Rev. bras. farmacogn. vol.27 número5

w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Isovitexin

as

marker

and

bioactive

compound

in

the

antinociceptive

activity

of

the

Brazilian

crude

drug

extracts

of

Echinodorus

scaber

and

E.

grandiflorus

Claudia

Lea

Strada

_,

_Karoline

_da

_C.

_Lima

_,

_Virginia

_C.

_da

_Silva

_,

_Reginaldo

_V.

_Ribeiro

_,

Eliana

F.G.

de

C.

Dores

_,

_Evandro

_L.

_Dall’Oglio

_,

_Guillermo

_{Schmeda-Hirschmann}

_,

Carlos

A.

Carollo

_,

_Domingos

_T.O.

_Martins

_,

_Paulo

_Teixeira

_de

_Sousa

_Júnior

a_{DepartamentodeQuímica,UniversidadeFederalMatoGrosso,Cuiabá,MT,Brazil}

b_{DepartamentodeCiênciasBásicasemSaúde,FaculdadedeMedicina,UniversidadeFederaldeMatoGrosso,Cuiabá,MT,Brazil} c_{InstitutodeQuímicadelosRecursosNaturales,UniversidaddeTalca,Talca,Chile}

d_{DepartamentodeFarmáciaeBioquímica,UniversidadeFederalMatoGrossodoSul,CampoGrande,MS,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received6February2017

Accepted9May2017

Availableonline12August2017

Keywords:

Chapéu-de-couro Flavonoids

Isovitexinmarker

Fingerprint

Antinociceptiveactivity

a

b

s

t

r

a

c

t

Echinodorusscaber Ratajand Echinodorusgrandiflorus(Cham. &Schltdl.) Micheli,Alismataceae,are popularlyusedtorelieveinflammatorycomplaints andasdiuretic.Astudyontheantinociceptive effectandselected markercompounds inelevenextractsfrom differentlocationswas undertaken andtheirantinociceptiveeffectwasassessed.ThefingerprintswerecomparedbyHPLC-DADandthe contentofvitexin,isovitexin,isoorientinandvitexin-2-O-rhamnosideweredetermined.Allsamples pre-sentedantinociceptiveactivityreducingthewrithesby36.4–62.5%and47.4–79.8%at10and50mg/kg, respectively;indomethacin(5mg/kg)reducedwrithesby82.6–90.1%.Thecontentoftheflavonoids

C-glycosides,however,presentedastrongvariation.Isovitexinandisoorientinwerefoundinallthe samples,withcontentrangingfromtracesto14.70␮g/mgand2.12–84.27␮g/mgextract,respectively,

whilevitexin-2-O-rhamnosideoccurredinquantifiableamountsonlyin3outof11samplesranging from5.43to33.13␮g/mgextract;vitexinwasnotdetectedatallordetectedintraceamounts.

Accord-ingtothefingerprints,thesamplescouldbearrangedinfourmaingroups.Allelevenextractsshowed antinociceptiveactivity.Isovitexinwastheonlyflavonoidpresentinallsamplesandcanberegarded, actinginsynergywiththeothercompoundsornot,astheresponsiblefortheantinociceptiveactivity. Therefore,isovitexinisagoodchoiceaschemicalmarkerwhentheantinociceptiveactivityofE.scaber

andE.grandiflorusisinvestigated.

©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

The genus Echinodorus, Alismataceae, comprises about 45 species,occurringmainlyintropicalareasandabout25ofthem have been described in Brazil (Lehtonen, 2008; Matias et al., 2015). The best-known Echinodorus species used as medicinal plantinBrazilisEchinodorus scaberRataj,heterotypicsynonym ofEchinodorusmacrophyllussubsp.scaber(Rataj)R.R.Haynesand Holm-Niels,but alsoEchinodorus grandiflorus(Cham.&Schltdl.) Micheliis mistakenly collectedand used as a substitute ofthe officialcrudedrug.Bothplantsareknownas“chapéu-de-couro”,

∗ _{Correspondingauthor.}

E-mails:[email protected],[email protected](P.T.SousaJúnior).

“chá-mineiro”,“erva-de-pântano”,“erva-de-brejo”,amongothers. Aleafinfusionordecoctionisusedasanti-inflammatory, antinoci-ceptive,diuretic, hypotensiveand antirheumatic(Brandãoetal., 2009).

PreviouschemicalstudiesinE.scaberledtounusual nitrogen-containingclerodanediterpenes(Kobayashietal.,2000a,b,c).From themethanolicleafextractseveralcembranediterpenes(Manns andHartmannn,1993;Tanakaetal.,1997;Shigemorietal.,2002), phenolics(Tanus-Rangeletal.,2010),flavonoidsandcaffeicacid derivatives(Schnitzleretal.,2007)werereported.Acomparative studyoffiveEchinodorusspecieswasundertakenbyTanaka(2000)

usinghyphenated methods.Diterpenes, steroidsandfattyacids wereusedaschemicalmarkers.

