Rev. bras. farmacogn. vol.27 número3

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

Original

Article

Antispasmodic

activity

from

Serjania

caracasana

fractions

and

their

safety

Fabiana

L.

Silva

_,

_Joelmir

_L.V.

_da

_Silva

_,

_Juciléia

_M.

_Silva

_,

_Luiza

_S.A.

_Marcolin

_,

Viviane

L.A.

Nouailhetas

_,

_Massayoshi

_Yoshida

_,

_Pedro

_H.

_Vendramini

_,

_Marcos

_N.

_Eberlin

_,

José

M.

Barbosa-Filho

_,

_Paulo

_R.H.

_Moreno

a_{LaboratóriodeTecnologiaFarmacêutica,UniversidadeFederaldeParaíba,JoãoPessoa,PB,Brazil} b_{InstitutodeQuímica,UniversidadedeSãoPaulo,SãoPaulo,SP,Brazil}

c_{DepartamentodeSaúdeII,FaculdadedeFarmácia,UniversidadeNovedeJulho,SãoPaulo,SP,Brazil} d_{DepartamentodeSaúdeIII,FaculdadedeMedicina,UniversidadeNovedeJulho,SãoPaulo,SP,Brazil} e_{DepartamentodeBiofísica,UniversidadeFederaldeSãoPaulo,SãoPaulo,SP,Brazil}

f_{CentrodeBiotecnologiadaAmazônia,Manaus,AM,Brazil}

g_{ThoMSonMassSpectrometryLaboratory,InstitutodeQuímica,UniversidadedeCampinas,Campinas,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received30September2016

Accepted20December2016

Availableonline10February2017

Keywords:

Antispasmodicactivity

Compoundsisolation

GC–MS

Hemolyticassay

Ileumrat

Extracttoxicity

a

b

s

t

r

a

c

t

Inapreviousstudy,wereportedtheantispasmodicandgastroprotectiveeffectsoftheSerjania

cara-casana(Jacq.)Willd.,Sapindaceae,extract.Inthepresentstudy,weevaluatedtheLD50,hemolyticand

antispasmodicactivitiesofitsfractionsandcharacterizeditsmajorconstituentsbyisolationandGC–MS. Theanimals showednon-toxicsymptomswithoraldosesup to2000mg/kg, suggestingasafeoral administration.Furthermore,alowhemolyticactivitywasdetectedforthesaponinfraction. Antispas-modicactivityofthefractionswasevaluatedthroughcarbachol-inducedcontractionsinratileum.The hexanefractionwasthemostpotent(IC5068.4±5.9␮g/ml)followedbythedichloromethanefraction (IC50161.3.4±40.7␮g/ml).Butanolfractionwasthelesseffective(IC50219.8±60.3␮g/ml).The phyto-chemicalstudyoftheS.caracasanafractionsaffordedtheisolationoffriedelin,␤-amyrin,allantoinand quercitrin.ThisisthefirsttimethatthepresenceofallantoinandquercitrinintheSerjaniagenushasbeen reported.AmongtheisolatedcompoundsandthosecharacterizedbyGC–MS,␤-amyrinand␤-sitosterol werepresentinthemostactivefractions,hexaneanddichloromethane,andtheymayberelatedtoits antispasmodicactivity.Inaddition,spathulenolwasonlyfoundinthehexanefractionanditspresence mightjustifythehighestantispasmodicactivityobservedforthisfraction.

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

Introduction

Serjaniacaracasana(Jacq.)Willd.,Sapindaceae,isusedfor weav-ing baskets and rustic ropes, mostly because it is considered ichthyotoxic.ThisbeliefcomesfromitssimilaritywithS.lethalis, which is knownto contain hemolyticsaponins (Teixeira et al., 1984).Infact,allpreviouspublicationsaboutS.caracasana(Aragão andValle,1973;XavierandMors,1975;CordeiroandValle,1975; Maia-Braggioetal.,1978)usedS.lethalisinstead,asitwaslater confirmed(Teixeiraetal.,1984).

∗ Correspondingauthor.

E-mail:prmoreno@iq.usp.br(P.R.Moreno).

