Contents lists available atScienceDirect
Industrial
Crops
and
Products
j o u r n a l h o m e p 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
HPLC-DAD
profile
of
phenolic
compounds
and
antioxidant
activity
of
leaves
extract
of
Rhamnus
alaternus
L.
Kamal
Moussi
a,∗,
Balunkeswar
Nayak
b,
L.
Brian
Perkins
b,
Farid
Dahmoune
a,
Khodir
Madani
a,
Mohamed
Chibane
caLaboratoiredeBiomathématiques,Biophysique,Biochimie,etScientométrie,FacultédesSciencesdelaNatureetdelaVie,UniversitédeBejaia,06000
Bejaia,Algeria
bFoodScienceandHumanNutrition,SchoolofFoodandAgriculture,UniversityofMaine,Orono,ME04469,UnitedStates
cLaboratoiredeGestionetdeValorisationdesRessourcesNaturelles,AssuranceQualité,FacultédesSciencesdelaNatureetdelaVieetdesSciencesdela
Terre”,UniversitédeBouira,Algeria
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received8January2015
Receivedinrevisedform2June2015
Accepted4June2015
Keywords:
RhamnusalaternusL.
Phenoliccompounds
Freeradicalscavengingactivity
HPLC-DAD
a
b
s
t
r
a
c
t
ThisresearchinvestigatedthepolyphenolsprofilingandantioxidativepropertiesofRhamnusalaternusL. AcomparativeanalysisofthedefattedandelutedfractionsofmethanolextractfromleavesofR. alater-nusL.wasperformedonhigh-performanceliquidchromatographycoupledtodiodearraydetection.The totalphenolic,flavonoid,monomericanthocyanincontents,andantioxidantactivityofallthefractions oftheleavesextractwerequantified.Allfractionsshowedthepresenceofphenoliccompoundsand exhibiteddifferentlevelsoffreeradicalscavengingactivity.Sevenindividualphenoliccompoundsand anthraquinonesasagroupofphenoliccompoundsinthesampleswereidentified.Luteolin, quercetin-3-rhamnoside,p-coumaricacid,ferulicacid,gallicacid,andrutinwerethenewlyidentifiedcompounds intheextract,confirmedbythepresenceofkaempferolandanthraquinones.Knowledgeofthese com-poundsintheleavesextractwillhelpinformulatingpharmaceuticalproductsforvariousdiseases.
©2015ElsevierB.V.Allrightsreserved.
1. Introduction
Ethnomedicinehasbeenpracticed sinceprehistory by virtu-allyallhumanculturesaroundtheglobe,includingEurope,Asia, Americas,andAfrica(Slikkerveer,2006).NorthAfricahasoneof theoldestandrichesttraditionsassociatedwiththeuseof medic-inalplants (Batanouny etal.,2005),where Herodotus(430B.C) wrotethatmedicineinEgyptispracticedamongthemonaplan ofseparation,byusinganinfinitenumberofdrugsfrom medici-nalplants(RawlinsonandBlakeney,1910).Farther,NorthAfricais knownforitsillustrioussavantkingJubaIIofLibya(Mauritania), whodiedabouttheyear20AD,leavingatleastadozenscientific works.OneofhismostesteemedtreatiesisthatwhichPlinyand Galenattributedthetitle“Deherbaeuphorbia.¨,itisthevarietyof
∗ Corresponding author at: Laboratoire de Biomathématique, Biophysique,
Biochimie, et Scientométrie. Faculté desSciences de la Nature et de la Vie,
UniversitéAbderrahmane Mira,RouteTargaOuzemour,06000Bejaia.Algerie.
Fax:+21334214762.
E-mailaddresses:moussikamal2012@yahoo.fr(K.Moussi),
balunkeswar.nayak@maine.edu(B.Nayak),BPerkins@Maine.edu(L.B.Perkins),
farid.dahmoune@univ-bejaia.dz(F.Dahmoune),khodir.madani@univ-bejaia.dz
(K.Madani),chibane18156@yahoo.fr(M.Chibane).
medicinalplant,towhichhediscoveredthemedicinalpotential (Eugène-Humbert,1952).
InNorthAfrica,thereareabout10,000vascularplantspeciesand about70%foundinthewildhavemedicinal,aromatic,andother uses.InAlgeria,anestimatednumberof3164identifiedspecies arequalifiedastraditionalmedicinalplants(VasishtandKumar, 2004).Amongtheidentifiedplantspeciesontheearth,onlyasmall percentagehasbeenphytochemically investigatedandthe frac-tionsubmittedtobiologicalorpharmacologicalscreeningiseven smaller.Moreover,aplantextractmaycontainseveralthousand differentnaturalproductsandanyphytochemicalinvestigationof agivenplantwillrevealonlyanarrowspectrumofitsconstituents (Hostettmannetal.,2000).