The anti-inflammatory activityof E. scaber (E. macrophyllus) leaf extract in acute and sub-chronic models of inflammation

http://dx.doi.org/10.1016/j.bjp.2017.05.011

0102-695X/©2017SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://

Table1

Botanicalidentification,voucherspecimennumber,collectionplace,geographiccoordinatesandextractionyieldsofEchinodorusscaberandE.grandiflorussamples

investigated.

Plantspeciesandsample Collection placeandstate

Geographical coordinates(GPS)

Voucherherbarium number

w/wextraction yield%

E.grandiflorus–L1 Juína,MT 15◦56′70′′S

56◦₃₆′₀₇′′_W

31645 18.50

E.scaber–L2 Poconé,MT 16◦31′40′′S

56◦₄₃′₅₃′′_W

33635 12.70

E.grandiflorus–L3 Chapadados

Guimarães,MT

15◦₃₆′₃₀′′_S

56◦₀₃′₄₄′′_W

33637 13.80

E.grandiflorus–L4 DomAquino,

MT

15◦₄₈′₄₀′′_S

54◦₅₅′₀₂′′_W

33638 20.60

E.scaber–L5 Cuiabá,MT 15◦₄₂′₈₀′′_S

55◦₅₃′₂₀′′_W

33639 18.70

Echinodorussp.–L6 Comercial

sample, Chapadados Guimarães,MT

– – 41.50

E.scaber–L7 CampoGrande,

MS

20◦₃₀′₇₉′′_S

54◦₃₄′₈₇′′_W

33665 47.38

E.scaber–L8 CampoGrande,

MS

20◦₃₀′₇₉′′_S

54◦₃₄′₈₇′′_W

33665 41.31

E.scaber–L9 CampoGrande,

MS 20

◦₂₈′₂₈′′_S

54◦₃₄′₀₆′′_W

33665 50.21

E.scaber–L10 CampoGrande,

MS

20◦₂₉′₈₄′′_S

54◦₃₅′₅₂′′_W

33665 48.95

E.scaber–L11 CampoGrande,

MS

20◦₃₀′₀₂′′_S

54◦₃₆′₅₂′′_W

33665 43.32

StatesofMatoGrosso(MT)andMatoGrossodoSul(MS).

wasassessedbyTanus-Rangeletal.(2010)usinghydroethanolic extractin miceandrats.Potent acute,systemic andtopic anti-inflammatoryactivitywasfound.Thesameauthorsreportedthe isolationofisovitexinfromtheethylacetatefractionoftheextract andthedetectionofthecloserelatedvitexininthesample.

TheimmunosuppressiveeffectofE.scaberaqueousextractwas describedbyPintoetal.(2007).

AtoxicologicalstudyonaqueousextractofE.scaberwas under-takenby Lopes et al. (2000).The authors foundminor hepatic toxicitybutnogenotoxiceffectatthedailydoserecommendedin traditionalmedicine.However,Vidaletal.(2010)usingabacterial assay,foundmutageniceffectintheaqueousextractandsolvent partitionfractionsoftheplant.

Therefore,consideringtheimportanceof“chapéu-de-couro”to thelocalfolkmedicineandtheseveralscientificstudiesreported abovethatseemstoconfirmitstraditionaluse,itisimportantto developasimple andcheapmethodforthequalitycontrolof a possible“chapéu-de-couro”phytotherapicmedicine.So,theaimof thisworkistoperformHPLC-DADfingerprintanalysesin “chapéu-de-couro”samplescollectedindifferentsitesfromMatoGrosso andMatoGrossodoSulstates,andtoinvestigatethepossibleuse ofvitexin,vitexin-2-O-rhaminoside,isovitexinandisoorientinas chemicalmarkersinthecrudedrugspresenting antinociceptive activity.

Materialsandmethods

Chemicals

ThesolventsusedwereofACSgradefromVetec(RiodeJaneiro, Brazil),andfor HPLCanalysiswereofspectroscopicgradefrom Tedia(Fairfield,OH,USA).Ultrapure waterwasobtainedbythe Milli-Qsystem(Waters,Darmstadt,Germany). Aceticacidfrom Merck(Darmstadt,Germany)andNaClProquimios(RiodeJaneiro, RJ,Brazil).Indomethacin,sodiumbicarbonateandstandard com-pounds for the chromatographic analyses from Sigma–Aldrich (St. Louis, MO, USA). The purity of the standards used in the HPLC analysis was as follows: vitexin (>95%), isovitexin (98%),

vitexin-2-O-rhamnoside (>98%), catechin (>98%). Isoorientin (≥97%)wasisolatedfromleavesofE.scaber,anditsidentitywas confirmedby1_Hand13_{CNMRanalysesonaVarianMercury300}

(PaloAlto,CA,USA),inagreementwiththeliterature(Pengetal., 2005).

