Anthelmintic activity of Cymbopogon martinii, Cymbopogon schoenanthus and Mentha piperita essential oils evaluated in four different in vitro tests

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VeterinaryParasitology183 (2011) 103–108

ContentslistsavailableatScienceDirect

Veterinary

Parasitology

j our na l h o 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

Anthelmintic

activity

of

Cymbopogon

martinii

,

Cymbopogon

schoenanthus

and

Mentha

piperita

essential

oils

evaluated

in

four

different

in

vitro

tests

L.M.

Katiki

a,∗

, A.C.S.

Chagas

b

, H.R.

Bizzo

c

, J.F.S.

Ferreira

d

,

A.F.T.

Amarante

e

a_IZ_–_Instituto_de_Zootecnia,_Rua_Heitor_Penteado_56,_CEP_13460-000,_Nova_Odessa,_SP,_Brazil b_Embrapa_{– Pecuária}_Sudeste,_Rod._Washington_Luiz_km_234,_CEP_13560-970,_São_Carlos,_SP,_Brazil c_Embrapa_–_{Agroindústria}_de_Alimentos,_Av._das_Américas_29501,_CEP_23020-470,_Guaratiba,_RJ,_Brazil d_USDA-ARS_–_Appalachian_Farming_Systems_Research_Center,₁₂₂₄_Airport_Rd.,_Beaver,_WV_25813,_USA e_UNESP_–_Universidade_Estadual_Paulista,_Departamento_de_{Parasitologia,}_IB,_CEP₁₈_618-970,_Botucatu,_SP,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received22March2011

Receivedinrevisedform30June2011 Accepted4July2011

Keywords:

Cymbopogon Menthapiperita Essentialoil

Invitrotest Sheep Haemonchus

a

b

s

t

r

a

c

t

Anthelminticresistanceis aworldwide concernin smallruminantindustry andnew plant-derivedcompoundsarebeingstudiedfortheirpotentialuseagainstgastrointestinal nematodes.Menthapiperita,CymbopogonmartiniiandCymbopogonschoenanthus essen-tialoilswereevaluated againstdevelopmentalstagesoftrichostrongylidsfromsheep naturallyinfected(95%Haemonchuscontortusand5%Trichostrogylusspp.)throughthe egghatchassay(EHA),larvaldevelopmentassay(LDA),larvalfeedinginhibitionassay (LFIA),andthelarvalexsheathmentassay(LEA).Themajorconstituentoftheessential oils,quantifiedbygaschromatographyforM.piperitaoilwasmenthol(42.5%),whileforC. martiniiandC.schoenanthusthemaincomponentwasgeraniol(81.4%and62.5%, respec-tively).InallinvitrotestsC.schoenanthusessentialoilhadthebestactivityagainstovine trichostrongylidsfollowedbyC.martini,whileM.piperitapresentedtheleastactivity. Cym-bopogonschoenanthusessentialoilhadLC50valueof0.045mg/mlinEHA,0.063mg/mlin

LDA,0.009mg/mlinLFIA,and24.66mg/mlinLEA.Theanthelminticactivityofessentialoils followedthesamepatterninallinvitrotests,suggestingC.schoenanthusessentialoilcould beaninterestingcandidatefornematodecontrol,althoughinvivostudiesarenecessaryto validatetheanthelminticpropertiesofthisoil.

1. Introduction

Gastrointestinal nematodes in livestock are usually controlled by commercial anthelmintics. However, few commercial anthelmintics are available for veterinary use due to reduced effectiveness caused by emerging

∗ Correspondingauthorat:RuaHeitorPenteado,56,CEP13460-000, NovaOdessa,SP,Brazil.Tel.:+551934669400;fax:+551934661279.

E-mailaddresses:[email protected], [email protected]

(L.M.Katiki).

drug-resistantparasite strains (Molan et al., 2002). For this reason, losses in weight gain and high morbidity andmortalityareaconsequenceofthoseparasite infec-tions. Haemonchus contortus is the most prevalent and pathogenic nematode found in small ruminants in the tropics.Manyalternativestrategiestocontrolnematodes havebeenstudiedsuchasadequatenutrition,selectionof resistantanimals,integratedpasturemanagement,useof nematophagusfungus,andnewanthelminticcompounds derived from plants. The search for new solutions to chemicaltreatmentsisnowadaysaworldwidenecessity toachieve more sustainablecontrol. There is increased

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evidence indicating that some bioactive plants might possess anthelmintic properties and, thus, represent a promising alternative to commercially available drugs

(BrunetandHoste,2006).

