w ww . e l s e v i e r . c o m / l o c a t e / b j p
Original
Article
Isolation
of
quinoline
alkaloids
from
three
Choisya
species
by
high-speed
countercurrent
chromatography
and
the
determination
of
their
antioxidant
capacity
Gilda
G.
Leitão
a,∗,
Joao
Paulo
B.
Pereira
a,
Patricia
R.
de
Carvalho
b,
Denise
R.
Ropero
c,
Patricia
D.
Fernandes
b,
Fabio
Boylan
caInstitutodePesquisasdeProdutosNaturais,UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro,RJ,Brazil
bLaboratóriodeFarmacologiadaDoredaInflamac¸ão,InstitutodeCiênciasBiomédicas,UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro,RJ,Brazil cSchoolofPharmacyandPharmaceuticalSciences,TrinityBiomedicalSciencesInstitute,TrinityCollegeDublin,Dublin2,Ireland
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received13October2016 Accepted22January2017 Availableonline2March2017
Keywords:
Quinolinealkaloids
Countercurrentchromatography Anhydroevoxine
Choisyine ROS
a
b
s
t
r
a
c
t
ChoisyaternataKunth,C.ternatavar.sundanceKunthandthehybridChoisya‘Aztec-Pearl’arethreerelated speciesbelongingtotheRutaceaefamily.Ethanolextractswerepreparedfromtheleavesofthesethree speciesandevaluatedinrelationtotheirantioxidantactivityusinginvitroandexvivomodels.The ethanolextractsbelongingtothethreespeciesproducedaveryhighantioxidantprofileasevidenced bytheDPPHradicalscavengingactivity,thedeterminationoftotalphenolicsandflavonoidequivalent. ThegenerationofreactivespeciesofoxygeninleukocytesstimulatedwithLPSwasdramaticallyreduced whenthethreeethanolextractswereused.Thealkaloidsanhydroevoxineandchoisyinewereisolated fromtheethanolextractofC.ternatausingHEMWat(4:6:5:5)asthesolventsystembymeansof high-speedcountercurrentchromatography.Thiswasthefirsttimequinolinealkaloidswereisolatedfrom thisspeciesusingHSCCC.Thesecompoundswerealsoassayedfortheircapacitytoinhibitthegeneration ofROSinleukocytesstimulatedbyLPSandtheresultsalsosuggestedthattheyarereactiveoxygenase inhibitors.
©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
ChoisyaternataKunth,knownasMexicanorange orMexican orangeblossom,iswidelycultivatedinBritainsince1825(Muller, 1940;Benson,1943).Thereisalsothegoldenleafyvariety,whichis calledC.ternatavar.sundance.Choisyadumosavar.arizonicaisalso knownasMexican-orangeorstarleafandtoMexicansas“sorilla” or“zorillo”. Thesetwo speciesare consideredtoxictothe live-stock,bearingtoxiccompounds(Dayton,1931)thatwerenotyet identified.C.ternataandC.dumosavar.arizonicawereartificially crossedin1982(Lancaster,1991)togeneratethehybridChoisya
‘Aztec-Pearl’,whichhasbiggerflowersthanitsparentsbutisnot asrobustinstatureasthem.Previousphytochemicalstudieson
C.ternatahaveindicatedtheimportanceofitsnon-volatile quino-linealkaloids.Therearesevenmainalkaloidsthatarewidespread intheRutaceaefamilyandthatwerefoundinspeciesofChoisya: skimmianine,kokusaginine,7-isopentenyloxy-c-fagarine,evoxine,
∗ Correspondingauthor.
E-mail:ggleitao@nppn.ufrj.br(G.G.Leitão).
choisyine,platydesminiummethosaltandbalfourodinium methos-alt(Johnsetal.,1967;Grundonetal.,1974;Boydetal.,2002).Boyd etal.(2007)haveisolatedseventeenquinolinealkaloidsfromC. ter-nata,includingseverallevelsofoxidationduringtheirbiosynthesis. DifferentspeciesofRutaceaehavealreadybeenusedastonic, febrifuge,againstinflammatoryandmicrobialprocessesandinthe treatmentofmalaria(Cruz,1995;DeMouraetal.,1997;Gonzaga etal.,2003;MoreiraandGuarim-Neto,2009;Jullianetal.,2006). Severalchemicalcompoundsisolatedfromdifferentspeciesofthis familycouldaccountforthepharmacologicalpropertiesobserved fortheextractsbutoneclassisprominent.Thequinolinealkaloids mentioned beforecan beresponsible forsomeoftheidentified activities.
