Total phenolic content, antioxidant and antibacterial activities of fruits of Eucalyptus globulus cultivated in Algeria

ContentslistsavailableatSciVerseScienceDirect

Industrial

Crops

and

Products

j o u r n al hom ep a 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

Total

phenolic

content,

antioxidant

and

antibacterial

activities

of

fruits

of

Eucalyptus

globulus

cultivated

in

Algeria

Lila

Boulekbache-Makhlouf

,

Sakina

Slimani,

Khodir

Madani

FacultyofNatureandLifeSciences,3BSLaboratoryA.MiraUniversity,Bejaia06000,Algeria

a

r

t

i

c

l

e

i

n

f

o

Received21January2012

Receivedinrevisedform9April2012 Accepted9April2012 Keywords: Eucalyptusglobulus Fruits Phenoliccontent Antioxidantactivity Antibacterialactivity

a

b

s

t

r

a

c

t

CrudeextractfromfruitofEucalyptusglobulus(E.globulus)wasscreenedforitsinvitroantioxidantand antibacterialproperties.Antioxidantactivitywasmeasuredbytwomethods,namelythereducingpower andlipidperoxidationinhibition.Antibacterialactivitywasdeterminedbyusingdiscdiffusionmethod againstthreebacteria(Staphylococcusaureus:ATCC6538,Bacillussubtilis:ATCC6633andKlebsiella pneu-moniae:E47).Theextractexhibitedmoderateinhibitionoflipidperoxidationoflinoleicacidemulsion (51.34±0.72%)andhighreducingpower(IC50=39.52␮g/mL).Italsoexhibitedstrongantibacterial

activ-ityagainstB.subtilisandS.aureuswithminimuminhibitoryconcentration(MIC)valuesof30␮g/mLand 80␮g/mL,respectively.TheseresultssuggestthatfruitsofE.globulushaveinterestingantibacterialand antioxidantactivities.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Aromatic and medicinal plants are known to produce

cer-tainbioactivemoleculeswhichreactwithotherorganismsinthe

environment, inhibiting bacterial or fungal growth

(antimicro-bialactivity)(Senguletal.,2009).Thesubstancesthatcaninhibit

pathogensandhavelittletoxicitytohostcellsareconsidered

can-didatesfordevelopingnewantimicrobialdrugs.Sinceimmemorial

time,manhasusedvariouspartsofplantsinthetreatmentand

preventionofvariousailments.In recentyears,secondaryplant

metabolites,previouslywithunknownpharmacologicalactivities,

havebeenextensivelyinvestigatedasasourceofmedicinalagents

(Parekh and Chanda, 2007). Thus, it is anticipated that

phyto-chemicalswithadequateantibacterialefficacywillbeusedforthe

treatmentof bacterialinfections.It iswell-knownthat phenolic

compoundscontributetoquality andnutritionalvalueinterms

ofmodifyingcolor,taste,aroma, and flavorand alsoin

provid-inghealth-beneficaleffects(GöktürkBaydaretal.,2007;Doughari

etal.,2008).Theyalsoserveinplantdefencemechanismsto

pre-ventdamagebymicroorganisms,insects,andherbivores(Doughari

etal.,2008).

Inrecent years, researchhasfocusedonmedicinalplantsto

extract natural and low-cost antioxidant that can replace

syn-theticadditivessuchasbutylatedhydroxytoluene(BHT),butylated

∗ Correspondingauthor.Tel.:+2130552932738;fax:+21334214762. E-mailaddress:lilaboulekbachemakhlouf@yahoo.fr(L.Boulekbache-Makhlouf).

hydroxyanisole(BHA),propylgallateandterbutylhydroquinone

(TBHQ),thatmighthavetoxic,carcinogenicandabnormaleffects

onhuman(GöktürkBaydaretal.,2007).BHAandBHThavealso

beensuspectedofbeingresponsibleforliverdamage(Gülc¸inetal.,

2005).Therefore, thereisa growinginterestinthenatural

sec-ondaryplantmetabolitesandtheirpotentialuseasantioxidants

inthefoodandpharmaceuticalindustries.

E. globulus belong to the family of Myrtaceae. It is known

as the blue gum and it is also called the fever tree. Because

of its fast growth and increase in woody biomass, the genus

Eucalyptus is extensively cultivated in the Mediterranean area.

