Antioxidant capacity and phenolic contents of some Mediterranean medicinal plants and their potential role in the inhibition of cyclooxygenase-1 and acetylcholinesterase activities

ContentslistsavailableatScienceDirect

Industrial

Crops

and

Products

j o u r n al ho me p ag 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

capacity

and

phenolic

contents

of

some

Mediterranean

medicinal

plants

and

their

potential

role

in

the

inhibition

of

cyclooxygenase-1

and

acetylcholinesterase

activities

Nadia

Amessis-Ouchemoukh

a,∗

_,

_Khodir

_Madani

a

_,

_Pedro

_L.V.

_Falé

b

_,

M.

Luisa

Serralheiro

b

_,

_Maria

_Eduarda

_M.

_Araújo

b

a_{Laboratoryof3BS,FacultyofLifeandNatureSciences,UniversityofA.Mira,Bejaia06000,Algeria}

b_{UniversityofLisbon,FacultyofSciences,CentreofChemistryandBiochemistry,CampoGrandeEd.C8,1749-016Lisboa,Portugal}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received28October2013

Receivedinrevisedform

28November2013 Accepted2December2013 Keywords: Cyclooxygenase-1 Acetylcholinesterase Medicinalplants Phenoliccompounds Antioxidantactivities

a

b

s

t

r

a

c

t

Extracts of nine medicinal plants were screened for their anti-inflammatory activity using the

cyclooxygenase-1assayandtheiracetylcholinesteraseinhibitoryeffect.Theantioxidantactivitywas

assessedbyfourmethods:freeradicalsDPPH•(1,1-diphenyl-2-picrilhydrazyl),nitricoxideassay,

␤-carotenebleachingtestandmetalchelatingpower.Theamountsofdifferentphenoliccompoundswere

alsodetermined.Myrtuscommunis(leaves),Pistacialentiscus(leaves)andGlobulariaalypum(flowers)

presentedthehighestamountsoftotalphenoliccompoundswhiletheconcentrationsoftotalflavonoids,

flavonols,proanthocyanidinsandtotaltanninsvariedwithplantspecies.Marrubiumvulgare(leaves)gave

thebestinhibitoryactivityoftheenzymeCox-1withanIC50of0.082mg/mlwhichwasstatisticallynot

differentfromthestandardindomethacin(0.061mg/ml).Thebestanti-acetylcholinesteraseactivitywas

exhibitedbytheleafextractsofM.communis,P.lentiscusandEryngiummaritimum,92.38,73.84and

65.34%,respectively.IntheDPPHassay,P.lentiscusandM.communispresentedthebestactivityand

theirinhibitionswerenotdifferentfromeachother(*p<0.05)butweresignificantlydifferentfromthe

purestandardsrutinandBHA.Amongthetestedplants,Scillamaritimapresentedthebestnitricoxide

scavengingactivity.Inthe␤-caroteneassay,extractsofM.communisleavesandfruitsandP.lentiscus

leaveswerethemostpotentwith63.60,47.61and43.02%,respectively.Metalchelatingactivityassay

showedthatE.maritimumleavesandstemandM.communisleaveshadthebestchelatingpower,49.78,

32.32and35.98%,respectively.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Non-steroidanti-inflammatorydrugs(NSAIDs)areofhuge

ther-apeuticbenefitin thetreatmentof inflammatorydiseases since

they are widely used for the treatment of pain, inflammation

andfever (Vonkemanand vandeLaar, 2010).The main

mech-anismof action of these drugs is believed to bethe inhibition

ofthecyclooxygenaseenzymes:theconstitutive

cyclooxygenase-1 (Cox-1) and the inducible cyclooxygenase-2 (Cox-2) (Ulbrich

etal.,2002).Cyclooxygenase(Cox)isabi-functionalenzymethat

firstcatalyzestheadditionoftwomoleculesofoxygento

arachi-donicacidtoform thehydroperoxideprostaglandin G2 (PGG2),

thenreducesthehydroperoxidetothealcohol,PGH2,bya

per-oxidase activity. Prostaglandins (PGs) are important biological

mediators of inflammation, originating from biotransformation

∗ Correspondingauthor.Tel.:+21334214762;fax:+21334214762.

E-mailaddress:amessisnadia@yahoo.fr(N.Amessis-Ouchemoukh).

of arachidonic acid catalyzed by cyclooxygenase (Gierse et al.,

2008).The most common side effects associated withall

cur-rently available NSAIDs are gastrointestinal haemorrhagia and

ulceration (Dannhardt et al., 2000). These side effects during

anti-inflammatory therapy are caused by interferencewiththe

physiological propertiesofprostaglandins(Ulbrichet al.,2002).

Duringinflammatory responses,theactivationofphospholipase

A2inducesthemobilizationoffattyacids,inparticulararachidonic

acidfromthemembranelipidpool(Fioruccietal.,2001;Fawole

etal.,2010).ArachidonicacidisthenoxidizedbyCox-1or

Cox-2enzymes,leadingtotheproductionofprostaglandins(Fiorucci

etal.,2001).Inaddition,oxidantssuchasreactiveoxygenspecies

(ROS)generatedfromactivatedneutrophilsandmacrophageshave

beenreportedtoplay animportantrole inthepathogenesis of

various pain-related diseases, including neurodegenerative

dis-orders,likeAlzheimer’sdisease(AD)(GibsonandHuang, 2005;

Fawoleet al., 2010). Thisdiseaseis frequent in elderly people,

asaresultofmalfunctioningofdifferentbiochemicalpathways.

ThedrugsapprovedfortheADtherapyactbycounteractingthe

0926-6690/$–seefrontmatter © 2013 Elsevier B.V. All rights reserved.

Table1

Botanicalandcommonnames,families,voucherspecimens,plantpartsandmedicinalpropertiesoftheinvestigatedplants.