Previouslyourgrouphasshown thatextractsobtainedfrom
C. ternataand two of theirquinolinealkaloids aswellas com-poundspresentinitsessentialoilpossessantinociceptiveactivity (Radulovi ´cetal.,2011;Pinheiroetal.,2014).Yowtaketal.(2011) hasshown a relationshipbetween spinalneuropathic pain and oxidativestress.
In this study, a high-speed countercurrent chromatography (HSCCC)methodwasdevelopedforthefractionationoftheethanol
http://dx.doi.org/10.1016/j.bjp.2017.01.003
extractofleavesofthreespeciesofChoisya;C.Aztec-Pearl,C. ter-nataandC.ternatavar.sundance.Countercurrentchromatography (CCC)isaformofliquid-liquidpartitionchromatographywhere thestationaryphaseis heldinside thecolumnwithouttheuse of a solid support (Conway,1990). In modern CCC equipment, thecolumncanrotate inoneaxis,generatinghydrostatic equi-libriumofthetwoimmiscibleliquidphases(thestationaryphase andthemobilephase),oritcanrotateinaplanetarymotion(two rotationaxes),generatingahydrodynamicequilibriumofthetwo phases.HSCCCmachineshave hydrodynamicequilibriumofthe twoliquidphasesandhavebeenlargelyusedforthefractionation andpurificationofnaturalproducts(Leitãoet al.,2012; Friesen etal.,2015).Manydifferentsolventsystemshavebeenusedfor thepurificationofalkaloidsusingmodernhydrostaticor hydro-dynamicequipment.Ionizable compoundslike alkaloidscanbe purified using a technique calledpH-zone refining CCC, devel-opedbyItointhe1990s(ItoandMa,1996),amodificationofthe CCCtechniquethatusesacidsorbasesasretainersand/oreluters. Ina studyfromFangetal. (2011)acompilationof several sol-ventsystemsforthepurificationofalkaloidsfromherbs,byboth HSCCCandpH-zone-refiningCCCispresented.Inthatreviewitis reportedtheseparationof94alkaloidsfrommorethanthirty dif-ferentplantsourcesbyconventionalHSCCCusingthirteendifferent solventsystems.Theauthorsreportthatmorethan67%ofthe alka-loids werepurified withhexane–ethylacetate–methanol–water (theso-called HEMWatsystem)and CHCl3–MeOH–H2O.In fact,
thesesolventsystems representtwo versatilefamilies that can beusedincountercurrentchromatography.Moreover,quinoline alkaloidshavenotyetbeenisolatedfromC.ternatabymeansof countercurrentchromatography.
SincespeciesofChoisyaandsomeoftheirisolatedcompounds havealreadyshownactivitytowardspinalneuropathicpain,we decidedto evaluatetheethanol extractsof these three species ofChoisyainrelationtotheircapacityofprotectingagainst reac-tiveoxygenspecies.anhydroevoxine(1),andchoisyine(2),isolated fromC.ternatainonestepHSCCCanalysis,werealsoevaluated.
Materialsandmethods
Chemicals
Solventsusedforplantextractionwereofanalyticalgradeand acquiredfromtheHazardMaterial Facilitiesstore (HMF-Trinity CollegeDublin)andthoseforcountercurrentchromatography sep-arationswereofHPLCgradepurchasedfromTediaBrazil(Riode Janeiro,Brazil).
Plantmaterial
FreshleavesofChoisyaternataKunth,thehybridC.Aztec-Pearl andC.ternatavar.sundance,Rutaceae,werecollectedinDublin, IrelandinSeptember2013andtheirvoucherspecimen(ref.TCD Hodkinson&Ropero01,02and03)weredepositedinthe Herbar-iumofTrinityCollege,Dublin.ThesampleswereidentifiedbyDr. TrevorHodkinsonfromtheDepartmentofBotany,TrinityCollege, Dublin.
Plantextraction
Theextractionofleaves(100g)ofC.ternata,C.Aztec-Pearland
C.ternatavar.sundancewasperformedusingSoxhletapparatus (80◦C)for72h.ThesolventusedfortheSoxhletextractionwas ethanol.Extract(ca.40geachplant)wasreducedtodrynessunder reducedpressureusingarotaryevaporatorinaheatingbath.