Menthapiperita,CymbopogonmartiniiandCymbopogon schoenanthusessentialoilswerechosen tobeevaluated against nematodes in in vitro tests because they have shownsomeinsecticideeffect.Invitrobioassayshavethe advantageofprovidinga simple,rapid,and inexpensive meansofprimaryscreeningofproductswithanthelmintic potential. M. piperita essential oil was active in killing insectsofstoredproducts(Shaayaetal.,1991),andhad larvicidalandmosquito-repellencyactivity(Ansarietal., 2000).C.martiniessentialoilwasactiveagainst Meloidog-yneincognita,asoilnematode,(Pandeyetal.,2000),and againstCaenorhabditis elegans(Kumaranet al.,2003).C. schoenanthusessentialoilwasactiveagainsttermites(Koba etal.,2007)andagainstthebruchidCallosobruchus macu-latus,whichisamajorpestofstoredgrains(Ketohetal.,

2002).

For this in vitro screening, different methods were employedtocomparetheresultsamongessentialoilsfrom differentplantspecies.Theevaluationofessentialoil emul-sionswasperformedontrichostrongylidsimmaturelife stagesbytheegghatchingassay(EHA),totesteggtoL1, larvaldevelopmentassay(LDA),usinglarvalstagesL1 to L3,larvalfeedinginhibitionassay(LFIA),usingL1stage,and larvalexsheathmentassay(LEA),usingL3stage.Therefore, thepurposeofthisstudywastoevaluatetheanthelmintic activityofthreeessentialoilsusingdifferentinvitroassays anddifferentlarvalstagesoftrichostrongylids.

2. Materialsandmethods

2.1. Sheepnematodes

Allearly life stages of trichostrongylids used in our workwere obtained fromsheep naturally infected and keptatEmbrapaPecuáriaSudeste(fecalcultureindicated 95%ofHaemonchuscontortusand5%Trichostrogylusspp.). Oncefeceswerecollected,testswereperformed,withsix replicates,tocomparethreeessentialoilsatthesame con-centrations.

2.2. Essentialoils

OilswerepurchasedfromWNFInd.eCom.Ltda(R.Dr. MarioPintoServa,64–SaoPaulo,SP,Brazil).M.piperita

oillotno.164,density(d)=0.919,C.martiniioillotno.081,

d=0.884andC.schoenanthusoillotno.10608,d=0.911. Essentialoilsweretestedin EHA, LDAand LFIAat con-centrationsrangingfrom0.018mg/mlto22.75mg/ml(C. schoenanthusand M. piperitaoil) andfrom 0.017mg/ml to 22mg/ml (C. martinii). In LEA, doses ranging from 18.2mg/ml to 136.5mg/ml for C. schoenanthus and M. piperita,anddosesrangingfrom17.6mg/mlto132mg/ml forC.martiniiwereevaluated.Toimproveemulsificationof essentialoilsinwater,solvents(0.5%DMSOor2%Tween 80) were added and solutions were mixed in a vortex shakeruntiloil,solvent,andwaterbecameastable emul-sion.

2.2.1. GC–MSanalysis

Analysis of the chemical composition of the essential oils were performed by gas chromatogra-phy coupled to mass spectrometry using an Agilent 5973N GC–MS system equipped with a HP5MS cap-illary column (5% diphenyl–95% dimethylsilicone, 30m×0.25mm×0.25␮m). The injector was set at

250◦Candtheovenprogrammedtogofrom60to240◦C at3◦C/min.Massdetectorwasoperatedinelectron ion-izationmode,at70eV.Heliumwasusedasthecarriergas ata flowrateof1.0ml/min.Samplevolumewas1.0␮L,

and consisted of 1%essential oilin dichloromethane. A splitratioof1:100wasused.Massspectrawerecompared withdata fromWiley6th editionlibrary.The retention indexes were calculated based ondata generated by a seriesofalkenes(C7–C26)injectedinthesamecolumnand conditionsspecifiedabove,andcomparedtothosefound intheliterature(Adams,2007).Identificationwasbased onboth massspectrumand retentionindex.Menthone, menthol, geraniol and geranial were also identified by injectionofauthenticstandards.