Family Species Localname Voucherspecimen Usedpart Traditionaluses

Myrtaceae Myrtus communis Chilmoune D-PH-2013-37-6 D-PH-2013-37-12 Leaves Fruits

Antiseptic,disinfectantdrugandhypoglycemiceffects, antimicrobial,tonicandbalsamicagent,Inhibitxanthine oxidaseactivity,anti-inflammatoryactivity

Apiaceae Eryngium maritimum Tabelyatut D-PH-2013-37-1 D-PH-2013-37-16 Leaves Stem

Diuretic,antiscorbutic,acytotonic,aurethritisremedy,a stoneinhibitor,anaphrodisiac,anexpectorant,an anthelmintic,antinociceptiveandanti-inflammatory activity.Usedforsnakebites,fevers,orfemalereproductive disorders

Anacardiaceae Pistacia lentiscus

Amadagh D-PH-2013-37-5 Leaves Antibacterial,andantiulceragent,treatmentofeczema, diarrheaandthroatinfections,anti-inflammatory, antipyreticandinsecticidalactivities

Globulariaceae Globularia alypum Taselgha D-PH-2013-37-7 D-PH-2013-37-17 Leaves Flowers

Hypoglycemic,laxativeanddiureticagent,treatmentof rheumatism,arthritis,hemorrhoids.Anti-inflammatory activity

Lamiaceae Marrubium

vulgare

Marruyet D-PH-2013-37-2 Leaves Treatmentofgastroentericalandrespiratorydiseases, antinociceptive,anti-inflammatory,hypoglycemicand insecticidaleffects.Tonic,aromatic,expectorant, diaphoreticanddiureticactivities

Liliaceae Scillamaritima Lebselwuchen D-PH-2013-37-14 Bulb Treatmentofheartinsufficiency,edemaandbadkidney performance,memory-enhancementandrodenticide. Anti-inflammatoryactivity,cardiotonic,diuretic

acetylcholinedeficit,thatis,theytrytoenhancetheacetylcholine level in the brain (Heinrich and Teoh, 2004). Acetylcholine is

involvedinthesignaltransferinthesynapses.Afterbeing

deliv-eredin thesynapses, acetylcholineishydrolyzedgivingcholine

andacetyl groupina reactioncatalyzedby theenzyme

acetyl-cholinesterase.ThemolecularbasisoftheAlzheimerdrugsusedso

far,takeadvantageoftheiractionasacetylcholinesteraseinhibitors

(AChEIs)(HeinrichandTeoh,2004;Ferreiraetal.,2006)butthese

drugs have beenreported tohave theiradverse effects

includ-inggastrointestinaldisturbances,hepatotoxicity,nausea,vomiting,

diarrhea,dizziness andproblems associatedwithbioavailability

(Schulz,2003;Mukherjeeetal.,2007),whichincreasesthe

inter-estinfindingbetteracetylcholinesteraseinhibitorsfromnatural

resources.

Considering the developing increasing demand for

plant-deriveddrugs,thenineselectedplants:Pistacialentiscus(leaves),

Myrtus communis (leaves and fruits), Globularia alypum (leaves

and flowers), Marrubium vulgare (leaves), Eryngium maritimum

(leaves and stem) and Scilla maritima (bulb) could be further

assessedandutilizedinviewoftheirhealthbenefiteffectswhere

someof them are reportedin Table 1. Moreover, accordingto

ourknowledge,therearenoreportsontheacetylcholinesterase

andcyclooxygenase-1inhibitoryactivitiesofthestudiedspecies

in Algeria. Therefore, the aims of the present investigation

weretoscreenfor antioxidantcapacitiesinthemedicinalplant

extracts,andtodeterminetheirinhibitoryeffectsonthe

acetyl-cholinesteraseandcyclooxygenase-1enzymes.Toelucidatetheir

oxidativeactions,theextractsweresubjectedtoarangeofinvitro

tests, includingthe metal chelatingpower, the ability to

scav-engeDPPH radical,the␤-carotenebleaching testand thenitric

oxideassay.Theamountsofantioxidantcomponents(total

phe-nolic compounds, flavonoids, flavonols, proanthocyanidins and

total tannins) from the crude plant extracts were also

deter-mined.

2. Materialandmethods

2.1. Chemicals

Allchemicalsandreagentswereofanalyticalgradeandwere

suppliedfromSigma–AldrichQuímicaS.A.(Sintra,Portugal)and

fromSigma(representedbyAlgerianChemicalSociety,Setif,

Alge-ria).

2.2. Plantmaterialsandsamplespreparation

Ninemedicinalplantswereharvestedin2009fromtwo

loca-tions,inremoteareasinthesuburbsofTaghzouitandAboudaou,

BajaiaCity(Algeria).Thevariousdata(localname,medicinaluses,

usedparts of plant,methodofpreparation and administration)

were collectedfromlocal inhabitants havingknowledge of the

curativeproperties of theseplants. Botanicalidentification was

made by the member of laboratory of Botany (Faculty of Life

and Nature Sciences, University Abderrahmane Mira of Bejaia)

accordingto“Nouvellefloredel’Algérieetdesregionsdesertiques

et Meridionales” (Quezel and Santa,1963).Voucher specimens

(Table 1) were deposited at the Herbarium of Natural History

Museum of Aix-en-Provence, France. Fresh leaves, flowers and

stemswereair-driedinshadeatroomtemperatureandthebulbs

ofS.maritimawerepeeled,groundandthenfrozenandlyophilized

immediately.Afterdryingandlyophilization,plantmaterialwas

groundtoafinepowder(diameter<250_␮m)usinganelectricmill

(IKAR _{A11basic,Germany) and1gofthis powderwas}

exhaus-tivelyextractedbymacerationwith10mlofmethanol,atroom

temperaturefor24h.Inallcases,thesolutionswerefilteredand

concentratedtodrynessunderreducedpressureinarotary

evap-orator(40◦C).Dryextractswerestoredat−20◦_Cuntilused.

2.3. Determinationoftheamountsofphenoliccompounds

2.3.1. Totalphenolicscontent

Total phenolic compounds content was assayed using the

Folin–Ciocalteu reagent, following Singleton and Rossi method

(1965).Analiquot(1ml)ofdilutedsampleextract(0.3mg/ml)was

addedto500␮loftheFolin–Ciocalteureagentand6mlofwater.

Themixturewasshakenandallowedtostandfor5min,before

additionof1.5mlofNa2CO3(20%).Analiquotof1.9mlofdistilled

waterwasaddedandmixedthoroughly.Afterincubationindark

for2h,theabsorbanceat760nmwasread versustheprepared

blank.Totalphenoliccontentofplantpartswasexpressedas

mil-ligramsofgallicacidequivalentspergramofdryweight(mgGAE/g

DW) throughthecalibrationcurvewithgallicacid(y=0.9397x;

R2_=0.998).

2.3.2. Totalflavonoidscontent

Determinationof theflavonoids contentwasachievedusing

aluminumchloridereagenttothesolutioncontainingtheextract.

The absorbance was read at 430nm and concentrations of

flavonoids were deduced from a standard curve (y=41.686x;

R2_{=0.997)andcalculatedinmgquercetinequivalent(QE)/gdry}

weight(DW).

