The anthelmintic effect of plant extracts on Haemonchus contortus and Strongyloides venezuelensis

ContentslistsavailableatScienceDirect

Veterinary

Parasitology

j o ur na l ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / v e t p a r

The

anthelmintic

effect

of

plant

extracts

on

Haemonchus

contortus

and

Strongyloides

venezuelensis

Camila

O.

Carvalho

_,

_Ana

_Carolina

_S.

_Chagas

_,

_Fernando

_Cotinguiba

_,

_Maysa

_Furlan

_,

Luciana

G.

Brito

_,

_Francisco

_C.M.

_Chaves

_,

_Marília

_P.

_Stephan

_,

_Humberto

_R.

_Bizzo

_,

Alessandro

F.T.

Amarante

a_{UNESP–UniversidadeEstadualPaulista,DepartamentodeParasitologia,InstitutodeBiociências,CaixaPostal510,Botucatu,SP,CEP18618-000,Brazil} b_{EmbrapaPecuáriaSudeste,SãoCarlos,SP,Brazil}

c_{UNESP– UniversidadeEstadualPaulista,DepartamentodeQuímicaOrgânica,InstitutodeQuímica,Araraquara,SP,Brazil} d_{EmbrapaRondônia,PortoVelho,RO,Brazil}

e_{EmbrapaAmazôniaOcidental,Manaus,AM,Brazil} f_{EmbrapaAgroindústriadeAlimentos,RiodeJaneiro,RJ,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received10January2011

Receivedinrevisedform22June2011 Accepted30July2011

Keywords:

Gastrointestinalnematodes Folkmedicine

Plantextracts Control

a

b

s

t

r

a

c

t

Theindiscriminateuseofanthelminticshasresultedintheestablishmentofparasite resis-tance.Thus,thisstudyaimedtoevaluatetheinvitroantiparasiticeffectofplantextractson HaemonchuscontortusinsheepandtheinvivoeffectonStrongyloidesvenezuelensisinRattus norvegicus.TheplantextractsfromPipertuberculatum,Lippiasidoides,Menthapiperita,Hura crepitansandCarapaguianensis,producedatdifferentresearchinstitutions,werechemically analyzedandevaluatedthroughtheegghatchtest(EHT)andlarvaldevelopmenttest(LDT) inH.contortus.P.tuberculatum(150and250mgkg−1_{ofbodyweight)wasevaluatedforits}

anthelminticactiononR.norvegicusexperimentallyinfectedwithS.venezuelensis.Inthe EHT,theLC50andLC90oftheextractswererespectivelyasfollows:0.031and0.09mgmL−1

forP.tuberculatum,0.04and0.13mgmL−1_{forL.sidoides,0.037and0.10mgmL}−1 _forM.

piperita,2.16and17.13mgmL−1_{forH.crepitansand2.03×10}−6_and1.22×10−12_mgmL−1

forC.guianensis.IntheLDT,theLC50andLC90wererespectively:0.02and0.031mgmL−1

forP.tuberculatum,0.002and0.04mgmL−1_{forL.sidoides,0.018and0.03mgmL}−1_forM.

piperita,0.36and0.91mgmL−1_{forH.crepitansand17.65and1890mgmL}−1_forC. guia-nensis.TheextractofP.tuberculatumshowedthefollowingsubstances:piperamidesas (Z)-piplartine,(E)-piplartine,8,9-dihydropiplartine,piperine,10,11-dihydropiperine,5,6 dihydropiperlongumineandpellitorine.Themajorcompoundsoftheoilswerethymol (76.6%)forL.sidoides,menthol(27.5%)forM.piperitaandoleicacid(46.8%)forC. guia-nensis.Regardingtheinvivotest,neitherdoseofP.tuberculatumcausedanysignificant reduction(P>0.05)inwormburdenandfecaleggcountscomparedwiththecontrolgroup. WeconcludethattheextractsofP.tuberculatum,L.sidoidesandM.piperitahaveeffective activitywhentestedinvitro,butthedosesoftheextractofP.tuberculatumhavenoeffect whenemployedininvivotests.

© 2011 Elsevier B.V. All rights reserved.

∗ Correspondingauthor.Tel.:+551438116239;fax:+551438153744.

E-mailaddresses:cocarvalho@ibb.unesp.br,cocarvalhobio@yahoo.com.br(C.O.Carvalho).

