w ww . e l s e v i e r . c o m / l o c a t e / b j p
Original
Article
Operculina
macrocarpa:
chemical
and
intestinal
motility
effect
in
mice
Daniele
Michelin
Paganotte
a,e,∗,
Miriam
Sannomiya
b,
Daniel
Rinaldo
c,
Wagner
Vilegas
d,
Hérida
R.N.
Salgado
aaProgramadePós-graduac¸ãoemCiênciasFarmacêuticas,DepartamentodeFármacoseMedicamentos,FaculdadedeCiênciasFarmacêuticas,UniversidadeEstadualPaulista
“JúliodeMesquitaFilho”,Araraquara,SP,Brazil
bEscoladeArtes,CiênciaseHumanidades,UniversidadedeSãoPaulo,SãoPaulo,SP,Brazil
cDepartamentodeQuímica,FaculdadedeCiências,UniversidadeEstadualPaulista“JuliodeMesquitaFilho”,Bauru,SP,Brazil
dUniversidadeEstadualPaulista“JúliodeMesquitaFilho”,CampusdoLitoralPaulista,UnidadeSãoVicente,SP,Brazil
eFundac¸ãoHerminioOmetto,CentroUniversitárioHerminioOmetto,Araras,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received14July2015 Accepted7March2016 Availableonline2April2016
Keywords:
Batata-de-purga HPLC-UV-PDA Laxative
Operculinamacrocarpa
Phenolicacids
a
b
s
t
r
a
c
t
Operculinamacrocarpa(L.)Urb.,Convolvulaceae,isusedbythepopulationasalaxative.Inthiswork wedescribedtheisolationofthethreephenolicacidspresentinthehydroethanolic extractofthe O.macrocarparoots.Thequantificationofthecaffeic,chlorogenicacidsandofthenewcaffeicdimer inthehydroethanolicandinfusionextractswasperformedbyhigh-performanceliquidchromatography coupledphotodiodearraydetector.Theseanalysesshowedthehighercontentofthechlorogenic,caffeic andthenew3,4′-dehydrodicaffeicacidinhydroethanolicandhydroethanolicextractswithoutresinin whichinfusion.Theacidfoundingreaterquantityiscaffeicacidfollowedbythe3,4′-dehydrodicaffeic acid.Thelaxativeactivitywasevaluatedbydifferentexperimentalmodelsofintestinaltransitwiththe hydroethanolicandinfusionextracts,andtheresinfraction,caffeic,chlorogenicandferulicacids.The resultsshowedallextractsandcompoundstestedhadsignificantactivityintheexperimentalmodel tested.Theseresultsobtainedareessentialforthefuturedevelopmentofapharmaceuticalproductwith safetyandefficacy.
©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Thetherapybasedonplantsdatesbacktotheoriginsof human-ityand,hasalwayshadanimportantroleinthecommunityhealth over time.AccordingtotheWorld Health Organization(WHO), medicinalplantsarethebestsourcestoobtaindrugs(WHO,2002). MedicinalplantshaveanimportantroleintheBrazilianculture andtradition.Nowadays,themajorityoftheurbanpopulation,as wellastheruralcommunities,usephytomedicinesasmedicinal treatments although a few number of species were studiedby thechemicalandpharmacologicalpointofview.Inthissense,the evaluationofthetherapeuticpotentialandchemicalcomposition oftheseplantscancontributetothedevelopmentoftheBrazilian phytotherapy.Thesecurityandefficiencyintheuseof phytother-apics should be based on the existence of relevant scientific literaturegroundedonthedemonstrationofitspharmacological activity,clinicalefficacyanditstoxicity.Asforsecurity,the knowl-edgethatguaranteestheuseofmanyphytotherapicscomesfrom
∗ Correspondingauthor.
E-mail:danielemichelin@uniararas.br(D.M.Paganotte).
traditionalmedicineandtheethnomedicinalknowledgegathered forcenturies(Carvalho,2005).
Operculina macrocarpa (L.) Urb., Convolvulaceae, popularly known as “batata-de-purga” or “jalapa”, (syn. Ipomoea purga
Hayne)(Xavieretal.,1994)fromthenortheastofBrazil,andwidely used by population due to its laxative, purgative and depura-tiveactivityagainstskindiseasesandintheleucorrheatreatment (Matos,1982;Martinsetal.,2000).