2.3.3. Flavonolscontent

Totalflavonolsintheplantextractswereestimatedusingthe

methodreportedbyAdedapoetal.(2008).To2mlofsample,2ml

ofAlCl3 (2%)ethanoland 3ml(50g/L)sodiumacetatesolutions

wereadded.Themixturewasshakenandincubatedfor2.5hat

20◦C. Theabsorbance wasread at440nm.Totalflavonols

con-tentwasexpressedasmgof quercetinequivalents pergof dry

weight(mgEQ/gDW)throughthecalibrationcurvewithquercetin

(y=15.16x+0.0092;R2_=0.999).

2.3.4. Proanthocyanidinscontent

The concentrationof proanthocyanidins was determined by

butanol–HCl assay (Maksimovic et al., 2005). Extract of plants

(0.5ml)wasmixedwith3mlofbutanol–HCl(95:5;v/v)and0.1ml

ofironsulphate(2g/100ml).Themixturewasincubatedat90◦C

for1h.Theabsorbancewasdeterminedat530nm.Theamount

ofproanthocyanidinswasexpressedasmg(+)-catechinequivalent

(CE)g−1DW(y=2.078x−0.1488;R2_=0.999).

2.3.5. Totaltanninscontent

Totaltanninswereestimatedaccordingtotheprotocol

devel-oped by Hagerman and Butler (1978) on the basis of their

precipitation by a protein, bovine serum albumin (BSA). The

method is based upon the obtention of a colored complex

Fe++_{–phenolswhichcanbemeasuredspectrophotometricallyat}

510nm.Theresultswereexpressedasmgequivalentoftannicacid

(ETA)/gDW(y=0.5184x;R2_=0.999).

2.4. Cyclooxygenase-1assay

The Cox-1 (EC 1.14.99.1) activity was measured by

follow-ingspectrophotometricallyat611nmthereactionofoxidationof

TMPDwitharachidonicacid.Thisassaywasperformedaccording

toCopelandetal.(1994),withthefollowingmodifications.Briefly,

50␮lofCox-1enzyme(200units/cuvette)and100␮lofhematin

(3␮MinTris-buffer,pH8)asco-factorand1000␮lTris–HClbuffer

(100mM,pH8)and50␮lofDMSOpersamplewerepre-incubated.

Thisenzyme/co-factorsolutionwasaddedtothetestsolution

con-sistingof50␮lplantextractoraninhibitor,dissolvedinDMSOand

pre-incubatedfor3minat25◦C.Then200␮lofTMPDwasadded.

Thereactionwasinitiatedbyadditionof50␮lofarachidonicacid

totheenzyme/extractmixtureandcontentsweremixed

immedi-ately.Theinitialvelocityofthereactionwasmeasuredfollowing

thereactionofoxidationofTMPDat611nmfor20s.Thevelocities

observedatdifferentinhibitorconcentrationweredividedbythe

velocityobservedforenzymesamplespre-incubatedforthesame

timewith10%(v/v)inhibitorfreeDMSO,andthisratiowas

mul-tipliedby100toyieldpercentcontrolactivity.IC50valueswere

determinedbyregressionanalysis.

I(%)=100−



Vwithinhibitor

Vwithoutinhibitor



×100

2.5. Acetylcholinesteraseinhibition

Acetylcholinesteraseenzymaticactivitywasmeasuredbythe

Ellman test (Ferreiraet al., 2006). 400␮l of 50mM Tris buffer

(pH8),50_␮lofplantextractinmethanolwithdifferent

concen-trationsand 25␮l of an enzyme solution containing 0.26U/ml

wereincubatedduring15min.Subsequently,75␮lofasolution

ofacetylthiocholineiodide(AChI)15mMand475␮lof3mM5,5

-dithiobis[2-nitrobenzoicacid](DTNB)wereadded.Absorbanceof

themixturewasmeasuredat405nmwhenthereactionreached

theequilibrium.Acontrolmixturewasprepared,usingmethanol

insteadofextractandwasconsidered100%activity.Inhibition,in

%,wascalculatedinthefollowingway:

I(%)=100−



Vsample Vcontrol



×100

whereIisthepercentinhibitionofacetylcholinesterase,Vsampleis

theinitialvelocityoftheextractcontainingreactionandVcontrolis

theinitialvelocityofthecontrolreaction.AblankwithTrisbuffer

insteadofenzymesolutionwasused.Extractconcentration

pro-viding50%inhibition(IC50)wasobtainedbyplottingtheinhibition

percentageagainstextractsolutionconcentrations.

2.6. Antioxidantcapacity

2.6.1. DPPH•radicalscavengingactivity

The DPPH (1,1-diphenyl-2-picrilhydrazyl) radical scavenging

activityoftheplantextractswasdeterminedusingthemethod

proposedbyKatalinicetal.(2006).Aliquot(50␮L)ofthetested

samplewasplacedinacuvetteand2mlof6×10−5Mmethanolic

solutionofDPPHradical(freshlyprepared)wasadded.The

mix-turewas maintainedat roomtemperature for 16min thenthe

absorbancewasmeasuredat517nmagainst thecorresponding

blank.MethanolicsolutionsofpurecompoundsBHAandrutinwere

testedtoo.

ThepercentageinhibitionoftheDPPHradicalbythesamples

wascalculatedbythefollowingformula:

I(%)=



Ablank−Asample Ablank



×100

whereI(%)isthepercentageinhibitionoftheDPPHradical,Ablank

istheabsorbanceofthecontrolreaction(areactionwithallthe

reagentsexceptthesampleextract)andAsampleistheabsorbanceof

thesampleextract.Extractconcentrationproviding50%inhibition

(IC50)wasobtainedplottinginhibitionpercentageversusextract

solutionconcentrations.

2.6.2. Nitricoxideradicalscavengingactivity

Theabilityoftheextractstoscavengenitricoxidefree

radi-calswasdeterminedusing themethodreportedby Dastmalchi

etal.(2008).Inbrief,a0.5mlaliquotofextractorpositive

con-trol(quercetinandcatechin)dissolvedinKH2PO4–KOH(50mmol/l,

pH7.4)wasmixedwith0.5mlof(10mmol/l)sodium

nitroprus-sidesolution.Themixturewasincubatedat37◦Cfor2.5hunder

normallightcondition.Afterincubation,thesamplewasplaced

indarkfor20min.Thereafter,1mlofGriessreagent(1g/l

N-(1-naphtyl)ethylenediamineand 10g/lsulphanilamidedissolvedin

20ml/laqueousH3PO4)wasaddedandtheabsorbancewastaken

after40minat546nm.Thepercentageinhibitionwascalculated

usingthefollowingequation:

I(%)=



Acontrol−Asample Acontrol



×100

whereI(%)isthepercentageinhibitionofNOradical,Acontrolisthe

absorbanceofthecontrolreaction(areactionwithallthereagents

exceptthesampleextract)and A_sample istheabsorbanceofthe

sampleextract.