Itsleaveshavebeenused astraditionalremediesfor treatment

ofvariousdiseasessuchaspulmonarytuberculosis(Sherryland

Warnke, 2004), influenza (Hou et al., 2000; Hasegawa et al.,

2008), fungal infections (Takahashi et al., 2004) and diabetes

(Alison and Peter, 1998; Gallagher et al., 2003; Jouad et al.,

2003). Because of their antioxidant activity, leaf extracts of

E. globulus have been used as food additives [Notification No.

120 (16 April 1996), Ministry of Health and Welfare, Japan]

(Amakuraetal.,2009).

Recently,wereportedthecharacterizationofseveral

polyphe-nolicconstituentsinfruitsofE.globulusincludinghydroxybenzoic

acids,hydrolyzabletanninsandflavonols(Boulekbache-Makhlouf

et al., 2010).However, sofar,no datahave been publishedon

thebiologicalactivitiesofthefruitextractofthisplant.Asa

con-tinuation of our studies, we have investigated the antioxidant

andantibacterialcapacitiesofthephenolicextractofE.globulus

fruits.

0926-6690/$–seefrontmatter © 2012 Elsevier B.V. All rights reserved.

2. Materialsandmethods

2.1. Chemicals

AllchemicalswerepurchasedfromSigma(representedby

Alge-rianChemicalSociety,Setif,Algeria).Theantibacterialactivitywas

screenedagainsttwoGram-positivebacteria,S.aureus(ATCC6538)

andB.subtilis(ATCC6633),obtainedfromSaidalAntibioticaland

oneGram-negativebacteria,K.pneumoniae(E47),obtainedfrom

theFoodMicrobiologyLaboratoryofBiologicalFacultyofBejaia

University.

2.2. Samplepreparation

Fruits of E. globulus, Myrtaceae family, were collected from

plantsgrowninthearboretumofDerguinah(Bejaia,NorthEast

ofAlgeria).Sampleswerecleaned,driedinthedryingovenat40◦C

during5days,and groundtogranulometrylowerthan250␮m

priortoextraction.

2.3. Evaluationofmoisturecontentofthesample

Thermaldryingmethodwasusedinthedeterminationof

mois-turecontentofthesample.10gofsamplewereplacedinanoven

at105◦C for 3h. Themoisturecontent(MC) wascalculated by

expressingtheweightlossupondryingasafractionoftheinitial

weightofsampleused.MC (%)=W0/Wi×100,whereW0

corre-spondtothelossinweight(g)ondryingandWicorrespondtothe

initialweightofsample(g).

2.4. Extractionofphenoliccompounds

Extraction of phenolics was performed as described by

Boulekbache-Makhloufet al. (2010). 10g of dried powder was

extractedwith1000mLofacetone–water(700:300,v/v)

contain-ing0.5%aceticacid.Theprocessofextractioncontinuedforaweek

atroom temperature,using magnetic blender. Theextract was

filtered(Whatmanpaperno.4)andtheacetonewasevaporated

underreducedpressureinrotaryevaporationat40◦C.The

remain-ingaqueousphasewastreatedwithhexane(25mL×3)toremove

lipids,concentratedunderreducedpressure,andlyophilized.The

extractionyieldwascalculatedasfollows:Yield (%)=W1/W2×

100,whereW1wastheweightofextractafterlyophilizationand

W2wastheweightofthedriedpowderofplantmaterial.

2.5. Determinationofphenoliccomposition

Theamountoftotalphenolicsintheextractwasdetermined

usingtheFolin–Ciocalteureagentandgallic acidasstandard as

describedbySingletonandRossi(1965).

Thetotalflavonoidscontentwasdeterminedbythe

method-ology of Quettier-Deleu et al. (2000); that of flavonols was

determinedusingthemethodofAbdel-HameedEl-Sayed(2009),

andquercetinwasusedasstandard.

Tanninswereestimated spectrophotometricallyaccordingto

theprotocoldevelopedbyHagermanandButler(1978)andtannic

acidwasusedasstandard.Allanalyseswereperformedintriplicate

andthemeanvaluewascalculated.