Plantmaterial

TheplantswerecollectedinMay2011inelevendifferent loca-tionsfromtheMatoGrossoandMatoGrossodoSulstates,Brazil andwereidentifiedbyMSc.ValiJoanaPott.Voucherherbarium specimenshavebeendepositedattheCentralHerbariumofUFMS (Table1).Theaerialpartsoftheair-driedplantmaterialwereused fortheanalyses,accordingtothetraditionaluse.Thepowdereddry material(20–25mesh;5g)wasextractedwithEtOH–H2O(100ml;

70:30,v/v)atr.t.during4hunderstirring.Afterfiltration,the sol-ventwasevaporatedtodrynessunderreducedpressure,andthe remainingsyrupwaslyophilized.Theextractionswerecarriedout intriplicate.

Animals

TheantinociceptiveassayswerecarriedoutusingSwissalbino mice (20–35g) from the Central Animal House of UFMT. Ani-malswerekeptinpolypropylenecageswithfreeaccesstowater and commercialPurina®

(Labina, SãoPaulo, Brazil) at 25±1◦_C

and12h(light)/12h(darkness)cycles.Experimentswerecarried outaccording totheethicalprinciplesonAnimal Experimenta-tion,adoptedbytheBrazilianCollegeofAnimalExperimentation (COBEA).TheexperimentswereallowedbytheAnimalUseEthics

Committee (CEUA) of the UFMT under authorization number

23.108043016/10-6.

Totalphenoliccontent

The total phenolic content (TP) was determined by the

testtubesand250␮lFolin–Ciocalteureagentwereadded.The

mix-turewaslefttostandfor5min,and750␮lof20%sodiumcarbonate

solutionand5mldistilledwaterwereadded.After30minof incu-bationatroomtemperature(20◦_{C)theresultingabsorbancewas}

measuredat765nm.Thecalibrationcurvewasperformedwith cat-echin(concentrationsrangingfrom4mg/mlto15mg/ml)andthe resultswereexpressedasmgofcatechinequivalentsper100gof dryplantmaterial.

Totalflavonoidscontent

Determination of the total flavonoids content (TF) of the hydroalcoholicextractwasperformedasreportedpreviously,using theAlCl3colorimetricmethod(Simirgiotisetal.,2008).

Quantifica-tionwasexpressedbyreportingtheabsorbanceinthecalibration graphofvitexinusedasstandardflavonoid(from1to29␮g/␮l; R2=0.992).Resultsareexpressedasmgvitexinequivalents/gdry mass.

HPLCanalysesofmarkercompounds

HPLC analyses were carried out using Varian Pro Star 5.5 (PaloAlto,CA,USA)equipmentwithaquaternarypump(model

240), autosampler (model 410) and UV-DAD detector (model

330). The conditions were as follows: column HiChrom C-18

[250mm×4.6mm×5␮m] (Reading, UK), pre-column Kromasil

C-18 [3.0mm×4.6mm] (Bohus,Sweden), mobilephase CH₃CN (solventA),MeOH(solventB)and0.05%(v/v)aqueous trifluoro-aceticacid(TFA)solution(solventC);flowrate,1ml/min;injection volume,4␮l.Thegradientprogramusedwasasfollows:0–20min

(75%C:15%B:10%A);20–25min(65%C:20%B:15%A);20–25min (55%C:25%B:20%A);25–30min(75%C:15%B:10%A).Theanalyses weremonitoredbytheProStar5.5softwareandthespectrawere detectedusingthePoliView2000program. Thechromatograms wereobtainedat270nm,andspectrafromtheextractconstituents weremeasuredintherange218–400nm.Underourexperimental conditions,theretentiontimeofthereferencecompoundswere: isoorientin (8.6min), vitexin-2-O-ramnoside (11.2min), vitexin (11.6min)andisovitexin(12.4min).Calibrationcurvesforthe ref-erencecompoundswerebuiltusingtheinternalstandardmethod (catechinasinternalstandard),ineightconcentrationsforvitexin, isovitexinandvitexin-2-O-rhamnoside(0.25–20␮g/ml),andinten

concentrationsforisoorientin(7–100␮g/ml),injectingeach

con-centration intriplicate. Theinternal standard and theanalyzed compoundsweredissolved(1mg)separatelyinCH3CN:0.05%TFA