2.6.3. _{ˇ-Carotene–linoleic}acidbleachinginhibition

The ability of theextract to inhibit thebleaching of the

described byKhadri et al.(2010) witha slight modification.In

brief,2mg␤-carotenedissolvedin1mlchloroform,25␮loflinoleic

acidand200mgofTween40wereaddedandtransferredintoa

round-bottomflask.Oncethechloroformhadbeenremovedunder

nitrogen,50mldistilledwatersaturatedwithoxygenwasadded

theflaskwithvigorousstirringandtheresultingmixturewas

soni-catedfor30minforhomogenization.Thereafter,2.5mlaliquotsof

thisemulsionweretransferredtoaseriesoftubescontaining300␮l

ofextractdissolvedinethanolandwerefurthershaken.Absorbance

ofeachsamplewasrecordedat470nmimmediatelyafter

sam-plepreparation(t=0min).Afterthatthesampleswereplacedina

waterbathat50◦Cforaperiodof2h,togetherwithtwoblanks,

onecontainingantioxidants(BHTorgallicacid)asapositive

con-trolandtheotherwiththesamevolumeofethanolinsteadofthe

extracts.Theabsorbanceofeachsampleafterheating(t=120min)

wasmeasured.

Theantioxidantactivity(AA)oftheextractsunderinvestigation

wasexpressedas:

AA(%)=



1−



A(t=0)−A(t=120) A0(t=0)−A0(t=120)



×100

where AA is the antioxidant activity, A(t=0) is the absorbance

(470nm)ofthesolutionininvestigationat0min,A(t=120) isthe

absorbance(470nm)ofthesamesolutionatt=120min,A0(t=0)is

theabsorbance(470nm)ofthepositivecontrol(ethanolwithout

extract)sampleat0minandA0(t=120)istheabsorbance(470nm)

of a the positive control (ethanol without extract) sample at

t=120min.Extractconcentrationproviding50%inhibition(IC50)

wasobtainedplottinginhibitionpercentageversusextractsolution

concentrations.

2.6.4. Metalchelatingactivity

Theabilityoftheextracttochelateiron(II)wasassessedbythe

methodofCarter(1971).Inbrief,toa200␮lofdissolvedextract

wasadded 100␮l (2mmol/l)FeCl2·4H2Oand 900␮l methanol.

After5minincubation,thereactionwasinitiatedbytheaddition

of400␮l(5mmol/l)ferrozine.Aftera further10minincubation

period,theabsorbanceofthereactionmixturewasmeasuredat

562nm.Theratioofinhibitionofferrozine–Fe2+_{complexformation}

wascalculatedasfollows:

%inhibition=



Abscontrol−Abssample

Abscontrol



×100

whereAsamplewastheabsorbanceinthepresenceofthesample

ofextractsorthestandardEDTA.Theconcentrationatwhichthe

extractexerts50%ofitseffect(IC50)valueswasestimatedbya

linearregressionalgorithm.

2.7. Statisticalanalysis

Datawerepresentedasmean±standarddeviation(SD)ofthree

determinations.Statisticalanalyseswereperformedusinga

one-wayanalysisofvariance(ANOVA)inthesoftwareSTATISTICA5.5.

Differenceswereconsideredsignificantat*p<0.05.

3. Resultsanddiscussion

3.1. Determinationofphenoliccompounds

Phenolic compounds constitute one of the major groups of

compounds known to inhibit some molecular targets of

pro-inflammatorymediatorsininflammatoryresponsesandtoactas

primary antioxidantsor free radical terminators(Fawoleet al.,

2009).Forthesereasons,wehavetried todeterminetheirtotal

amounts.

Amonganalyzedplantextracts,extremelyhightotalphenolic

contentwasdetectedinM.communis(leaves),P.lentiscus(leaves)

andG.alypum(flowers)(Table2).Thelowesttotalphenoliclevel

wasdetectedin E.maritimum (stem).The leavesofM.

commu-niscontainedmorephenoliccompoundsthanthefruitsandthe

amounts offlavonolsand proanthocyanidinsin thesetwoparts

werenotfarfromeachother.SeveralworksfoundthatM.

com-munis,P.lentiscusandG.alypumwererichinphenoliccompounds

(Chryssavgietal.,2008;AidiWannesetal.,2010;Djeridaneetal.,

2010)andourresultsareinagreementwiththem.

TheleavesofE.maritimumandM.vulgarepresentedalmostthe

sameamountsofphenolics(*p<0.05).Thecolorimetricdosageof

flavonolsrevealedthattheextractscontainedsmallamountsof

thisgroup.Onwhatconcernsproanthocyanidinscontent,P.

lentis-cus(leaves),S.maritima(bulb)andM.communisfruitsandleaves

gave the best results. The amounts of flavonolsand

proantho-cyanidinsinG.alypum(leavesandflowers)didnotpresent any

significantdifferences betweenboth plant parts;and thesame

remarkfor proanthocyanidinscontentintheleavesofE.

mariti-mumandM.vulgarecanbedone.Table2showedalsothat the

amountsoftotalflavonoidsvariedwidelyintheinvestigatedplant

extractsandrangedfrom1.34to24.86mgEQ/gdryextract.Thebest

amountswereobtainedbyM.communisandE.maritimumleaves,

followedbyP.lentiscusandG.alypumleaves. Recently,withthe

useofreversed-phasehigh-performance liquidchromatography

hyphenatedtomassspectrometryelectrosprayionizationcoupled

toquadrupoletime-of-flight (RP-HPLC-ESI-QTOF-MS),we found

thatP.lentiscusleaveswererichinflavonoidsandphenolicacids

(Rodríguez-Pérezetal.,2013).

Anotherimportantgroupinvestigatedinthisstudywasthetotal

contentoftannins(Table2).P.lentiscusandM.communisgavethe

bestlevelsoftotaltannins.Ourresultsareinagreementwiththose

foundbyRomanietal.(2002)andCakir(2004)whodetermined

thattheseplantswererichintannins.