2.6. Antibacterialactivity

Theantibacterialactivityoftheextractwasmeasuredbya

dif-fusiontestusingMueller–Hintonagarpreviouslyinoculatedwith

1mLof18holdofbacterialsuspension(106_{CFU/mL)(Changwei}

etal.,2008).Sterilizedpaperdiscs(6mm)wereimpregnatedwith

20␮L of different concentrations of extract, (500, 1000, 1500,

2000␮g/mL)preparedinaqueousmethanol(80%),andplacedonto

nutrientagar.Theplateswereincubatedat4◦Cfor2htoallow

dif-fusionoftheactivecompoundsinthemedium(TaggandMcgiven,

1971).Negativecontrolswerepreparedusingthesamesolvent

employedtodissolvetheplantextract.Gallicandtannicacidsare

testedinthesameconditionsaspositivecontrols.Incubationof

plateswasperformedat 37◦C for 24h. Inhibitionzones inmm

(withoutdiscpaperdiameter)arounddiscsweremeasured.The

antibacterialactivitywasexpressedasthediameterofinhibition

zonesproducedbytheextractagainsttestmicroorganisms.The

experimentwasrepeatedintriplicateandthemeanofdiameterof

theinhibitionzoneswascalculated.

Minimuminhibition concentration(MIC) wasdeterminedas

described by Zampini et al. (2005). Different concentrations

(10–2000␮g/mL)ofextractorstandards(gallicandtannicacids)

weretested.1mLofeachsolutionwasmixedwith9mLofMuller

HintonmediumandpouredintosterilizedPetriplates.

Immedi-atelyaftersolidification,theplateswerespotinoculatedwith10␮L

ofsuspensioncontaining106_{CFU/mLofeachbacterium.The}

inoc-ulated plates wereincubated at 37◦C for 24h. The MIC values

weredeterminedasthelowestextractorstandardconcentration

atwhichnogrowthwasobserved.

Todeterminetheminimumbactericidalconcentration(MBC)

values,nutrientbrothtubeswereinoculatedwithasampletakenat

thespotoftheplateswhichdidnotshowanygrowth.Themixture

wasincubatedat37◦Cfor24h.Thelowestconcentrationofthe

extractorstandardswithnovisiblegrowthafterincubationwas

takenastheminimumbactericidalconcentration.

2.7. Antioxidantactivity

Thereducingpoweroftheextractwasevaluatedaccordingto

theprotocolofHseuetal.(2008).1mLofdifferentconcentrations

ofthesamples(25,50,75,100and125␮g/mL)wasmixedwith

phosphatebuffer(1mL,0.2M,pH6.6)andpotassiumferricyanide

[K3Fe(CN)6] (1mL,1%). The mixturewasincubated at50◦C for

20min.Trichloroaceticacid(TCA)(1mL,10%)wasaddedtothe

solutionwhichwasthencentrifugedfor10minat3000_×g.The

supernatantwasgatheredandmixedwithdistilledwater(1.5mL)

and FeCl3 (150␮L,0.1%),and theabsorbance wasmeasuredat

700nmandcomparedtothestandards(BHAand␣-tocopherol),

anyincreaseinabsorbanceissynonymousofanincreasein

reduc-ingpower.

Lipid peroxidation inhibition (LPI) activity was determined

usingthe␤-carotenebleachingassay,asdescribedbyChanetal.

(2009). 0.5mL of extract (4mg/mL) was added to 2mL of

␤-carotene/linoleicacidsolution.Themixturewasincubatedat50◦C

for60minalongwithtwocontrols,onecontainingtheantioxidant

BHA(positivecontrol)andtheotheronewithoutBHAorextract

(negativecontrol).Theabsorbancewasmeasuredat470nm.LPI

activityexpressedasAOA(%)wascalculatedasfollows:

Bleachingrateof␤-carotene (BR)= ln[(Ainitial)/Asample]

60 AOA (%)=





whereAinitialandAsampleareabsorbanceoftheemulsionbeforeand

1hafterincubation,andBRsampleandBRcontrolarebleachingrates

ofthesampleandnegativecontrol,respectively.