(10ml,1:1,v/v),theinjectionvolumewas4␮l.Thelinearityofthe

detectorforvitexin,vitexin-2-O-rhamnoside,isoorientinand iso-vitexinwasobtainedfromlinearplotsofcurves,built-upusingthe MicrosoftOfficeExcel2007program.Theconcentrationsofvitexin, vitexin-2-O-rhamnoside,isovitexin,andisoorientininleavesofE. scaberandE.grandifloruswerecalculatedfromthepeakareasusing theequationfor linearregressionobtainedfromthecalibration curve.Thedetectionlimit andquantification limitfor the com-poundswereasfollowin␮g/␮l,respectively:vitexin(0.29and0.9),

vitexin-2-O-rhamnoside(0.68and2.05),isovitexin(0.25and0.75) andisoorientin(1.55and4.7).Themethodwasvalidated (speci-ficity,linearity,detectionandquantificationlimits,accuracyand precision),accordingtotheliterature(Anvisa,2003).

PrincipalComponentAnalysis

Unsupervised Principal Component Analysis (PCA) was per-formedwithselectspeaksobtainedbymatchingfromHPLC-DAD date,alignedbyChromalignerplatform(Wangetal.,2010).The aligneddatawereanalyzedinMetaboAnalystplatform(Xiaetal., 2012).Sampleswere clustered intotwo groups based on their

geographicallocation:MatoGrosso(MT)andMatoGrossodoSul (MS)states.PCAwasperformedwiththelog-transformedand auto-scaleddata,atUFMS.

Antinociceptiveactivity

The antinociceptive activity was determined in mice as

describedbyKosteretal.(1959).Theexperimentswerecarried outemploying6–8animalspertreatmentgroup.Theextractswere administeredorallyatdosesof10or50mg/kgbodyweight,60min beforeintraperitoneal injectionof 0.6%acetic acidin 0.9% nor-malsaline(0.1ml/10gbodyweight)toinducethecharacteristic writhings.Vehicle(10ml/kgbodyweight,p.o.)andindomethacin (5mg/kgbody weight,p.o.)weredissolvedin 2%NaHCO3

solu-tionandweregiventomiceinthecontrolandreferencegroups, respectively.Themicewereobservedandcountedforthenumber ofabdominalwrithesandstretchingsinaperiodof0–30min.The responsesinthetreatedgroupswerecomparedwiththoseof ani-malsinthecontrolgroupandreferencecompound.Thepercentage ofinhibitionofthenumberofwrithingswascalculatedandresults arepresentedaspercentinhibition.

Statisticalanalyses

Theresultsareexpressedasmeans±S.E.M.Onewayanalysisof variance(ANOVA)wasusedforthecomparisonofmorethantwo meansfollowedbyStudent–Newman–Keulstest;pvalues<0.05 wereconsideredsignificant.GraphPadInStat®

version2.01 was usedfordataanalysis.

Results

Taxonomicauthentication

Elevensamples(L1–L11)from“chapéu-de-couro”werecollected

orpurchasedindifferentlocationsofMatoGrosso(MT)andMato GrossodoSul(MS)statesin2011,duringthefloweringtime.From theelevensamples,sevenwereconfirmedasE.scaber,whilethree turnedout tobe thecloserelated species E.grandiflorus and a commercialsampleremainsasEchinodorussp.Thew/wextraction yieldsfortheE.scabersamplesrangedbetween12.70and50.21% whileforE.grandiflorusthesolublecontentwas13.80and20.60%, respectivelyand41.50%fortheEchinodorussp.sample.Thebest extractionyields(41.31–50.21%)wereobservedfromthesamples collectedinMSsites(Table1).

Thetotalphenolic,flavonoidandC-glycosideflavones quantification

Thetotalphenolsandflavonoidcontent,aswellastheC-glycosyl flavonoid content of hydroethanolic extracts from “chapéu-de-couro”leavesare givenin Table2.Table3 providescalibration curvedata,obtainedusingstandardC-glycosideflavones.Thetotal phenolicconcentrationdidnotvaryappreciablyamongtheeleven analyzedsamples,rangingfrom283.25mg/g(Echinodorussp.,from MT)to330.00mg/g(E.scaberfromMS).However,greatvariation wasobservedregardingthetotalflavonoidconcentration,ranging from3mg/g(E.scaber,fromMS)to233.75mg/g(E.scaberfromMT). Thecontentof theC-glycosideflavonesalsopresented great variation(Table2).Vitexinwasnotdetectedinanybutoneofthe elevenanalyzed samples, whereas vitexin-2-O-rhamnoside was quantified in onlythree sites(all from MT), rangingfrom 5.43 to33.13␮g/mg,andisovitexinand isoorientinweredetectedin

allsampleswithconcentrationrangingfromtraces14.70␮g/mg

fortheformerand2.12–84.27␮g/mgforthelatter.An

Table2

Totalphenolic(TP),totalflavonoid(TF)(mgofcatechinandvitexinequivalentspergofdryplantmaterial,respectively),isovitexin,vitexin-2-O-rhamnosideandisoorientin

(␮gpermgofhydroethanolicextractsofEchinodorusscaberandE.grandiflorus).

