Antioxidant, antimicrobial and anti - quorum sensing activities of Rubus rosaefolius phenolic extract.

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ContentslistsavailableatScienceDirect

Industrial

Crops

and

Products

j ou rn a l h o m epa g e :w w w . e l s e v i e r . c o m / l o c a t e / i n d c r o p

Antioxidant,

antimicrobial

and

anti-quorum

sensing

activities

of

Rubus

rosaefolius

phenolic

extract

Brígida

D’Ávila

Oliveira

a

_,

_Adeline

_Conceic¸

_ão

_Rodrigues

a

_,

_Bárbara

_Moreira

_Inácio

_Cardoso

a

,

Ana

Luiza

Coeli

Cruz

Ramos

a

_,

_Michele

_Corrêa

_Bertoldi

b

_,

_Jason

_Guy

_Taylor

c

_,

Luciana

Rodrigues

da

Cunha

d

_,

_Uelinton

_Manoel

_Pinto

e,∗

a_School_of_Nutrition,_Federal_University_of_Ouro_Preto,_Ouro_Preto,_Minas_Gerais,_Brazil

b_Departament_of_Pharmacy,_Faculty_of_Pharmacy_and_{Biochemistry,}_Federal_University_of_Juiz_de_Fora,_Governador_Valadares,_Minas_Gerais,_Brazil c_Department_of_Chemistry,_Institute_of_Physical_and_Biological_Sciences,_Federal_University_of_Ouro_Preto,_Ouro_Preto,_Minas_Gerais,_Brazil d_Department_of_Food,_Federal_University_of_Ouro_Preto,_Ouro_Preto,_Minas_Gerais,_Brazil

e_Department_of_Food_and_Experimental_Nutrition,_Faculty_of_{Pharmaceutical}_Sciences,_University_of_São_Paulo,_São_Paulo,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received9November2015

Receivedinrevisedform22January2016 Accepted24January2016

Availableonline8February2016

Keywords: Antioxidantactivity Antimicrobialactivity Anti-quorumsensing Phenoliccompounds Centesimalcomposition

a

b

s

t

r

a

c

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Phenoliccompoundsareplantsecondarymetabolitesthatpresentmanybiologicaleffectsincluding antioxidantandantimicrobialactivities.Studieshaveshownthatthesecompoundscanalsoinhibit quorumsensingbacterialcommunication.Theaimofthisstudywastocharacterizethecentesimal composition,mineralandphenoliccontent,todeterminetheantioxidantandantimicrobialeffectas wellastheinhibitionofquorumsensingbythephenolicextractobtainedfromwildstrawberry(Rubus rosaefolius).CentesimalcompositionandmineralswereintherangeexpectedforfruitsoftheRubus generum,eventhoughFeandZnpresentedhigherlevels.Thephenoliccontentwas5902.89mgGAE/L, alsoapproachingthelevelsfoundforfruitsofRubussp.Theantioxidantactivitydeterminedthrough theABTSmethodwas162.4±5.6and120.8±1.5␮MTrolox/goffruitintheDPPHassay,indicatingan elevatedpotentialforABTSandmediumpotentialforDPPHmethod.Thephenolicextractwasableto inhibitalltheevaluatedbacteriapresentingMICsintherangeof491.90–1475.74mgGAE/L.Insub-MIC concentrations,thephenolicextractinhibitedallthephenotypestypicallyregulatedbyquorum sens-inginbacteria,includingviolaceinproduction,swarmingmotilityandbioﬁlmformation.Thephenolic extractofR.rosaefoliuspresentedantioxidant,antimicrobialandanti-quorumsensingactivitieswhich areinagreementwithpreviousstudieslinkingphenoliccompoundstotheseproperties.

1. Introduction

Thehealthbeneﬁtsoffruitsandvegetablesarewellestablished andtheyhavebeenshown toprovideessentialcomponentsfor the basic functionsof many living organisms (Sreeramulu and Raghunath, 2010). Among these components are vitamins and phenoliccompounds.Thosearesecondarymetabolitesimplicated inthedefenseagainstphytopathogens,radiationandantioxidant activitiesobservedinfruitsandvegetables(Tiveronetal.,2012; RoginskyandLissi,2005).Duetothepresenceofantioxidant com-pounds,fruitsandvegetablesexhibitprotectivefunctions,reducing therisksofnon-transmissiblechronicdiseasessuchascancerand

∗ Correspondingauthorat:UMP,Av.Prof.LineuPrestes580,B.14,05508-900São Paulo,SP,Brazil.Fax:+551126480677.