Plantsrepresentarichsourceofnaturalproducts,withalmost infinitemoleculardiversity(HostettmannandMarston,2002),of whichactiveingredientsofmedicinalplantsaremostlysecondary metabolites(Zengetal.,2013).Thephenoliccompoundsarelarge andheterogeneousgroupsofthesesecondarymetabolites,thatare distributedthroughouttheplantkingdom(Maestrietal.,2006). Phenoliccompoundsareamongstthemostdesirable phytochem-icalsduetoantimicrobial,antiviral,anti-inflammatoryproperties, andhighantioxidantcapacities(Ignatetal.,2011).Antioxidants aredefinedascompoundsthatcandelay,inhibit,orpreventthe http://dx.doi.org/10.1016/j.indcrop.2015.06.015
oxidationofoxidizablematerialsbyscavengingfreeradicals. Phe-nolicshavebeenconsideredpowerfulantioxidantsinvitro(Daiand Mumper,2010).Manyeffortshavebeenmadetoprovideahighly sensitiveandselectiveanalyticalmethodforthedeterminationand characterizationofphenolic compounds.It isveryimportantto understandtheprofileofphenoliccompoundsinmedicinalplants byusinghighlyeffectiveextractionsandanalyticalmethods(Ignat etal.,2011).
RhamnusalaternusL.isafleshy-fruited,shrubofthe Mediter-ranean region (Ben Ammar et al., 2005). One of the species commonlyusedinreforestationprograms,due toitsfruit char-acteristics and ability to survive in xeric environments, which representsanimportantwaterandnutrientsourceforbirdsand smallmammals(Guliasetal.,2004).Ithastraditionallybeenused aslaxative;purgative;hypotensive;anditisalsousedfortreatment ofthedermatological,ocular,burning,odontological,hepatic(Ben Ammaretal.,2008;BenAmmaretal.,2005),psychological, depres-sion,andgoiterproblems(MatianddeBoer,2011).Thewoodhas beenalsousedascosmeticforsplithair(Lardos,2006)andasa dyeingagentfor wool(Guarrera,2006).Inaddition,ithasbeen reportedthattheleavesextractsofR.alaternusshowedthehighest antimutageniclevelinabacterialassaysystem,highxanthine oxi-daseinhibition(BenAmmaretal.,2008)andinhibitionofbothrat intestineandpurifiedporcinelivercarboxylesterase(Stockeretal., 2004).
Asaprominentlocalmedicinalplant,R.alaternusL.hasbeen widelydistributed intheNorthernAlgeria. Theleavesareused locallytoinKabyliabydecoctionaspurgativeandlaxative,andalso usedfreshfortreatmentofjaundice.Thepurposeofthisstudywas (i)defattingandfractionationofmethanolextractfromleavesofR. alaternusL.(ii)toinvestigateprofilingofthephenoliccompounds includingphenolicacids,flavonoids,andanthocyaninsby HPLC-DAD, and (iii) assessing total phenolic compounds, flavonoids, monomericanthocyaninsusingcolorimetricmethods,and antiox-idantactivityusingaradicalscavengingassayofallfractionsof defattingandfractionationstepsofmethanolleavesextract.
2. Materialsandmethods
2.1. Chemicals
Acetonitrile, hexane, acetic acid, sodium carbonate, Folin–Ciocalteau reagents,aluminium trichloride (AlCl3), potas-siumchloride,andsodiumacetatetrihydratewerepurchasedfrom FisherScientific(FairLawn,NJ,USA).EthylacetateandDPPH (2,2-diphenyl-1-picrylhydrazyl) were obtained from Sigma–Aldrich (St.Louis,MO,USA).Thephenolicstandards,i.e.,catechin,caffeic acid,quercetin-3-rhamnoside,p-coumaricacid,gallicacid,ferrulic acid,luteolin,apegenin,vanillicacid,and(–)epigallocatechinwere purchasedfromSigma–Aldrich(St,Louis,MO,USA).Chlorogenic acidandrutinwerepurchasedfromAcrosorganics(ThermoFisher Scientific,FairLawn,NJ,USA)andnaringeninandkaempferolfrom Sigma–Aldrich(Gillingham,Dorset,UnitedKingdom).Allsolvents usedforthisworkwereofHPLCgrade.
2.2. Preparationofmethanolextract
R. alaternus L. leaves were collected in March 2013, from Hengued,Adekar,North-WestpartofBejaiainKabylia (Algeria) (latitude:36◦4315.46Nand longitude: 4◦3454.15E) and kept fordryingundera forcedairovenat60◦Cuptomoisture con-tentofabout3.25%(determinedbydryweightinovenat105◦C untilconstantweight)(Spignoetal.,2007).Thedriedmaterialwas crushedtopreparepowder,whichwasmilledthrougha1mmsieve (finalpowder size<1mm).Fiftygrams of powderwere
macer-atedwith500mLofmethanolfor72hatroomtemperature.After filtrationthroughafilterpaper,methanolwasentirelyremoved usingarotaryevaporator(Buchi,Flawii,Switzerland)at45◦C(at 337mbar).Thedryextractwasweighedandthenre-dissolvedin methanoltoobtainasolution,withknownconcentrationwhich wasdesignatedas crude extract(CE).The extractivevalue was calculatedondryweightbasisfromtheformulagivenbelow: %extractivevalue(yield%)= WeighttakenforextractionWeightofdryextract ×100
2.3. Defattingandfractionationofextract
Allthestepsofextraction,defatting,andfractionationare sum-marizedinFig.1.Fiftymillilitersofcrudeextractweresubjectedto entireevaporationofthemethanolonarotaryevaporator(Buchi, Flawii,Switzerland)at45◦C(at337mbar).Theresultingdryextract wasdissolvedin90mLofUltrapurewater(Milli-Q)toobtain aque-oussolutionandwasdefattedseveraltimes,withthesamevolume ofhexane,untila clearsolutionofhexaneresulted.Thehexane inthesolutionwasevaporatedusingarotaryevaporatortoget dryextractat45◦C(at335mbar).Thedryextractwasre-dissolved in40mLofmethanolanddesignatedashexanefraction(HF).The insolublefractionformedbetweenhexaneandaqueoussolution waswashed severaltime withultrapurewater (Milli-Q),dried, dissolvedin25mLofmethanolandisdesignatedasintermediate fraction(INT).