AccordingtoKohlietal.(2010)inAyurveda,O.turpethumhas beenincludedinthegroupof‘tenpurgativeherbs’.Thepresence ofresinglycosidesinConvolvulaceaefamilyhasbeenestablished associatedwiththelaxativepropertiesoftheherbaldrugs.Resin glycosideswereclassifiedintotwogroups:jalapinandconvolvulin. The jalapin group presents the commonstructure of a macro-lactonewithoneacylatedglycosidicacidwhiletheconvolvulin groups areoligomersof glycosidicacids (Pereda-Miranda etal., 2006).Previouslywe havereportedtheintestinalmotilityfrom thedichloromethane,ethylacetateandresidualfractions,andthe powderpreparationofO.macrocarpa(MichelinandSalgado,2004). Xavieretal.(1994)isolatedfromtheleavesofthis speciestwo
C-flavonoids. Despitethepopularuseofthis specieit is funda-mentalameticulousstudyaboutthechemicalcompositionandits
http://dx.doi.org/10.1016/j.bjp.2016.03.003
laxativeeffect.Thepresentstudyistherefore,aimedtotheisolation thephenoliccompoundspresentsinthehydroethanolicextractof rootsoftheO.macrocarpaandtheevaluationofthelaxativeeffectof theextracts,resinfractionandthecompoundsisolatedofthisplant.
Materialandmethods
Plantmaterial
Operculinamacrocarpa(L.)Urb.,Convolvulaceae,wascollected inthegardenofMedicinalPlantsUniararas,andtheexsiccatewas identifiedand includedin thecollection oftheESA-Agriculture SchoolLuizdeQueirozHerbarium,DepartmentofBiological Sci-encesESALQ/USP,registrationnumberESA114652.
Therootswerepreviouslyshavedanddriedinanairheater,at atemperatureof45◦Cuntilitwasreachedaconstantweight,and thentheywerecrushedinaballgrindertoreducethesizeofthe particles.
Chemicals
Caffeicacid,chlorogenicacidandferulicacid,bothsolidstate, werepurchasedfromSigma(St.Louis,MO,USA).Analyticalgrade aceticacidwaspurchasedfromRiedel-deHaën(Seelze,Germany). Methanol(HPLCgrade)waspurchasedfromMerck (Darmstadt, Germany).HPLC-grade water (18Mcm)wasobtainedusing a DirectQ5Milli-Qpurificationsystem(MilliporeCo.,Bedford,MA, USA).
Extraction
Thepowderoftheroots(200g) ofO.macrocarpa was previ-ouslymoistenedwithethanol70◦GLandmaintainedduring2h. Then,thismixturewastransferredintothepercolatorand com-pletedwith2lofthesolvent.Thereductionofthesolventvolume wasobtainedbytheutilizationofa rotatoryevaporator.During thisprocesswasobservedtheprecipitationofayellowishbrown solid,theresinfraction(5g;2.5%ofyield).Thismaterialwas sep-aratedbydecantation.Afteralltheprocesswereobtained36gof dryhydroethanolicextract(EEtOH;18%ofyield).
Preparationoftheinfusion
Thedryandpulverizedroots(200g)wereheatedandboiledfor 10minin2lofwatertoextracteffectivecomponents,andremoved. Afterfiltration,theinfusionextractwasconcentratedinarotary evaporatoranditwasfrozenandlyophilized,furnishing46.2gof theinfusionextract(23%ofyield).
Chromatographicmethods
ExtractchromatographicprofilebyHPLC-UV-PDA
The analysis of the chemical profile of the O. macrocarpa
extractswereperformedinaHighPerformanceLiquid Chromato-graphwitha Photodiode ArrayDetector(HPLC-UV-PDA),brand VarianTM modelProStar210/330.Theseparationwasperformed ona reverse phase Luna C18 column (250mm×4.6mm, 5m) (PhenomenexTM),equipped witha PhenomenexSecurityGuard (4mm×3mm,5m).Samplesandpatternswereinjectedthrough aRheodyneTM7125injectorwitha20lloop.