1. Introduction

Widelydistributedaroundtheworld,Haemonchus con-tortus,agastrointestinalnematodeusuallyfoundinsmall ruminants,causeslargeeconomiclossestolivestock breed-ers by causing appetite depression, damages in gastric functionandalterationsin totalproteincontent,energy andmineralmetabolism(Fox,1993).Themain prophylac-ticmethodusedagainstthisparasitehasbeenanthelmintic treatments.However,thewidespreadandindiscriminate administrationof anthelminticshasresulted in parasite resistance.Thefirstcaseofresistancetoanthelminticswas accuratelydescribedbyDrudgeetal.(1964).Thereafter, manystudiesreportingdecreasedanthelmintic effective-nesshavebeenpublished.

Anthelminticsderivedfromplantscanbean alterna-tivefor the treatmentof parasitic infections (Akhtaret al.,2000).Researchin thefield of medicinalplantsis a goodsourceofknowledgeregardingthepotentialaction ofplantextractsoncertaindiseasesandpests.Asaresult, thisareaofstudyhaswitnessedimpressivedevelopment relatedtohumanandanimalhealth.Therearereports indi-catingantiparasiticeffectsofsomeplantspecies,suchas Pipertuberculatum,Lippia sidoides,Mentha piperita,Hura crepitansandCarapaguianensis.Themaincharacteristics oftheseplantsaredescribedinthefollowingparagraphs.

ThecommonnameofP.tuberculatum (Piperaceae)in Brazilis “pimentalonga” or“pimento-d’arta”. ThePiper genusisdistributedinbothhemispheresintropicaland subtropicalregions(JaramilloandManos,2001).Ithasbeen thesubjectofstudiesthathavenoteditsinsecticidal, fungi-cidal,andtrypanocidalactions(Mirandaetal.,2002;Scott etal.,2008; Freire-de-Limaet al.,2008).The toxicityof PiperaduncumtothecattletickRhipicephalus(Boophilus) microplushasalsobeenreported(Silvaetal.,2009).

NativeofnortheasternBrazil,L.sidoides(Verbenaceae) ispopularlyknownas“alecrim-pimenta”,“estrepa-cavalo” and“alecrim-bravo”. Scientific studieshave revealedits effectsagainstsomebacteria(Aguiaretal.,1984;Baraand Vanetti,1998),Leishmania(Oliveiraetal.,2009a)andAedes aegyptilarvae(Carvalhoetal.,2003).

Inturn,M.piperita(Lamiaceae),whichcomesfromthe Mediterraneanregionandisknownaspeppermint,is culti-vatedasahybridofMenthaaquaticaL.andMenthaspicataL. acrosstheworld.Itexhibitsantiseptic,antibacterial, fungi-cidal,antispasmodicandstimulantactions(Mimica-Duki ´c etal.,2003;McKayandBlumberg,2006).

H. crepitans (Euphorbiaceae) is naturally distributed throughoutCentralandSouthAmerica,fromCostaRicato theAmazon.InBrazil,itispopularlyknownas“assacu” or“ac¸ac¸u”(Brondani,2006).Itsseedsandsapwere for-merlyusedasapurgativeandalsoasapopularmedicine totreat elephantiasis,leprosy,rheumaticfever,swelling and intestinal parasites (Francis, 1990). The latex has beenobservedtohaveaneffectonlarvaeoftheticksR. (Boophilus)microplusandR.sanguineus(Brondani,2006).

Finally,C.guianensis(Meliaceae)isatreethatis gener-allyfoundbothinCentralandinSouthAmerica,popularly knownas“andiroba”.Theessentialoilextractedfromthis plant is used industrially in theproduction of candles, shampoos,soapsandrepellents(PastoreJuniorandBorges,

1998,1999).Somestudieshavereportedvarious effects producedbythisplant,suchasanti-allergicandanalgesic effects(Penidoetal.,2006a),acaricidalaction(Fariasetal., 2009),anti-inflammatoryeffect(Penidoetal.,2006b)and insectrepellentaction(Miotetal.,2004;Mendonc¸aetal., 2005).Furthermore,theteapreparedwithC.guianensis’s barkandflowersisusedbothasananthelminticand heal-ingagentinhumans(Boufleuer,2004).

Considering the advances made in this research in recent years, this study aimed to evaluate the in vitro antiparasitic action of five plants (P. tuberculatum, L. sidoides,M.piperita,H.crepitansandC.guianensis)against H.contortus,and theinvivo actionagainstStrongyloides venezuelensisinrats.