2.2.2. GC-FIDanalysis

Forquantification,theoilswereanalyzedinanAgilent 7890Agaschromatographequippedwithaflame ioniza-tiondetectorandaHP5capillarycolumn(5%diphenyl–95% dimethylsilicone, 30m×0.32mm×0.25␮m). Hydrogen

wasusedasthecarriergasata flowrateof1.5ml/min. Allotherparameterswerethesameasdescribedabove. Resultswerereportedinrelativepercentageofpeakarea.

2.3. Invitrotests

2.3.1. Egghatchingassay(EHA)

Apre-establishedprocedurewasfollowedforthisassay

(Bizimenyeraetal.,2006)aftersomemodifications.About

5goffeces,directlycollectedfromtherectum,weremixed withwarmwater(37◦C)andfilteredthroughsieveswith aperturesof1mm,105␮m,55␮m,and25␮m,thelatter

retaining theeggs.Recoveredeggswereadded to satu-ratedNaClsolution,centrifugedat3000rpmfor3minand thefloatingeggswerecollectedusingthe25␮msieveand

washedwithdistilledwater.Onehundredeggsin20␮l

dis-tilledwaterwereaddedtothetreatments(water,Tween80 at2%,ortheessentialoiltested).Allconcentrations, posi-tive(water+Tween80at2%),andnegative(distilledwater) controlshadsixreplicatesandwereperformedin24-well plates.Plateswereincubatedat26◦Cfor48handreadin aninvertedmicroscopetocounteggsandL1larvae.

2.3.2. Larvaldevelopmentassay(LDA)

FollowingBizimenyeraetal.(2006),withsome modifi-cations,onehundredeggswereaddedintothewellswith distilledwaterinatotalvolumeof200␮l,incubatedfor

24hat27◦CtoobtainL₁ larvae.Toeachwellcontaining thetreatment(water,dimethylsulfoxideat0.5%(DMSO), essentialoil),weaddednutritivemedium(Escherichiacoli, yeast extract,Amphotericin-B) according toHubert and

Kerboeuf, 1992. All concentrations, the positive control

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Table1

CL50(mg/ml)andconfidencelimitsofCymbopogonschoenanthus,MenthapiperitaandCymbopogonmartiniiessentialoilsinegghatchassay(EHA),larval developmentassay(LDA),larvalexsheathmentassay(LEA)andlarvalfeedinginhibitionassay(LFIA)againstgastrointestinalnematodesofsheep.

C.schoenanthus M.piperita C.martinii

EHA 0.04(0.03–0.05) 0.26(0.24–0.28) 0.13(0.11–0.14)

LDA 0.06(0.05–0.07) 0.26(0.23–0.30) 0.15(0.14–0.16)

LEA 24.66(19.44–29.46) 61.93(55.81–68.58) 28.17(26.28–30.33)

LFIA 0.009(0.009–0.018) 0.07(0.06–0.08) 0.03(0.03–0.04)

incubatedat27◦Cforfourdays,whenplateswerereadin aninvertedmicroscopetocountallL3 andundeveloped larvae.

2.3.3. Larvalfeedinginhibitionassay(LFIA)

Eggs plus distilled water were kept in a Petri dish covered and incubatedat27◦C for24h.ActiveL₁ were recoveredbyBaermannizationusinga25␮msieve.Ina 1500␮lEppendorftube,onehundredL1larvaewereadded tothetreatments(water,DMSO0.5%andessentialoil).This solutionwasprepreparedin sixreplicates.Asexample, a concentrationof 22.75mg/ml=225␮l C. schoenanthus essential oil+45␮lDMSO+8130␮ldistilled water were mixed in a vortex shakerand 1400␮lwere distributed insixEppendorftubeandthen,100␮lsolutionwith100 L1 was addedto each tubeto complete 1500␮l.Tubes wereincubatedhorizontallyat24◦Cfor2h.Thework pro-ceeded indarkfromthispointtotheendofprocedure. E.colimarkedwithfluoresceinisothiocyanatewasadded inavolumeof20␮landincubatedhorizontally,covered with aluminum foil for 24h at 24◦C. Tubes were cen-trifugedat6000rpmfor1min,and800␮lofsupernatant wasremoved.Alllarvaefromthebottomwereexamined underafluorescencemicroscope,countingallnematodes that had fedon E.coli (luminous intestine). Thereafter, countingwasperformedunderanopticalmicroscope.All concentrations,positive(water+DMSO0.5%),andnegative controlsweredonewithsixreplicates(Álvarez-Sánchez

etal.,2005).