Plantspeciesandsample TP(mg/g) TF(mg/g) Isovitexin(␮g/mg) Vitexin-2-O-rhamnoside (␮g/mg)

Isoorientin(␮g/mg)

E.grandiflorus–L1 290.00 25.50 nq nd 2.12

E.scaber–L2 292.50 198.00 0.72 13.46 21.82

E.grandiflorus–L3 296.75 39.25 1.06 nd 22.24

E.grandiflorus–L4 296.00 145.5 0.62 nq 21.54

E.scaber–L5 324.00 233.75 nq 5.43 14.34

Echinodorussp.–L6 283.25 95.00 2.44 33.13 84.27

E.scaber–L7 308.75 141.75 6.75 nq 12.22

E.scaber–L8 298.50 61.25 14.70 nq 34.27

E.scaber–L9 287.50 100.50 9.22 nq 18.10

E.scaber–L10 330.00 123.00 6.99 nq 35.54

E.scaber–L11 308.75 3.00 5.71 nq 16.89

CollectionplacesL1–L5andL7–L11arefromtheMatoGrosso(MT)andMatoGrossodoSul(MS)states,respectively;L6referstoacommercialsample(pleaseseeTable1);

nq,detected,butnotquantified(belowquantificationlimit);nd,notdetected.

Table3

Linearregressionequation,correlationcoefficient,detectionandquantificationlimitsforvitexin-2-O-rhamnoside,vitexin,isovitexinandisoorientin.

Standard Conc.range(␮g/ml) Linearregression(y=a+bx) Correlationcoefficient(r2) Detection limit(␮g/ml)

Quantification limit(␮g/ml) Vitexin-2-O

-rhamnoside

0.50–8.00 y=0.0280+0.1051x 0.9967

0.68 2.05

8.00–20.00 y=0.1651+0.0877x 1.0000

Vitexin 0.25–8.00_8.00–20.00 y_y=₌0.0136_0.574++_0.15620.2242_xx _1.00000.9994 0.29 0.90

Isovitexin 0.25–8.00_8.00–20.00 y_y=₌0.0072_0.3473++0.2512_0.2073x_{x 0.9997}0.9996 0.25 0.75

Isoorientin _{40.00–100.00}7.00–40.00 y_y=₌8.3363_7.109x+6.1229x 0.9982 1.55 4.70

−36,274 0.9997

theEchinodurussp.analyzed; whileE.grandiflorussamples pre-sented only traceamounts or none of vitexin-2-O-rhamnoside andvery smallamountsof isovitexin(traces–1.06␮g/mg) but appreciablecontent ofisoorientin (2.12–22.24␮g/mg), a differ-entpattern can be observed toE. scaber,also presenting trace amountsofvitexin-2-O-rhaminoside(exceptsites2and5;13.46 and 5.43␮g/mg respectively)along with higher concentrations ofisovitexin (traces– 14.70␮g/mg)and highconcentrations of isoorientin(12.22–35.54␮g/mg).ThecommercialEchinodorussp. sample presented very high concentration of isoorientin and vitexin-2-O-rhaminoside (84.27 and 33.13␮g/mg respectively) andamoderateamountofisovitexin(2.44␮g/mg).

Antinociceptiveactivity

Oraladministrationoftheextractselicitedantinociceptionin theanimalsatbothselecteddosesof10and50mg/kg.Invehicle treated groups,injection ofacetic acidcaused an intense noci-ceptiveresponse in mice, ranging from 38.9±2.5 to 40.4±4.0 writhings(Table4).

Pretreatmentof theanimalswithhydroethanolic extractsof “chapéu-de-couro”(10mg/kg),resultedininhibitionofwrithings, attainingthelowestvalue(36.4%,p<0.001)withE.scaber–L5and

highestvalue(62.5%,p<0.001)withE.scaber–L10,whencompared

tothevehiclegroup.Whena50mg/kgextractdosewasemployed, reductioninnumberofwrithingswasgreaterthanatthelower dose,reachingthelowestvalue(47.4%,p<0.001)withE.scaber– L1,andthehighestvalue(79.8%;p<0.001)withE.scaber–L7,when

comparedtothevehiclegroup.

Animalsthatreceived5mg/kgindomethacin,thestandarddrug forthistest,hadthehighestpercentageinhibitionofnociceptive response,withvaluesrangingfrom82.6%to90.1%(p<0.001).