2. Materialsandmethods

2.1. Plantmaterial

Theextractsfromthefiveplantspecieswereproduced inthelaboratoriesofseveralinstitutions.TheInstituteof ChemistryofPaulistaStateUniversity,SãoPaulostate, pro-videdthecrudeextractpreparedfrom18kgofleavesofP. tuberculatumcultivatedingreenhouseatAraraquara,São Paulo.The green leavesweredried in a stovewithhot aircirculationandthermostatizedat40◦_{Cduring4days.} Aftergrindingthem,theresultingmaterialwassubjected toextractionwithethylacetateandethanol(3:1),using 9L ofthismixtureforeachextraction. Thisprocesswas repeatedfour timeswithan interval of7daysbetween extractions.Thetotalweightoftheextractobtained cor-respondedto5.5%offreshplantmass(Cotinguibaetal., 2009).

TheessentialoilsfromL.sidoidesandM.piperitawere obtainedattheEmbrapaWesternAmazonResearch Sta-tion fromplants cultivated in Manaus, Amazonasstate, Brazil.TheleavesofL.sidoidesandM.piperitawerecutat groundlevelandplacedinafreezeruntilextraction.After separation oftheleaves,two samplesof20gwereused todeterminemoisturebydryingan ovenat65◦_{Cfor 3} days.Twoothersamplesof100geachwereusedtoextract theessentialoilbyhydrodistillationinaClevengertype apparatusfor3h.

H.crepitanslatexwascollectedinthetreeslocatedin thecityofPortoVelho,Rondôniastatebyemployeesof EmbrapaRondônia.TheseedoilofC.guianensiswas pro-ducedandacquiredinthelocalmarketofPortoVelho.

2.2. Chemicalanalyzes

TheactivesubstancesfromP.tuberculatumusedinthe presenttrialwerepreviouslydescribedbyCotinguibaetal. (2009).

anHP-5MScapillarycolumn(5%diphenyl,95% dimethyl-silicone,30m×0.25mm;filmthickness0.25␮m).Helium was used as the carrier gas (1.0mLmin−1_{), with} injec-tion of 1.0mL of a 1% solution of the essential oil in dichloromethaneinaninjectorheatedto250◦_C, operat-ing in split mode(splitratio 1:100). Oventemperature wasvariedfrom60to240◦_Catarateof3◦_Cmin−1_.The massdetectorwasoperatedinelectronionization(70eV) withthemassanalyzermaintainedat150◦_C,the ioniza-tionsourceat220◦_{Candtransferlineat260}◦_C.Toobtain thequantification, theessential oilswere alsoanalyzed in an Agilent 7890A chromatograph (Agilent Technolo-gies, Delaware, USA) equipped with a flame ionization detector (FID) kept at 280◦_{C and fitted with an} HP-5 capillary column(5%diphenyl–95%-dimethyl silicone; 30m×0.32mm;filmthickness0.25␮m).Thesame injec-tionandchromatographicconditionsabovewereapplied, buthydrogenwasusedasthecarriergas,at1.5mLmin−1_. Theresultswereindicatedthroughrelativearea(%area). Linearretentionindiceswerecalculatedbyinjectionofa seriesofn-alkanes(C7–C26)inthesamecolumnand con-ditions stated for GC-FID analyses.Identification of the essentialoils’componentswasdonebycomparisonofboth massspectraandretentionindiceswiththeWiley6th edi-tionspectraldatabaseandliteraturedata.

FortheH.crepitanslatexanalysis,protein

electrophore-sis in polyacrylamide gel was performed, using the

PROTEAN II xi Cell system from BIORAD, according to themethodologyproposedbyLaemmli(1970).The elec-trophoresis was performed over a period of 7h and a voltageof 100V.Proteins fromgelsremainedovernight in astainingsolutionofacetic acid10%(v/v),methanol 40%(v/v)andCoomassiebrilliantblueR2501%(v/v).The gelwasdestainedinasolutioncontaining10%aceticacid (v/v)and40%methanol(v/v),withsolutionrenewedevery 30minuntilobtainingaclearrevelation.Thecalculationof molecularweightproteinfractionswasperformedbythe constructionofstandardcurves,withmolecularmarkers againsttheirrespectivedistancesinthegel.