Equipment
HSCCC fractionationswere performedonthe98mlcoil of a QuattroHT-Prepcounter-currentchromatograph(AECS,Bridgend, UnitedKingdom)equippedwithtwobobbinscontainingtwo poly-tetrafluoroethylenemulti-layercoilseach(Bobbin1containsthe 26mlandthe224mlcoils,1.0mmi.d.and3.2mmi.d.respectively; Bobbin2containsthe95mlandthe98mlcoils,both2.0mmi.d.). Therotationspeedisadjustableupto865rpm.TheHSCCC sys-temswereconnectedtoaconstantflowpumpJascoPU-2089SPlus (JapanSpectroscopicCorporation,Japan).A5mlsampleloop(low pressuresampleinjectionvalve,Model5020,Rheodyne)wasused toinjectthesample.Separationswereperformedatroom temper-ature.
HPLCanalysis
TheHPLCsystemconsistedof a WatersAllianceSeparations moduleequippedwithatemperatureprogrammableautosampler andWaters2996PDAdetector.TheLCseparationwasperformed onareversedphasecolumnC18(250mm×4.6mm,5m)from
ThermoScientific,usingmobilephaseA(water)andmobilephaseB (acetonitrilewith1%formicacid)inagradientprogramwithaflow of0.8ml/min:0–30min:10%B;30–32min:100%B;32–35min: 10%Band35–38min:10%B.Thevolumeofasingleinjectionwas 10l.UV/visspectrabetween190and400nmwererecorded.
ChoiceofthesolventsystemforCCCfractionations
SmallamountsoftheethanolextractsofleavesofeachChoisya
species were dissolved in separate test tubes containing the hexane–ethylacetate–methanol–water(HEMWat)solventsystem in the ratios 10:5:5:1, 1:1:1:1, 6:4:6:4 and 4:6:5:5. Then the test tubes were shaken and the sample was allowed to par-tition between the two liquid phases. Equal aliquots of each phasewerespottedsidebysideonsilicagelTLCplates(Merck, Art.5554,Germany)developed withthesolventsystem chloro-form:methanol:water(9:1:1).Theresultswerevisualizedunder UVlight(254and365nm)followedbysprayingTLCplateswith vanillin(2%inmethanol)andsulfuricacid(1%inmethanol).
HSCCCfractionations
Preparationofthetwo-phasesolventsystemandsamplesfor injection
The selected solvent system (hexane:ethyl acetate: methanol:water 6:4:5:5) was thoroughly equilibrated in a separationfunnelatroomtemperature.Thetwophaseswere sep-aratedshortlybeforeuseanddegassedbysonicationfor10min. Thesamplesolutionswerepreparedbydissolvingthesample(ca. 500mgofeachChoisyaextract)inthesolventmixtureof2.5ml stationaryphaseand2.5mlmobilephaseofthesolventsystem usedfortheHSCCCseparation.
HSCCCseparationprocedure
2ml/min,whiletheapparatuswasrotatedat860rpm,ata con-stanttemperatureof25◦C.Thesamplesolution(ca.500mgofeach Choisyaextractin5mlbiphasicsystem)wasinjectedintothe col-umnafterhydrodynamicequilibriumwasreached.Thestationary phaseretentionSfforthesolventsystemineachfractionationwas around84–87%.Fractionsof4mlwerecollectedinatotalof80in eachrun.
Determinationoftheantioxidantactivity
Theantioxidantactivitywasperformedontheethanolextracts ofleavesofChoisyaAztec-Pearl,C.ternataandC.ternatavar. sun-dance.Theantioxidantactivitywasinvestigatedusingfourdifferent assays:DPPHfreeradicalscavengingactivitymicroassayusinga 96-wellplate,quantificationoftotalphenolicsanddetermination oftotalflavonoidcontentsandfinallytheproductionofreactive oxygenspecies.
Antioxidantactivity,useof2,2-diphenyl-1-picrylhydrazyl(DPPH)
Theradicalscavengingeffectoftheplantextractswas deter-mined using the DPPH methodology. Here, the hydrogen free radicalsdonationabilityoftheplantextractswasmeasuredfrom thebleachingofthepurplecoloredethanolsolutionofDPPH.The assaywasbasedonthestudydonebyMensoretal.(2001).
Assayfortotalphenolics
Thetotalphenoliccontentoftheextractswasdetermined fol-lowing theFolin–Ciocalteumethodcarried outby Gursoyetal. (2009),onlyadaptedtoamicroscale(Medina-Remonetal.,2009).