SomeSerjaniaspeciesareusedinfolkmedicinesuchasS.comata

(rheumatism),S.lethalis(kidneypain,anti-inflammatory),S.erecta

(gastricpain,anti-inflammatory),S.marginata(gastricpain)andS. triquetra(diuretic)(ChávezandDelgado,1994;Guarin-Netoetal., 2000;Agraetal.,2008;Péricoetal.,2015).Basedonthesepopular uses,ourgrouphaspreviouslydemonstratedthattheS.caracasana

hydroethanolicextractshowedagastroprotectiveeffectby inhib-itinggastriculcerinductionandaninvitroantispasmodicactivity (Silvaetal.,2012).

Inthisstudy,wefurtherevaluatedtheantispasmodicactivity toidentifythecompoundsresponsibleforthiseffect.Theextract safety was initially evaluated through the determination of its medianlethaldose(LD50)andthesearchforhemolyticsaponins inthebutanolfraction.

http://dx.doi.org/10.1016/j.bjp.2016.12.002

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

Materialsandmethods

Chemicalmaterials

StandardsampleofsaponinsofQuillajasaponariawasakind courtesyofProf.Dr.CarmenQueiroga.Thecitratedbovineblood waspurchased attheUSP VeterinaryHospital(USP-Brazil). Sil-ica gel 60 F254 TLC aluminum sheets were purchased from MerckCompany.Allothersreagentsandsolventswereanalytical grade.

Plantmaterial

The aerial parts of Serjania caracasana (Jacq.) Willd., Sapin-daceae,werecollectedatthebaseofPicodoJabre(7◦₁₅′_34.27′′ S,37◦₂₃′_8.53′′_{W),Paraiba,Brazilduringfructificationperiod(June,} 2009)byDr.JoseanF.Tavares(UFPB).Theplantmaterialwas identi-fiedbyProf.Dr.MariadeFátimaAgra(UFPB).Avoucherspecimen (No.M.F.Agraetal.,6963)wasdepositedintheHerbariumProf. LauroPiresXavier(JPB),atthesameUniversity.

Phytochemicalanalysis

Spectroscopicpropertiesoftheisolatedcompounds

NMR analysis was performed in an Agilent INOVA-500 (500MHz)spectrometer.Highresolutionmassspectrawere per-formedonaMicroTOFLCmassspectrometerfromBrukerDaltonics andIRspectrawasrecordedwithaBomeminstrument.The struc-turesoftheisolatedcompoundsweredeterminedby1_Hand13_C NMR spectroscopy,using one and two-dimensionaltechniques, togetherwithIRandHRMSdatabycomparisonwithdata previ-ouslypublishedforthesecompounds.

Gaschromatography–massspectrometry(GC–MS)analysis

The hexane and CH2Cl2 fractions had their chemical com-position analyzed by gas chromatography–mass spectrometry (GC/MS),performedusingaShimadzuGCMS-QP2010Ultrasystem, withafusedsilicacapillarycolumncoatedwith5% polyphenyl-siloxane/95% dimethylpolysiloxane (Rxi®-5ms,10m×0.10mm ID×0.10␮mfilmthickness), electronionizationsystem operat-ingat70eVwithaninterfacetemperatureof 260◦_{C,scan time} of 0.1scans/s and acquisition mass range of 35.0–500.0Da. For theGCanalysis,theinjectiontemperaturewassetat250◦_C,the oventemperatureprogram was100◦_{C for 1min,100–290}◦_{C at} 15◦_{C/min,maintaining290}◦_{Cfor15minwithheliumasacarrier} gas(0.42ml/min).Thecompoundidentificationwasperformedby comparingthemassspectrawiththeWileylibraryandliterature data(Adams,2007)togetherwiththeco-injectionofstandards.The relativecompositionwasobtainedfromtheelectronicintegration measurementsusingflameionizationdetection(300◦_C)without takingintoaccountrelativeresponsefactors,inthesameconditions describedabove.

Extractpreparation

Thepowderedair-driedaerialpartsofS.caracasana(1916g) were extracted exhaustively with 96% aqueous ethanol solu-tionat roomtemperature.The combinedextractswerefiltered and concentrated under reduced pressure at 40◦_C afford-ing a hydroethanolic extract (202.84g; 10.6% extraction yield) (EtOH).