Discussion

Thisworkperformedfingerprintanalyses,aswellasflavonoid quantification on samples of the popular phytotherapic drug namedas“chapéu-de-couro”.Thesampleswerecollectedin dif-ferentplacesfromMatoGrossoandMatoGrossodoSulStatesin Brazil,tosearchforphytochemicaldifferences,andforchemical markersinthecrudedrugspresentingantinociceptiveactivity.

ThedosesofE.scaberandE.grandiflorusextractsin antinocicep-tivetestsmodelswerechosenbasedonmurinepilotexperimentsof ourlabwiththeaceticacidtest,andfrompreviousliteraturereports onother inflammatory or antinociceptive models.The classical methodofWhittle(1964),usingaceticacid,waschosenbecauseit isasimple,rapidandcheaptest,beinganon-selectivemodelthat servesforbothperiphericandcentralanalgesia,withoutaltering consciousness.Indomethacinwasusedasapositivecontrolsince itisaclassicalstandardnon-steroidaldrugthatassuresits speci-ficityasaninhibitorofcyclooxygenase(Whittle,1964;Cabral-Silva, 2013).

Despitethedistinctprofileofthetwostudiedspeciesand

Echin-odorussp.(commercialsample)regardingflavonoidcontent,the

amountoftheC-glycosylatedflavonoidscanbesuccessfullyused inthequalitycontrolifa“chapéu-de-couro”drugistobe com-mercialized.ThePCA(Fig.1)showsthatthegeographicallocation ofthespeciesisthemaindifferentiatingfactorinthepolar sec-ondarymetabolism.Inthelastyearsseveralauthorsshowedthat edaphoclimatic factors may substantially change metabolomics composition(Gobbo-NetoandLopes,2007).

Table4

EffectoforaladministrationofEchinodorusscaberandE.grandiflorushydroethanolicextractsonaceticacid-inducedwrithesinmice.

Extracts Dose(mg/kg) No.ofwrithes %inhibition

Vehicle – 38.9±2.5 –

E.grandiflorus–L1 10 21.0±2.2

b _46.0

50 20.4±2.6b 47.4

E.grandiflorus–L3 10 15.9±1.7

b _59.2

50 13.0±3.6b 66.5

E.scaber–L10 10 14.6±2.6

b _62.5

50 9.4±3.3b 75.7

Indomethacin 5 5.3±0.9b 86.3

Vehicle – 40.1±3.0b –

E.scaber–L2 10 19.0±2.7

b _52.7

50 14.1±2.1b 64.8

Echinodorussp.–L6 10 21.3±4.2

b _46.9

50 18.1±3.6b 54.8

Indomethacin 5 4.8±1.1b 88.0

Vehicle – 40.4±4.0 –

E.scaber–L7 10 23.4±3.9

a _42.1

50 8.2±1.7b 79.8

E.scaber–L8 10 18.7±4.7

b _53.8

50 8.6±2.5b 78.6

E.scaber–L9 10 19.8±2.3

b _51.0

50 12.6±1.6b 68.9

Indomethacin 5 4.0±1.2b 90.1

Vehicle – 40.3±1.7b –

E.grandiflorus–L4 10 19.0±2.8

b _52.9

50 15.7±3.0b 61.1

E.scaber–L5 10 25.7±4.1

b _36.4

50 15.0±2.0b 62.8

E.scaber–L11 10 22.8±2.5

b _43.4

50 10.3±1.8b 74.5

Indomethacin 5 7.0±1.1b 82.6

Resultsarepresentedasmeanvalues±S.E.M.forn=6–8animals/group.OnewayANOVAfollowedbytheStudent–Newman–Keulstest.

a_{p<0.01vsvehicle.}

b_{p<0.001vsvehicle.}

P

C2 (19.3%)

30

20

10

0

–10

–40 –20 0 40 60

PC1 (45.5%) 20 Scores Plot

a

b

c

d

E. grandiflorus - L4

E. scaber - L5

E. scaber - L8 Echinodorus - L6

0 5 10 15 20 25 Minutes

Fig.2.RepresentativeHPLCchromatogramsoftheEchinodorusscaberandE.grandiflorussamplesfromtheBrazilianPantanal.(A)E.grandifloras:L4(representativefrom

collectionplacesL1,L3,andL4fromMTstate);(B)E.scaber:L5(representativefromplacesL2andL5–MTstate);(C)Echinodorussp.–L6(commercialsample);(D)E.scaber:L8