2.3.4. Larvalexsheathmentassay(LEA)

ActiveL3larvaefromcoproculturewereseparatedusing a 25␮m sieve. They were concentrated by

centrifuga-tionat6000rpmfor2mintoprepareasolutionwith100 L3/100␮l.OnehundredL3larvaewereaddedtothe treat-ments(water,Tween80at2%andessential oil).TheL3 larvaewereexposedtoemulsionofessentialoilsduring 3hat22◦C,centrifugedat6000rpmfor2min,removed supernatant,andaddeddistilledwatertocleanthelarvae fromessentialoil.Thisprocedurewasrepeatedtwicewith 1200␮lwith1200larvaekeptasresidualatthebottomof

centrifugetubes.Onehundredlarvaewereaddedtoeach welland1400␮lofbleachsolution(150␮ldomesticbleach

with6%sodiumhypochloritedilutedin15.625mlofwater) wasaddedintowellscontaining100L3.Atevery10min, theexsheathmentwasstoppedwithiodinesolution.The gradual exsheathmentalong 60min shouldbefoundin controlgroups.Allconcentrations,positive(water+Tween 80at2%)andnegativecontrolsweretestedwithtwo repli-cates(Alonso-Diazetal.,2008).

2.4. Statistics

Thecalculationoftheextractlethalconcentration(LC) intheinvitrotestswasperformedbyfittingregression usingnormalandlogisticdistribution,withtheparameters estimativeoftheseequationsobtainedbymaximum like-lihood.TheprocedureusedwastheSASProbittoestimate theLC50andLC99withtheindependentvariables(dose) transformedbynaturallogarithm(logdose).

3. Results

In theEHA,C. schoenanthusessential oilshowedthe lowest LC50 value (0.045mg/ml) when compared to C.

martiniiandM.piperitaessentialoils,andthisresultwas closetotheLC50valueobtainedfortheLDA(0.063mg/ml). TheLFIAindicatedthat theL1 werevery sensitivetoC.

schoenantusoiland requiredless essential oiltoinhibit their feeding activity. C. schoenanthus essential oil had LC50of0.009mg/ml,whiletheLEAdemonstratedthatL3 wereveryresistantand higherconcentrationsof essen-tialoils were needed.In the LEA, C. schoenanthus LC50 presentedthelowestvalue,24.66mg/ml,whilethe high-estwas61.93mg/ml forM. piperita.In all invitro tests

C.schoenanthusessentialoilhadthebestactivityagainst ovinetrichostrongylidsfollowed byC. martini,while M. piperitapresentedtheworstresults(Table1).The same tendencyinessentialoileffectivenesswasfoundforthe

LC99inEHA,LEA,andLDA(Table2).

Thesensitivityofimmaturelarvalstagestosolventswas tested(Table3).Inordertomakeanemulsionof essen-tialoilsandwater,Tween80wasusedinbothEHAand LEAdue tothetoleranceof eggsand L3 tothis solvent. However,Tween80wasnotusedineitherLFIAorLDA

Table2

CL99(mg/ml)andconfidencelimitsofCymbopogonschoenanthus,MenthapiperitaandCymbopogonmartiniiinegghatchassay(EHA),larvaldevelopment assay(LDA),larvalexsheathmentassay(LEA)andlarvalfeedinginhibitionassay(LFIA)againstgastrointestinalnematodesofsheep.

C.schoenanthus M.piperita C.martinii

EHA 0.27(0.18–0.36) 1.0(0.81–1.27) 0.61(0.45–0.88)

LDA 0.27(0.18–0.36) 0.91(0.72–1.27) 0.35(0.35–0.44)

LEA 54.23(40.22–151.69) 240.24(187.55–347.52) 56.32(48.48–72.77)

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Table 3

InhibitoryactionofsolventsDMSOat0.5%orTween80at2%expressed bypercentagesandits(standarddeviation)inegghatchassay(EHA), larvaldevelopmentassay(LDA),larvalexsheathmentassay(LEA)and lar-valfeedinginhibitionassay(LFIA)againstgastrointestinalnematodesof sheep.

Test DMSO(0.5%) Tween80(2%)

EHA 4.79(1.55)%

LDA 12.35(3.37)%

LFIA 14.53(5.24)%

LEA 3.23(0.24)%

becauseitresultedinhighmortalityincontrolgroupsand, wassubstitutedbyalesstoxiccompound,DMSO.