Extractfractionationandcompoundisolation

TheEtOHextract(200g)wassuspendedinMeOH–H2O(70:30, v/v) mixture, and it wassubsequently fractionedwith hexane, CH2Cl2 andBuOH.Eachsolventfractionwasthenevaporatedto drynessunderreducedpressuretogivehexane(31.53g;15.8%),

CH2Cl2 (21.9g;10.96%)andBuOH(44.50g;22.2%)fractions.The CH2Cl2andBuOHfractionswereseparatelysubjectedtoCCon sil-icagelusingstepgradientsofhexane–EtOAcandEtOAc–MeOH. Columnfractionsthatpresentedonlyonemajorcompoundwere resubmitted tothe samechromatographic separation to obtain thepurifiedcompounds.CH2Cl2fractionyieldedfourcompounds (friedelin(1),␤-amyrin(2),stigmasteroland␤-sitosterol),while BuOHfractionaffordedonecompound(␤-sitosterolglucoside).A remainingpartoftheBuOHfractionwassuspendedwith300ml ofMeOH.Thesolutionwasfilteredandinsolublepartreserved. Diethyletherwascarefullyaddedintothesolutionuntilsome pre-cipitationstarted,and thenitwasplacedin thefreezer(−22◦_C for24h).Theprecipitateformedwasremovedbyfiltration,and reserved.Thisprocesswasrepeatedtwice.Thepooledprecipitates weresolubilizedwithMeOHandconcentratedunderreduced pres-sureat40◦_{C,togiveBuOH-1fraction.Theremainingorganicphase} wasevaporatedunderreducedpressuretodrynesstogive BuOH-2fraction.TheBuOH-2fractionwassubjectedtoCConsilicagel usingstepgradientsofCHCl3–MeOHandafterfixedsolventsystem (CHCl3–acetone–formicacid,75:16.5:8.5(v/v))(Ikedaetal.,1991) toobtain27fractionscombinedaccordingtotheirTLCprofilesinto fifteenmajorfractions(Fr1–Fr15).FromfractionFr7obtained allan-toin(3)asprecipitateaftersolventdrying.FractionFr8waspurified bypreparativeTLC(CHCl3–MeOH,80:20(v/v))togivequercitrin (4).

Friedelin(1).5.6mg,0.03%yield;amorphouspowder;IRmax (KBr)cm−1_{:3000,2848,1714;}1_{HNMR(500MHz,CDCl}

3):1.16(3H,

s,H-28),1.03(3H,s,H-27),0.99(3H,s,H-26),0.98(3H,s,H-30),0.93 (3H,s,H-29),0.86(3H,slap,H-23),0.85(3H,s,H-25),0.70(3H,s, H-24).13_{CNMRdatawereconsistentwiththosepreviouslyreported} (Akihisaetal.,1992).

␤-Amyrin(2).169.8mg,1.05% yield;white powder;IRmax (KBr)cm−1_{:3299,2946,2852,1034;}1_{HNMR(500MHz,CDCl}

3): 5.16 (1H, t, J=3.6Hz, H-12), 3.20 (1H, dd, J=11.15, 4.5, H-3), 1.11 (3H, s, H-27), 0.97 (3H, s, H-23), 0.94 (3H, s, H-26), 0.91 (3H, s, H-24), 0.80 (3H, s, H-28), 0.76 (3H, s, H-25). 13_{C NMR} datawereconsistentwiththosepreviouslyreported(Diasetal., 2011).

Stigmasterol. 37.8mg, 0.24% yield; white powder;1_{H NMR} (500MHz;CDCl3):5.33(1H,sl,H-6),5.13(1H,dd,H-23),5.00(1H,

dd,H-22),3.50(2H,m,H-3).13_{CNMRdatawereconsistentwith} thosepreviouslyreported(Kojimaetal.,1990).

␤-Sitosterol. 21.4mg, 0.13% yield; white powder; 1_{H NMR} (500MHz;CDCl3):5.33(1H,sl,H-6),3.50(2H,m,H-3).13CNMR datawereconsistentwiththosepreviouslyreported(Kojimaetal., 1990).