E-mailaddress:[email protected](U.M.Pinto).

cardiovascular diseases (Jonville et al., 2011; Faller and Fialho, 2010;Scalbertetal.,2005).

Thephenoliccompounds,besideshavingantioxidantcapacity, arealsocapableofinhibitingthegrowthofmicroorganisms accord-ingtotheirconcentration(Martinsetal.,2015;Pinhoetal.,2014). Thesecharacteristicsjustifythegrowinginterestofthefood indus-tryfornaturalproductscontainingthesecompounds.

Itiscommonlybelievedthatfooddeterioration,atleastinpart, isregulatedbyamechanismofcell-to-cellcommunicationknown asquorumsensing(BaiandRai,2011).Thissystemisimportant fordeﬁning bacterialactivitysince itregulatesimportant cellu-lar functionssuchassporulation,bioﬁlmformation, bacteriocin production,conjugation,competence,virulencegeneexpression, pigmentandbioluminescenceproduction,amongothers(Hammer andBassler,2003;Smithetal.,2004).Byusingquorumsensing inhibitors,mainlyof natural origin,virulencefactors couldalso beinhibitedinpathogenicmicroorganismsbesidesaffectingfood deteriorationphenotypes(Kalia,2013).

http://dx.doi.org/10.1016/j.indcrop.2016.01.037

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The species Rubus rosaefolius is original from the Himalaia, WesternAsiaandAustralia,belongingtothegroupofraspberryand blackberry(Souzaetal.,1995).Theroundthin-skinnedfruitfrom R.rosaefolius,popularlyknownas“amora-do-mato”(wild straw-berry)inBrazilhasagreeablecolors,whichvariesfrompurpleto darkredasripe,appreciablearomaand ﬂavor.Thefruitis rich inanthocyanins,mainlyperlagonidinsandcyanidins,whichhave beenrelatedtoitsbiologicalandtherapeuticproperties (Bowen-Forbesetal.,2010; Campbellet al.,2015;Santoni etal., 2015). Eventhoughstudiesconcerningthephenoliccontent,antioxidant, antimicrobialandanti-quorumsensingactivitiesarescarce,results obtainedwithraspberryandblackberry,whichbelongtothegenus Rubus,provideanindicationfortheseactivities.Therefore,theaim ofthisstudywastoevaluatetheaforementionedactivitiesofthe phenolicextractobtainedfromthefruitsofR.rosaefolius.

2. Materialandmethods 2.1. Phenolicextractpreparation

Fruits of R. rosaefolius were collected in the city of Ouro Preto—MG,herborizedandavoucherspecimendepositedinthe ProfessorJoséBadiniHerbariumattheFederalUniversityofOuro Preto(Oliveira,B.D.,28763).

Fruitswerewashedand sanitizedwithsodiumhypochloride at50mg/Lfor15min.Theseedsweremanuallyremovedandthe pulpwashomogenizedandkeptfrozenat₋₂₀◦Cuntiluse. Phe-noliccompoundswereextractedaspreviouslydescribed(Bertoldi, 2009).Thetermphenolicextractusedfrequentlywithinthisreport hasnorelationtothesolventsystemusedtoobtaintheextract. Phenolwasnotusedasanextractionsolventandinsteadtheterm phenolic extractis usedtodescribe anextract rich inphenolic compounds.Thetotalphenoliccontentoftheextractswas deter-minedbytheFolin–Ciocalteuassay(ShahidiandNaczk,1995)and expressedasmgofgalicacidequivalentperL(mgGAE/L).

2.2. Antioxidantactivity(ABTSandDPPH)

TheantioxidantactivityofthephenolicextractfromR. rosae-foliuswasdeterminedbyusingtheABTS+radical,accordingtothe methodologydescribedbyRuﬁnoetal.(2007).Forthe standardiza-tionoftheresults,troloxwasusedasanexternalstandardandthe resultsexpressedasantioxidantactivityin␮MTroloxequivalent pergramoffreshpulp.

Theantioxidantcapacitywasalsodeterminedaccordingtothe DPPHmethoddescribedbyBrand-Williams etal.(1995).Trolox wasalsousedasanexternalstandardinthisassayandtheresults expressedas␮MTroloxequivalentpergramoffreshpulp.

2.3. Mineralcontentdetermination

The content of potassium, calcium, iron, copper, zinc and chlorideoftheextractwasanalyzedbyTotalReﬂectionX-ray Flu-orescence(TXRF).TheanalysiswasperformedonaS2-PICOFOX instrument (BrukerAXS, Berlin, Germany) witha molybdenum anodeoperatingat50kVand 750mA.A solutionof theextract indistilledwaterwasmadeand10␮Lofthesolutionwas trans-ferredontoquartzdiscsanddried.Theresultswereobtainedby integrationof thesignalon a BrukerSpectrasoftware (version 6.1.5.0)withquantiﬁcationbeingperformedbymeansof compar-ingwithinternalstandardsofknownconcentration.Theminerals wereexpressedasmg/goffruit.