(representativefromcollectionplacesL7toL11fromMSstate).Peakidentities:(1)catechin(internalstandard),retentiontime:5min,max324and279nm;(2)isoorientin*,

retentiontime:8.6min,max348and269nm;(3)swertiajaponin**,retentiontime:9.2min,max344and269nm;(4)vitexin-2-O-rhamnoside,retentiontime:11.2min,

max334and269nm;(5)vitexin*,retentiontime:11.6min,max335and269nm;(6)isovitexin*,retentiontime:12.4min,max335and269nm;(7)caffeoyl/feruloylacid

derivatives,retentiontime:18.2min,max327,290shand245nm;(8)caffeoyl/feruloylacidderivative,retentiontime:25.1min,max328,290shand243nm.*Identity

confirmedbyco-injectionofstandards.**IdentityconfirmedbyNMRdata,UVspectrumandcomparisonwithliterature(Schnitzleretal.,2007;Pengetal.,2005).

effectsuggestthatthecrudedrugfromMSpresentabetterprofile regardingthetraditionalindicationsofuse.Therelatedspecies,E. grandiflorus,withthesamecommonnameanduse,showedsimilar antinociceptiveeffectthanthatobservedfortheMTcollectionsof

E.scaber.

The analgesic and anti-inflammatory activity of extracts obtainedfromtherhizomesofE.grandifloruswasreportedbyDutra etal.(2006)andthehypotensiveeffectoftheleafethanolextract onspontaneoushypertensiveratswasalsodescribed(Lessaetal., 2008).

Garciaetal.(2010)publishedacombinedphytochemicaland anti-inflammatorystudyofE.grandiflorusleavesextractandfound activityatthehighestdoseof1g/kg,toohightoexplainthe tradi-tionalindicationsofuse.

Themaindifferencesofthepresentworkwithpreviousstudies istheethnopharmacologicalapproach,usingthecrudedrug follow-ingthetraditionalindicationsofuseandthedosesusedwhichare lowerthaninthestudyofDutraetal.(2006).Inaddition,ourwork wasundertakenusingalargernumberofplantsamples,including materialfromdifferentlocationsandpurchasedatlocalmarkets. Inthisway,acloserpictureofthecrudedrug“chapéu-de-couro”is obtained.

AllextractswerecomparedbyHPLClookingforcharacteristic compoundsinthepolarextracts.Accordingtothefingerprints,our samplescanbearrangedinfourmaingroups,twoofthem represen-tativeofE.scaberfromMTandMSandathirdsetinagreementwith

E.grandiflorus,beingthefourthsamplethecommercialEchinodorus

sp.Thechromatogramsrepresentingthedifferentprofilesare pre-sentedinFig.2.Table5presentstheretentiontime,theUVdata andthetentativeidentificationofthemainphenoliccompounds foundintheanalyzed“chapéu-de-couro”leafextracts.

TheHPLCchromatogramsrepresentativeofallofourE.scaber

samplesfromMSshowstwomaincompoundsidentifiedas isoori-entinandisovitexin(Fig.2,traceD;peaks2and6,respectively). However,asubsetfromthesamplescanberecognizedby com-paringtheoccurrenceoftwocompoundselutingatretentiontime (Rt)of18.2and25.1min(peaks7and8),respectively,both pre-sentingUV spectrawith maximaat327, 290sh and 245nm, in agreementwithcaffeoyl/feruloylacidderivatives(Schnitzleretal.,

Table5

IdentificationofphenoliccompoundsinEchinodorusscaberandE.grandiflorus

hydroethanolic”extractsbyHPLC-DAD.

Peak Rt(min) max(nm) Tentativeidentificaction

1 5.0 324,279 Catechin(internalstandard)

2 8.6 348,269 Isoorientina

3 9.2 344,269 Swertiajaponinb

4 11.2 334,269 Vitexin-2-O-rhamnosidea

5 11.6 335,269 Vitexina

6 12.4 335,269 Isovitexina

7 18.2 327,290sh,245 Caffeoyl/feruloylacidderivativeb

8 25.1 328,290sh,243 Caffeoyl/feruloylacidderivativeb

Rt:Retentiontime;HPLC-DAD:HighPerformanceLiquidChromatographywith

DiodeArrayDetector.

a_{Identityconfirmedbycoinjectionofstandards.}

b_{IdentityconfirmedbyUVspectrumandcomparisontotheliterature(Schnitzler}

etal.,2007).

2007).Fig.2,traceB, representing samplesL2 and L5 from MT

showsisoorientin(peak2)asthemainconstituentand

vitexin-2-O-rhamnoside(peak4)assecondarycompound,differentlyfrom theMSplants,whichshowverylowcontentofthiscompound.The unidentifiedcrudedrugsampleofEchinodorussp.purchasedinMT (L6inFig.2,traceC)isclosetoE.scaberfromMTaccordingtothe

phenoliccompoundspatternandshowedthehighestcontentin vitexin-2-O-rhamnosidefromallthesamplesinvestigated.