C-18Sep-PakVac35-cc(Waters,Milford,MA,USA)columnwas sequentiallyrinsedwith50mLofethylacetate,acidifiedmethanol (0.01%v/vHCl),andacidifiedwater(0.01%v/vHCl),andcharged separatelywith20mLofdefattedaqueoussolutionofextract.The columnwasthenwashedwith60mLofacidifiedUltrapurewater (Milli-Q)(AW),toremoveanyorganicsugarsandacidsremaining inthesolution(fractionAW),followedbyelutionwith60mLof ethylacetateandwasdesignatedasfractionEA.Thefinalelution wasmadewith60mLofacidifiedmethanolandwasdesignated asfractionAM(Lacombeetal.,2012).Theethylacetatefraction EAcontainedasmallamountofwater,resultingfromtheprevious elutionwithacidifiedwater,whichledtotheseparationofethyl acetatesolutiondesignatedasEA1andwatersolutiondesignedas EA2.
ThesolventsofthefractionsHF,EA1,AMwereremoved com-pletelyonarotaryevaporator(Buchi,Flawii,Switzerland)at45◦C (at240mbar).FortheaqueoussolutionsAWandEA2,thesolvents wereremovedbylyophilization(VirTis,NewYork,USA).Alldry extractsHF,EA1,AM,AW,EA2,andINTfractionsweredissolvedin HPLCgrademethanolandweresubjectedtoHPLCanalysis, phyto-chemicaldosages,andantioxidantactivity.
2.4. Determinationoftotalphenoliccompounds
The total phenolic content of the standards, crude extract (2mg/mL)andallfractions(INT,HF,AW,EA1,EA2,andAM) with-outdilutionweredeterminedbyFolin–Ciocalteu’sphenolmethod followingtheproceduresofVeliogluetal.(1998).Briefly,20L of standard or samples were mixed separately with 150L of Folin–Ciocalteu reagents(previously diluted 1:10 withdistilled water)andallowedtostandat22◦Cfor5min.Afterincubation, 150Lofsodiumbicarbonate(6g/100mL)wereaddedandafter 90minat22◦C,theabsorbancewasread ontheBMGLABTECH FLUOstarOmegaplatereader(Germany)at725nmagainstablank. Theexperimentwascarriedoutintriplicateandtheconcentration oftotalphenoliccompoundsintheextractwereexpressedinmg gallicacidequivalent(GAE)pergramplantpowder(PPW)i.e.,(mg GAE/gPPW).
Washed with hexane several times 50 ml of crude extract(50 mg/mL)
Entire evaporation of methanol
Dry extract
Dissolved in 90 ml of Ultrapure water
Aqueous solution(AQ)(90 ml)
Insoluble material Hexane solution 20 ml of aqueoussolution(AQ)
Washed several times with ultrapurewater
Fraction INT (25 ml)
Evaporation of hexane
Fractionation through C-18 Sep-Pak Vac 35cc
Ethyl acetate solution + water
Acidified methanol solution Acidified
water solution
Ethyl acetate phase
Water phase
Evaporationof
ethyl acetate lyophilization
Dry material Dry material Dry material
Dry material
Dry material Dry material
lyophilization
Evaporation of methanol Re-suspended in methanol Fraction HF (40 ml) Fraction AW (10 ml)
Fraction EA1 (10 ml)
Fraction EA2 (10 ml)
Fraction AM (10 ml)
HPLC and phytochemical analysis
Fig.1. SchematicdiagramoffractionationofextractofRhamnusalaternusL.leaves.
2.5. Determinationoftotalflavonoid
The total flavonoid content was determined using the alu-miniumtrichloridemethod(Adedapoetal.,2008).Briefly,150Lof 2%aluminiumtrichloride(AlCl3)inmethanolweremixedwiththe samevolumeofastandardorcrudeextract(2mg/mL)orfractions (INT,HF,AW,EA1,EA2,andAM).Absorptionreadingsat430nm weretakenafter10minagainsta blankusinga BMGLABTECH FLUOstar Omega platereader. The experiment was carried out intriplicateand thetotal flavonoidcontentswereexpressedas milligramquercetin-3-rhamnosideequivalent(QE)pergramplant powder(PPW)i.e.,(mgQE/gPPW).