2.8. Statisticalanalysis

Allexperimentswereconductedin triplicateandresultsare

expressed as mean±standard deviation (SD). Analysisof

Table1

AntibacterialactivityoffruitsextractofE.globulus. Microorganisms Inhibitionzone(mm)

Extract Gallicacid Tannicacid S.aureus 8.67±0.58 13.67±0.58 10.00±0.00 B.subtilis 5.5±0.5 1.00±0.00 5.33±1.15

K.pneumoniae ND ND ND

ND:notdetermined.

determinationofmoistureandphenoliccontents.Statistical

analy-sisoftheantioxidantandantibacterialactivitieswasperformedby

analysisofvariancewithtwofactorsinthesoftwareSTATISTICA5.5

Fr.IC50valueweredeterminedbyregressionanalysis.Differences

wereconsideredtobesignificantatp<0.05.

3. Resultsanddiscussion

3.1. Moisturecontentofsample,extractionyieldandphenolic

contents

Moisturecontentof sample is 57.14±0.59%and the

extrac-tion yield is about 30.83%. Fruits extract of E. globulus was

rich intotal phenolics and tannins(464.71±1.52mg GAE/g CE,

332.05±9.31mgGAE/gCE,respectively),butpoorinflavonoids

(2.99±0.01mgQE/gCE),andflavonols(2.3±0.22mgQE/gCE).

3.2. Antibacterialactivity

Table1presentsdiametersofinhibitionzonesexertedbythe

extractandthetwostandardstowardstestedmicroorganisms.E.

globulusfruitsextractwaseffectiveagainstthetwoGram-positive

strains(S.aureus,B.subtilis)butnoactivitywasobservedagainst

theGram-negativestrain(K.pneumoniae).Higherinhibitionwas

detectedagainstS.aureus,whichisoneofthemostcommonof

theGram-positivebacteriacausingfoodpoisoning.Theactivityof

eucalyptfruitextractislowerthanthatofgallicandtannicacids.In

thecaseofB.subtilis,tannicacidgavecomparableinhibitionzone

(5.33±1.15mm)comparedtothatofextract(5.5±0.5mm)while

gallicacidexhibitedonlyweakactivity(1.00±0.00mm)against

thisstrain.

ThesensitivityofS.aureustofruitextractofE.globulusis

con-sistentwithpublisheddataabouteucalyptspecies,buttheresults

aredifficulttocomparebecauseliteratureassayswerecarriedout

atdifferentconditions.Takahashiet al.(2004)founda

remark-ableantibacterialeffectofextractsofleavesofE.maculataandE.

viminalisagainstS.aureus.Khanetal. (2009)have reportedthe

antistaphylococcolactivityofleafextractofE.globulus(diameter

ofinhibitionzonesrangedfrom16to25mm).Egwaikhideetal.

(2008)havereportedaninhibitoryeffectofmethanol,hexaneand

ethylacetateextractsofE.globulusagainstS.aureus.Otherstudies

haverevealedthesensitivityofthisstraintoseveralgeneraof

Myr-taceaefamily.Theextractsofleavesofguava(Psidiumguajava)and

cloves(Syzgiumaromatica)showedinhibitoryeffectsongrowthof

S.aureus,withinhibitionzonesrangingfrom10to20mmandfrom

21to30mm,respectively(AhmadandBeg,2001).

Quantitative evaluation of the antibacterial activity of the

extractofE.globulusfruitand ofthestandardswascarriedout

againstselectedmicroorganisms;theMICsofthetestedsamples

arepresentedinTable2.

MICvaluesoftheeucalyptextractandtannicacidwerelower

forB.subtiliswhilegallicacidwasamorepotentinhibitorofS.

aureusandmuchlessefficientagainstB.subtilis.Indeed,extracts

ofleavesandbarkofE.globulushavebeenreportedtobeeffective

againstB.subtilis(MIC=100;6250␮g/mL,respectively)(Cruzetal.,

2005;Khanetal.,2009).Anotherstudy,hasreportedtheeffective

antibacterialcapacityofextractofleavesofthisplantagainstthe

samestrain(MIC=500ppm)(Houetal.,2000).

Aligiannis et al. (2001) have proposed a classification of

plant extracts on the basis of their MIC values: strong

inhi-bition:MIC<500␮g/mL;moderateinhibition:600␮g/mL<MIC<

1500␮g/mLand lowinhibition:MIC>1600␮g/mL. Onthebasis

of this classification, the fruit extract and thetested standards

exerta stronginhibitory activityonS.aureus (MIC=80␮g/mL).