Approximately20mg of each EEtOH and infusion were dis-solvedin 1mlof methanol/water(1:1, v/v).For theremoval of possiblelipophiliccompounds,eachsolutionwaspurifiedbysolid phaseextraction(SPE),usingPhenomenexStrataC18 cartridges (500mg of stationaryphase), previously activatedwith 5ml of methanoland equilibratedwith 5ml ofHPLC-grade water. The
sampleswereelutedfromcartridgesusing5mlof amixtureof methanol/water(1:1,v/v).Thesampleswerethenfilteredthrough a0.45mpolytetrafluoroethylene(PTFE)filterandaliquotsof20l weredirectlyinjectedintoHPLC.
Themobile phase compositions usedwere water (eluentA) andacetonitrile(eluentB),bothcontaining0.05%trifluoroacetic acid.Thegradientprogrammewasasfollows:28–40%B(20min), 40–70%B(1min),70–100%B(10min)and100%(9min).Totalrun timewas40min.Theflow-rateofthemobilephasewas1.0ml/min. StarLCWorkstationsoftwarewasusedbothfortheoperationofthe detectorandfordataprocessing.
Isolationofphenolicacids
The EEtOHextract (4g) was fractionatedby gel permeation columnchromatography.ThecolumnwaspackedwithSephadex LH-20(57cm×3.0cmi.d.)andsoakedwithmethanol.Thecolumn
wasthenelutedwiththesamesolventmixtureyielded156 frac-tions(5ml each one).AfterTLCanalysis,similarfractionswere combinedtoyield28subfractions.
Part of subfraction 17 (25mg) was refrationated on a sil-icagelcolumnusingthemixtureofchloroform/methanol/water (43:37:20, v/v, organicphase) asmobile phase. This procedure furnishedawhiteprecipitated(13mg,caffeicandprotocatechuic acids).Theothercompoundwasobtainedfromthesubfraction25 (50mg)bycolumnchromatography(CC)usingasmobilephasethe mixtureofethyl acetate/aceticacid/water(15:0.5:0.1, v/v).This procedurefurnisheda whiteprecipitated(14mg,thenew com-pound3,4′-dehydrodicaffeicacid).
Quantitativeanalysisofphenolicacids
The quantitative analysis of phenolic compounds in the
O.macrocarpaextractandinfusionwasperformedby HPLC-UV-PDAusingtheconditionsdescribeinExtractchromatographicprofile byHPLC-UV-PDAsection.
Theseexperimentspermittedthedeterminationofthe chloro-genicandcaffeicacids,andcaffeicdimer.Theidentificationwas performedbycomparisonretentiontime,byspikingwithknown standards,andbycomparisonwithpreviouslyisolatedcompounds underthesameconditions.Methodsusingexternalstandardswere usedtoquantifyeachcompound.
These curves were obtained from seven stock solutions (1000g/ml) in the range of concentrations between 1 and 100g/ml.Thedilutionsweremadeinwater/methanol(1:1,v/v), andwereinjectedintheHPLC-UV-PDA.Allexperimentsweremade intriplicate.Theanalyticalcurveshadgoodlinearityinthe concen-trationrangestudiedandpresentedcorrelationcoefficients(r)with valuesabove0.999,whichsuggestsasagoodcorrelationbetween theareasandtheconcentrationsstudied.
NMRanalysis
The1H NMR and13C NMR 1Dand 1H NMR2D-NMR13C g -HMBCexperimentswereobtainedusingaBruker® spectrometer
at300MHzfrequency(7.0T)andonaVarianInova®
spectrome-terat500MHzfrequency(11.7T).Thesamplesweredissolvedin dimethylsulfoxide(DMSO-d6)containingTMSforchemical refer-ence.
Direct-injectionESI/MSandESI/MS/MSanalysis
The ESI analysis was carried out on a LCQ FLEET Thermo Scientific®equippedwithaniontrapanalyzersystem:datawere
acquiredusingXcalibur2.1.0.2.40software.Thesource tempera-turewassetat250◦C,andthesourcevoltagewasconstantat3.5kV. Nitrogenwasusedassheathandnebulizergasat5l/minand10psi. Heliumwasintroducedintothesystematanestimatedpressure of6×10−6mbartoimprovetrappingefficiency,andwasprovided
thefragmentationamplitudevariedbetween0.6and0.9V;theMS operatedinthenegativeionmodewithascanrateof13,000u/s. SampleswereinfusedintotheESIsourcebyuseofasyringepump ataflow-rateof5l/min.
