉S ₀R ₁₇S

5 0R ₂₀S ₀R ₁₆S ₀R ₁₁1 ₉R ₁₇S ₀R ₁₉S ₉R ₂₅S ₁₆S ₁₈S

AMP10 40S _{nt 25}S ₂₅S ₂₀S ₃₄S ₀R

TE20 36S _{nt 0}R ₂₈S ₃₆S ₂₅S ₂₆S

R,resistant;S,susceptible;I,intermediate;nt,nottested;AMP10,ampicillin(10␮g);TE20,tetracycline(20␮g).

Table2

Percentagebiofilmreductionfollowingexposureto100–500␮gml−1_{offourcompoundsisolatedfromF.natalensissubsp.Natalensis.}

Compound Percentbiofilmreduction(%)a

E.coli ATCC35218 P.aeruginosa ATCC27853 S.aureus ATCC43300

100 250␮gml−1 _{500 100 250␮gml}−1 _{500 100 250␮gml}−1 ₅₀₀

1 −315.62 −1456.10 −1176.99 −64.04 28.16 18.05 −445.34 −338.40 −437.78

2 −783.27 −1168.21 −1151.57 59.65 8.59 19.45 −1.81 −330.97 −381.66

3 −149.72 −1346.58 −1071.16 39.07 −26.45 −38.54 −54.09 −302.98 −474.39

5 −593.81 −1220.89 −1189.00 54.69 −6.49 −48.49 −51.30 −395.19 −416.02

a_{BiofilmreductioncalculatedaccordingtoPittsetal.(2003).Negativevaluesareindicativeofanincreaseinattachment/biofilmformation.}

fromF.natalensissubsp.natalensisinthisworkcouldberesponsible fortheantibacterialactivityexperiencedbythosewhousetheplant for medicinalpurposes since mostof them have demonstrated promisingantibacterialactivity.Decreasedadhesionwith>100% biofilmreductionwasdemonstratedbyallthecrudepolarextracts againstresistantbacterialstrains.Theisolatedcompounds exhib-itedstrain-specificanti-adhesionpotential,withbiofilmreduction againstP.aeruginosa,butnotE.coliorS.aureus.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Confidentialityofdata. Theauthorsdeclarethattheyhave fol-lowed theprotocolsof theirworkcenter onthe publicationof patientdata.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authors’contributions

GVAandHBcollectedtheplantmaterials.Allexperimentalwork wascarriedbyGVAunder thesupervision ofNK. Antimicrobial

andanti-adhesionanalysiswasdoneunderthesupervisionofHC. GVAandNKwrotethemanuscriptwithcontributionsfromHCand HBintheirfieldsofexpertise.Alltheauthorshavereadthefinal manuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

TheauthorsarethankfultotheResearchOffice,Universityof KwaZulu-Natal(UKZN),WestvilleCampus,Durban,SouthAfricafor adoctoralresearchgrant.GVAwasawardedaPhDstudyfellowship (studyleave)bytheUniversityofIlorin,KwaraState,Nigeria.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2017.07.004.

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