Theextractandtannicacidalsohaveastronginhibitoryactivity

on B. subtilis (MIC=30; 50␮g/mL, respectively), whereas

gal-lic acid exerts just moderate inhibitory activity on this strain

(MIC=600␮g/mL).

TheMBCvaluesoffruitsextractagainstS.aureusislessthan

that of B.subtilis (Table2). Thecomparison of MICs and MBCs

valuesallowsabetterevaluationofantibacterialeffectof

bioac-tivecompounds.AccordingtoBiyitietal.(2004),asubstanceis

bactericidalwhentheratioMBC/MIC≤2,andbacteriostaticifthe

ratio MBC/MIC>2. Based onthesedata, theextract ofeucalypt

fruit and gallic acidexert bactericidal effects against S. aureus

(MBC/MIC=1and1.4,respectively),whiletannicacidexertsa

bac-teriostaticeffectonthesamebacterium(MBC/MIC≈3.33).Thefruit

extractandtannicacidshowabacteriostaticeffectagainstB.

sub-tilis(MBC/MIC≈13.33and40, respectively),whereas gallicacid

exhibitsabactericidaleffectagainstthisbacteria(MBC/MIC≈1.33).

Resultsfromthisstudysuggestthatphenoliccompoundsare

responsibleoftheantibacterialactivityofextractof E.globulus

fruits.Numerousworkshavereportedtheantibacterialeffectsof

thesemetabolitesagainstawiderangeofbacteria(AhmadandBeg,

2001;Rodríguezetal.,2009).Ithasbeenreportedthathydrolyzable

tanninshavepotentantibacterialeffectsonvariousbacteria

includ-ingB.subtilisandS.aureus(Tagurietal.,2006;Rodríguezetal.,

2009).Houandcolleagueshavereportedtheantibacterialactivity

ofeucaglobulinandtellimagrandinisolatedfromE.globulusleaves,

againstS.aureus(MICvaluesare,1000and31ppm,respectively),

indicatingthattheobservedactivityofourextractcouldbedueto

itsrichnessinhydrolyzabletannins(Boulekbache-Makhloufetal.,

2010).

Phenoliccompounds canactattwo differentlevels:thecell

membraneandcellwallofthemicroorganisms(Tagurietal.,2006).

Theycaninteractwiththemembraneproteinsofbacteriabymeans

of hydrogenbonding through theirhydroxyl groupswhich can

resultinchangesinmembranepermeabilityandcausecell

destruc-tion.Theycanalsopenetrateintobacterialcellsandcoagulatecell

content(Tianetal.,2009).Phenoliccompoundsareknowntobe

synthesizedbyplantsinresponsetomicrobialinfection(Doughari

etal.,2008;Senguletal.,2009);itisthereforelogicalthattheyhave

beenfoundinvitrotobeeffectiveantimicrobialsubstancesagainst

awidearrayofmicro-organisms(Cowan,1999).

Thisstudyshowsthat E.globulus fruitsextract waseffective

againstthe twoGram-positive strains(S.aureus,B.subtilis)but

noactivitywasobservedagainsttheGram-negativebacteria(K.

pneumoniae).Thisisconsistentwithpreviousstudiesreportingthat

Gram-negativebacteriaaremoreresistanttoantimicrobialsthan

Gram-positivemicroorganismsduetotheirouter

lipopolysaccha-ridemembrane(Khanetal.,2009;Al-Zoreky,2009).

3.3. Antioxidantactivity

Thereducingpowerofdifferentconcentrationsofextract(25,

50,75,100,125␮g/mL)wascomparedwiththatofBHAand

␣-tocopherol(Fig.1).Thereducingcapacityofallsampleswasdose

dependent.ThefruitextractandBHAshowedsignificantlyhigher

activitythan_{␣-tocopherol}(p<0.05).IC50valueofextractwas

cal-culated, it wasabout39.52␮g/mL, significantlylower (p<0.05)

Table2

MICandMBCforfruitsextractofE.globulusandstandards.

Microorganisms Extract Gallicacid Tannicacid Extract Gallicacid Tannicacid

MIC(␮g/mL) MBC(␮g/mL)

S.aureus 80 50 90 80 70 300

B.subtilis 30 600 50 400 800 >2000

Fig.1. ReducingpoweroffruitsextractofE.globulus,␣-tocopherolandBHA.