Animals
TherewereusedSwissmice(Musmusculus),male,30daysof age,between20and30gofweight,adaptedtotheexperimental animalfacilityfor5daysbeforethebeginofthebiologicaltrials.The animalshadfreeaccesstofoodandwater,andwerekeptinroom temperatureof20±1◦C,controlledhumidityandaphotoperiodof 12hlight/darkness.Thedayofthetrialtheanimalswereinfasting for3h,receivingwateradlibitum.
The animalsused are from theBioterism Centreof the São PauloStateUniversity,Botucatu.Theprocedureswerepreviously approvedbytheEthicsResearchCommitteeoftheSãoPauloState University,Araraquara,approvalnumber25/2006.
Preparationofsoliddispersionfromtheresin
Consideringtheinsolubilityoftheresininwater,asolid dis-persionwaspreparedfororaladministrationinmiceinthetestof intestinalmotility.
Thesolid dispersionwasprepareddissolvingtheresin inan ethanolicsolutionofpolivinilpirrolidone(10%).Theethanolwas evaporatedinrotaryevaporatorat50◦C.Thefinalconcentrationof thesoliddispersionwas12.5mg/mlofresin.
Intestinaltransitwithactivecharcoal
Theanimalswererandomlyseparatedingroupsoftenanimals each,andreceivedthetreatmentbyoralgavageadministration. Acontrolgroupreceivedsalinesolution.After45min,theanimals receivedasuspensionofactivecharcoal10%inasolutionof5% ara-bicgumand0.5ml/animal,bygavageadministration.After45min, themiceweresacrificedinaCO2chamber,anditwasperformed animmediateextirpationoftheintestinefromthepylorustothe beginningofthecaecum.Thereby,itwasmadethemeasurementof thetotallengthofthesmallintestineandthedistancetravelledby theactivecharcoalsuspension.Theresultswereexpressedin per-centageofthetotallengthofsmallintestine.Theintestineswere weightedindividuallyinananalyticalbalance.
Theactivityintheintestinaltransitwasdeterminedaccording toJanssenandJageneau(1957)andWongandWay(1981).
Intestinaltransitwithmodifiedactivecharcoal(Maronaand Lucchesi,2004)
Theanimalswererandomlyseparatedingroupsofteneachone, andreceivedtheoraltreatmentbygavageadministration.Acontrol groupreceivedsalinesolution.After90min,theanimalsreceived asuspensionofactivecharcoal10%inanarabicgumsolution5% and0.3ml/animal,bygavageadministrationaswell.After60min, theanimalshadaccessagaintofoodandwater.
Themicewereobservedfor4heach5min,anditwastimed howlongdidittakethemtoeliminateofthefirstfaeceswithactive charcoal.
Intestinaltransitinmetaboliccage
Theanimals were randomlyseparated in groups often and received the oral treatment by gavage administration. A con-trolgroupreceivedsalinesolution.Theanimalswereplacedina
metaboliccage,andafter4hthefaeceswereweightedand com-paredtothecontrolgroup.
Statisticalanalysis
Theexperimentalresultswereexpressedin mean±standard
deviation.ThestatisticalanalysiswascalculatedbyStudentt-test (p<0.05)(DeMuth,1999).