2.6. Determinationoftotalmonomericanthocyanin
Thetotal monomericanthoyanincontentwasdeterminedby thepHdifferentialmethodwithpH1.0buffer(potassiumchloride, 0.025M)andpH4.5buffer(sodiumacetate,0.4M)(Lee,2005).The crudeextractandallfractions(INT,HF,AW,EA1,EA2,andAM)were dilutedwithpH1.0andpH4.5buffers.350Lofbufferedsamples atpH1andpH4.5wereaddedseparatelytoappropriate
micro-platewellsand readingswererecorded, usingaBMG LABTECH FLUOstarOmegaplatereader(Germany).Theabsorbancewasthen measuredat520and700nm.Theexperimentwascarriedoutin triplicateandtheconcentrationofanthocyaninwascalculatedas follows:
Totalmonomericanthocyanin(cyd−3−glu,mg/L) =x=A×MW
×DF×103
×l
whereA=(A520nm–A700nm)pH1.0–(A520nm–A700nm)pH4.5;MW (molecularweight)=449.2g/molforcyanidin-3-glucoside (cyd-3-glu);DF=dilutionfactor establishedinD;l=pathlengthincm;
=26900molarextinctioncoefficient(L×mol−1×cm−1)for cyd-3-glu;and103=factorforconversionfromgtomg.2.7. Determinationofantioxidantactivity
DPPHisastableradicalwithadeeppurplecolor.Reactionwith otherradicals,electrons,orhydrogenatomsleadstolossofcolor at515nmand lossoftheEPRfreeradicalsignal(Schaichetal., 2015).Theantioxidantactivityofcrudeextract andfractionsof
Table1
Totalphenolics,totalflavonoids,totalmonomericanthocyaninscontents,andantioxidantactivityofRhamnuslaternusL.leavesextractandfractions.
Fraction Totalphenolic contents (gGAE/mL) Totalflavonoids (gQE/mL) Totalmonomeric anthocyanins (mgc3g/L) Antioxidantactivity (%)DPPHinhibition) Crudeextract CE 155.60±0.77a2 60.22±5.76a3 0.0±0.0 66.83±3.88a3 Fractionsafter defatting INT 165.51±7.87a1 156.39±9.02a1 0.0±0.0 90.36±0.45a2 HF 245.53±11.58a2 163.29±12.55a1 0.0±0.0 80.14±0.52a1
FractionsthroughC–18 EA1 317.47±0.65b2 103.18±5.96b2 0.0±0.0 90.81±2.46b1
EA2 78.91±7.72b4 13.53±0.57b3 0.0±0.0 25.93±5.41b2
AM 712.58±4.35b1 705.30±2.04b1 0.0±0.0 86.04±0.09b1
AW 204.88±4.14b3 0.00±0.00b4 0.0±0.0 12.60±1.24b3
CE:crudeextract;INT:intermediatefraction;HF:hexanefraction;EA1:ethylacetatefraction1;EA2:ethylacetatefraction2(containingwater);AW:aqueousfraction (containingorganicsugars);AM:acidifiedmethanolfraction(seeFig.1fordetailedseparationmethod);GAE:gallicacidequivalent;QE:quercitinequivalent;C3G:
cyanindin-3-glucoside.a,barenotationsforcomparisonbetweensamplesand1-4arenotationsforthelevelofstatisticaldifferences.
theextractwasdeterminedbymeasuringtheDPPHfreeradical scavengingactivity(Dudonneetal.,2009).TheDPPH•solutionin methanol(6×10−5M)wasprepared,and300Lofthissolution wasmixedseparatelywith10Lofcrudeextractandallfractions (INT,HF,AW,EA1,EA2,andAM)inflatbottomwells,byusingBMG LABTECHFLUOstarOmegaplatereader(Germany)setat37◦C.After 20minofincubation,adecreaseintheabsorbanceat517nmwas measured(Asample).Acontrolcontaining10Lofmethanolinthe DPPH• solutionwaspreparedanditsabsorbancewasmeasured (Acontrol).DPPHisafreeradicalthatproducesablue–violet solu-tioninmethanol.Inthepresenceofantioxidantcompounds,DPPH radicalisreduced,producinganon-colormethanolsolution.The experimentwascarriedoutintriplicate.Radicalscavengingactivity wascalculatedusingthefollowingformula:
%inhibition= AbscontrolAbs−Abssample
control ×
100
whereAbscontrolwastheabsorbanceofcontrolandAbssamplewas theabsorbanceofsample.
2.8. HPLC-DADanalysis
Theanalysisforphenoliccompoundswasperformedonthe Agi-lent 1200seriesrapidresolution liquid chromatograph(Agilent Technologies,SantaClara,CA)consistingofavacuumdegasser,an auto-sampler,abinarypumpanddiode-arraydetection(DAD) sys-tem.DataanalysiswasperformedwithAgilentHPLCChem-Station software. This instrument was equipped with a Phenomenex Prodigy5ODS-2column(4.60mm×250mm,5micron,CA,USA). Sampleswere centrifugedat2655×g for10minprior toHPLC injection.Simultaneous monitoringwasperformedfor determi-nationat254,280,300,520,and640nm;andspectraldatawere collectedfrom200to700nm.Thecolumntemperaturewasset at30◦C,andtheinjectionvolumewas20L,withaflowrateof 0.5mL/min.Acidifiedwater(6%aceticacid,v/v)and acetonitrile wereusedasmobilephasesAandB,respectively(Chirinosetal., 2008).Thegradientwasprogrammedasfollows:0–40min,0–25% B;40–80min,25–85%B;80–90min,85–100%B;90–95min,100%B. Allidentifiedphenoliccompounds(phenolicacidsandflavonoids) werequantifiedwithexternalstandards,usingcalibrationcurves. Thestandardresponsecurvewasalinearregressionfittedtovalues obtainedateachconcentrationwithintherangeof12.5–200g/mL forphenolicacidsand20–350g/mLforflavonoids.