Concerning the lipid peroxidation activity, the addition of

extractandBHApreventedthebleachingof␤-carotene.Theextract

ofE.globulusfruitshowedmoderateantioxidantactivity

(inhibi-tionof51.34_±0.72%)comparedtothatofBHA,thedifferencebeing

statisticallysignificant(p<0.05).␤-Caroteneinthismodelsystem

undergoesrapiddiscolorationintheabsenceofanantioxidant.This

isbecauseofthecoupledoxidationof␤-caroteneandlinoleicacid,

whichgeneratesfreeradicals.Inthisstudy,theextractoffruitof

E.globuluswasfoundtolimit␤-carotenebleachingbyneutralizing

thelinoleate-freeradical.

TheantioxidantcapacityobservedwiththeextractofE.globulus

fruitsisprobablyduetoitshighcontentinphenoliccompounds.

Activity ofputativeantioxidantshasbeenattributed tovarious

mechanisms,bindingoftransitionmetalioncatalystsand

reduc-tivecapacity(Hayounietal.,2007).Theactivityoffruitsextract

mayberelatedtothepresenceofcompoundswithhigh

molecu-larweight,especiallyhydrolyzabletanninswhichwerethemain

compoundsdetectedinthisextract(Boulekbache-Makhloufetal.,

2010).Indeed,thisclassoftanninshasbeenreportedtohavepotent

antioxidativeactivities(Amakuraetal.,2009).Eylesetal.(2004)

haveestimatedthesuperoxidedismutase-likeactivityofcrudeE.

globulusextractsobtainedfromwoundedwoodusingthe

water-solubletetrazolium saltassay; theyconcluded thatthefraction

whichcontains hydrolyzabletanninsshowedthe highestlevels

ofantioxidantactivity.Inaddition,ithasbeenreportedthatthe

antioxidantactivityofgallotanninsincreasedwithincreasing

num-berofgalloylgroups(Tianetal.,2009).Thus,antioxidantactivity

ofourextractwasdueprobablytopresenceofgallotanninswith

O-dihydroxylgroupssuchaspentagalloylglucose,whichismainly

responsiblefortheirhydrogendonatingabilities.Antioxidant

activ-ityofthisextractmayalsoduetothepresenceofellagicacidwhich

wasdetectedinourpreviousstudyinfruitsextract(

Boulekbache-Makhloufet al., 2010).Cruz et al. (2005) have reportedthat a

veryremarkableantioxidantactivitywasdeterminedforextract

ofwoodofE.globulus,whichhadellagicacidasitsmain

pheno-liccomponent.Thishydroxybenzoicacidhasbeenreportedtobe

veryeffectiveinprotectinglipidsandLDLfromoxidationandan

effectiveinvivometalchelatingagent(Cruzetal.,2005).

4. Conclusion

E.globulus has been used as traditional remedies for

treat-mentofvariousdiseases anditsleaveshavebeenusedasfood

additive.Inthis studywe reportfor thefirsttime, the

antioxi-dantandantibacterialactivitiesofafruitextractofthisplant.The

acetonicextractofE.globulusfruits,thatwasshowntocontain

phe-noliccompoundsandespecially hydrolyzabletannins,exhibited

highreducingpowerandmoderatelipidperoxidationinhibition

activity.Thisextract exhibited also strongantibacterialactivity

againstthetwoGram-positivestrains:S.aureusandB.subtilis.

References

Abdel-HameedEl-Sayed,S.,2009.Totalphenoliccontentsandfreeradical scaveng-ingactivityofcertainEgyptianFicusspeciesleafsamples.FoodChemistry114, 1271–1277.

Ahmad,I.,Beg,A.Z.,2001.Antimicrobialandphytochemicalstudieson45Indian medicinalplantsagainstmulti-drugresistanthuman pathogens.Journalof Ethnopharmacology74,113–123.

Aligiannis,N., Kalpotzakis, E.,Mitaku, S.,Chinou, I.B.,2001. Composition and antimicrobialactivityoftheessentialoilsoftwoOriganumspecies.Journalof AgriculturalandFoodChemistry49,4168–4170.