2.4. Centesimalcomposition

Thisexperimentwasperformedwiththepulpofthefruitby quantifyingthecontentofmoisture,ash,totallipids,totalsoluble solutes,proteins,carbohydratesandﬁbersaccordingtotheAOAC (1995),AOAC(2003),AOAC(2005)methodologiesandIALInstitute (2005).Thecarbohydratecontentwascalculatedbysubtractingthe protein,totallipids,moistureandashfrom100%.

2.5. Antimicrobialactivity

The antimicrobial activity of the phenolic extract, in dif-ferent concentrations, was evaluated by plate diffusion assay andbydeterminingtheminimalinhibitoryconcentration(MIC). The tests were performed against Gram negative bacteria Escherichia coli ATCC10536, Aeromona hydrophila IOC/FDA110, Pseudomonas aeruginosa ATCC15442, Pseudomonas ﬂuorescens NCTC10038,Salmonellaspp.(Laboratorystock),Serratiamarcescens UFOP-001 (Laboratory stock), Hafniaalvei ATCC11604, and the GrampositivesStaphylococcus aureusATCC6538P,Listeria mono-cytogeneATCC7644and BacilluscereusATCC 11778,allof great relevance in foodmicrobiology. Thetests were alsoperformed againstChromobacteriumviolaceumATCC6357whichwasusedin thequorumsensingbioassays.

For the plate diffusion assay, the procedures described by Salawuetal.(2011)werefollowed,whileMICwasperformedas previouslydescribedbyWiegandetal.(2008).Allquorumsensing assayswereperformedinsub-MICconcentrationswithno mea-surableinterferenceonthebacterialgrowth,asdeterminedbythe enumerationofcolonyformingunitspermL(CFU/mL)inLBagar. 2.6. Anti-quorumsensingactivity

Thequorumsensinginhibitoryeffectofthephenolicextractwas evaluatedinC.violaceumATCC6357,A.hydrophilaIOC/FDA110-36 andS.marcescensUFOP-001.Thestrainswereculturedat28and 30◦CinLuria-Bertani(LB)broth,accordingtoPintoetal.(2007). 2.6.1. QuorumsensinginhibitioninC.violaceum

Initially, an agar diffusion assay was performed according to the method described by Tan et al. (2012), with modifica-tions. The test was performed with C. violaceum ATCC6357 at 30◦C. Toeach well,20␮L of phenolic extract in different con-centrationswereadded.Distilledsterilizedwaterandkanamycin (100␮g/mL—Sigma–Aldrich)wereusedascontrols.Thequorum sensinginhibitoryactivityinC.violaceumwasverifiedbythe forma-tionofaturbidhalo,indicatedbybacterialgrowth,withnopigment productionaroundthewellonapurplebackgroundontheplate. 2.6.2. QuantificationofviolaceinproductioninC.violaceum

TheassaywasperformedasdescribedbyTanetal.(2012),with modiﬁcations.C.violaceumwascultivatedat30◦Candtheassay wasperformedintesttubes.Thepositivecontrolforquorum sens-inginhibitionusedwas ((Z-)-4-Bromo-5(bromomethylene-2(5h)-furanone)—C30(Sigma–Aldrich),atconcentrationof39.4␮M.The percentage inhibitionof violaceinproduction wascalculatedby usingthefollowingformula:

%inhibitionofviolaceinproduction

=(ODofcontrolat585nm−ODoftreatmentat585nm/ ODofcontrolat585nm)×100

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Table1

CentesimalcompositionofR.rosaefoliuspulp(g/100gofpulp).

Proteins 0.66±0.001 Lipids 0.02±0.01 Ash 0.57±0.12 Moisture 80.22±0.15 Carbohydrates 18.53±0.07a Fibers 4.97±1.08

Totalsolublesolutes(◦_Brix) _10.4_±_0.5

Numbersrepresentthemeanvaluesoftriplicates±standarddeviation.

a_Value_obtained_by_the_difference_{(100%—protein,}_lipid,_moisture_and_ash

con-tent).

2.6.3. Swarmingmotilityassay

Theswarmingmotilityassaywasperformedasdescribedby Huberetal.(2003)withA.hydrophilaandS.marcescensstrains. 2.6.4. BioﬁlmformationinC.violaceum,S.marcescensandA. hydrophila

This experiment was performed according to the protocol describedbyConwayetal.(2002).