2.9. Statisticalanalysis
Theexperimentalresultswereexpressedasmeans±standard deviation for triplicate measurements. Correlation analysis of antioxidantactivity(Y)versusthetotalphenoliccontent(X)and flavonoidcontent(X)wascarriedoutusingthecorrelation
pro-graminMicrosoftExcelsoftware.Differencesbetweenfractions weredeterminedbyANOVAprocedures,followedbytheTukey’s honestly significantdifference(HSD)at P<0.05 usingstatistical software(SASVersion7.0,Trialversion8.0.7.1).
3. Resultsanddiscussion
3.1. Quantificationofphenoliccompounds
Solublephenoliccompoundscanbeisolatedeasilyfromplant tissue by extraction into methanol (Vermerris and Nicholson, 2007),whichisthemostsuitablesolventduetoitsabilitytoinhibit the action of polyphenol oxidase, that causes the oxidation of polyphenols(LimandQuah,2007).Themethanolextractivevalue (yield)fromleavesofR.alaternusL.was22%ofplantpowder(PPW). Thetotalphenolicandflavonoidcontentsofcrudeextract(CE)were 155.60±0.77gGAE/mLand60.22±5.76gQE/mL,respectively (Table1).Consideringtheextractionyield,theresultsrepresent 17.13±0.09mgGAE/gPPWand6.63±0.63mgQE/gPPW, respec-tively.
Determinationofthetotalmonomericanthocyanincontentsby thepHdifferentialmethodwasbasedontheanthocyaninstructural transformation,thatoccurswithachangeinpH(coloredatpH1.0 andcolorlessatpH4.5)(Lee, 2005).Theanalysisofmonomeric anthocyaninshowednochangeincolororabsorbancebetweenpH 1.0andpH4.5,whichindicatestheabsenceofmonomeric antho-cyaninincrudeextract(CE)ofR.alaternusL.
Plantcrude extractsusuallycontainlargeamountsof carbo-hydratesand/orlipoidalmaterial.Theconcentrationofphenolics in the crude extract may be low. To concentrate and obtain polyphenol-rich fractions before analysis, strategies including sequential extraction or liquid–liquid partitioning and/or solid phase extraction,basedonpolarityandacidityhavebeen com-monlyused. In general,elimination of lipoidal material can be achievedbywashingthecrude extractwithnon-polarsolvents, suchashexane,dichloromethane,orchloroform(DaiandMumper, 2010).However, in thepresent study, defatting step gave rise to three phases, HF,INT, and AQ (aqueous solution). The non-phenolic compounds were eliminated, but also the phenolic compoundswerefoundinHFandINTfractions,withthe quanti-tiesof245.53±11.58and165.51±7.87GAEg/mL,respectively (Table1).Consideringthedilution factor,theseamounts repre-sent3.44±0.16and1.45±0.07%oftotalphenoliccontentincrude extract(CE),respectively.Theflavonoidcontentwas66.51±5.11 and94.49±5.45%oftotalphenoliccontentinHFandINT, respec-tively.
ReversephaseC18cartridgeshavebeenmostwidelyusedin phenolic compounds separation (Nayak et al., 2011). After the aqueous sample was passed through preconditioned C18 car-tridges,thecartridgeswerewashedwithacidifiedwatertoremove
Fig.2. Linearcorrelationoffreeradicalscavengingactivity(Y)intermsof%DPPHinhibitionversusthetotalphenoliccompounds(TPC)(X)andversusflavonoidcontent
(X)inascendingorderconcentrations(a)and(b)forcrudeextract(CE),intermediatefraction(INT)andhexanefraction(HF),(c)and(d)forelutedfractionsthroughreverse
phaseC-18Sep-Pakcolumn.
sugar, organicacids, and other water-solubleconstituents (Dai andMumper,2010;Lacombeetal.,2012).Thepolyphenolswere thenelutedwithabsolutemethanoloraqueousacetone(Daiand Mumper,2010).Monomericphenolicacidswerethenelutedwith ethylacetatefollowedbyelutionofanthocyaninsand proantho-cyanidinswithacidifiedmethanol(Lacombeetal.,2012).
Theelutionofdefattedsolution(aqueoussolution(AQ))by C-18 Sep-Pakshowedthepresence of phenolic compoundsin all fractionsofEA1, EA2,AM,andAW.Thetotal phenolic contents of24.16±0.04, 6.01±0.59,54.24±0.33 and 15.59±0.22%were foundin EA1, EA2, AM, and AW fractions, respectively, with a highamountofelutioninAMfraction,whichisconstitutedwith 98.98±0.29%offlavonoidoftotalphenoliccontentinAMfraction. Itwasobservedthatasignificantamountofphenoliccompounds werelostduringwashingoftheC18columnwithacidifiedwater. Theflavonoidswerepresentinallelutedfractionsofextractexcept inAW.Theresultsconfirmedtheabsenceofanthocyanininall frac-tions,eveninthefractionthatelutedwithacidifiedmethanol(AM) (Table1).