Resultsanddiscussion
The phytochemical study of the EEtOH extract from the O. macrocarparesultedintheisolationofprotocatechuicacid(1), caf-feicacid(2)andthenewcompound3,4′-dehydrodicaffeicacid(3). The1HNMRanalysisofthewhitecrystalrevealedthepresence ofamixtureoftwocompounds.Themajorcompoundwas identi-fiedbythepresenceofthetwodoubletsat7.02ppm(J=2.5Hz,1H) andat6.76ppm(J=8.5Hz,1H)andadoubledoubletat6.95ppm, (J=8.5 and 2.5Hz, 1H), Thiscouplingpattern of three aromatic hydrogenssuggestedtheexistenceof1,3,4-trisubstitutedbenzene. Thetwolargedoubletat6.17ppm(J=16.0Hz,1H)andat7.38ppm (J=16.0Hz,1H)indicatedtransolefinichydrogens.Thesedata char-acterizedthepresenceofthecaffeicacid(2).The13CNMRdata corroboratethiscompoundasthecaffeicacid(Dürüstetal.,2001). Thesamearomaticsubstitutionpatternwasobservedforthe minor compound 1. The signals at 7.34ppm (d, J=2.5Hz, 1H), 7.29ppm(dd,J=8.5and2.5Hz,1H)and6.78ppm(d,J=8.5Hz,1H). The13CNMRspectrumrevealedthepresenceof7signalsbeingone ofthemcorrespondingtoacarbonylcarbonat168.7ppm.The spec-traanalyzeofthetwo-dimensionalHMQCandHMBCexperiments allowtheidentificationoftheminoritycompoundas protocatech-uicacid(1)(Yuetal.,2006).
OH HO
CO2H
2
OHCO2H
HO
1
Analyzingthe1HNMRspectraofanotherphenolicacid,showed thepresenceofhydrogensignalswithchemicalshiftsfairly con-stantandsimilarcouplingstothoseofthecaffeicacid.However, theyareduplicated.Throughtheintegrationofthesignalsalong withanalysisof13CNMRspectraandtwo-dimensionalHMBCand HMQCexperimentsitwaspossibletoidentifythenewsubstance asthecaffeicdimerboundbypositions3oftheunityIwith4′of theunitII.Thesignalsat7.04ppm(d,J=2.5Hz,1H),6.76ppm(d,
J=8.5Hz,1H)and6.90ppm(m,1H)referstohydrogensH-2,H-5 andH-6,respectivelythearomaticringofunitI.Theseassignments canbemadebythecorrelationsobservedintheHMBCspectrum. ThehydrogensofthetransdoublebondofthatunitIcanbeassigned by thesignals at 6.24ppm (d, J=16.0Hz, 1H) and 7.48ppm (d,
J=16.0Hz,1H),consecutively.Theconfirmationthatthesesignals belongingtoUnitIweregivenbycorrelationsbetweenthesignalat 7.48ppmand6.24ppm(H-␣andH-)withasignalofacarbonyl at166.1ppm,aswellasthecorrelationbetweenprotonofthe doublebond(7.48ppm)withC-6at121.0ppm.Thesecorrelations allowedtheunambiguousassignmentoftheunitI.Thearomatic partoftheunitIImaybeascertainedthroughsignalsat7ppm(d,
Table1
PercentageofdistancetravelledbytheactivecharcoalintheintestineofmicetreatedwithOperculinamacrocarpa.
Treatment Dose(mg/kg) Distancetravelledbytheactivecharcoal(%) Weightoftheintestin(g)
Control(saline) 10ml/kg 72.01±8.15 2.3983±0.25
Infusionextract 1000 83.74±12.20* 2.2830±0.28
Infusionextract 500 75.34±7.43 2.3139±0.31
EEtOHwithoutresin 1000 91.23±13.22* 2.4160±0.44
Resinfraction 400 60.62±5.79 2.5684±0.28
* p<0.05;n=10.
correlationtothedoublebondoftheunitIIofthecaffeicdimer. Sim-ilarlytotheunitI,itwaspossibletoassignallsignalsthroughthe analysisofthe1Hand13CNMRspectraaswellastwo-dimensional experiments(HMBCandHMQC).Toestablishthelinkageposition ofthetwocaffeicunitieswasmeasuredandalongrangecarbon hydrogencorrelationforH-2(unityI)→C-4(unityII)observed.In
ordertocorroboratethisstructurewerealizedtheESI-MS experi-ment.Thisexperimentrevealedthepresenceofapseudomolecular ionm/z341[M−H]−.WhileintheMS2showedabundantionsat m/z179(100)and135(90)whichaccordswiththecaffeicacid.In comparisonourdatawiththosereportedbyShaheenetal.(2011) forthe3,3′-didehydrodicaffeicacidweconfirmedthepresenceof the3,4′-dehydrodicaffeicacid(3)(Plazoni´cetal.,2009).