2.7. Statisticalanalysis

Assayswereperformedintriplicateandtheresultsexpressed asaveragewithstandarddeviation.Thesigniﬁcance(p<0.05)was obtainedbytheANOVAtestandTukeytestbyusingtheGraphPad Prismversion5.00softwareforWindows(SanDiego,Califórnia, USA).

3. Resultsanddiscussion 3.1. Centesimalcomposition

ThecentesimalcompositionoftheR.rosaefoliuspulpisshown onTable1.

R.rosaefoliuspulppresentedahighcontentofmoisture, carbo-hydratesandtotalsolublesolutes,whilepresentingalowprotein, ashandlipidcontent.Thesearecommoncharacteristicsofmost commerciallyconsumedfruitssuchasapples,oranges,mangoes andothers(Silveiraetal.,2011).AstudyperformedbyHirschetal. (2012)withthreecultivarsofRubusssp.showedverysimilarresults formostoftheparametersevaluatedinthepresentstudy; how-ever,theirlipidcontentwasconsiderablyhigher(0.15g/100g).We believethatthisdifferencecouldbeaccountedforbythe differ-entmethodologiessinceHirsch etal.used coldextractionwith reagentsthatpresentdifferentpropertiesfromthosedescribedby AOAC(1995).Guedesetal.(2014)evaluatedthechemical compo-sitionofR.rosaefoliusandfoundamoisturecontentsimilartothe presentstudy.

3.2. Traceelements

TotalreﬂectionX-rayﬂuorescencespectroscopy(TXRF)isa non-destructive,rapidandmultielementalmethodthatprovidesallthe resultsinasinglemeasurementofthesample.Forthisreason,TXRF wasemployedtoquantifytraceelementsinthephenolicextract. TraceelementquantitiesfoundinthepulpofR.rosaefoliuswere determinedforpotassium,calcium,iron,copper,zincandchlorine (Table2).

Themostabundantelementpresentwaspotassium,followed bychloride,ironand zinc.Theseresultsareofgreat nutritional importance, since according to therecommendations fromthe WorldHealthOrganization(2003),thedailyintakeofironandzinc wouldbe45mgand11mg,respectively.Thus,theconsumption

Table2

SpectroscopyresultsdeterminedbyTotalReﬂectionX-rayFluorescenceTXRFof traceelementsfoundinR.rosaefolius,inmg/ginthepulpoffruit.

Metals Fruitpulp(mg/g)

Potassium(K) 0.965 Calcium(Ca) 0.185 Iron(Fe) 0.155 Copper(Cu) NF Zinc(Zn) 0.039 Chlorine(Cl) 0.563 NF—notfound.

ofR.rosaefoliusfruitscouldcontributetoreachtherecommended nutrientintake.

Tothebestofourknowledgetherearenoreportsdescribing theuse ofTXRF to evaluatethetrace elementcomposition for thisfruit.However,KinuppandBarros(2008)evaluatedthemacro andmicronutrientsbyinductivelycoupledplasmaoptical emis-sionspectrometry(ICP-OES),for69nativespeciesfromthesouth ofBrazil,includingR.rosaefolius.Theauthorsfoundhigher con-tentsforcalciumandpotassiumthanthepresentstudy.Foriron, thecontentwaslower(0.0053mg/g)intheirstudyandforzinc, thevaluesweresimilar.Thesedifferencescouldbeduetomany factorsincludinggeneticpropertiesofthespecies,climate condi-tions,soilcompositionandthedegreeofmaturationuponharvest (Martínez-Ballestaetal.,2010).

Despitetheimportanceof consumingmineralsfor ahealthy humandiet,thereislittleinformation onthepresenceof these constituentsinfruits,evenforthosehighlyconsumed (Martínez-Ballestaetal.,2010;KinuppandBarros,2008).Thetraceelements are widely distributed in nature and are important for the healthyfunctioningofthehumanbody,participatinginenzymatic reactions,bone formation,coagulation,cicatrizationand oxygen transport(Martínez-Ballestaetal.,2010;KinuppandBarros,2008). Besides,theorganismantioxidantdefenseandthecontroloffree radicalproductionareinﬂuencedbymanyminerals(Barbosaetal., 2010;Kouryetal.,2007;Mafraetal.,1999).Thus,thepresenceof thesemicronutrientsinfruitsinsigniﬁcantlevelsisofgreat impor-tance forquenching radicals and contributing totheprotective actionofourdiet.