E.maritimum andM.vulgare leavesdidnot presentany

sig-nificantdifferences inthetotal tannins contentand theselater

compoundsrepresentedapproximatelythehalfcontentoftotal

tannins.G.alypumflowerscontainedmoreamountsoftanninsthan

flavonoidsanditsleavespresentedtheinversecase.Itisimportant

tonotethatforalltheinvestigatedplantextracts,

proanthocyani-dinscontentswereverylowcomparedtothetotaltanninscontent,

thisresultmaysuggestthattheplantscontainedmorehydrolysable

tanninsthanthecondensedones.

Variation in the amounts of phenolic compounds could be

attributedtoseveralreasons.Thesolubilityofphenoliccompounds

isactuallygovernedbythetypeofsolventused,thedegreeof

poly-merizationofphenolics,aswellasbytheinteractionofphenolics

withotherfoodconstituentsandformationofinsolublecomplex.

For that purpose, methanol was recommended and frequently

usedfortheextractionofphenolics(Galvezetal.,2005).Indeed,

thephenoliccontents ofaplantdependona numberof

intrin-sic(genetic)andextrinsic(environmental,handlingandstorage)

factors(Rapisardaetal.,1999;Fratiannietal.,2007).

3.2. Cyclooxygenase-1assay

From nine plant extracts, only four have presented

anti-inflammatoryactivity(Fig.1).Percentagesofinhibitionobtained

ataconcentrationof0.033mg/mlfromtheseplants,indecreased

order,wereM.vulgareleaves(68.15%),G.alypumflowers(61.05%),

E.maritimumleaves(8.16%)andG.alypumleaves(5.33%),

respec-tively.NoinhibitioninthistestwasfoundwiththeextractofM.

communis(leavesandfruits),S.maritima(bulbs),P.lentiscusleaves

andE.maritimum(stem)atthisconcentration.

M.vulgareleavesandG. alypumflowershavegiven thebest

Table2

Totalphenolics,flavonoids,flavonols,proanthocyanidinsandtanninscontentinthemedicinalplantextracts.

TPC(mgGAE/gDW) Flavonoids(mgQE/gDW) Flavonols(mgQE/gDW) Proanthocyanidins (mgCE/gDW)

Totaltannins (mgTAE/gDW) Myrtuscommunis(leaves) 285.73±2.28a _{24.868±0.415}a _7.38±0.04b,c _19.10±0.12d _{117.670±1.543}a Pistacialentiscus(leaves) 238.33±3.28b _{19.578±0.326}b _7.29±0.24c _39.29±0.61a _{100.694±2.409}b

Globulariaalypum(flowers) 156.97±1.14c _1.346±0.023h _3.12±0.41e _14.07±0.83e _7.459±0.223f

Globulariaalypum(leaves) 102.29±0.50e _{16.592±0.799}c _3.26±0.13e _14.18±0.14e _9.388±2.357f

Scillamaritima(bulb) 127.90±1.49d _2.159±0.069g _2.08±0.06f _27.41±0.23b _{11.703±0.589}e

Myrtuscommunis(fruits) 91.34±3.92f _7.170±0.083e _4.24±0.14d _22.13±0.34c _{28.807±0.803}c,d

Eryngiummaritimum(leaves) 43.83±1.,32g _{24.309±0.262}a _7.63±0.22b _11.97±0.14f _{33.179±1.391}c Marrubiumvulgare(leaves) 40.57±1.91g _{10.249±0.083}d _10.48±0.19a _12.28±0.13f _{32.407±0.386}c

Eryngiummaritimum(stem) 16.49±0.19h _3.505±0.061f _1.04±0.02g _10.12±0.10g _{17.747±21.050}d,e

Averages±standarddeviationwasobtainedfromthreedifferentexperiments.GAE,gallicacidequivalents;QE,quercetinequivalents;CE,catechinequivalents;TAE,tannic acidequivalents;DW,dryweight.MeansfollowedbythesameletterarenotdifferentaccordingtoANOVA(analysisofvariance)(*p<0.05).Theresultsaresortedindecreasing order:a>b>c>d>e>f>g>h. 68.15a 61.05b 8.16d 5.33d 50.00c 0 10 20 30 40 50 60 70 80 90 100 Marrubium vulgare leaves Globularia alypum flowers

Eryngium maritimum leaves

Globularia alypum leaves

Indomethacin

C yclooxyge nas e-1 i nh ib itory activity (% )

Fig.1.Cyclooxygenaseinhibitoryactivityofthedifferentplantextracts (concen-trationofplantextractsis33␮g/ml).Datawereanalyzedusingone-wayANOVA (analysisofvariance).Differentletter(s)indicatethevaluesaresignificantly differ-ent(*p<0.05).

beattributedtotheirpolyphenoliccompounds (i.e.tanninsand proanthocyanidins).Thesecompoundsareknowntoinhibitsome moleculartargetsofpro-inflammatorymediatorsininflammatory responses(Fawoleetal.,2010).Whereas,theresultsobtainedfor

E.maritimumandG.alypumleavesweretoolowercomparedto

thetwoprecedingplantsandtheywerestatisticallynotdifferent

fromeachotherat(*p<0.05).Inaddition,theanti-inflammatory

activityobtainedfromtheflowersofG.alypumwasmuchhigher

comparedtoitsleaves,whichleadstosaythattheinhibitoragents

werepresentintheflowerspart.

Fromtheobtainedresults,theIC50valuesweredeterminedfor

M.vulgareleavesand G.alypum flowersaswellasthestandard

indomethacin.Theanti-inflammatoryactivityofthesampleswas

foundtoincreasedose-dependentlyandtheresultsweredepicted

inTable3.

M.vulgareleavesextracthaspresentedapotentactivitywithan

IC50of0.082mg/mlwhichwasstatisticallynotdifferentfromthe

chemicalstandardindomethacin(0.061mg/ml).

Intheliterature,severalreportshavedescribedthatM.vulgare

andG.alypumcontainedsomeactivecomponentssuchas

pheno-liccompounds,flavonoids,coumarins,phenylethanoidglycosides,

iridoidsand monoterpenes. Someauthors(Sahpazet al., 2002;

Berrouguietal.,2006;Taskovaetal.,2006;Dussossoyetal.,2011)

affirmthatthisvarietyofmoleculescouldberesponsibleforthe

cyclooxygenaseinhibition.

Sincetheextractsofmedicinalplantsunderstudywererichin

polyphenols,flavonoidsandtannins,thedatafromTable2

repor-tingtheevaluationofthesetwoclassofcompoundssupportedthe

inhibitionofCox-1.