Assayfortotalflavonoids
Thedeterminationofthetotalflavonoidcontentoftheextracts wasdone based onthe methodology adapted by Gursoyet al. (2009).
Determinationoftheproductionofreactiveoxygenspecies(ROS)
Leukocyteswerecollectedfromasubcutaneousairpouch cre-atedinthedorsalareaofamouseforthemodelofcellmigration inducedbycarrageenan andstoredin vials(106 cells) ina
vol-umeof 1ml. Cellswerefurther incubatedat 37◦C and 5% CO2 for 1h. Crude ethanol extracts of the three Choisya species or theisolatedcompoundswerethen addedtothecells ata con-centrationof1,10and30g/mlandincubatedfor30minmore
usingthesame conditions.Cells werethen treated with10nM phorbol myristateacetate (PMA) and re-incubatedat thesame conditions for another45min.Twomicroliters ofa solutionof 2′-7′-dichlorodihydrofluoresceindiacetate (DCF-DA) wasadded, followedbyanotherincubationperiodof30minatthesame condi-tions.TheDCF-DApermeatedrapidlythroughthecellmembrane. Onceintheintracellularcavityitishydrolysedbythestearases beingconvertedinto2′-7′-dichlorofluorescin(DCFH),whichis a non-fluorescentcompoundimpermeabletothecellmembrane.In thepresenceofanyROS,DCFHisoxidizedinsidethecellforminga fluorescentcompound:2′-7′-dichlorofluorescin(DCF),whichstays intheintracellularcavity(SrivastavaandGonugunta,2009). Flu-orescencewasmeasuredbyflowcytometryintheFL-1channel.
Resultsanddiscussion
Countercurrent chromatography (CCC),a liquid–liquid parti-tionchromatographytechniquewithnosolidsupport,isidealfor theseparationofcomplex naturalproductmixtures anditsuse in this area of interest keeps growingeach day (Friesen et al., 2015).Themajorchallenge ofthetechnique isthe selectionof thecorrectsolventsystem,whichcanaccountfor90%ofthetime spentontheanalysis(Ito,2005).Severalstrategieshavebeen pro-posedinthelastyearsforafastsolventsystemselection(Friesen andPauli, 2007;CostaandLeitão,2010;Yinetal.,2010;Leitão et al.,2012; Lianget al., 2015) Literature reportshexane–ethyl acetate–methanol–water,usuallyreferredtoasHEMWat,asthe mostusedsolventsysteminCCCseparations(Leitãoetal.,2012). Thisisbecauseitcoversalargerangeofpolarities,beingusefulin thepurificationofeitherlowtomediumpolaritycompoundsas wellasmediumtohighpolarityones.So,thissolventsystemwas ourchoiceforthepurificationofthethreeextractsofChoisya:C.
Aztec-Pearl,C.ternataandC.ternatavar.sundance.Severalratiosof theHEMWatsolventsystemweretestedforthefractionationofthe extracts:HEMWat10:5:5:1,1:1:1:1,6:4:6:4and4:6:5:5,beingthe lattertheonethatgavethebestresults,affordingKvaluesoftarget compounds(thealkaloids)near1.Twoalkaloids,anhydroevoxine,
1(0.5%yield)andchoisyine,2(1.1%yield),wereisolatedinasingle HSCCCstepfromC.ternataleaves.Choisyinewasalsoisolatedfrom
C.Aztec-PearlandC.ternatavar.sundance.However, anhydroevox-inecouldnotbeisolatedaspurecompoundsinonestepfromthese twospecies.Thiscouldbeduetodifferencesinthecomplexityof thematrix(theethanolextract),whichwaspreviouslyobserved byourgroupwhenoptimizingtheisolationofverbascosidefrom
LantanaandLippiaspecies(Leitãoetal.,2015)Thesetwoalkaloids werefurtheridentifiedbyHPLCintheethanolextractoftheother twospecies(datanotshown).
Anhydroevoxine (1)was isolated as a white crystal and its structurewasundoubtedlyestablishedbymeansofMSandNMR analysisandcomparisonwiththeliteraturedata(Akhmedzhanova etal.,1975).