3.3. Phenoliccomposition

Thephenoliccontentfoundintheextractwas5902.89±7.42mg GAE/L(thephenolicextracthadanequivalentof3300gofpulp/L). Thisamountisequalto177.26mgGAE/100gofpulpwhichfalls withintherangefoundforotherfruitsfromtheRubusgenerum (Zielinskietal.,2015;Ferreiraetal.,2010).

Rufinoetal. (2010)suggesteda classificationschemewhere thephenoliccontentoffruitscouldbegroupedaslowifthe con-centrationwas<100mgGAE/100goffruit,mediumfortherange 100–500mgGAE/100gandhighfor>500mgGAE/100g.Basedon this classification, R.rosaefolius would beclassifiedas havinga mediumcontentofphenoliccompounds.Itisimportanttonote that fruits from the same species could beclassified in differ-entcategoriessince thephenoliccontentcanvary accordingto maturation,cultivationpractices,location,harvestconditionsand storage(Soares,2002).Themethodsusedtoextract the pheno-liccompoundscanalsointerfereinthequantificationsincesome otherreducingcompoundsfromnon-phenolicorigincanalsobe extracted.Contrastingwithmanypublishedstudiesinwhichthe extractswereobtainedbymacerationwithsolvents,followedby filtration(Bowen-Forbesetal.,2010;Ferreiraetal.,2010;Zielinski etal.,2015;Sariburunetal.,2010), inthecurrentstudyseveral interferingcomponentswereeliminatedbythesolidphase extrac-tion,includingreducingsugars,solubleproteinsandacidswhich

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Table3

AntioxidantactivityofphenolicextractsfromthepulpofR.rosaefoliusdetermined bytheABTSandDPPHmethods.

Antioxidantactivity

ABTS(␮MTrolox/goffruit) DPPH(␮MTrolox/goffruit)

162.4±5.6a _120.8_±_1.5a

a_Values_represent_the_mean_of_triplicates_±_standard_deviation.

minimizethepossibilityforoverestimationofthephenolic con-tentandtheantioxidantactivityoffruits(Rijkeetal.,2006;Dias etal.,2010).

Thereisnorecommendeddailyintakefortheconsumptionof phenoliccompounds,despiteitsimportantroleinthehumandiet. ItisestimatedthattheaverageintakebyBraziliansis48,3mg/day (FallerandFialho,2010).Sincefruitsandvegetablesarethemajor sourcesofthesecompounds,andbasedonthefactthattheWHO recommendsconsumptionof400goffruitsandvegetablesdaily (about5portions),theBrazilianpopulationdoesnotconsume ade-quateamounts(Brasil,2010).

3.4. Antioxidantactivity

Table3presentstheresultsfortheantioxidantactivityofthe phenolicextractofR.rosaefolius.Nostudiesreportingthe antiox-idantcapacityofR.rosaefoliuswerefound.However,therehave beenreportsonotherfruitsfromthegenerumRubus.Forinstance, Sariburunetal.(2010)evaluatedthemethanolicextractof rasp-berryfruits(RubusidaeusL.)andblackberryfruits(Rubusfruticosus L.)andfoundvaluesrangingfrom64.14to153.70␮MTrolox/gof fruitfortheDPPHassayandfrom64.36±1to117.07±0.94␮M Trolox/goffruitfortheABTSmethod.Ontheotherhand,Souza etal.(2014)foundthattheantioxidantactivityofR.idaeusfruits were6.27±0.02␮MTrolox/gbytheABTSmethods,whileC¸ekic¸ andÖzgen(2010)foundvaluesrangingfrom8.9␮MTrolox/gto 21.5_␮M Trolox/ginthesameassay.Our studyrevealedthat R. rosaefoliuspresentedagoodantioxidantactivityasrevealedbyboth methodsandthecomparisonwiththeliterature.

Inthepresentwork,ahigherantioxidantactivitywasobserved with the ABTS assay, which could be related to the charac-teristics of this method since ABTS can be a better measure for hydrophilic and lipophilic compounds and those withhigh pigmentation (Floegel et al., 2011). It is worth mentioning that most of the values reported in the literature are from extracts dissolved in organic solvents, which differ from the current study that focused on the phenolic extracts which are likely to contain anthocyanins like cyanidin-3-glucoside, pelargonidin-3-glucoside, pelargonidin-3-rutinoside, cyanidin-3-(2g-glucosylrutinoside),cyanidin-3-sophoroside,cyanidin, peoni-din,malvidin,pelargonidin, and delphinidin (Rijkeet al., 2006; Bowen-Forbes et al.,2010; Guedes et al., 2014).Therefore, the

Table5

Minimalinhibitoryconcentration(MIC)ofphenolicextractfromR.rosaefolius (val-uesexpressedasmgofGAE/Lofextractandequivalentextractdilution).