3.2. Antioxidantactivity
TheantioxidantactivityusingscavengingofDPPHfreeradicals wascorrelatedwiththetotalphenolic,flavonoidcontentsofcrude extractandallfractions(INT,HF,AW,EA1,EA2,andAM).Thefree radicalscavengingactivityshowedthattheINTfractionhadthe higheractivity,witharateof90.36±0.45%,followedbyHFwith 80.14±0.52%andCEwith66.83±3.88%.Fortheelutedfractions derivedfromtheaqueoussolution(AQ),thehigheractivityoffree radicalscavengingwasobservedinEA1 fraction(90.81±2.46%) followed by AM (86.04±0.09%), EA2 (25.93±5.41%) and AW
(12.60±1.24%)fractiondespitethehigherquantitiesoftotal phe-nolicandflavonoidcontentsinAMfractioncomparedwithEA1 (Table1).Correlationanalysis(Fig.2)of freeradicalscavenging activityshoweddifferentlevelsoflinearpositivecorrelationwith totalphenolicandflavonoidcontentsinallfractions.Furthermore, thecorrelationresultsoftheantioxidantactivity(Y)versustotal phenolic(X)andflavonoid(X)incrudeextract(CE),INTandHF fractions(Fig.2aandb)suggestthatonly17.52%ofthe antioxi-dantcapacitydependonthetotalphenoliccontents.However,for flavonoid,theantioxidantactivitydependencewas87.36%,with partialaccordance tothestatisticallysignificantdifferencelevel (Table1).Theantioxidantactivityoftheelutedfractionsderived fromtheaqueoussolution(AQ)showedadependenceonthetotal phenoliccontent(Fig.2c)andflavonoid(Fig.2d),witharateof 72.43%and62.97%,respectively,whichwasinpartialaccordance withthestatisticallysignificantdifferenceleveloftotalphenolic contentandwithnoaccordanceforflavonoidcontent.
Inpreviousstudies,ithasbeenreportedthattheantioxidant activitiesofmanyplantextractswereproportionaltotheir phe-noliccontent,suggestingacausativerelationshipbetweenthem (Nayak etal., 2013), which ismainly due totheredox proper-tiesofphenoliccompounds,whichallowthemtoactasreducing agents,hydrogendonors,andsingletoxygenquenchers.Theymay alsohaveametalchelatingpotential(Javanmardietal.,2003).The antioxidantactivityisstronglydependentonthemodel system inwhichitisevaluated,andasingleanalyticalassaytomeasure theantioxidantactivitymaybeinadequate.Thepolarityofboth theextractingandthereactingmediumduringtheassaysis usu-allydeterminedfortheprotectionagainstoxidation(Moureetal., 2001).Forthis,theevaluationofantioxidantsmustbefollowed byassayswithothersystems,aslipidsinmicellarsystems,bulk
Table2
Identificationofindividualphenoliccompoundsandanthraquinonesasagroupofphenoliccompoundsat280nmusingHPLC-DADsystem.
Nameofphenolics Compoundnotations Retentiontimes Maximumabsorbance(200–700nm)
Rutin 1 38.03 256,355 Antraquinones 2 a a Quercetin-3-rhamnoside 3 54.55 231,255,371 Kaempferol 4 59.34 232,265,336 Gallicacid 5 8.73 232,270 p-Coumaricacid 6 36.20 214,234,312 Ferulicacid 7 39.52 220,226,238,323 Luteolin 8 54.34 230,253,347
a17peaksofanthraquinoneswithadifferentretentiontimesandabsorbance.Identificationofspectraabsorbanceofanthraquinonesbycomparingwithbibliography
(Markovi ´cetal.,2008).
oils,foods,etc.,becausethemorepotentprotondonorisnot nec-essarily thebestantioxidantin differentsystems(Franco etal., 2008).However,phenoliccompoundsdonotcontributeto100%of antioxidantactivityinplantextracts.Somecontributionmayalso comefromthepresenceofotherantioxidantsecondary metabo-lites,suchasvolatileoils,carotenoids,andvitamins(Javanmardi etal.,2003).Moreover,ithasbeenreportedthatthecontentof chlorophyllscontributedtotheantioxidantcapacityandstudies haveshownthatchlorophyllsactaselectrondonors,reducingfree radicalssuchasDPPH(Fernandez-Orozcoetal.,2011).Thestudy concludedthatthesolubilityofphenoliccompoundsderivedfrom defattingandfractionationintheevaluatedantioxidantsystem, qualitativeeffectofphenoliccompoundsindifferentfractions,the presenceofchlorophyllinCE,INT,andHFandtheeventual pres-enceofothermetabolitesmayberesponsiblefortheantioxidant capacity.
3.3. DeterminationofphenoliccompoundsHPLCanalysis
TheHPLC-DADmethoddevelopedforthis studyachievedan appropriate separation among the fourteen standards and the methodvalidationdataobtainedforthesevenindividualidentified phenoliccompoundsandanthraquinonesasagroupofphenolic compoundsat280nmisshowninTable2.HPLCprofileofall frac-tionsis summarizedinTable3andchromatogramsrecordedat 280nmfor allfractionsareshown inFig.3.Thepeaksof inter-estwere identified by theirretention time and UV–vis spectra insamplesandstandards.TheHPLCprofileofallfractionsofthe resultingsolutionsofthedefatting(INTandHF)andfractionation oftheretainedcompoundsofaqueoussolution(AQ)bytheC-18 Sep-Pak(EA1,EA2,AW,AM)showedseveralpeakscorresponding todifferentphenolic compoundswithquantitativeand qualita-tivedifferenceofidentifiedcompounds.Presenceofhighamounts oftotalphenolicandflavonoidcontentsinAMfraction(Table1) were confirmed by HPLC analysis with reference to the peaks recorded(36–53min), withveryhighabsorbance(Fig.3F),with characteristicspectraabsorbanceofflavonoids,withclearspectra forpeakswithretentiontimesof36.885,43.458,and52.638min (Supplement Fig.1a). For the qualitative, theidentified pheno-liccompoundswererutin(HFandEA2),quercetin-3-rhamnoside (HF,INT,and EA1,)kaempferol (HF,INT,EA1,EA2, and AM), p-coumaricacid(EA1),ferulicacid(EA1),gallicacid(AW),luteolin (AM),antraquinones(HF,INT,EA1,EA2,andAM)andintheend chlorophyll(HFandINT)asnophenoliccompounds.