Whitecrystallinesolid;Rf:0.52(chloroform:methanol:water;
9:1:1); mp: 88–92◦C; [˛]20
D 0.0 (c 0.009, CH2Cl2); EIMS for
C18H19NO5 m/z;330[M+H]+and352[M+Na]+;1HNMR(CDCl3,
400MHz)ı:3.32(1H,t,J=10.92,H-2′),4.11(3H,s,OCH3–C-8),4.29 (1H,m,H-1′),4.41(3H,s,OCH
3–C-4),7.02(1H,d,J=2.4,H-9),7.22
(1H,d,J=9.0,H-6),7.57(1H,d,J=2.4,H-10),7.96(1H,d,J=9.0,H-5);
13CNMR(CDCl
3,100MHz)ı:18.9(CH3,C-4′),24.6(CH3,C-5′),58.3
(CH,C-3′),59.0(OCH
3,C-4),61.6(OCH3,C-8),61.7(CH,C-2′),69.4
(CH2,C-1′),102.3(CH,C-9),104.6(C,C-3),114.8(CH,C-6),118.1
(CH,C-5),141.6(C,C-8a),143.1(CH,C-10),143.2(C,C-8),151.1(C, C-7),155.5(C-4a),157.1(CH,C-4),164.3(C-2).
Choisyine(2)wasisolatedasanamorphousyellowishpowder anditsstructurewasundoubtedlyestablishedbymeansofMSand NMRanalysisandcomparisonwiththeliteraturedata(Frolovaand Kuzovkov,1963).
Yellowish amorphous solid; Rf: 0.44
(chloro-form:methanol:water; 9:1:1); mp: 88–92◦C; [˛]20
Table1
CorrelationbetweenstudiedparametersDPPHantioxidantactivitywithtotalphenolicandflavonoidequivalentofeachethanolextract.
Plantspecies DPPH(EC50)(g/ml) Phenolics(gGAEs/mgextract) Flavonoids(gGAEs/mgextract)
ChoisyaAztec-Pearl 25.84±0.005 338.33±0.002 0.59±0.003
Choisyaternata‘Sundance’ a 713.33±0.012 182.77±0.005
Choisyaternata 9.32±0.002 655.00±0.014 37.42±0.003
aEC
50valueisundefined.ConcentrationofEC50valuetoolowtoallowforinterpretationbutisindicativeofhighpotency.EC50forGinkgobilobaintheDPPHassaywas
38±0.005.Eachassaywasperformedintriplicate.
CH2Cl2);EIMSforC18H19NO5m/z;330[M+H]+and352[M+Na]+; 1HNMR(CDCl
3,400MHz)ı:1.28(3H,s,H-4′),1.41(3H,s,H-5′),
2.02(1H,s,H-1′),3.72(1H,m,H-3′),3.95(3H,s,OCH3–C-7),4.33 (3H,s, OCH3–C-4), 4.78(1H, t,J=9.5, H-2′), 6.97(1H,d, J=2.4,
H-9),7.18(1H,s,H-8),7.51(1H,d,J=2.4,H-10);13CNMR(CDCl 3,
100MHz)ı:24.1(CH3,C-4′),26.1(CH3,C-5′),33.7(CH2,C-1′),55.7
(OCH3,C-7),58.8(OCH3,C-4),71.9(CH,C-3′),90.5(CH,C-2′),93.08
(C,C-3),102.7(C,C-4a),104.6(C,C-7),106.4(CH,C-8),118.7(C, C-5),142.5(C,C-8a),144.5(C,C-6),148.3(C,C-4),156.5(CH,C-4), 164.3(C-2).
Antioxidantactivityoftheethanolextractofthethree
Choisyaspecies
DPPH,totalphenolicsandflavonoidequivalent
TheethanolextractsofleavesofChoisyaAztec-Pearl,C.ternata
var.sundanceandC.ternataweretestedforantioxidantactivityand thencompared(Table1).TheDPPHfreeradicalscavengingactivity usingUV–visspectrophotometrywasfirstemployed,followedby totalphenolicsandflavonoidequivalent.Itisclearfromthe val-uesthattheethanolextractsofleavesfromtheChoisyaspecies havemuchgreaterantioxidantactivitythantheethanolextractof
Ginkgobiloba,usedaspositivecontrol.Negativecontrolconsistedof onlyethanolandDPPH.Therefore,accordingtothisstudy,allthree samplesaremorepotentantioxidantsthantheG.bilobastandard. The antioxidant activity of phenolics and their derivatives dependonthenumberandpositionofhydroxylgroupsboundto thearomaticring,thebindingsitesandthemutualpositionsof hydroxylgroupsinthearomaticringandthetypeofsubstituent (Ricoetal.,2013).Thehighesttotalphenoliccontentwasseenin theextractsofC.ternataAztec-Pearl(713.33gGAE/mgextract).