␤-Sitosterolglucoside.3.2mg,0.04%yield;amorphous pow-der; 1_{H NMR (500MHz,Py-d}

5): 5.32(1H, sl, H-6), 5.04 (1H, d, H-1′_{),4.55(1H,map,H-6}′_{),4.28(1H,sap,H-4}′_),3.99(1H,m, H-3),2.70/2.45(1H,m,H-4),0.98(3H,d,H-21),0.94(3H,sl,H-19), 0.91(3H,sap,H-26),0.87(3H,dap,H-29),0.84(3H,d,H-27),0.64 (3H,s,H-18).13_{CNMRdatawereconsistentwiththosepreviously} reported(Kojimaetal.,1990).

Allantoin(3).6.5mg,0.08%yield;colorlesscrystal;IRmax(KBr) cm−1_{:3439, 3344,3224,3063,1782,1659;} 1_{HNMR (500MHz,} DMSO-d6): 10.51(1H,br s,H-1), 8.03 (1H,s, H-4),6.91(1H, d,

J=13.5Hz,H-3),5.78(2H,s,H-8),5.24(1H,d,J=13.5Hz,H-6).13_C NMRdatawereconsistentwiththosepreviouslyreported(Sripathi etal.,2011).EI-HRMS(negative-ionmode)m/z:157.03722[M−H]− (C4H6N4O3requires157.0315).

Toxicologicalassays

Medianlethaldose(LD50)

MaleSwissmice(35–40g)wereseparatedinthreegroupsoffive animalsforeachtreatment(n=5).Animalsfromeach experimen-talandcontrolgroupswerehostedincagesmaintainedatconstant roomtemperature(22±1◦_{C)andsubjectedtoa12/12hlight/dark} cyclewithaccesstofoodandwateradlibitum.Proceduresinvolving animalsandtheircarewereperformedinconformitywithOECD 420(2001),adoptedinourlaboratory,andincompliancewith cur-rentinternationallyacceptedinstructionsforthecareoflaboratory animalsandethicalguidelines.Furthermore,clearancefor conduct-ingthestudywasobtainedfromtheEthicsCommitteeofAnimal UseoftheNovedeJulhoUniversity(approval#AN0003/11).

Inordertodeterminetheoralmedianlethaldose(LD50)ofthe EtOHextract,thistestfollowedthemethoddescribedforMillerand Tainter(1944)withsamemodifications.Thecontrolgroupreceived thevehicle(0.1%Tween20indistillatedwater).Twodoses(1000 and2000mg/kg,inavolumeof1ml/g)weregivenorally,bygavage, totwogroupsofmiceforthedeterminationofLD50.Theanimals wereobservedduringthefirst180minafterthetreatmentandafter 24,48and72hforanytoxicsignsandsymptoms.

Qualitativeassayofhemolyticsaponins

Thequalitativehemolyticactivityofthesaponinswastested withbovinebloodreagentasdescribed bySharmaetal.(2012) withsomemodifications.Briefly,aliquotsof1mlofcitratedbovine bloodwerewashedthreetimeswith9mlofsaline(0.9%;w/vNaCl) followedbycentrifugationat180×gfor5min.Thecell suspen-sionwasfinallypreparedbydilutingthepelletto3%(v/v)insaline solutiontoobtainthebloodreagent.Also,tovisualizethesaponins acomparativesprayreagent(Liebermann–Burchardreagent)was prepared(WagnerandBladt,1996).

Theassaywasperformedwitha10␮laliquotofsaponinsfrom

Quillajasaponaria(SQ)andBuOHfraction(conc.20mg/ml, solubi-lizedinMeOH–H2O,70:30(v/v))appliedinTLCplateswhichwere elutedwiththesolventsystemCHCl3–MeOH–TFA0.5%(60:40:5 (v/v))to6.5cmfromtheorigin.Aftertheelution,theTLCplates wereairdried.