3.3. Acetylcholinesteraseinhibitoryactivity

Medicinalplantextractsweretestedtodeterminetheir

abil-ityasacetylcholinesteraseinhibitorsandresultsweredepictedin

Fig.2.Foreachplant,theinhibitioncapacityshowedthe

follow-ingorder:M.communisleaves>P.lentiscusleaves>E.maritimum

leaves>G.alypumflowers>M.vulgareleaves>S.maritimabulb>M.

communisfruits>G.alypumleaves.

Ataconcentrationof0.5mg/mlforeachextract,thepercentage

inhibitionofacetylcholinesteraserangedfrom26to92%.Thebest

inhibitoryactivitywasexhibitedbytheleafextractofM.communis

followedbytheleavesofP.lentiscusandE.maritimumwhichwere

notsignificantlydifferent(*p<0.05).ExtractsofM.vulgareleaves,

G.alypumflowersandS.maritimabulbhavepresentedabout50%of

inhibitionoftheenzymeatthetestedconcentration.AsG.alypum

leaves,theextractofM.communisfruitspresentedthesame

capac-ityofinhibition,butinfact,thistwoplantsshowedlowervalues

thanalltheotherextracts.Inaddition,thefruitsofM.communis

werelesspotentthanitsleaves.ConcerningE.maritimumstem,it

wasnotpossibletotestitsextractduetoappearanceofturbidity

inthetestconditions.

Ourresultswerewithintherangeofthevaluesfoundinthe

lit-eraturefortheinhibitionofthisenzymewithplantextracts(Orhan

etal.,2009)andwerehigherthanthoserecentlyreportedforother

LamiaceaeandFumariaceaespecies(Mukherjeeetal.,2007;Loïzzo

etal.,2010;Adewusietal.,2011).Theresultsobtainedforthethree

firstplantextracts(citedinorder)werebetterthanthosefoundfor

thestandardgalantamin(48.80%at1mg/ml)usedintheworkof

Orhanetal.(2004).

Inthisstudy,AChEinhibitoractivityofextractswasfoundto

increase dose-dependently,theresultsexpressedas IC50 values

werecalculatedfromtheregressionequationsobtainedfromthe

activityof samplesatdifferentconcentrations.Theresultswere

presentedinTable3.

TheIC50valuesfoundforM.communisandP.lentiscusleaves

werehigherthanthosefoundforthestandardcompounds

(chloro-genicacid,rutin,hyperoside,isoquercitrinandquercitrin)usedby

Hernandezetal.(2010).

The obtained results showed that eight methanolic plant

extractscontainedsomelevelofinhibitoryactivityagainstAChE,

thismaysuggestthatorganicsolventswereabletoextractmore

activecompoundswithpossibleAChE inhibitoryactivity.These

results werein concordancewith those found by Orhan et al.

(2004)whousedacetoneextractsandobtainedvaluesof

inhibi-tion(13.25to96.89%at1mg/mlconcentrationoftheextract),as

wellasAdewusietal.(2011)whousedDCM:MeOHextractsand

obtainedvaluesofinhibitionrepresentedbyIC50(from0.03mg/l

to1mg/ml).

The best inhibitory activity obtained with M. communis, P.

Table3

Cyclooxygenase-1assay,acetylcholinesteraseactivity,radicalscavengingactivity,␤-carotenebleachingassayandmetalchelatingpowerrepresentedbyIC50(mg/ml).

Cox-1IC50 DPPHIC50 AChEactivity ␤-Caroteneinhibition Metalchelatingactivity

Myrtuscommunis(leaves) 0.003±0.000d _0.03±0.00f _0.07±0.00h _1.24±0.03b

Pistacialentiscus(leaves) 0.008±0.000d _0.01±0.00f _0.12±0.00g _ND

Globulariaalypum(flowers) 0.122±0.006a _0.004±0.001d _0.53±0.05c _0.26±0.02e _ND

Globulariaalypum(leaves) 0.031±0.004c _0.82±0.05a _0.51±0.03b _ND

Scillamaritima(bulb) 0.028±0.003c _0.62±0.03b _0.17±0.01f _ND

Myrtuscommunis(fruits) 0.005±0.001d _0.81±0.02a _0.12±0.01g _ND

Eryngiummaritimum(leaves) 0.043±0.005b _0.42±0.03d _0.31±0.02d _1.17±0.05c

Marrubiumvulgare(leaves) 0.082±0.004b _0.083±0.004a _0.52±0.03c _0.25±0.00e _1.41±0.01a

Eryngiummaritimum(stem) 0.087±0.006a _{ND 0.43±0.03}c _0.43±0.00d

Indomethacin 0.061±0.009c

Gallicacid ND 0.83±0.04a _ND

BHT 0.01±0.00i _ND

EDTA 0.016±0.00e

Galantamine 0.21±0.02e

Averages±standarddeviationwasobtainedfromthreedifferentexperiments.MeansfollowedbythesameletterarenotdifferentaccordingtoANOVA(analysisofvariance) (p<0.05).ND,notdetermined.Nocorrelationbetweenactivityandconcentrationwasobtained.IC50ofgalantamineisexpressedin␮g/ml.

43.25c 45.62c 73.84b 65.34b 92.38a 26.03d 42.45c 29.98d 0 10 20 30 40 50 60 70 80 90 100 Marrubium

vulgare leaves alypum flowers Globularia lentiscus leaves Pistacia maritimum Eryngium leaves

Myrtus

communis leaves alypum leaves Globularia Scilla maritima bulb communis fruits Myrtus

Acetylcholinesterase activity (%)

Fig.2. Acetylcholinesteraseinhibitoryactivityofthedifferentorganplantextracts.Datawereanalyzedusingone-wayANOVA(analysisofvariance).Differentletter(s) indicatethevaluesaresignificantlydifferent(*p<0.05).

their chemical composition which mainly contain flavonoids, phenolic acids and tannins, as well as, the possible synergis-ticinteractionbetweenthesecomponents.Severalstudieshave shownthatflavonoidsandotherphenoliccompoundspossess anti-acetylcholinesterase activity(Hostettmann et al., 2006; Fawole etal.,2010;Hernandezetal.,2010).So,inourstudy,correlation

coefficientswerefoundbetweenacetylcholinesteraseactivityand

totalflavonols,proanthocyanidinsandphenoliccontents,0.66,0.53

and0.53,respectively.