ThiswasfollowedbytheC.ternatavar.sundanceextractsandthen theC.ternataextracts.
Flavonoidsare phenolic compounds that could be responsi-ble for the antioxidant capacity of medicinalplants. According to theresults in this study, the highest amountof flavonoids, 182.77±0.005equivalentofquercetin,wasfound,asexpected,for
C.ternatavar.sundance,thegoldenleafyspecies.
FromtheanalysisofTable1,itcanbeseenthattherewasa distinctcorrelationbetweenthestudiedparameters(total pheno-liccontent,flavonoidequivalentandDPPHscavengingactivity)for
ChoisyaAztec-Pearl,C.ternataandC.ternatasundanceextracts.The scavengingactivitymeasuredbytheDPPHshowedaverygood cor-relationtothequantityofphenolsandflavonoidsineachofthese extracts.
ROSproduction
Thegenerationofreactivespeciesofoxygenwasmeasuredin leucocytesbythemethoddescribedpreviously.Itisobservedthat
C.ternataandC.ternatavar.sundancesimilarlyprotected leuco-cytesfromgeneratingfreeradicalswhenstimulatedwithPMAby 83%bothat10g/mlandby95%and92%at30g/ml,respectively.
However,inthismodelC.Aztec-Pearlconfersaslightlyhigher pro-tectionofthecellsafterstimulationwithPMA;ataconcentration
of10g/mltheprotectionisaround98%andataconcentration
of 30g/ml it goes up to99%. According tothis model,
anhy-droevoxineprotectsthecellsby71%at30g/mlwhilechoisyine
confers64%protectionatthesameconcentration.Inleukocytes, ROSaremainlygeneratedbyNADPHoxidase(Saueretal.,2001) atargetofproteinkinaseC,whichisactivatedbyPMA(Karlsson etal.,2000).ThePMA-inducedROS formationwassignificantly reducedbyChoisyaspeciesat10mM,reflectingitsknown radical-scavengingproperties(Loetal.,2002).Incontrast,Choisyaspecies didnotinterferewithPMA-activatedROSgenerationupto10mM, excludingunspecificanti-oxidantproperties.Theseresultssuggest thatC.ternata anditsalkaloidscouldinhibitROSgenerationor production.ThisstudyalsoshowedthatCyathulaprostata exhib-itedanti-inflammatoryandantinociceptiveactivitiesandthiswas mediated byother pathwaysbut not bya direct action onthe oxidativemetabolism(Imbrahimetal.,2012).Poeckeletal.(2008) observedthattheactionofRosmarinusofficinalisandSalvia offici-nalis,aswellasoftheirmainconstituents,thediterpenescarnosol andcarnosic acidinrelationtotheiranti-inflammatoryactivity hadastrongcomponentlinkedtotheirantioxidantcapacity.The evaluationofthegenerationofreactiveoxygenspeciesin leuko-cytesstimulatedbyPMA,followedbytheuseofthesediterpenes andtheplantextractsshowedthestronglinkbetweentheir anti-inflammatoryactionandthespecificantioxidantcapacity.
ItcouldthusbeconcludedthatthethreespeciesofChoisya eval-uatedpossessedaveryhighantioxidantpotentialasillustratedby
invitroandexvivoassaysusedinthisstudy.Thesamegoesfor thetwoquinolinealkaloidsisolatedfromC.ternata.It wasalso thefirsttimethatquinolinealkaloidswereisolatedbymeansof countercurrentchromatographyfromthisspecies.
Authors’contribution
GGLandJPBPdidHSCCCseparationsandchoiceofsolvent sys-tems.PRC,DRR,PDFandFBdidantioxidantexperiments.FBalso performedHSCCCseparations.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
TheauthorsfromBrazilwanttoacknowledgethegrantsfrom CNPq and FAPERJ. The authors from Ireland wish to acknowl-edgetheHighEducationAuthority’sProgrammeforResearchin Third-LevelInstitutions Cycle 5’sfunding support for TBSI and toreinforcetheimportanceofSFIProgrammeISCA-Brazil(Grant no.SFI/13/ISCA/2843)thatcontributedtothecollaborativework betweenBrazilandIreland.
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