2.3. Invitroassays

2.3.1. Egghatchtest(EHT)

EggsofH.contortuswereobtainedfromfeces,directly collectedfromtherectumoftwolambs(Ovisaries) exper-imentallyinfected withthestrain H.contortus Pratânia, which hasshown anthelminticresistance to all classes ofdrugsavailablecommerciallyinBrazil(Almeidaetal., 2010).Theeggsweretakenfromfecesaccordingtothe methodologydescribed byBizimenyeraetal.(2006),an adaptationoftheoriginalmethodproposedbyColesetal. (1992). Feces were mixed with distilled water and fil-teredthrough100,56,30and25␮maperturesieves.In thelast sieve,theeggswereretainedand washedwith distilled water and centrifuged in Falcon tubes (50mL) at 3000rpm/5min filled with water. Then the super-natant wasremovedand aNaCl saturatedsolutionwas added. This solution was once again centrifuged under

the same conditions and the supernatant was washed

through a 25␮m sieve. The eggs collected were put inside a wine glass toallow sedimentation for 30min.

Theeggcountwasperformedin fivealiquots,andthen 100eggsinsuspensionwereadded perwellto24-well plates. The plantextracts were dilutedin a solutionof Tween 80 (3%) to obtainthe concentrations, and incu-bated in wells (0.3mL per well) for 48h at 25◦_{C. The} concentrations were determined following the ratio of 2 in at least six replicates for each concentration. The highestandthelowestconcentrationsevaluatedforeach plant extract were as follows: 0.313–0.0195mgmL−1 for P. tuberculatum, 1.25–0.039mgmL−1 _{for L. sidoides,} 0.156–0.0098mgmL−1 _{forM.piperita,2.5–0.156mgmL}−1 for H.crepitans and 10–0.625mgmL−1 _{for C. guianensis.} Afterincubation,Lugol’siodinesolutionwasaddedinthe wellstostopthehatchingprocess.ThenumberofL1larvae andeggsperwellwasthencountedusingareverse micro-scopeunder100×magnification(Olympus,modelCK-2, Japan).Thenegativecontrolcontained3%Tween80and thepositivecontrolwaspreparedwith0.025mgmL−1_of albendazole,inbothcasesinsixreplicates.

2.3.2. Larvaldevelopmenttest(LDT)

Eggs were recovered according the above

proce-duresandtheLDTwasperformedfollowingthemethod described byHubert and Kerboeuf (1992).In summary, 0.2mLofeggsuspension,containingapproximately100 eggs, wasadded perwell,in 24-well plates.They were incubatedfor24hat27◦_{CtoobtainL1.Afteregg} hatch-ing,90␮Lofculturemedium(containingEscherichiacoli and yeast extract) was added to each well, followed by the test extract. The concentrations were deter-mined following the ratio of 2 from 5␮gmL−1 _to 0.0003␮gmL−1 _{(i.e.,5, 2.5, 1.25␮gmL}−1 _{and soon) in} at leastsix replicatesfor each concentration.The high-est and the lowest concentrations evaluated for each plantextractwereasfollows:0.313–0.0195mgmL−1_for

P. tuberculatum, 0.0195–0.0003mgmL−1 _{for L. sidoides,} 0.156–0.0098mgmL−1_{forM.piperita,2.5–0.0078mgmL}−1 forH.crepitansand5–0.039mgmL−1_{forC.guianensis.The} plateswereincubated for6 daysat25◦_{C, andthen the} differentialcountfromL3,L2andL1wasperformed.A solu-tionof0.5%DMSOwaspreparedasnegativecontroland 0.64mgL−1_{ofivermectinaspositivecontrol,insix} repli-cates.

In both in vitro tests, the lowest concentration was determined when thehatching and larval development weresimilartothecontrol.Thehighestconcentrationwas basedonthesolubilityorontheturbiditythatlimitsits readability.

2.4. Invivoassay

Table1

LC50andLC90obtainedintheegghatchtest(EHT)forPiper tubercula-tum,Lippiasidoides,Menthapiperita,HuracrepitansandCarapaguianensis extractsonHaemonchuscontortus.

Species LC50(mgmL−1_{) LC90(mgmL}−1₎

P.tuberculatum 0.031(0.029–0.033) 0.09(0.08–0.10)

L.sidoides 0.04(0.038–0.053) 0.13(0.12–0.14)

M.piperita 0.037(0.035–0.039) 0.10(0.09–0.11)

H.crepitans 2.16(1.60–3.40) 17.13(8.61–57.21)

C.guianensis 2.03×10−6_(3.78 ×

10−17_–4.86 ×10−4₎

1.22×10−12_(2.53 ×

10−34_–7.66 ×10−8₎

Thenumbersinparenthesesrefertovaluesofupperandinferior confi-dencelimits95%.