Bacteria Extract concentrationin mgGAE/L Equivalentextract dilution A.hydrophila 491.90 1:14 B.cereus 491.90 1:14 C.violaceum 491.90 1:14 E.coli 737.87 1:8 H.alvei 491.90 1:14 L.monocytogenes 1475.74 1:4 P.aeroginosas 491.90 1:12 P.ﬂuorescens 491.90 1:14 S.aureus 421.63 1:14 S.marcescens 737.87 1:8 Salmonellaspp. 737.87 1:8

results described for extracts in the literature are less speciﬁc andrepresenttheantioxidantcapacityofbothphenolicand non-phenoliccompounds.

3.5. Antimicrobialactivity

Theresultsfortheplatediffusionassaywiththephenolicextract obtainedfromR.rosaefolius,atdifferentconcentrations,areshown inTable4.

AsobservedinTable4,thephenolicextractinhibitedallthe evaluatedbacteria,atleastattwoconcentrations.Themost sen-sitive bacterium was P. fluorescens which was inhibited in all testedconcentrationsandpresentedthegreatestinhibitionzone (14.2±0.11mm).Asitisoftendifficulttocomparestudieswith plantextracts,Alvesetal.(2000)suggestedaclassificationscheme whereextractswithhalos<9mmwereclassifiedasinactive,from 9–12mm,partiallyactive,from13–18mmactive,and>18mmvery active.Ifwefollowthisclassificationscheme,thepresentextracts werepartiallytoactiveagainstthetestedbacteria.

Onceagain,nostudieswerefoundfortheantimicrobialactivity ofR.rosaefoliusfruits.However,Zeidanetal.(2013)studiedthe antimicrobialactivityoftheethanolicandmethanolicextractsfrom leavesandfruitsofR.sanguineus,whichisarelatedspecies,against strainsofpathogenicE.coli,P.aeruginosa,S.aureus,B.cereusand Candidaalbicans,bytheplatediffusionassay.Theyfoundthatall theorganismsshowedsomedegreeofsensitivitytotheleavesand thefruits(halosvariedfrom7±0.5mmto22±0.5mm).

Complementingtheinhibitorypotentialofthephenolicextract ofR.rosaefolius,theresultsfortheMICarepresentedonTable5.

StudiesdeterminingtheMICofanytypeofextractprepared withR.rosaefoliushavenotbeenreported.However,Mauroetal. (2002)evaluatedtheantimicrobialacitvityoftheaqueousextract obtainedfromtheroot,stemandleavesofR.sanguineus which inhibitedS.aureus,P.aeruginosa,E.coliandC.albicans.Azevedo,

Table4

GrowthinhibitionbydifferentconcentrationsofphenolicextractfromR.rosaefolius,measuredasinhibitionzonearoundthewells(mm),bytheplatediffusionassay.

Bacteria Concentration(mgGAE/L)

5902.8 2951.4 1475.0 983.8 737.8 A.hydrophila 10.5±0.10 10.2±0.05 – – – B.cereus 11.5±0.10 9.8±0.05 – – – E.coli 11.0±0.14 11.0±0.02 – – – H.alvei 13.2±0.15 8.2±0.05 5.5±0.01 – – L.monocytogenes 11.0±0.04 12.0±0.02 – – – P.aeroginosas 13.0±0.16 12.5±0.02 – – – P.ﬂuorescens 14.2±0.11 11.8±0.05 9.2±0.05 9.2±0.05 7.2±0.05 S.aureus 12.0±0.00 9.0±0.15 – – – Salmonellaspp. 13.0±0.04 12.0±0.15 10.0±0.05 – –

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Fig.1.PlatediffusionassayforthequorumsensinginhibitioninC.violaceumbydifferentconcentrationsofthephenolicextractfromR.rosaefolius.(A)Kanamycin (100␮g/mL)—controlforgrowthinhibition,(B)steriledistilledwater—negativecontrol,(C)5902.8mgGAE/L,(D)2951.4mgGAE/L,(E)1475.7mgGAE/Lofphenolicextract. (Forinterpretationofthereferencestocolorinthetext,thereaderisreferredtothewebversionofthisarticle.)

(2011)studiedtheantimicrobialactivityoffruitsofR.fruticosus ﬁndinginhibitionagainstS.entericaserovarEnteritidis.

3.6. QuorumsensinginhibitioninC.violaceum(platediffusion assay)

Fig.1presentstheresultsfor thequorumsensing inhibition assayinC.violaceumusingdifferentconcentrationsofthephenolic extract.Theinhibitioncanbeseenbytheformationofaturbidhalo, onaclearbackgroundaroundthewell,onapurpleplate,resulting fromtheinhibitionofviolaceinproduction.