13CNMR(DMSO-d
6,125MHz)forthe3,4′-dehydrodicaffeicacid was:ıforunityI:114.7(C-␣),114.8(C-2),115.8(C-5),121.0(C-6), 125.5(C-1),145.5(C-3),144.6(C-),148.2(C-4),166.1(COOH).For theunityII:114.2(C-␣),114.6(C-2),115.7(C-5),121.3(C-6),125.6 (C-1),145.5(C-3),144.9(C-andC-4),148.2(C-4),165.6(COOH).
HO2C OH
O OH
CO2H
3
9' 8'
7' 1' 6'
5' 4'
2' 3'
3 4
5
6
1
7 8
9 2
Evaluationofthelaxativeactivity
SincethepopulationusesO.macrocarpaasalaxative,this activ-itywasevaluatedusingthreeexperimentalmodels.
Thefirstmodelevaluatedwastheonethatusesactivecharcoal asmarker in order toobserve theintestinal transit. The activ-ityisdeterminedbythedistancetravelledbythemarkerinthe smallintestine, compared tothecontrol group.Thismethod is widelyusedtoevaluatetheactivityofvegetalextracts(Michelin andSalgado,2004;Figueiredoetal.,2005;Salgadoetal.,2006).
Thesecondmodel,proposedbyMaronaandLucchesi(2004), usesthesamemethodologyasthefirstone;however,inthismodel theanimalsarenotsacrificed,andcanbeusedlateroninanother experiment,factthathelpswiththewellbeingoftheanimal.
Inthethirdmodel,animalsareplacedinametaboliccageand theactivityisdeterminedanalyzingthefaeces,differentlyofthe
othertwomodels.Inthismodel,theanimalscanalsobeusedagain inotherexperimentssincethereisnoneedtosacrificethem.
Accordingtoliterature,untilnowitisthefirststudyevaluating theactivityofO.macrocarpaintheintestinaltransit,exceptbythe preliminarystudy,previoustothisone,carriedoutbyourresearch group(MichelinandSalgado,2004).
Intestinaltransitwithactivecharcoal
Thistestwascarriedoutin1957byJanssenandJageneau,andit wasevaluatedtheinhibitionofthegastrointestinalpropulsionwith acharcoalsuspensioninmice.Then,WongandWay(1981) eval-uatedtheeffectofaspirinandacetaminophenintheinhibitionof thegastrointestinalpropulsioninducedbymorphineinmice.The trialwasperformedaccordingtothesecondexperimentalmodel developedby JanssenandJageneau(1957)and Wongand Way (1981).
Theinfusionextractandthestandardsofthecaffeic,chlorogenic andferulicacidwereevaluatedusingthisexperimentalmodel.
The results shown in Table 1, demonstrate the significant increaseoftheintestinalmotility,whencomparedtothecontrol group,causedbytheinfusionextractusingadosageof1000mg/kg. Usingadosageof500mg/kgtheresultappearedtobenot signifi-cant.
The EEtOH extract was previously tested using a dosage of 1000mg/kg,andtheresultsshowedasignificantincreaseofthe intestinalmotilityofmice(69.99%)whencomparedtothecontrol group(47.87%)(MichelinandSalgado,2004).Thelow solubility presentedbytheresininwaterimpliedthepreparationofasolid dispersiontotestinmice,howeveritdidnotshowsignificant activ-ityintheconcentrationused.
TheevaluationoftheEEtOHextractaftertheprecipitationofthe resinatthedoseof1000mg/kghasshowedasignificantincrease oftheintestinalmotility.Despitethehigherquantityof1–3 com-pounds,weresubmittedtothesametest.Thethreeacidsshowed asignificantincreaseoftheintestinalmotilityinmicewhen com-paredtothecontrolgroup(Table2).
Theacidsweretestedindosagesof10and20mg/kg.Thecaffeic acidpresentedactivityinthedosageof20mg/kg,andtravelleda distanceof86.80%ofthesmallintestine,whileinthecontrolgroup ittravelledadistanceof81.53%.Theferulicacidshowed signifi-cantactivityinthetesteddosages,travelling86.43and91.45%of thesmallintestinesusingthedosagesof10and20mg/kg, respec-tively.Finally,theresultsobtainedforthechlorogenicacid,were
Table2
Percentageofthedistancetravelledbytheactivecharcoalinthesmallintestineofmicetreatedwithcaffeic,ferulicandchlorogenicacids.