Oxygenatedmonoterpeneswerethemajorconstituents of the essential oils tested. M. piperita oil presented 29 compounds and had 42.5% menthol, followed by 27.4% menthone as major constituents. C. martinii oil presented 11 compounds and had 81.4% of geraniol and 10.1% isomenthyl acetate, and C. schoenanthus oil presented 28 compounds and had 62.5% geraniol, fol-lowed by 12.5% geranial and 8.2% neral and 3.4% beta-caryophyllene(Table4).

4. Discussion

Theobjectiveofthisstudywastoevaluatethree essen-tialoilsusing fourdifferentin vitrotests.The EHA and LDAarethemostwidelyemployedinvitromethodsfor detectionofanthelminticresistanceinovinenematodes underfieldconditions(Váradyetal.,2009).TheLFIAwas successfultodetectanthelminticresistancetomacrocyclic lactones and imidazothiazoles (Álvarez-Sánchez et al.,

2005).TheLEAwasextensivelyusedtoconfirmeffectof tanninrichplantextractsanditsinhibitoryprocessonL3

(BrunetandHoste,2006).TheLFIAandLDAarenot

cur-rentlyemployedininvitrotestshoweverthosetestscanbe usedasacomplementofotherinvitromethods.Allinvitro

testsareusuallyinterpretedbyusingLC50values(Várady

etal.,2009).

Inthisstudy,C.martinii,C.schoenanthusandM.piperita

essentialoilspresentedhighinvitroactivityagainstsheep trichostrongylids.Theresultsobtainedinvitroherewere superiortootheroilstestedpreviously.Forinstance, Euca-lyptusglobulusessentialoilinhibited99.3%egghatching and98.7%larvaldevelopmentatconcentrationsof21.75 and43.5mg/ml(Macedoetal.,2009).Ocimumgratissimum

essential oilinhibited 100% egg hatching at

concentra-tion of 0.5% (Pessoa et al., 2002).Croton zehntneri and

Lippia sidoides essential oils inhibited egg hatching in morethan98%at1.25mg/mlandlarvaldevelopmentin over98%at10mg/ml(Camurc¸a-Vasconcelosetal.,2007).

Chenopodiumambrosioidesessentialoilinhibited100%egg hatchingat1.33␮l/ml(Ketzisetal.,2002).TheLC50of

Euca-lyptusstaigerianaessentialoilintheEHAwas0.324mg/ml andLC50intheLDAwas1.702mg/ml(Macedoetal.,2010). Thosevaluesarehigherincomparisontoourresultsthat showedLC50inEHAof0.04;0.26and0.13mg/mlandLC50 inLDAof0.06;0.26and0.15mg/mltoC.schoenanthus,M. piperitaandC.martiniiessentialoils,respectively.

Terpenes are a chemical class of chemicals found in essential oils. C. schoenanthus had approximately 20 constituents, beingrich in geraniol, geranial, and neral. Terpenoid compoundsareknowntobeactiveagainst a rangeoforganismsandthesynergyofseveralterpenoids can be effective on several targets because they are a

Table4

PercentageofcompositionofCympobogonmartinii,CympobogonschoenanthusandMenthapiperitaessentialoilsobtainedbygaschromatographycoupled tomassspectrometry.