Anthocyaninsareresponsibleforthered,purple,andbluehues presentinfruit,vegetables,andgrains;have typicalabsorbance inthevisibleband400and600,withamaximumaround520nm (Lee,2005).Inthepresentstudy,theresultsofcolorimetricmethod fortotalmonomericanthocyaninwereconfirmedbyrecordingthe chromatogramsofallfractionsat520nm,whicharecharacterized bytheabsenceofpeaks.
Anthraquinone derivatives are a group of phenolic com-pounds, present in plants, usually in theform of oxidised 1,8-dihydroxyanthronewitha specificsubstitutionpattern(Kosalec etal.,2013).Emodin isabiologicallyactive,naturally-occurring anthraquinonederivativefromplant(Suchetaetal.,2011),presents spectraabsorbanceinmethanolbetween200and580nm,with absorption maxima (Amax) located at 203.10, 221.72, 253.59, 263.85, 289.43, and 436.83nm (Markovi ´c et al., 2008). In the presentstudy,17peakswithdifferentretentiontimesand absorp-tionmaximaweredetected(Table3)andconstitute6.18%ofthe areaoftotalfractions,withtypicalspectraofanthraquinones,for example,thepeakwithretentiontimesat75.88min,which repre-sents20.98%oftheareaofHFfraction,presentsspectraabsorbance between200and510nm,withabsorptionmaxima(Amax)located at204,230,253,266,288,and440nm(SupplementFig.1.b).
Chloroplasticpigmentsarelipophiliccompounds( Fernandez-Orozcoetal.,2011)andchlorophyllsasnophenoliccompound(in allknownforms)havetwomajorabsorptionbandsinthevisible range,“red”and“blue”andabsorptionmaxima(Amax)for Chloro-phyll“a”and“b”inacetonearelocatedat662.10and645.50nm, respectively(Zvezdanovi ´cetal.,2009).Inthiscontext,theINTand lipophilicHFfractionspresentthepeaksatdifferentretentiontimes (between88and92min)withtypicalspectraofchlorophyll (Sup-plementFig.1.c).
Polyphenols are hydrophilic (Fernandez-Orozco et al., 2011) andinordertoeliminatethelipophiliccompoundssuchaslipids, carotenoids,andchlorophyllfromtheplantextract,hexanewas used(DaiandMumper,2010).In contrast,theresultsof colori-metric method for total phenolic and flavonoid contents were confirmedinHPLCprofileofINTand HFfractions,bythe pres-enceofpeakswithspectracharacteristicofphenoliccompounds. Indeed,substantialquantitativelosseswererecordedupon defat-ting.60.88%ofanthraquinones,53.47%ofkaempferoland49.43% ofquercetin-3-rhamnosidewerelost,whichrepresentsa distribu-tionrespectivelyinINTandHFwith46.93and13.96%;21.46and 32.01%;12.43and37.00%.Forfractionsderivedfromtheaqueous solution(AQ)(C-18Sep-Pakelutedfractions),thesecompounds arepresentrespectivelyinEA1,EA2,andAMwith32.94,4.43,and 1.75%foranthraquinones,33.13,7.62,and5.79%forkaempferol and50.57%onlyinEA1forquercetin-3-rhamnoside.Unlikethese results,thefractionsderivedfromtheaqueoussolution(AQ),100% ofrutin(withtracesinHF)wasfoundinEA2;100%ofluteolinin AM;100%ofp-coumaricacidandferulicacidpresentinEA1and 100%ofqualitativelossesofgallicacidwasrecordeduponwashing (AW).
Around 12.85% of the total areaof total peaks detectedby HPLC-DADat280nmofidentifiedcompoundsforallfractionsis constitutedby48.06% ofanthraquinones,26.12% ofkaempferol, 9.46%ofluteolin,7.97%ofquercetin-3-rhamnoside,5.53%of gal-licacid,1.26%ofp-coumaricacid, 0.99%offerulicacid,0.34%of rutinand0.27%ofchlorophyll.
K. Moussi et al. / Industrial Crops and Products 74 (2015) 858–866 Table3
Quantificationofidentifiedindividualphenoliccompoundsandanthraquinonesasagroupofphenoliccompoundsat280nmusingHPLC-DADsystem.