G5– L. sidoides essential oil(150mgkg−1_{) and G6 – L.} sidoidesessentialoil(250mgkg−1_{).Infectionintensitywas} determinedbycountingthenumberofeggspergramof feces(EPG)ondays1,2,3,4and6afterthefirstdayof treatmentandbycountingthenumberofparthenogenetic femalewormsfoundinthefirst-thirdportionofthesmall intestine.Toobtainadultworms,theupperthird ofthe smallintestine wasremovedfromtherats13days after infection,cutlongitudinallyandincubatedinsaline solu-tion(0.9%NaCl)for4hat39◦_{C(NakaiandAmarante,2001).} Theaimofthistestwastoevaluateinratstheextracts thatshowedgreatest activityinthein vitrotests.Thus, itwouldbepossibletoobtainanindicationofthemost active extract in an in vivo model for future testing in sheep.AlthoughtheessentialoilofM.piperitashowedgood resultsintheinvitrotests,itwasnotpossibletoperform theinvivotestwithitduetothesmallamountofextract thatwasprovidedbythepartnerinstitution.

2.5. Statisticalanalysis

IntheEHTandLDT,theefficacyofeachtreatmentwas determinedbasedonhatchingordevelopmentpercentage accordingtothefollowingequation: Inhibition(%)=100 (Ptest/Ptotal),where,Ptestreferstothenumberofeggsinthe EHTornumberoflarvaethatdidnotdevelopuntilL3inthe LDTandPtotalcorrespondstothenumberofeggs+L1(EHT) ornumberofL1+L2+L3(LDT).

The50%lethalconcentration(LC50),i.e.,effective con-centrationtokill50%oftheeggsorlarvae,wasdetermined byProbitanalysis(SASInstitute,2003).

Fortheinvivotests,thevalueswerelogtransformed [log(x+1)]andsubjectedtoanalysisofvariance.The aver-ageswere compared bythe Tukeytest at 5%using the Minitab®_{statisticalsoftware.}

3. Results

Threeofthefiveextractstested–M.piperita,L.sidoides andP.tuberculatum–exhibitedsatisfactoryresultsbythe EHT(Fig.1),withlowLC50values(Table1).Thepositive controlwas100%effectiveininhibitingegghatchingand thenegativecontrolhadeffectivenessof3.5%.

AccordingtotheLDT,allextractsprovidedsatisfactory resultswiththeexceptionoftheextractofC.guianensis, whichdidnotprovideeffectiveinhibition(Fig.2).Thedata ontheLC50 are shown in Table 2.The positive control

Table2

LC50andLC90obtainedinthelarvaldevelopmenttest(LDT)forPiper tuberculatum,Lippiasidoides,Menthapiperita,HuracrepitansandCarapa guianensisextractsonHaemonchuscontortus.

Species LC50(mgmL−1_{) LC90(mgmL}−1₎

P.tuberculatum 0.020(0.0197–0.0206) 0.031(0.029–0.033)

L.sidoides 0.002(0.0016–0.0025) 0.004(0.003–0.007)

M.piperita 0.018(0.0166–0.0186) 0.03(0.03–0.04)

H.crepitans 0.36(0.33–0.40) 0.91(0.80–1.05)

C.guianensis 17.65(7.39–93.01) 1890(257–105,990)

Thenumbersinparenthesesrefertovaluesofupperandinferior confi-dencelimits95%.

presented100%inhibitionoflarvaldevelopmentandthe negativecontrol5.43%.

H.crepitansshowedbetterresultsintheLDT,100% inhi-bitionataconcentrationof2.5mgmL−1_{,whileatthissame} concentrationtheinhibitionintheEHTwasonly16.84%. Incontrast,C.guianensisdidnotshowinhibitoryeffecton thedevelopmentofeggsandlarvae.Atthehighest con-centrationevaluated(10mgmL−1_{),only8.52%inhibition} wasobservedintheEHT,whileintheLDT(5mgmL−1_)the inhibitionwas39.74%.

Cotinguibaetal.(2009)performedqualitative identifi-cationofthemainsubstancesintheP.tuberculatumextract and indicated thepresenceofpiperamides, suchas(Z )-piplartine,(E)-piplartine,8,9-dihydropiplartine,piperine, 10,11-dihydropiperine, 5,6-dihydropiperlongumine and pellitorine.TheessentialoilsofL.sidoidesandM.piperita wereanalyzedbygaschromatography-massspectrometry andpresentedastheirmaincomponentsthymol(76.6%) and menthol(27.5%),respectively.TheoilofC. guianen-sis wasevaluated and presented oleic acid(46.8%) and palmiticacid(39.0%)asitsmajorconstituents(Table3).