C.martinii C.schoenanthus M.piperita

Geraniol 81.4 Geraniol 62.5 Menthol 42.5

Isomenthylisomenthylacetate 10.1 Geranial 12.5 Menthone 27.4

Linalool 2.6 Neral 8.2 1.8-Cineole 4.6

Geranial 2.1 Citronelol 3.6 menthylacetate 4.6

Trans-ocimene 1.7 (E)-beta-caryophyllene 3.4 Limonene 2.0

(E)-beta-caryophyllene 0.8 Geranylacetate 2.0 Pulegone 1.8

Cis-ocimene 0.4 Linalool 1.3 (E)-beta-caryophyllene 1.6

Myrcene 0.4 Delta-cadinene 0.9 4-Terpineol 1.3

Limonene 0.3 Caryophylleneoxide 0.6 Beta-pinene 0.9

Neral 0.2 Citronelal 0.5 Alpha-pinene 0.6

Nerol 0.1 6-Methyl-5-heptenone 0.5 p-Cymene 0.4

Alpha-humulene 0.4 Isomenthol 0.4

Elemol 0.4 Sabinene 0.2

Alpha-cadinol 0.4 Isopulegol 0.2

Epi-alpha-cadinol 0.3 Alpha-terpineol 0.2

Beta-elemene 0.3 Piperitone 0.2

Decanal 0.3 Neo-menthylacetate 0.2

Geranylformiate 0.2 Caryophylleneoxide 0.2

Eugenol 0.2 3-Octanol 0.1

Gamma-humulene 0.2 Sabinenecis-hydrate 0.1

Alpha-muurolene 0.2 Linalool 0.1

Notidentified 0.7 Isomenthylacetate 0.1

Beta-bourbonene 0.1

Beta-elemene 0.1

Alpha-humulene 0.1

GermacreneD 0.1

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complexmixtureofcompoundsthatcaninteractwith mul-tiplemoleculartargetsonvarious developmentalstages of theparasite(Marie-Magdeleineet al.,2009).So, it is quitereasonabletoconsiderthatthemajorconstituentsof eachplantspecies,asdetectedbygaschromatography,had somebiologicalactivityinvitroagainsttrichostrongylids inthepresentstudy.Becausegeraniolwasthemain com-ponent in both Cymbopogon species, which had better anthelminticeffectsthanMentha(devoidofgeraniol),we canhypothesizethatgeraniolmightbeofpotentialinterest for in vivo tests. However, the concentration of geran-ioldoes not preclude thepotentialsynergistic effect of geranialandneral,presentathigherconcentrationsinC. schoenanthus,theessentialoilwiththebestanthelmintic activity.

The insolubility of essential oils and many of their constituents in aqueous media is likely toimpair their performanceinsusceptibilitytests,andattemptsto over-comethisproblemhavebeenmadebyusingtensio-active agents such as Tween 20 and Tween 80 (Juven et al., 1994). Although Tween 80 has low toxicity to nema-todes(4.79%inhibitionhatchabilityand3.23% inhibition exsheathment) compared to Tween 20, it caused toxic-ityonLFIAandLDAassays.DMSO,whichislesstoxicto L1 larvaethanTween80,wasusedforbothLFIA(14.53% inhibition feeding activity) and LDA (12.35% inhibition development).Thosesolventscanworkasbioenhancers,as theyhavetheabilitytoincreasethebioavailabilityofdrugs byincreasingtheirtransportacrossmembranes,increasing anthelminticeffect.Thus,thetypeandconcentrationof sol-ventsrequiredtomakeanemulsionshouldbeconsidered beforetestsareperformed.Dependingontheorganism, thesolventscanbehighlylethal.Nematodesinimmature formareverysensitivetoarangeofcompounds,including tapwater(vonSamsonHimmelstjernaetal.,2009).Because ofthissensitivity,itwasnoticedthatisimportanttokeep goodcontrolbyremovingallpossibleagentsthatcan neg-atively affect the regular life cycle of trichostrongylids. Regardingwatersolubility,althoughdesirable,itmightnot berequiredinsomecases.Forinstance,testsindogsusing carbontetrachlorideagainsthookwormsshowedthatthe efficacyofthetestedcompoundsincreasedassolubilityin waterdecreased(Bennet-JenkinsandBryant,1996).

Anotherpoint ofdifficultyfoundininvitrotestswas thecontamination.Álvarez-Sánchezetal.(2005)usedthe LFIAtodetectanthelminticresistancetoivermectinand levamisoleandreportedthatsuchassayoffersthe advan-tageofsimplicityandrapidityincomparisontotheLDA. LDA takes a long time to be performed and problems related tocontaminationsfrequentlyoccur. These prob-lemsreflectedinfewerlaboratoriesusingtheassayasthe primary screen for activityin vitro (Jackson and Hoste, 2010).Inourworkwedealtwithbothbacterialandfungal contaminationbyusingsteriletechniquesasmuchas pos-siblebecausedevelopmentstagescouldnotstandhigher quantitiesofantibioticsthanfoundinnutritivemedium

(HubertandKerboeuf,1992).Inaddition,insteadofseven

daysofincubationat23◦C(Bizimenyeraetal.,2006),we increase temperatureto27◦Cand decreased incubation timetofivedays.Thismodificationallowedlarval devel-opmentathigherratesandmorereproducibleresults.It

alsodecreasedfungalandbacterialcontaminationinthe plates.