3.4. Determinationofantioxidantcapacities

3.4.1. DPPH•radicalscavengingactivity

In this study, antioxidant capacity was determined by the

DPPH• radicalscavengingactivity.Thismethodwaschosendue

tothefact thatDPPHhastheadvantageof beingunaffectedby

certainside reactions, suchas metalion chelatingand enzyme

inhibitionas reportedbyAmarowiczetal.(2004).Asshown in

Fig.3,all thetestedplants presentedmore than50% ofradical

inhibition, except E. maritimum which showed 48.94%. These

resultsrevealedthat considerableantiradical componentswere

extractedfromthedifferentplantparts.Differencesinpolarityand

thusdifferentextractabilityoftheantioxidativecomponentsmay

alsoexplainthedifferencesinantioxidantactivityoftheextracts.

P.lentiscusandM.communishavepresentedthebestactivityand

theirinhibitionwerenotdifferentfromeachother(*p<0.05)but

weresignificantlydifferentfromthepurestandardsrutinandthe

syntheticcompound BHAwhich is usedas afoodpreservative.

TheabilityoftheleafextractsofP.lentiscusandM.communisto

scavengefreeradicalscouldbeattributedtotheirhighphenolics

contents. Amarowiczetal.(2010) showedamarkedantiradical

activity of tannins. Results from Table 2 revealed that these

plants contained highlevels of tannins and proanthocyanidins.

Severalworkshavereportedthesameresults(Romanietal.,2002;

Chryssavgietal.,2008;AidiWannesetal.,2010).

TheleavesofM.communisandE.maritimumpresentedhigher

antioxidantactivitythantheirfruitsandstem,respectively.While

theflowersofG.alypumweremorepotentthanitsleaveswith

anIC50=4␮g/ml(see Table3).Thescavengingpotencyofthese

later washigherthan foundbyDjeridaneet al.(2010) withan

IC50=8.7mg/ml.Theobtainedresults(Table2)alsoshowedthat

thisplantcontainedagoodamountofflavonoidsandtannins.A

lotofworkconfirmedourresults(Djeridaneetal.,2006;Es-Safi

etal.,2006).Thecorrelationcoefficientbetweenphenolics(tannins

andproanthocyanidins)andthe%oftheDPPH•scavenging

activ-itywassignificant(r=0.63forboth),indicatingthatpolyphenolics

mayplayanimportantroleinfreeradicalscavenging.

3.4.2. Nitricoxideradicalscavengingactivity

Crude extractsof the studiedplants werechecked for their

inhibitoryeffectonnitricoxideproduction.Theobtainedresults

94.89b _94.18b 97.33a 96.95a 88.75c 84.76d 71.26e 56.31f 53.77g 52.04h 48.95i 0 10 20 30 40 50 60 70 80 90 100

BHA Rutin Pistacia lentiscus (leaves) Myrtus communis (leaves) Myrtus communis (fruits) Globularia

alypum (flowers)

Globularia alypum (leaves) Scilla maritima (bulb) Eryngium maritimum (leaves) Marrubium vulagre (leaves) Eryngium maritimum (stem)

DPPH

•radical reducing power (%)

Fig.3. DPPHradicalscavengingactivityoftheplantextractswith100␮g/ml.Datawereanalyzedusingone-wayANOVA(analysisofvariance).Differentletter(s)indicate

thevaluesaresignificantlydifferent(*p<0.05).

Amongthetestedplants,S.maritimapresentedthebest

scav-engingactivityfollowed byE.maritimum(leaves),M.communis

(fruits)andG.alypum(flowers)with32.06,22.07,15.54and14.41%,

respectively.Otherplantshavepresentedlessthan10%ofthe

activ-ity.S.maritimabulbhavepresentedthehalfactivityofthepure

standardcatechinandquercetin.Accordingtoourresults,thisplant

wasfoundtocontaintotal tanninsand proanthocyanidinsmore

thanflavonoids, and consequently, thesecompounds may

con-tributetoitsmarkedactivity.M.communis(fruits)andG.alypum

(flowers)havenotpresentedanysignificantdifferencesbetween

them(*p<0.05).Theywerefoundtohavebetteractivitythantheir

leaveswhichcontained morephenolic compounds.Despite the

highcontentofphenoliccompounds,P.lentiscusandM.

commu-nisleaveswerelessactive.VariationsintheNO•radical-scavenging

activitybetweentheseplantsweredirectlyrelatedtothestructural

characteristicsandtheamountofphenolicconstituentspresentin

plantmaterials.Also,interactionbetweenvariousphytochemicals

contributedtotheoverallscavengingactivityofplantextracts.

3.4.3. Antioxidantactivityintheˇ-carotene–linoleatemodel

system

Thepotentialoftheextractstopreventorminimizetherapid

discolorationof the␤-carotenewaspresented inFig.5.Ascan

be seen, the antioxidant activity was observed in all screened

plantextracts.Antioxidantactivityincreasedwithincreasingofthe

extractconcentrations.Ataconcentrationof0.10mg/mlthe

activ-ityrangedfrom3.35to63.60%.M.communis(leavesand fruits)

andP.lentiscus(leaves)extractsdemonstratedthebestabilityto

inhibitthebleachingof␤-carotenebyscavenginglinoleate-derived

freeradicals.Itisimportanttonotethattheantioxidantactivityof

theextractsofM.communisfruitsandP.lentiscusleaveswere

sta-tisticallyindistinguishable(*p<0.05)fromeach other.Thesame

observationwasfoundfortheextractsofG.alypum flowers,M.

vulgareandE.maritimumleavesat(*p<0.05),buttheinhibitory

activitywasverylowcomparedwiththefirstplants.These

sim-ilaractivities mayimply thepresenceof approximatelysimilar

amountsoflipophilicantioxidantsinallthreespecies.Different

organsfromthesameplantweretested,aswasthecaseofG.

aly-pum,M.communisandE.maritimum.Theresultsobtainedindicated

thattheextractsobtainedfromtheleavespresentedhigher

inhibi-tionthanotherorganswhichleadstosaythatlipophilicantioxidant

weremoreimportantinthispart.Accordingtoourliteraturesearch,

there arelimited numbersofreports onS.maritima (bulb)and

E.maritimum(leavesandstem).Sincetheactivitiesofthesetwo

speciesevaluatedherearenotavailableintheliterature,data

pre-sentedherecouldbeassumedasthefirstreport.