OneofthedevelopedTLCplateswasimmersedfor20sinaglass dishcontainingthe3%bloodreagentfreshlyprepared.Afterthis time,theTLCplatewasremovedandheldverticallyfor30s,and subsequentlyimmersedinsalinefor30s. Finally,this TLCplate washeldverticallyforcompletedryingandfurthervisualization ofthehemolyticspots.Thehemolyticspotswerecomparedwith thedeveloped TLC sprayed with Liebermann–Burchardreagent (Sharmaetal.,2012).

Quantitativeassayofhemolyticsaponins

Thequantitativehemolyticactivityofthesaponinswas eval-uated with a bovine blood cell suspension by turbidimetry as describedby Xieetal. (2008)withsomemodifications. Briefly, aliquotsof10mlofbloodwerewashedthreetimeswithsaline solu-tionbycentrifugationat180×gfor2min.Thecellsuspensionwas finallypreparedbydilutingthepelletto5%(v/v)insalinesolution. TheSQand BuOHfractionsamplesweresolubilized initially insalineto25mg/ml.Forthe assay,180␮loffreshly prepared 5% blood cell suspension was mixed with 20␮l of sample or controlsolution.Thesampleconcentrationrangedfrom820.0to 16.4␮g/mlforBuOHfraction,andfrom500.0to5␮g/mlforSQ. Themicroplatewasincubatedfor30minat37◦_{Candcentrifuged} at70×g for10min.Analiquotofeachsupernatant (75␮l)was transferredtoaflat-bottommicroplateandthefreehemoglobin was measuredat 540nm (Silveira et al., 2011). Saline and SQ (100␮g/ml)wereconsideredasminimalandmaximalhemolytic controls.Theconcentrationinducing50%ofmaximumhemolysis

(HC50)wascalculated bynon-linerregression (GraphPadPrism 5.01).Eachexperimentincludedtriplicatesforeachconcentration. Theresultsofthequantitativehemolyticactivityarepresentedas mean±SDandtheotherresultsasmean±SEM.

Pharmacologicalassay

Antispasmodicactivityonileumisolatedrat

Ileumstrips wereisolatedfromrat following the methodol-ogydescribed by Walker and Wilson(1979) and suspended in organbaths(5ml)containingmodifiedKrebsphysiological solu-tion,consistingof(mmol/l):NaCl117.0;KCl4.7;NaH2HPO4·H2O 1.2;MgSO4·7H2O1.3;CaCl2·2H2O2.5;NaHCO325.0;glucose11.0; pH7.4(SunandBenishin,1994).Furthermore,theclearancefor conductingthestudywasobtainedfromtheEthicsCommitteeof AnimalUseoftheFederalUniversityofSãoPaulo(approval#CEUA 4195060514/14).

Thehexane,CH2Cl2andBuOHfractionsweredissolvedinitially in0.01%CremophorELanddilutedinMilliQwatertoobtainthe stocksolutionof10mg/mlwhichwasstoredat0◦_C.

Thetissuesweremaintainedunder1gtension,bubbled con-tinuouslywithO2at37◦C.Theywereattachedtoforceisometric transducers and connectedto a datasystemAQCD(AVS Proje-tos,Brazil).FollowingcontrolcontractionswithKCl (40mmol/l) or carbachol (1␮mol/l), and washing with a fresh Krebs solu-tion,tissuestripswereexposedtoconcentrationgradientranging from 500 to 9␮g/ml of hexane, CH2Cl2 or BuOH fractions for 15min(WalkerandWilson,1979),thenstimulatedagainwiththe previousreferredconcentrationofcarbachol.Theantispasmodic activityofthesampleswasexpressedasmaximum contraction (Emax value) obtained in the presence of the distinct fraction relativetothemaximumcontractionintheirabsence(control). The concentration of fraction that reduces to 50% a maximal responseforanagonist(IC50)valuesweredeterminedfrom indi-vidual concentration–response curves by non-linear regression (Jenkinsonetal.,1995).

Statisticalanalysis

Statisticalsignificancebetweencontrolandexperimentalgroup wasevaluatedusingeitherStudent’st-testorone-wayANOVA fol-lowingDunnett’sMultipleComparisonTest.Datawereconsidered significantwhenp<0.05.