Different LC50 values found between assays can be attributedtothesensitivityofeachstage.Eggsaremore resistantthanL1 duetoits hardand resistantshell.On theotherhand,L3weremoreresilientduetotheirdouble sheath.L1 wasthemostsensitivestagedue toits phar-ynxthatismoresensitivetotheparalysiscausedbydrugs thanthe axialmuscles (Molanet al., 2002).These facts leadtohigheror lesservolumesofactivecompound to achieveLC50foreachtest.Váradyetal.(2007)usedthe cri-terionofLC99concentrationtoevaluatethesensitivityto thiabendazoleunderfieldscreening.Thoseauthorsfound inegghatchandlarvaldevelopmentassaythe compari-sonofLC50valueswasnotsignificantlydifferent,however theLC99concentrationisabletodifferentiatesusceptible group,susceptibleheterozygotegroupandresistantgroup intestforresistancediagnosis.OurLC99resultsinEHA,LDA andLEAshowedthesamepatternofactivityfoundinLC50 values.

For trichostrongylids, the L3 exsheathment is a key processinthelifecyclebecauseit isthetransitionstep betweenthefreelivingandtheparasiticstages(Hertzberg etal.,2002).Studiesonthekineticsoflarvaeexsheathment haveemphasizedthatanydisturbingfactorsortoxic com-poundsmightreducetheparasiteestablishmentinthehost

(Dakkaketal.,1981).

Theinvitromethodsprovidemeanstoscreenrapidlyfor potentialanthelminticactivitiesofdifferentplantextracts andtoanalyzethepossiblemechanismsinvolvedinthe interactionsbetweenactivecompoundsandparasites.C. schoenanthusshowedthebestanthelminticactivityinvitro. Thus, basedon theLC50 of24.66mg/ml obtainedfor C.

schoenanthusinLEA,anapproximatedoseof1.18–2.45gof oil/kgbodyweight(BW)wouldprovidea50%reductionin exsheathmentandwormreductionconsideringananimal of40kgand2–4labomasalvolume.However,sometimes theeffectinvitroorina differentanimalsystemcanbe lowerthanwhentestedinthetargethost.Arecent illus-trationofthispointistheworkwithorangeemulsionoil, where600mgoftheoilemulsionperkgBWcaused7%and 62.6%wormreductioningerbilswithasingledoseordaily forfivedays,respectively(Squiresetal.,2010).However, whentheseauthorstestedtheemulsionwith600mgof orangeoilperkgBWinsheepinfectedwithH.contortus,it resultedina97.4%reductioninfecaleggcount(adultworm reductionwasnotevaluated).Althoughencouraging,these resultsmust beinterpreted withcautionbecauseofthe highdosesofthepreparation(40%orangeterpenes,20% Valenciaorangeoil,4%polysorbate80,and1.5%hydrogen peroxide)required for anthelminticeffects.The authors mentionedthatfewlambspresentedtoxicitysignssuchas headshakingandfeedaversion.Thesesymptomsmaybe aggravatediftheactivecomponent(s)has(ve)alowLD50. Inthecaseoftheorangeoilsused,theauthors(Squires etal.,2010)reportedthat>95%wasd-limonene,whichhas ahighLD50(5000mg/kg).

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content, and which metabolites are being generated. Besidesnematocidaleffect,plantextracts/compoundsare testedfortheirabilitytoimpairegghatchingandlarval developmentfromfecesofinfectedanimalstreatedwith thoseplantextracts.Desiredeffectscanresultinreduced re-infectionandlighterwormloadsleadingtodecreased pasture contamination levels (Ketzis et al., 2002; Max, 2010). In vivo tests, problems withabsorption through thegastrointestinal tract, and compound solubility and stabilityafteroralintakearethemainobstaclesin devel-opingherbalformulationswithgoodbioavailability and anthelminticefficacy.AccordingtoStepeket al. (2007), giventhesensitivitytopH,itisnotsurprisingthatplant enzymesforinstance havelower efficacyagainst stom-achnematodesinsituthanagainstthoseresidingfurther downthegastrointestinaltract.It isnecessaryto evalu-ateadditional parameters onongoing research,suchas performancemeasurements,indicatorsofimmunity,and behavioralobservationswhenconsideringthepotentialof suchplants(Athanasiadouetal.,2007).

Thus,albeit encouraging,ourpositive resultswithC. schoenanthus have to be interpreted with caution and testedinvivotoconfirmorrefuteourinvitroresults,and withintherealmofhost–parasiteinteractivephysiology, biochemistry,compoundavailabilityortoxicity.

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