TheHPLCprofileoftheE.grandiflorusextractscorrespondingto “chapéu-de-couro”(L1,L3andL4–Fig.2,traceA)weresimilarto

thosereportedbyGarciaetal.(2010)forasamplefromthesame speciescollectedintheParanástateinsouthernBrazil.Peaks2and 3fromoursampleshowthesameUVspectrumsuggestingthatthey areflavonoidsfromthesamestructuralgroup,namelyisoorientin andapparentlyswertiajaponin.

InastudyofthephenolicconstituentsfromE.grandiflorusssp.

The main flavonoids occurring in the polar hydroethanolic extractsof“chapéu-de-couro”areC-glycosylflavonoidsdifferingin thenumberoffreehydroxyand/ormethoxygroupsintheflavonoid ring,namelyisoorientin,swertiajaponin,vitexin-2-O-rhamnoside andisovitexin.

VitexinisaC-glycosylflavoneusedasachemical markerfor

Passifloraspecies(Dhawanetal.,2004).TheC-glycosylflavonoids foundintheplanthave beenassociatedwithantiinflammatory (Sridharetal.,2006)andantinociceptiveeffect(Kumaretal.,2012). Isoorientinispresentinhighconcentrationsinallextractsof “chapéu-de-couro”(Table2);thisflavoneisalsofoundinspecies ofPassifloraandisfrequentlyassociatedwithanti-inflammatory andantinociceptiveactivities.However,thiscompoundcannotbe consideredthemainresponsiblefor theantinociceptiveactivity of“chapéu-de-couro”,sincetheantinociceptive testsresponded randomlytoisoorientinconcentrations.Swertiajaponinwas ten-tatively identified(Table5)only in E.grandiflorus extractsand, therefore,cannotbethemainresponsiblefortheantinociceptive activity.

Vitexin-2-O-rhaminosideisoftenfoundinPassifloraspecies.In thepresentworkthiscompoundwasquantifiedonlyinextractsof

E.scaberfromMTaswellasthecommercialsample(Table2).In extractswherethisflavonewasnotdetected,however,therewas considerablepercentageofpaininhibition,indicatingthat vitexin-2-O-rhaminosidecannotbethemainresponsiblebytheanalgesic activitypresentedby“chapéu-de-couro”samplesanalyzedinthis work.

IsovitexinwasdetectedinallextractsfromE.scaber,in con-centrationrangingfromtraces to14.60␮g/mg;higheramounts

weredetectedinsamplesfromMS(Table2).Adose-dependent antinociceptiveactivitycouldbeobserved,whencomparing iso-vitexinconcentrationandthepercentageofinhibitionofE.scaber

extracts(Table4).Thesametrendwasobservedwhenanalyzing

E.grandiflorussamples.AscommercialEchinodorusandPassiflora

speciessharethesameC-glycosylflavonoidconstituents,new stud-iesontheanxiolyticeffectof“chapéu-de-couro”areofinterest. RecentreportsontheeffectofPassifloraextractsonthecentral nervoussysteminclude theisoorientinand isovitexin (Li etal., 2011).

OveralltheHPLCmethodpresentedallowedaclearandrapid differentiationofE.scaberanditssubstituteE.grandiflorusandis suitableforcomparativeanalysesofEchinodorussamples.

Thecollectionsdisplayingbetterantinociceptiveeffectshows thehigherisovitexincontentandalsobetterw/wyields,making theMSsamplesthebettersourceforthecrudedrug.Further stud-iesshouldbecarriedout,includingtheharvestingsourcesofthe crudedrugforcommercialpurposestoproviderecommendations onqualitycontrolbasedontheactiveconstituentsandextraction yields.Thus,isovitexincanberegarded,atleastinpart,as respon-siblefortheantinociceptiveactivityandseemstobea suitable chemicalmarkerfor“chapéu-de-couro”crudedrug.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat

nopatientdataappearinthisarticle.

Authors’contributions

CLE,KCLandELDparticipatedinphytochemicalanalysis.VCS participatedininterpretationofNMRspectraldata.EFGCD partici-patedinHPLCanalysis,acquisitionandinterpretationofdata.RVR andDTOMparticipatedintheevaluationofantinociceptive activ-ity,acquisitionandinterpretationofdata.CACparticipatedinPCA analysis,acquisitionandinterpretationofdata.PTSJauthoredthe projectandsuperviseditsexecutionandthedraftingofthispaper andGSHparticipatedinthediscussionofthedatapresentedherein aswellasthedraftingofthemanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WearegratefultoINCT-ÁreasÚmidas(INAU)/CNPq,Centrode PesquisasdoPantanal(CPP-MCTI)andCAPESforscholarshipsand financialsupport.WearealsogratefultoM.Sc.IsaneteGeraldini CostaBieskiforvaluablehelpinplantcollection.

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