IntheevaluationoftheextractofH.crepitans,the pres-enceoftwobandswasobserved,a stronglycoloredone correspondingtothepolypeptidechainofmassbetween 36.5and 49.5kDaandweaklystainedpolypeptidechain correspondingtoamassbetween36.5and28.8kDa.From themassof28.8kDa,therewasdiffusestainingwith spe-cificstainingfortheproteinthatdiffusedthroughtheend ofthegel.Thepresenceofproteinmaterialwithmolecular massabove200kDawasobservedontopofthegel.

TheinvivoassaywasperformedwiththeextractsofP. tuberculatumandL.sidoides.Theinvivoassayconducted withtheextractofP.tuberculatumshowedthattherewas nosignificantreductionin theEPG(Fig.3)andinadult parasiteaverages incomparison withthecontrolgroup (P>0.05)(Fig.4).ConcerningtheextractofL.sidoides,there wassedativeactiononratsthatreceivedbothdoses.Asa result,theadministrationofthethreeconsecutivedosesof thisextractwasnotpossible,sofecalexamswerealsonot performed.Consideringadultparasites,therewere statisti-caldifferencesbetweenthetreatmentgroups(G5showed areductionof74.4%andG6of76.8%)andsorbitolcontrol group(Fig.4).

4. Discussion

a-Fig.1.MeanpercentageofinhibitionofHaemonchuscontortusintheegghatchtest(EHT)performedwithextractsofPipertuberculatum,Lippiasidoides, Menthapiperita,HuracrepitansandCarapaguianensis.

Vasconcelosetal.(2007)evaluatedinvitrotheanthelmintic potentialoftheessentialoilofthesameplantagainsteggs andlarvaeofH.contortusandobtainedmeaningfulresults: attheconcentrationof0.62mgmL−1_{therewas94.88%egg} hatchinginhibition.TheLC50was0.40mgmL−1intheEHT and2.97mgmL−1_{intheLDT.Weobserved100%inhibition} ofegghatchingataconcentrationof0.625mgmL−1 _and theLC50was0.04mgmL−1 intheEHTand0.02mgmL−1 intheLDT.ThesedifferencesbetweentheLC50ofthetwo studiesispossiblyduetothecompositionoftheessential oils.Inthepresentstudy,thymolaccountedfor76.6%of theL.sidoidesessentialoilwhilethissubstanceonly rep-resented59.65%oftheoiltestedbyCamurc¸a-Vasconcelos etal.(2007).Themainelementof theessential oilofL.

sidoidesisthymolandtherearereportsofitsantimicrobial activity(Helanderetal.,1998;Nostroetal.,2007), mol-luscicidalactivity(Singhetal.,1999)andlarvicidalactivity (Carvalhoetal.,2003).Camurc¸a-Vasconcelosetal.(2007) conductedtestswiththymolandobtainedresultsvery sim-ilartothatfoundwiththeessentialoil,whichsuggeststhat thiscompoundmayhaveovicidalandlarvicidalaction.

TheessentialoilofM.piperitaalsoprovidedinteresting resultsin vitro.The action of menthol, oneof the con-stituentsofthisoil(includedintheterpenoidsclass)has alsobeenreportedinsomestudies.Júnior(2003)reported theinsecticidalactivityofsometerpenoids,whichmightbe involvedintheinhibitionorretardationofgrowth, matu-rationdamage,reducedreproductivecapacityorappetite

Table3

PercentagecompositionofLippiasidoides,MenthapiperitaandCarapaguianensisessentialoilsobtainedbygaschromatography/massspectrometry.