Inthiswork,wefoundthatplantextractshavingpotentialto

preventor minimizethe rapid discoloration of the ␤-carotene

containhighphenolicamountsandcoefficientcorrelationbetween

thesecompoundsandtheactivitywasveryhigh(r=0.75).Some

authorshavealsofoundacorrelationbetweenthem(Veliogluetal.,

1998;Ozsoyetal.,2008).Thepresenceofaphenolicantioxidant

canhindertheextentof␤-carotenedestructionby“neutralizing”

thelinoleatefreeradical(i.e.utilizingitsredoxpotential)andany

otherfreeradicalsformedwithinthesystem.Further,interactions

may occurbetween theextract components. The synergism of

64.71a _63.66a 32.06b 22.07c 15.54d _14.41d 9.31e _9.23e 7.88e 3.75f 0 10 20 30 40 50 60 70 80 90 100

Catechin Quercetin Scilla maritima (bulb) Eryngium maritimum (leaves) Myrtus communis (fruits)

Globularia alypum (flowers) Eryngium maritimum (stem) Myrtus communis (leaves) Marrubium vulgare (leaves) Pistacia lentiscus (leaves) NO Rad ical scave n gi ng activity (% )

Fig.4.Nitricoxidescavengingactivityoftheplantextractswith0.5mg/ml.Datawereanalyzedusingone-wayANOVA(analysisofvariance).Differentletter(s)indicatethe

97.29a 43.02c 63.60b 47.61c 13.38e 3.35f 4.52f 12.01e 19.77d 11.73e 0 10 20 30 40 50 60 70 80 90 100 BHT Pistacia lentiscus leaves Myrtus communis leaves Myrtus communis fruits Eryngium maritimum leaves Eryngium maritimum stem Globularia alypum leaves Globularia alypum flowers Scilla maritima bulb Marrubium vulgare leaves

-ca

rotene bleaching inhibition (%)

Fig.5.␤-carotenebleachinginhibitionofthemethanolicplantextracts.Datawereanalyzedusingone-wayANOVA(analysisofvariance).Differentletter(s)indicatethe

valuesaresignificantlydifferent(*p<0.05).

15.13c,d,e 4.30e 2.77e 35.98b,c 5.44e 32.32b,c,d 49.78b 13.05c,d,e 9.55d,e 21.51c,d,e 97.99a 0 10 20 30 40 50 60 70 80 90 100

Metal chelating powe

r (%)

Fig.6.Metalchelatingpowerofthedifferentmedicinalplantextracts.Datawereanalyzedusingone-wayANOVA(analysisofvariance).Differentletter(s)indicatethevalues

aresignificantlydifferent(*p<0.05).

polyphenolics,withoneanotherand/orothercomponentspresent

inanextractmaycontributetotheoverallobservedantioxidant

activity.

3.4.4. Metalchelatingactivity

TheformationofFe2+_{–ferrozinecomplexisnotcompletedin}

thepresenceofplantextractsandstandards.At0.5mgml−1

con-centration,thepercentageofmetalchelatingcapacityoftheplants

rangedfrom2.77to49.78%(Fig.6).

Mostoftheplantextractsinterferedwiththeformationof

fer-rousandferrozinecomplex,suggestingthattheyhavechelating

activityandcancaptureferrousionformingamorestable

com-plexthanferrozine.Thehighestferrousion-chelatingpowerwas

observedinextractofE.maritimum(stemandleaves),followedby

theleavesofM.communis.Theseresultscouldbeattributedtotheir

total phenolics and proanthocyanidins content suggesting their

abilityasaperoxidationprotectorthatrelatestotheironbinding

capacity.Shyuetal.(2009)havefoundhighcorrelationbetweenthe

contentsofphenoliccompoundsandferrousionchelatingpower

(r2_>0.83).

TheabsorbancedecreasedlinearlyinthepresenceofM.vulgare

andS.maritimaextractsaswellasthestandards(rutinandtannic

acid),whichindicatedthattheformationofFe2+_{–ferrozinecomplex}

wasnotcompleted.Nosignificantdifference(*p<0.05)between

themforironchelation.ThechelatingactivityoftheleavesofM.

communisandE.maritimumaswellasitsstemwassignificantly

higher(*p<0.05)thanthatoftheofthestandardtannicacidand

rutinanddemonstrateamarkedcapacityforironbinding.

ExceptE.maritimum,inallspecieswereleavesandanotherpart

oftheplantwasinvestigated,theleavesshowedalwaysthe

high-estantioxidantactivityinthistestindicatingthatistheorganof

theplantwereantioxidantsaccumulated.Amongalltheextracts

screened in this study, G. alypum (leaves and flowers) and M.

communisfruitsdisplayedthelowestferrousion-chelatingpower

despitetheirphenoliccontentsandtheextractofP.lentiscuswas

totallyinactive.

4. Conclusion

From the present work, we can concludethat among plant

proanthocyanidinsweredetectedinM.communis(leaves),P.

lentis-cus(leaves)andG.alypum(flowers).Theamountsoftotalflavonoids

variedwidelyintheinvestigatedplantextractsandwerehigherin

M.communisandE.maritimumleaves.Thesefindingsmayconfirm

theinterestingpotentialoftheseplantsasavaluablesourceof

natu-ralbioactivemolecules.Percentagesofcyclooxygenase-1inhibition

showedthatMarrubiumvulgareleavesextracthadthebestandthe

potentactivitywhilethebestanti-acetylcholinesteraseactivitywas

exhibitedbytheextractofM.communis,P.lentiscusandE.

mariti-mumleaves.Inthe␤-carotenebleachingtest,M.communis(leaves

andfruits)andP.lentiscus(leaves)extractsdemonstratedthebest

inhibitionactivitywhilethehighestferrousion-chelatingpower

wasfoundwithE.maritimum(stemand leaves)andM.

commu-nisleaves.Thehighantioxidantactivityexhibitedbytheselected

plantsintheseassayssuggeststhattheyhaveapotentialforuse

infoodsparticularlythosecontainingemulsifiedoils.Almostall

thetested plantspresented morethan50%of radicalinhibition

andP.lentiscusandM.communishavepresentedthebest

activ-ity.TheNO•scavengingactivityshowedthatS.maritimapresented

thebestscavengingactivityandpresentedthehalfactivityofthe

purestandardcatechinandquercetin.Onthewhole,itis

interest-ingtonotethatthestudiedplantshave,infact,propertiesthatmay

suggestapplicationsinpharmaceuticalindustryandfood.

There-fore,theobtainedresultsareusefultofurtherresearchsuchasthe

identificationofspecificphenoliccompoundsresponsibleforthe

acetylcholinesteraseandcyclooxygenase-1activitiesandtoallow

tostudytheirstructure–functioninteractionswiththeseenzymes.

Acknowledgements

We wish to thank the Algerian Ministry of High Education

andScientificResearchforsponsoringthisworkandMr.

AMES-SISDjamel(Taghzouit,Seddouk,Bejaiacity)forprovidingplants

material.

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