Resultsanddiscussion

Medianlethaldose(LD50)

Inourearlierstudy,weobservedtheS.caracasanaEtOHextract significantly inhibited KCl pre-contracted ileum, indicating an interestinginvitroantispasmodicactivity(Silvaetal.,2012). How-ever,duetothepossibletoxicitytomammalsthatcouldberelated tothepresenceofhemolyticsaponins,asreportedforotherSerjania

A

B

0.6 0.6

Rf Front

0.4

0.2

0.1

Start

1 1

2 2

0.4

0.3

Fig. 1.TLC of BuOH fraction of Serjania caracasana aerial parts (1) and

of rich fraction saponins of Quillaja saponaria (SQ) (2). Solvent system:

chloroform–methanol–trifluoroaceticacidaqueoussolution0.5%(60:40:5,v/v).

Detection:bloodreagent3%(A);Liebermann–Burchardreagent(B).

andhydroethanolicextractfromS.marginata,wheremicetreated withasingleoraldoseof5000mg/kghadnosignsoftoxiceffects inanacutetoxicitystudy(Arrudaetal.,2009;Péricoetal.,2015).

Qualitativeandquantitativeassaysofhemolyticsaponins

DespitethelowacutetoxicityofS.caracasanaEtOHextract,the presenceofhemolyticsaponinsinSerjaniaextractsshouldbe inves-tigated.Thesecompoundsareconsideredharmfulnotonlybecause oftheiracutetoxiceffects, asreportedtothesaponinsisolated fromS.lethalis,serjanosidesA,BandC,thatshowedtoxicacute effectsinratsandmice(Teixeiraetal.,1984).Also,chronic admin-istrationofsaponinsinanimalsisknowntoaffecttheirgrowth oralteringtheirpalatabilityand,consequently,foodconsumption oralteringthedigestionprocessandabsorption(Oleszek,1996). TheTLCchromatogramoftheS.caracasanaBuOHfractionsprayed withLiebermann–Burchardreagent(Fig.1B)showedthepresence ofthreemainpurplezones,characteristicofsaponins,atRf’s0.3,

0.4and 0.6, withthelatterasthemajor component.The com-parisonofthisTLCprofilewiththatobtainedaftersprayingwith theBloodreagent(Fig.1A)indicatedthattheobservedsaponins werenotabletocausehemolysisintheTLCassayinthe concen-trationtested.Incontrast,thepositivecontrol(SQ)showedsome whitespots(Rf=0.1and0.2)againstapinkbackground(Fig.1A),

characteristic of hemolysis. However, SQ also presented two othercompounds thatwerenon-hemolytic compounds(Rf=0.4

and 0.6), typically saponins for Liebermann–Burchard reagent (Fig.1B).

Similarly,inthequantitativehemolyticactivity,theBuOH frac-tion exhibited a low hemolysis rate(∼2%) until themaximum concentrationtested(820␮g/ml)whencomparedwiththepositive control (SQ) (100% at 100␮g/ml) (Fig. 2). Once at the maxi-mumconcentrationtestedthehemolysisrateswereunder50%of hemolyticactivity,theBuOH fractionHC50valuewasestimated as>1000␮g/ml.Thepositivecontrol(SQ)showedhighhemolytic activity (HC50=24.03±1.03␮g/ml) under the same conditions. Thus,differentthanwouldbeexpectedforanichthyotoxicspecies, ourresultsindicatedthatS.caracasanasaponinscouldbe consid-eredasnon-hemolytic.

Antispasmodicactivityonileumisolatedrat

As S. caracasana demonstrated low toxicity and almost no hemolyticeffect, we decided toinvestigate which extract frac-tionwouldberesponsiblefortheantispasmodicactivitypreviously reported(Silvaetal.,2012).