L.sidoides % M.piperita % C.guianensis %

␣-Tujene 0.3 ␣-Tujene t Oleicacid 46.8

␣-Pinene 0.1 ␣-Pineno 0.8 Palmiticacid 39

Mircene 1.1 3-Methyl-cyclohexanone 0.1 ␣-Copaene 2.3

␣-Terpinene 0.7 Sabinene 0.4 Stearicacid 1.7

o-Cimene 6.3 ␤-Pinene 1.3 ␣-Cubebene 0.5

Limonene 0.4 Mircene 0.6 n.i. 8.7

1.8-Cineol 0.7 3-Octanol 0.1

␥-Terpinene 2.0 o-Cimene 0.1

Ipsdienol 0.6 Limonene 3.5

Umbelulone 0.2 1,8-Cineole 2.1

4-Terpinenol 1.0 cis-␤-Ocimene 0.1

␣-Terpineol 0.2 ␣-Terpinene 0.1

Timil-methyl-ether 1.0 cis-Sabinenehydrate 0.2

Thymol 76.6 Terpinolene 0.1

␣-Copaene 0.4 Linalool 0.1

␤-Caryophylene 5.0 neo-Isopulegol 0.1 Aromadendrene 0.4 p-Ment-3-en-8-ol 0.1

␣-Humulene 0.3 Menthone 11.0

Ledene 0.3 Menthofuran 22.5

␦-Cadinene 0.3 Menthol 27.5

Caryophylleneoxide 0.7 4-Terpineol 1.0

iso-Menthol 0.2

␣-Terpineol 0.3

Pulegone 12.8

Piperitone 0.6

neo-Menthylacetate 0.7 Menthytlacetate 12.5

trans-␤-Caryophyllene 0.5 Mintlactone 0.1

t,traces;n.i.,notidentified.

suppression,allofwhichcancauseinsectmortality.The activityoftheessentialoilofM.piperitaoneggsandlarvae ofH.contortusislikelyrelatedtoallorsomeoftheactivities describedabovefortheterpenoids.

RegardingtheoilextractedfromseedsofC.guianensis, therewasnoovicidalorlarvicidalactivityatthe concentra-tionstested.Onedifficultyfacedinhandlingthisoilinthe

invitrotestswithgastrointestinalnematodeswasits solu-bilization.Itwouldbenecessarytoincreasetheamountof solvent,buttheparasitesareverysensitive,whichexplains thelowconcentrationsusedinthisstudy.Itwasnecessary toincreasetheconcentrationsofC.guianensisandH. crepi-tansconsiderablytoobtainbetterresultsintheEHTand LDTandtoreachtheLC50.Sothestandarddeviationfor thesetwospecies,andespeciallyforC.guianensisinthe LDT,presentedlargevariability.

The latex extracted from H. crepitans did not show anyovicidalactivity,butwaseffectiveintheLDT.Inthe EHT, it was not possible to assess this latex in higher concentrationsbecausetheextract possesseddarkcolor

and contained many particles, making the subsequent

visualization and countingof larvae and eggs impossi-ble.Brondani(2006)analyzedtheactivityofthelatexof this plant oninfective larvae of ticks(R. microplus and R. sanguineus) and observed mortality rates above 95% atallconcentrations.Someconstituentsofthelatexare lectin,creptin(bothglycoproteins)andhurin(proteolytic enzyme)(Brondani,2006).Therearestudiesthatindicate theharmful action of proteases onthe cuticle of some

nematodes(Stepeketal.,2004).Forinstance,Jaffé(1943) comparedtheactionofhurin(fromthelatexofH.crepitans) withpapain(comingfromthesapofFicus)onAscaris lum-bricoidesandearthworms.Theresultsshowedthatpapain digestedbothspecies, whilehurindigestedearthworms but notA.lumbricoides,althoughitcaused deathof this species. Lectin, which is alsopresent in thelatex of H. crepitans,actsbybindingspecificallytocarbohydratesand otherresiduesofglycoconjugatesonthecellsurface.Its potentialhasbeenreportedasaninsecticideagainstsome

Fig.4.MeannumberofStrongyloidesvenezuelensisadultwormsrecoveredfromtheinitialthirdofintestineofratstreatedwithLippiasidoidesessential oil(LS1– 150mgkg−1_{andLS2– 250mgkg}−1_{)andPipertuberculatumextract(PT1– 150mgkg}−1_{andPT2–250mgkg}−1_{).Meanswithdifferentlettersare} significantlydifferentbytheTukeytest(P<0.05).

species,bybindingtotheperitrophicmembrane(the acel-lularchitinstructurethatlinesthedigestivetractofsome ColeopteraandLepidoptera),interferinginthefeeding pro-cess(Fitchesetal.,2001).Takingintoaccounttheactionof lectin,weassumedthatitmaybeconsumedbytheL1 lar-vae,leadingtoinhibitionoftheirdevelopmentuntiltheL3 stage.Therewasanincreaseininhibitionofdevelopment withincreasingextractconcentration.

InrelationtotheextractofP.tuberculatum,satisfactory resultswerefoundinboththeEHTandtheLDT.