Treatment Dose(mg/kg) Distancetravelledbytheactivecharcoal(%) Weightofthesmallintestin(g)
Control(saline) 10ml/kg 81.53±9.75 1.5632±0.19
Caffeicacid 10 79.70±13.05 1.6160±0.15
Caffeicacid 20 86.80±10.99a 1.6400±0.37
Ferulicacid 10 86.43±16.63a 1.4864±0.09
Ferulicacid 20 91.65±13.60a 1.5282±0.17
Chlorogenicacid 10 78.62±9.34 1.3862±0.07
Chlorogenicacid 20 86.08±7.16a 1.4385±0.20
Table3
Elimination time ofthe faeces with marker in mice treatedwith Operculina
macrocarpa.
Treatment Dose(mg/kg) Timetoeliminatethefaeces
withmarker(min)
Control(saline) 10ml/kg 194.6±30.37
EEtOHextract 1000 156.4±23.29a
Infusionextract 1000 65.1±4.70a
ap<0.05;n=10.
Table4
WeightofthefaeceseliminatedbymicetreatedwithOperculinamacrocarpa.
Treatment Dose(mg/kg) Weightofthefaeces(g)
Control(saline) 10ml/kg 1.80±0.23
EEtOHextract 1000 2.01±0.60a
Infusionextract 1000 3.98±0.71a
ap<0.05;n=10.
significantinthedoseof20mg/kg,havingtravelledadistanceof 86.08%.
Basedontheseresults,itcanbesuggestedthatphenolicacids havestimulant activityoftheintestinalmotility,sincetheyare presentintheEEtOHandinfusionextractsofO.macrocarpa,where thisactivitywasalsofound.
Intestinaltransitwithmodifiedactivecharcoal(Maronaand Lucchesi,2004)
In this model were evaluated the EEtOH and the infusion extractsofO.macrocarpa.Thismodelofintestinaltransitalsouses activecharcoalas marker,however itwasmodified in2004by MaronaandLucchesitoimprovethewellbeingoftheanimal,since inthismodeltheanimalsarenotsacrificedandcanbeusedagain inotherexperiments.
Theresultsdisplayed inTable3,showasignificantdecrease oftheeliminationtimeofthemarkerwhenitiscomparedtothe controlgroup.
Intestinaltransitinmetaboliccage
In this model were evaluated the EEtOH and the infusion extractsofO.macrocarpa.Itisimportanttohighlightthatinthis model,animals areplaced in a metaboliccageand theactivity isdeterminedbytheweightofthefaeceseliminatedduringthe observationperiod,thusanimalsarenotsacrificedandcanbeused againinotherexperiments.
Table4showstheresults.Itcanbeobservedasignificant dif-ferenceoftheweightofthefaecesbetweenthetreatedgroupsand thecontrolgroup,indicatingthattheEEtOHandinfusionextracts ofO.macrocarpaalsohavestimulantactivityoftheintestinetransit inthismodel.
200
150
100
1 (14.4 min)
3 (27.0 mn) 4
(27.6 min)
5 (27.9 min)
2 (15.2 min)
50
0
5 10 15 20
Time (min)
25 30 35
Fig.1.HPLCseparationchromatogram,inanalyticmode(210nm)oftheEEtOH extract of Operculina macrocarpa. 1. clorogenic acid; 2. caffeicacid; 3. 3,4′ -dehydrodicaffeicacid;4and5.Notidentified.
20
15
1 (14.4 min) 2 (15.2 min)
3 (27.1 mn) 4 (27.5 min) 5
(28.0 min)
10
Response (mA
U)
5
0
5 10 15 20
Time (min)
25 30 35
Fig.2.HPLCseparationchromatogram,inanalyticmode(210nm)oftheinfusion extract of Operculina macrocarpa. 1. clorogenic acid; 2. caffeicacid; 3. 3,4′ -dehydrodicaffeicacid;4and5.Notidentified.