Thephenolic extractinhibitedtheproductionofviolacein,as canbe observedin Fig.1(C) which showsno colorproduction, butbacterialgrowthcanbeobservedbytheturbidityofthehalo. Contrasting withthisresult, theantibioticKanamycininhibited bacterialgrowth,sinceaclear,transparenthalowasformedaround thewell.

Zhang et al. (2014) studied phenolic compounds from Rosa rugosaextractsand alsoencounteredcolorless,butturbidhalos indicatinginhibitionofquorumsensing.Otherplantextractshave alsoshownsimilarresultsinthisassay,asobservedforgarlic, gera-nium,lavender,rosemaryandeucalyptus(Bodinietal.,2009;Del Monteetal.,2015;Khanetal.,2009;Szabóetal.,2010).

3.7. QuantiﬁcationofviolaceinproductioninthepresenceofR. rosaefoliusphenolicextract

Thephenolicextractwaseffectiveatinhibitingviolacein pro-ductionatalltestedconcentrationsasshowninFig.2(p<0.05). Thenumberofviablecellscountedafter24hofincubationdidnot differbetweentreatmentsandthecontrol(absenceofextract) indi-catingthattherewasinhibitiononlyagainstviolaceinproduction (quorumsensinginhibition)andnotagainstgrowthofthestrain (Fig.2).

Theresultshighlightthatviolaceininhibitionbythephenolic extracts,especiallyat118.60mgGAE/L, inhibited88.6%,avalue higherthanthepositive controlforthis experiment(furanone), whichinhibited68.6%.Theextractataconcentrationof78.70mg GAE/Ldidnotdifferstatisticallyfromfuranone(p>0.05),whileat

0 1 2 3 4 5 6 7 8 9 10 0 10 20 30 40 50 60 70 80 90 100 Control Furanone 118,6 78,7 59,02 29,51 L og CF U m l -1 % V iol ace in In hi bit ion

Extract of concentration mg GAE/L % Violacein Inhibition Count CFU/mL

a

b

c c

d

Fig.2. PercentageinhibitionofviolaceinproductionbythephenolicextractofR. rosaefoliusatdifferentconcentrationsandevaluationofmicrobialgrowth(LogCFU mL-1)after24hincubationinthepresenceoftheextract.Thecontrolconsistedof LBmediumaddedof200␮LofsteriledistilledwaterandfuranoneC-30(quorum sensinginhibitioncontrol)at39.4␮M.Averagesfollowedbythesamelettersdonot differstatistically(p<0.05).

theotherconcentrations,inhibitionwasobserved,butatalevel lowerthanfromfuranone.

NostudieswerefoundontheeffectofR.rosaefoliusfruitsor anyotherpartsoftheplantontheproductionofviolaceinoron anyotherquorumsensingcontrolledphenotype.Likewise, stud-iesshowingthequorumsensinginhibitoryeffectoffruitextracts are scarce. However, studies have been performed withplants in general, includingmedicinal plants, withthe aim of ﬁnding newantipathogenicandtherapeuticcompounds(Al-Hussainiand Mahasneh,2009;Adonizioetal.,2006;Kalia,2013;DelMonteetal., 2015).Itisestimatedthatabout10%oftheterrestrialﬂorapresents somekindofactivitythatcouldbeexplored,butonly1%ofthis biodiversityhasbeenevaluated(Al-HussainiandMahasneh,2009). 3.8. EffectofR.rosaefoliusphenolicextractontheswarming motilityofS.marcescensandA.hydrophila

Swarming motility was evaluated in two important strains whicharecommonlypresentinrefrigeratedproducts(Pintoetal.,

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Fig.3.SwarmingmotilityagainstS.marcescens.(A)Control,S.marcescensinLBagar(B)LBagaraddedofphenolicextractofR.rosaefoliusintheconcentrationof236.11mg GAE/L.(Forinterpretationofthereferencestocolorinthetext,thereaderisreferredtothewebversionofthisarticle.)

Fig.4.SwarmingmotilityagainstA.hydrophilaIOC/FDA110-36.(A)Control,A.hydrophilainLBagar(B)LBagaraddedofphenolicextractofR.rosaefoliusintheconcentration of236.11mgGAE/L.