The Piperaceae contains several plants with insecti-cidal effects, especially thePiper genus,which contains species withsecondarymetabolitessuchaslignans and amides,usedintheirdefenseagainstherbivores. Piplar-tine,identifiedbyDuhandWu(1990)asoneofthetoxic componentsofP.arborescens,hasdemonstratedcytotoxic activityoncells.Bezerraetal.(2005)comparedthemitotic activityofpiperineand piplartineagainst differentcells andobservedamorepotenteffectofpiplartine.Piperine, inturn,hasantiparasiticactivity,asobservedbyRibeiro etal.(2004)againstepimastigotesandamastigotesof Try-panosoma cruzi.Also, Freire-de-Lima etal.(2008) noted thatpiperinecausedadelayincellcycleofthatparasite.

Thebestresultsfromthisstudywerewiththeextracts of P. tuberculatum (EHT with LC90=0.09mgmL−1 and LDT withLC90=0.03mgmL−1)and L.sidoides(LDTwith LC90=0.03mgmL−1).Theseresultsweresignificantwhen comparedtothoseobservedfortheotherplantextracts evaluatedagainstH.contortus.Spigeliaanthelmiashowed 100%inhibitionintheEHTand81.2%intheLDTata con-centrationof50mgmL−1_{(Assisetal.,2003).Macieletal.}

(2006) observed better efficacy of ethanol seed extract (100%inhibitionat1.56mgmL−1_{)andleafextract(98.24%} at12.5mgmL−1_{)ofMeliaazedarachoneggsofH.contortus} andtheethanolextractofleaves(91.64%at50mgmL−1₎ onthelarvaeofthesamespecies.Macedoetal.(2010),in turn,analyzedtheeffectofEucalyptusstaigeriana essen-tialoilandobservedinhibitoryeffectoneggs(99.27%at 1.35mgmL−1_{)andlarvae(99.20%at5.4mgmL}−1_).Cocos

nuciferashowed 100%of inhibition at 5mgmL−1 _{in the} EHTand99.77%at80mgmL−1_{intheLDT(Oliveiraetal.,}

2009b).The potential larvicidal activity of the aqueous extract (97.3% inhibition at 150mgmL−1_{) and ethanol} extract (99.6% inhibition at 60mgmL−1_{)of Anacardium}

humileleavesongastrointestinalnematodesinsheepwas reportedbyNeryetal.(2010).

Some studies have analyzed the antiparasitic action ofplantextractsonparasites ofratsormiceandsheep. Amongthese,BorbaandAmorim(2004)studiedtheaction ofextractsofChenopodiumambrosioidesL.ontheoxyurids SyphaciaobvelataandAspiculuristetraptera,obtaining neg-ativeresultsforallconcentrationstested.Thissamespecies wasevaluatedinratsagainstStrongyloidesvenezuelensis and showed efficacy in reducing the EPG(75.89%) and the number of adult parasites (86.31%) at 400mgkg−1 (Bernardes, 2006). In field trials with sheep, Camurc¸ a-Vasconcelosetal.(2008)administeredtheessentialoilof L.sidoidesatconcentrationsof230and283mgkg−1 _and observedareductionintheEPGcountintheevaluations conducted7and14daysafterthetreatment:38%and30% and45%and54%,respectively.

Inourevaluationwithrats,asignificantreductionwas observedinthenumberofadultparasites atbothdoses tested (150 and 250mgkg−1_{) compared to the control} group,treatedwithsorbitol.Thehighestmeannumberof EPGwasrecorded7or8daysafterinfectionofratswith S.venezuelensis,afterwhicheggoutputshowed progres-sivereduction.Therefore,thetrendinEPGvaluesobserved in thesorbitolgroupreflects a naturalreduction of the eggelimination.Duetoitssedativeeffect,observedafter thefirstadministrationof theoil, wechosenotto per-formthefollowingtwotreatmentstopreventtheanimals’ death.Invivotestsareneededtoevaluatetheeffectofplant extractswithsignificantresultsinvitroonparasites. How-ever,thepossibletoxiceffectsonthetargethostsshould beperformedearlier.

actioninrats.Nevertheless,futurestudieswithP. tuber-culatumextractsandM.piperitaandL.sidoidesoilswillbe necessarytounderstandtheirabsorptionandmetabolism inratsandsheep.

Acknowledgments

Wegratefullyacknowledgethetechnicalassistanceof AndrineM.C.Navarro,CésarC.BassettoandLetícia Bos-chini.ThisworkreceivedfinancialsupportfromFAPESP (SãoPauloStateResearchFoundation).CamilaO.Carvalho hasagrantfromFAPESP.

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