In order to relate the pharmacological activity observed with the metabolites found in this plant was performed the chromatographic profile by HPLC-UV-PAD from theEEtOH and infusionextractsof O.macrocarpa.TheUVdatashowedintense bands inthespectral range between190–220and 260–280nm confirmingthepresenceoffive phenolicacidsin theseextracts (Saldanhaetal.,2013)(Figs.1and2).Foreachcompound assign-ment,we usedthecomparisonof retentiontime ofthesample chromatogramwiththoseofstandards,additionallywecomparing theabsorptionspectraforpeaksobtainedwiththoseofstandards analyzedunderthesamechromatographicconditions.Itwas per-formedbyco-injectionoftheauthenticpatternsorthemetabolites isolatedfromthisplant(Figs.1and2).Thecompounds4and5 observed inthechromatographic profileof EEtOHand infusion extractswerenotidentified,butcouldbequantifiedthroughthe sameprofiledisplayedontheUVabsorptionspectra.According tothequantificationofphenolicacidsintheEEtOHextractthere wasagreatercaffeicacidconcentration(4.7mg/g),followedbythe
Table5
QuantityofphenolicacidsdeterminedintheextractsofOperculinamacrocarpa.a
Substances Concentration(mg/g)±standarddeviation
EEtOHextract EEtOHwithoutresin Infusionextract
Caffeicacid,2 4.7±2.9 5.1±4.1 2.4±3.1
Chlorogenicacid,1 1.1±5.1 1.1±9.8 0.4±5.5
3,4′-Dehydrodicaffeicacid,3 1.9±2.9 1.5±1.7 0.4±2.9
NI1 1.2±2.4 1.0±4.2 0.3±1.5
NI2 2.4±1.2 1.9±4.7 0.3±3.5
3,4′-dehydrodicaffeicacid(1mg/g)andchlorogenicacid(1.1mg/g) (Table5).TheEEtOHextractwithouttheresinshowedahigher concentrationofcaffeicacid(5.1mg/g)andalowerconcentration of3,4′-dehydrodicaffeicacid(1.5mg/g).Itwaspossibletoobserve thesamelevelofchlorogenicacidinbothEEtOHextractasinEETOH extractwithoutresin.Theinfusionhasasignificantreductioninthe contentofbothquantifiedacids.
Conclusions
ThephytochemicalanalysisoftheEEtOHextractoftheroots ofO.macrocarpaguidedtotheisolationandidentificationofthe caffeicandprotocatechuicacidsandthenew3,4′-dehydrodicaffeic acid.
The EEtOH and infusion extracts were subjected to HPLC-UV-PDA chemical profiling and 5 principal peaks (1–5) were detected(Figs.1and2).Thecaffeicandchlorogenicacidsand3,4′ -dehydrodicaffeicacidwerequantifiedinO.macrocarpaextracts. Thecaffeicacidwasfoundtobethecharacteristiccomponentof thesesextracts.
Theresultsobtainedwiththethreeexperimentalmodelstested showedthelaxativeactivitywithincreasingtheintestinalmotility ofallextractsofO.macrocarpa.Thecaffeic,ferulic,chlorogenicacids andthe3,4′-dehydrodicaffeicacidevaluatedshowedthelaxative action.Thiseffectisdirectlyrelatedtothecontentofcaffeic, chloro-genicacidsandthe3,4′-dehydrodicaffeicacidintheextract.The correlationbetweenthephenolicacidsstructuresandtheintestinal motilitywecansuggestthatactivityinvolvetheskeletonwiththe conjugatedacidportionand/orthephenolicwithoutnecessarily withtheorto-dihydroxisystem.Ourresultsconfirmthetraditional useofthisspeciealaxative.
Ethicaldisclosures
Protectionofhumanandanimalsubjects. Theauthorsdeclare thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).
Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.
Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.
Authors’contributions
DMPcontributionincludedtheexecutionofintestinalmotility effectin mice, theisolation of the compoundsand elaboration the paper. MS contribution included the identification of the compounds,analyzing the resultsand preparing the paper.DR contributionincludedtheanalysischromatographicby HPLC-UV-PDA.WVandHRNScontributionincludedtotheprovisionofall
laboratoryequipmentandinfrastructureforthedevelopmentof allexperimentalpartofthephytochemicalandpharmacological testsofthiswork.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
FAPESPandCNPq–Brazil.
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