TheantispasmodicactivitywasanalyzedfortheS.caracasana

hexane,CH2Cl2andBuOHfractions.Fig.3(A)–(C)showsthe over-allinhibitoryeffectofthesefractionsonratileumcontractions. Thehexane fractionsignificantly inhibitedthemaximumileum contractionsattheconcentrationsof27,81,243and500␮g/ml (Fig.3A),presentingtherespectiveamplitudedecreasevaluesof 66.1±7.1,47.5±3.8,24.5±2.4and23.5±7.3%(Fig.3A),whilefor theCH2Cl2fractionasignificantinhibitionstartedatthe contrac-tionsof81,243and 500␮g/ml,withEmax valuesof62.7±12.6, 47.7±7.9and 20.2±3.7%respectively (Fig.3B).In contrast,the BuOHfractionwasonlyabletosignificantlyreducetheintensity ofthecontractionsat243and500␮g/mlwithrespectiveEmaxof 44.9±13.1and25.8±5.5%(Fig.3C).

Theseresultsindicatedthattheantispasmodicactivityis dis-tributed in all S. caracasana fractions. However, the n-hexane fractionwasmorepotentthanthecrudeEtOHextract,thatwas 46%attheconcentrationof81␮g/ml(Silvaetal.,2012).TheCH2Cl2 fractionwaslessactiveshowingsimilarresultstothoseobtained withthecrudeextract,andtheBuOHfractionshowedevenless activitythantheextract.Thus,allthefractionswereinterestingto searchfornaturalantispasmodiccompounds.

Phytochemicalanalysis

Inordertodeterminethepossiblecompoundsresponsiblefor theantispasmodicactivity,CH2Cl2andBuOHfractionswere sub-mittedtocolumnchromatography.Fromthesefractions,wecould

100

A

B

75

50

25

0

0 100 200

[SQ] µg/ml

Hemolysis

, %

Hemolysis

, %

300

0

0 200 400 600

[BuOH] µg/ml

800 1000 2

4 6 8 10

100

A

B

C

50

25

0

CCh 9 27 81 243 500

[Hexane] µg/ml

Contr

actions

, %

100

75

50

25

0

CCh 9 27 81 243 500

[CH2CI2] µg/ml

Contr

actions

, %

100

75

50

25

0

CCh 9 27 81 243 500

[BuOH] µg/ml

∗∗∗

∗∗∗ ∗∗∗ ∗∗∗

∗∗∗ ∗∗∗ ∗∗∗ ∗∗

∗∗

Contr

actions

, %

Fig.3. Effectofhexane(A),CH2Cl2 (B)orBuOH(C)fractionsofSerjania

cara-casanaaerialpartsonpre-contractedileumbycarbachol1␮M(n=3).One-way

ANOVAfollowedbyDunnett’sMultipleComparisonTest:**p<0.01;***p<0.001

(control×fraction).

isolateandcharacterizesevencomponents,twooleanane triter-penes:friedelin(1)and␤-amyrin(2),threesteroids:stigmasterol, ␤-sitosteroland␤-sitosterolglucoside,oneuratederivative: allan-toin(3)andoneflavonol:quercitrin(4).

Table1

CompoundscharacterizedinthehexaneandCH2Cl2fractionsofSerjaniacaracasana

aerialparts.

Hexanefraction CH2Cl2fraction

Compound RT(min) % RT(min) %

Spathulenol(5) 5.8 4.2 – –

6,10,14-Trimethyl-2-pentadecanone 7.3 1.7 7.3 1.8

Methylpalmitate 7.7 1.7 7.7 2.7

Ethylpalmitate 8.1 6.2 8.1 8.6

␤-Sitosterol 14.7 1.2 14.7 2.1

␤-Amyrin(2) 15.1 60.0 15.1 51.0

Total 75.0 66.2

O

H H H

1

HO

H

2 H

H

H N

N H O

N H

O O

NH₂

3

O HO

OH

O

O

OH

OH

O HO

HO

OH

4

Additionally,thetwomostactivefractions,hexaneandCH2Cl2, hadtheirchemicalcompositioncomparedbyGC–MS,indicatinga similarchemicalcompositionforthesefractions(Fig.4).Themass spectraobtainedallowedtocharacterizesixcompounds(Table1) representing79.8%and66.6%ofthetotalofcompoundsdetectedin theGC–MS.Thetwofractionsshowedthesamemajorcomponents, ␤-amyrin(2)(60.0%and51.0%)andethylpalmitate(6.2%and8.6%), respectivelyforhexaneandCH2Cl2fractions.Additionally,inthe hexanefractionwasfoundspathulenol(5)inarepresentative quan-tity(4.2%).Thissesquiterpeneisacommonessentialoilcomponent inseveralplantspecies,ithasshownanantispasmodicactivityin uterusringscontractionmodelinducedbyKCl(Perez-Hernandez etal.,2008).Thepresenceofspathulenolexclusivelyinthehexane fractionmightjustifythehighestantispasmodicactivityobserved incomparisontotheotherfractions.