2007).TheresultspresentedinFigs.3and4showthatthe pheno-licextractwasabletoinhibitswarmingmotilityinbothbacteriaas wellasprodigiosinproductioninS.marcencens(redpigment),both ofwhichareregulatedbyquorumsensing(Packiavathetal.,2014)

ThisworkistheﬁrsttoprovideresultsontheeffectofR. rosae-foliusphenolicextractonswarmingmotilityinbacteria.Bakkiyaraj etal.(2012)havealsoevaluatedtheswarmingmotilityinhibition againstS.marcescenswithbioactivecompounds extractedfrom coralreefs.Theyobservedthatuponexposuretothecompounds, therewasinhibitiononmotilityaswellasonprodigiosin produc-tion,asobservedinthepresentstudy.Inanotherstudy,Packiavathy etal.(2012)foundthateugenol,asecondarymetaboliteobtained fromsomeplants,presentedinhibitoryactivityagainstswarming motilityonProteusmirabilis,P.aeruginosaPAO1andS.marcescens. Thequorumsensinginhibitionexhibitedbythephenolicextractis likelytoberelatedtotheirstructuralsimilaritywithknown autoin-ducermolecules(Bakkiyarajetal.,2012;Packiavathyetal.,2012; Wangetal.,2006).

3.9. BioﬁlmformationinA.hidrophila,C.violaceumandS. marcescens

AsobservedinFig.5,allconcentrationsoftheextractthatwere testedpresentedbioﬁlminhibitory capabilityagainstthetested strains,whencomparedtothecontrol(p<0.05),whichwassetto 100%.TheAHLsignalingmoleculeshavealreadybeenreportedas havinganessentialroleonbioﬁlmformation(Lazar,2011;Tarver,

2009;Geskeetal.,2005).AsstatedbyKalia(2013)thequorum sens-inginhibitoryeffectobservedinthepresentstudycouldberelated totheinterferenceontheautoinducermolecule,eitheronits pro-ductionordegradationorevenbycompetingtothebindingsiteon thequorumsensingreceptorproteins.Morestudiesarerequired inordertoidentifythemodeofactionofthephenoliccompounds aswellastodeterminewhichofthesecompoundsorhowmanyof thoseactuallypresentactivity.

Natural compoundslike penicillic acid, patulin, ajoenefrom garlicand p-coumaricacidhavealreadybeenshowntointeract withbacterialquorumsensing,sometimesstimulatingor inhibit-ingthesystem(Lazar,2011;Prihaetal.,2014).Lately,berrieshave beenstudiedforthepresenceofanti-quorumsensingcompounds sincetheypresentagoodconcentrationofphenolics(Prihaetal., 2014).Forinstance,astudyhasshownthatanexudatefrom cran-berrycontainingmanyidentiﬁedphenoliccompoundswastested againststrainsofVibrioharveyiinhibitingbioluminescenceupto 57%(Feldmanetal.,2009).Interestingly,thesesamephenolic com-poundscan befoundin fruitsof thegenerumRubus, especially cyanidin-3-glucoside,which iscommonlyfoundinR.rosaefolius (Bowen-Forbesetal.,2010;Leeetal.,2012).

4. Conclusions

ThephenolicextractfromR.rosaefoliusdisplayedgreat bioac-tivepotentialdue to thepresence ofphenolic compounds that demonstratedantioxidant, antimicrobialandanti-quorum

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sens-Fig.5. EffectoftheR.rosaefoliusphenolicextractonbioﬁlmformationagainstdifferentbacteria.(A)C.violaceum;(B)S.marcescens;(C)A.hydrophila.Averagesfollowedby thesameletterdonotdifferstatistically(p≥0.05).

ingactivities.Withrespecttoitsmineralcomposition,iron and zinchavebeenfoundinthefruitandthosecanpositively inﬂu-encemetabolicreactionsrelatedtotheantioxidantaction,which canfurtherbeimprovedbyphenoliccompounds.The consump-tionofthisfruitisencouragedsinceitpresentsgoodantioxidant activityandtraceelementswhichcontributetothereductionofthe riskofnon-transmissiblechronicdiseases.BerrieslikeR.rosaefolius canbeagoodsourceofnewbioactivecompoundswithquorum sensinginhibitorypotentialwhichcouldbefurtherexploredasa meanstoproduceantivirulencedrugsandnewadditivesforthe foodindustry.

Acknowledgements

U.M.P.acknowledgesagrantfromCNPq-Brazil (486240/2013-4).B.D.O.andA.C.R.thankCAPES-Brazilforprovidingscholarship. B.M.I.C.acknowledgesscholarshipfromCNPqandA.L.C.C.R.thank FederalUniversityofOuroPreto(PIP´ısprogram).Theauthorswould liketothankDr.RobsonJosédeCássiaFranco(UFOP)forproviding accesstotheTXRFinstrumentandforinsightfulscientiﬁc discus-sions.

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