Alison,M.G.,Peter,R.F.,1998.AntihyperglycemicactionsofEucalyptusglobulus (Eucalyptus)areassociatedwithpancreaticextra-pancreaticeffectsinMice. JournalofNutrition128,2319–2323.

Al-Zoreky,N.S.,2009.Antimicrobialactivityofpomegranate(PunicagranatumL.) fruitpeels.InternationalJournalofFoodMicrobiology134,244–248. Amakura,Y.,Yoshimura,M.,Sugimoto,N.,Yamazaki,T.,Yoshida,T.,2009.Marker

constituentsofthenaturalantioxidanteucalyptusleafextractfortheevaluation offoodadditives.Bioscience,Biotechnology,andBiochemistry73,1060–1065. Biyiti,L.F.,Meko,D.J.L.,AmvamZollo,P.H.,2004.Recherchedel’activité

antibactéri-ennedequatreplantesmédicinalesCamerounaises.PharmacologieetMedecine TraditionelleenAfrique13,11–20.

Boulekbache-Makhlouf,L.,Meudec,E.,Chibane,M.,Mazauric,J.P.,Cheynier,V., Sli-mani,S.,Henry,M.,Madani,K.,2010.Analysisofphenoliccompoundsinfruit ofEucalyptusglobuluscultivatedinAlgeriabyhigh-performanceliquid chro-matographydiodearraydetectionmassspectrometry.JournalofAgricultural andFoodChemistry58,12615–12624.

Chan,E.W.C.,Lim,Y.Y.,Wong,S.K.,Lim,K.K.,Tan,S.P.,Lianto,F.S.,Yong,M.C.,2009. Effectsofdifferentdryingmethodsontheantioxidantpropertiesofleavesand teaofgingerspecies.FoodChemistry113,166–175.

Changwei,A.O.,AnpingLiAbdelnaser,A.E.,Tran,D.X.,Shinkichi,T.,2008.Evaluation ofantioxidantandantibacterialactivitiesofFicusmicrocarpaL.fil.extract.Food Control19,940–948.

Cowan,M.M.,1999.Plantproductsasantimicrobialagents.ClinicalMicrobiology Reviews12,564–582.

Cruz,J.M.,Domínguez,H.,Parajó,J.C.,2005.Anti-oxidantactivityofisolatesfrom acidhydrolysatesofEucalyptusglobuluswood.FoodChemistry90,503–511. Doughari,J.H.,El-mahmood,A.M.,Tyoyina,I.,2008.Antimicrobialactivityofleaf

extractsofSennaobtusifolia(L.).AfricanJournalofPharmacyandPharmacology 2,007–013.

Egwaikhide,P.A.,Okeniyi,S.O.,Akporhonor,E.E.,Emua,S.O.,2008.Studieson bioac-tivemetabolitesconstituentsandantimicrobialevaluationofleafofEucalyptus globulus.AgriculturalJournal3,42–45.

Eyles,A.,Davies,N.W.,Mitsunaga,T.,Mihara,R.,Mohammed,C.,2004.RoleofE. glob-uluswoundwoodextractives:evidenceofsuperoxidedismutase-likeactivity. ForestPathology34,225–232.

Gallagher,A.M.,Flatt,P.R.,Duffy,G.,Abdel-Wahab,Y.H.A.,2003.Theeffectsof tra-ditionalantidiabeticplantsoninvitroglucosediffusion.NutritionResearch23, 413–424.

GöktürkBaydar,N.,GülcanÖzkanb,G.,Yas¸arc,S.,2007.Evaluationoftheantiradical andantioxidantpotentialofgrapeextracts.FoodChemistry18,1131–1136. Gülc¸in,I.,Berashvili,D.,Gepdiremen,A.,2005.Antiradicalandantioxidantactivityof

totalanthocyaninsfromPerillapankinensisdecne.JournalofEthnopharmacology 101,287–293.

Hagerman,A.E.,Butler,L.G.,1978.Proteinprecipitationmethodforthe quantita-tivedeterminationoftannin.JournalofAgriculturalandFoodChemistry26, 809–812.

Hasegawa,T.,Takano,F.,Takata,T.,Niiyama,M.,Ohta,T.,2008.Bioactive monoter-peneglycosides conjugatedwithgallic acidfromthe leavesof Eucalyptus globulus.Phytochemistry69,747–753.