Fractions280nm TotalareamAUas Identifiedcompounds Time(min) Area(mAUas) Area(%)infraction Quantity(g/gPPW)
HF 59858.500 Rutin 38.80 90.30 0.15 UQa Antraquinonesa 53.7555.5158.00 58.6065.3866.60 75.8886.7293.33 68.80 557.90104.80 574.90561.90 879.4012,559.90 3539.50207.40 0.12 0.93 0.18 0.96 0.94 1.4720.985.91 0.35 – Quercetin-3-rhamnoside 54.41 780.00 1.30 43.35 Kaempferola 59.45 3490.20 5.83 – INT 103010.000 Quercetin-3-rhamnoside 54.55 3993.30 3.88 272.63 Antraquinonesa 58.72 75.66 3152.10 6598.30 3.06 6.41 – Kaempferola 59.45 8960.60 8.70 – Obtainedfractionsby C-18Sep-PakVac fractionation AW 13594.100 Gallicacid 8.66 6973.10 51.30 470.50 p-coumaricacid 36.48 1391.80 1.82 19.79 Ferulicacid 39.78 1097.80 1.44 28.45 Quercetin-3-rhamnoside 54.49 4052.20 5.30 276.84 EA1 7,650,000.000 Antraquinonesa 55.54 58.72 61.74 75.85 3659.1 9404.60 1510.80 2510.00 4.79 12.30 1.98 3.28 – Kaempferola 59.46 6883.60 9.00 Rutin 38.83 283.30 4.71 24.06 AE2 6016.595 Antraquinonea 58.59 181.00 3.01 – Kaempferola 59.40 124.70 2.07 – AM 602701.000 Antraquinonea 53.82 7157.00 1.19 – Luteolina 54.29 10480.10 1.74 –
Fig.3.Identificationofselectedindividualphenoliccompoundsandanthraquinonesasagroupofphenoliccompoundsindifferentfractionsofextracti.e.(A)HF,(B)INT,
(D)EA1,(E)EA2,and(F)AMfromleavesofR.alaternusL.Thephenoliccompoundswereidentifiedat280nmusingaHPLC-DADsystem.Phenoliccompoundsidentifiedwere
Rutin(1),antraquinone(2),quercetin-3-rhamnoside(3),kaempferol(4),gallicacid(5),p-coumaricacid(6).ferulicacid(7),andluteolin(8).Phenoliccompoundsofother
peakswereeitherunknown.
At present, the composition of phenolic compounds in R. alaternus L. is still limited. From the previous studies, it is known that the extracts from R. alaternus L. are rich in compounds as anthraquinones and flavone heterosides (Stocker et al., 2004). In addition, four anthraquinine agly-cones (emodin, chrysophanol, alaternin, and physcion) were isolated (Ben Ammar et al., 2008; Izhaki et al., 2002) from the above-ground parts of the plant, with emodin being the most abundant of these (Izhaki et al., 2002). Moreover, the leaves extracts showed the presence of various quantities of anthraquinones;coumarins;tannins;andflavonoids(BenAmmar etal.,2005), askaempferol3-O-isorhamninoside, rhamnocitrin-3-O-isorhamninoside, rhamnetin-3-O-isorhamninoside, apigenin (BenAmmaretal.,2009),kaempferolandquercetin(BenAmmar etal.,2008,2009).Incomparison,thepresenceofanthraquinones
andkaempferolwasconfirmed;apigeninwasabsent;andluteolin, quercetin-3-rhamnoside,p-coumaricacid,ferulicacid,gallicacid, andrutinarethenewlyidentifiedcompoundsinR.alaternusL.The previousstudiesshows,thattheanthraquinonederivativespresent inplantsexhibitlaxativeeffects(Kosalecetal.,2013).Infact,theuse ofR.alaternusL.intraditionalmedicineaslaxativemayberelated principallytothepresenceofanthraquinones.
4. Conclusion
Inthisstudy,HPLC-DADanalysisofmethanolextractofleaves shows thecharacterizationofsix newphenolic compounds.All fractionsobtainedindefatting(INTandHF)andfractionationby C-18Sep-Pakcartridges(EA1,EA2,AW,AM)ofmethanolextract ofR.alaternusL.showedthepresenceofphenoliccompoundswith
qualitativeandquantitativedifferences.Inordertoeliminatethe lipoidalmaterialusinghexane,considerablequalitative,and quan-titativelossesofphenoliccompoundswereobservedintheINTand HFfractions.InthewashingstepofC-18Sep-Pakcartridges con-tainedaqueoussolution(AQ),inordertoremovesugarandorganic acids;theresultingacidifiedwatershowstheeliminationof phe-noliccompounds.Forantioxidantsactivity,allobtainedfractions exhibitedfreeradicalscavengingactivity.Anthraquinonescanbe consideredasmultipotentantioxidants.Inadditiontotheir antiox-idantactivity,theyareshowntopossessotherbiologicalactivities, includingantibacterial, antiviral,antifungal, anticancer (Kosalec etal.,2013),andpossesspotentantimicrobialactivityagainstskin infectingpathogenicorganisms(Suchetaetal.,2011).R.alaternus L.,byitsrichnessofanthraquinones,canbeapotentialcandidate forinvestigationandtherapyasantimicrobialandanticancer.
Acknowledgements
WeacknowledgeKatherineDavis-DenticiandMikeDougherty fortheirtechnicalsupportinanalyzingsomeofthephenolic com-pounds.TheauthorsalsothankKaitlynE.Feeneyforherhelpin conductingHPLCanalysis.
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.indcrop.2015.06. 015
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