HO H

H

H H

5

In a recent study, Coutinho et al. (2015) reported the seed fatty-acidcompositionsforsixteenSapindaceaespecies.Serjania

species,includingS.caracasana,presentedhighlevelseicosenoic acidand other palmitic acidesters. In ourstudy, palmitic acid methylandethylesterswerefoundinbothhexane andCH2Cl2 fractions.Friedelin(1)and␤-amyrin(2),togetherwithother triter-penes,aretypicallyisolatedwithinthegenusSerjania,suchasthe presenceof1and2reportedinS.salzmanniana(Barbosa-Filhoetal., 1988).In thisstudy,wedetectedforthefirst timeina Serjania

2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 1.0

2.0 3.0 4.0 5.0 6.0 7.0 8.0

(x100 000) TIC

2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 1.0

2.0 3.0 4.0 5.0

(x100 000) TIC

8.1

7.3 7.7

10.7

10.8 11.9

15.1

14.714.8 16.216.5 15.5 15.1

7.3 7.7 8.1

14.7 15.5 16.1

16.4

A

B

Fig.4.GC–MSchromatogramofhexane(A)andCH2Cl2(B)fractionsofSerjaniacaracasanaaerialparts.

Accordingtoourresults,S.caracasanasaponinsandquercitrin arenotthemainmetabolitesresponsiblefortheantispasmodic effect observed, but they may contribute to the overall effect observed in the crude extract. In similar studies, it has been observedthatthefractionsenrichedwithsaponinsandflavonol derivatives,includingquercitrin,showedsignificantantispasmodic activityintheacetylcholinemodel(Truteetal.,1997).

In conclusion,ourfindings demonstrated thatalthough ofS. caracasanaisconsideredanichthyotoxicspecies,thisproperty,if present,cannotberelatedtothepresenceofhemolyticsaponins. Thetoxicityformammalspecieswasnotconfirmedbytheacute toxicitytest,requiringcomplimentarystudiesonthesubject.In addition,future studiesshouldbecarried outwiththeisolated compoundstoverifytheirspasmolyticactivity.

Authors’contributions

FLS(Ph.D.student)contributedbyconductingthe phytochem-icallaboratorywork,thehemolyticevaluation anddraftingthe paper.JLVScontributedbyconductingthepharmacologicalassays, teachingJMSandLSAMintheconductingofthepharmacological assaysandbycriticalreadingofthemanuscript.VLANsupervised thepharmacologicalworkandcontributed tocritical readingof themanuscript.MYcontributedwiththecharacterizingofmany isolatedsecondarymetabolites.PHVcontributeddeterminingthe EI-HRMSofallantoin.MNEsupervisedthelaboratoryworkand con-tributedtocriticalreadingofthemanuscript.JMBFdesignedthe phytochemicalworkandcontributedguidingandsupportingthe plantcollectionandthelaboratorywork.PRHMcontributed sup-portinganddesigningthephytochemicalworkandthehemolytic

assays,supervisedthelaboratoryworkandcontributed to criti-calreadingofthemanuscript.Alltheauthorshavereadthefinal manuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Ethicalresponsibilities

Protectionofhumanandanimalsubjects. Theauthorsdeclare thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftheresponsibleClinicalResearchEthicsCommitteeandin accordancewiththoseoftheWorldMedicalAssociationandthe Helsinki Declaration.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearsinthisarticle.

Righttoprivacyandinformedconsent.Theauthorsdeclarethat nopatientdataappearsinthisarticle.

Acknowledgments

TheauthorsaregratefultoCNPq/RENORBIOandCAPES/Brazil forfinancialsupportandresearchfellowships.

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