Hayouni,E.A.,Abedrabba,M.,Bouix,M.,Hamdi,M.,2007.Theeffectsofsolventsand extractiononthephenoliccontentsandbiologicalactivitiesinvitroofTunisian QuercuscocciferaL.andJuniperusphoeniceaL.fruitextracts.FoodChemistry105, 1126–1134.

Hou,A.J.,Liu,Y.Z.,Yang,H.,Lin,Z.W.,Sun,H.D.,2000.Hydrolyzabletanninsand relatedpolyphenolsfromEucalyptusglobulus.JournalofAsianNaturalProducts Research2,205–212.

Hseu,Y.C.,Chang,W.H.,Chen,C.S.,Liao,J.W.,Huang,C.J.,Lu,F.J.,Hia,Y.C.,Hsu,H.K., Wu,J.J.,Yang,H.L.,2008.AntioxidantactivitiesofToonasinensisleavesextract usingdifferentantioxidantmodels.FoodandChemicalToxicology46,105–114. Jouad,A.,Maghrani,M.,ElHassani,R.H.,Eddouks,M.,2003.Hypoglycemicactivity ofaqueousextractofEucalyptusglobulusinnormalandstreptozotocin-induced diabeticrats.JournalofHerbs,Spices&MedicinalPlants10,462–464. Khan,R.,Islam,B.,Akram,M.,Shakil,S.,Ahmad,A.,Manazir,A.S.,Siddiqui,M.,

Khan,A.U.,2009.Antimicrobialactivityoffiveherbalextractsagainstmultidrug resistant(MDR)strainsofbacteriaandfungusofclinicalorigin.Molecules14, 586–597.

Parekh,J.,Chanda,S.V.,2007.Invitroantimicrobialactivityandphytochemical anal-ysisofsomeIndianmedicinalplants.TurkishJournalofBiology31,53–58. Quettier-Deleu,C.,Gressier,B.,Vasseur,J.,Dine,T.,Brunet,C.,Luyckx,M.,Cazin,

M.,Cazin,J.C.,Bailleul,F.,Trotin,F.,2000.Phenoliccompoundsand antioxi-dantactivitiesofbuckwheat(FagopyrumesculentumMoench)hullsandflour. JournalofEthnopharmacology72(1–2),35–42.

Rodríguez,H.,Curiel,J.A.,Landete,J.M.,DelasRivas,B.,DeFelipe,F.L., Gómez-Cordovés,C.,Manche ˜no,J.M.,Mu ˜noz,R.,2009.Foodphenolicsandlacticacid bacteria.InternationalJournalofFoodMicrobiology132,79–90.

Sengul,M.,Yildiz,H.,Gungor,N.,Cetin,B.,Eser,Z.,Ercili,S.,2009.Totalphenolic con-tent,antioxidantandantimicrobialactivitiesofsomemedicinalplants.Pakistan JournalofPharmaceuticalSciences22,102–106.

Sherryl,E.,Warnke,P.H.,2004.Successfuluseofaninhalationphytochemicalto treatpulmonarytuberculosis.Phytomedicine11,95–97.

Singleton, V., Rossi, J.A., 1965. Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. American Journal of EnologyandViticulture16,144–158.

Tagg,J.R.,Mcgiven,A.R.,1971.Assaysystemforbacteriocins.AppliedMicrobiology 21,943–944.

Taguri,T.,Tanaka,T.,Kouno,I.,2006.Antibacterialspectrumofplantpolyphenols andextractsdependinguponhydroxyphenylstructure.Biologicaland Pharma-ceuticalBulletin29,2226–2235.

Takahashi,T.,Kokubo,R.,Sakaino,M.,2004.Antimicrobialactivitiesof eucalyp-tusleafextractsandflavonoidsfromEucalyptusmaculate.LettersinApplied Microbiology39,60–64.

Tian,F.,Li,B.,Ji,B.,Zhang,G.,Luo,Y.,2009.Identificationandstructure–activity relationshipofgallotanninsseparatedfromGallachinensis.LWT–FoodScience andTechnology42,1289–1295.

Zampini,I.C.,Vattuone,M.A.,Isla,M.I.,2005.AntibacterialactivityofZuccagnia punctata Cav. ethanolic extracts. Journal of Ethnopharmacology 102, 450–456.

View publication stats View publication stats