w ww.e l s e v i e r . c o m / l o c a t e / b j p
Original
Article
In
vitro
activity
of
the
essential
oil
from
Hesperozygis
myrtoides
on
Rhipicephalus
(Boophilus)
microplus
and
Haemonchus
contortus
Caroline
V.V.
Castilho
a,
Rafaela
R.
Fantatto
b,
Yousmel
A.
Gaínza
c,
Humberto
R.
Bizzo
d,
Nancy
S.
Barbi
a,
Suzana
G.
Leitão
a,
Ana
Carolina
S.
Chagas
e,∗aFaculdadedeFarmácia,UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro,RJ,Brazil
bFaculdadedeCiênciasFarmacêuticasdeAraraquara,UniversidadeEstadualPaulista,Araraquara,SP,Brazil cCentroNacionaldeSanidadAgropecuaria,SanJosédelasLajas,Mayabeque,Cuba
dEmbrapaAgroindústriadeAlimentos,Guaratiba,RJ,Brazil eEmbrapaPecuáriaSudeste,SãoCarlos,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received7June2016 Accepted4August2016
Availableonline17September2016
Keywords:
Phytotherapy Medicinalplants Essentialoils Pulegone Acaricides Anthelmintics
a
b
s
t
r
a
c
t
Commercialantiparasiticshavebeenthemaintooltocontrolparasites,butduetotheresistance devel-opment,plantextractshavebeenwidelyinvestigatedtofindnewmolecules.Thepresentstudyaimed toinvestigatetheinvitroacaricideandanthelminticactivitiesoftheessentialoilfromtheaerialpartsof
Hesperozygismyrtoides(A.St.-Hil.exBenth.)Epling,Lamiaceae.Theessentialoilwasobtainedby
hydrodis-tillationanalyzedbyGC-FIDandGC-MS.Fourtestswereconductedinvitrotoscreentheantiparasitic actionoftheessentialoil.TheevaluationonRhipicephalus(Boophilus)micropluswasperformedwiththe adultimmersiontestatconcentrationsrangingfrom0.391to25mg/mlandthelarvalpackettestfrom 3.125to100mg/ml.ForHaemonchuscontortustheegghatchtestwasperformedfrom0.012to25mg/ml andthelarvaldevelopmenttestfrom0.003to0.4mg/ml.TheLC50andLC90valueswerecalculatedby Probit.Themaincomponentsidentifiedintheessentialoilwereisomenthone(47.7%),pulegone(21.4%), limonene(7.7%),isomenthylacetate(6.8%)andneoisomenthol(3.9%).InthelarvalpackettesttheLC50 andLC90were13.5and21.8mg/ml,respectively.Inegghatchtest,theLC50andLC90were0.249and 0.797mg/ml,respectively,whileinthelarvaldevelopmenttestwere0.072and0.167mg/ml, respec-tively.ThisisthefirstreportoftheH.myrtoidesevaluationagainstthoseparasites.Theanthelmintic resultsproveditsefficacyonH.contortusencouragingnewresearchwithafocusontheirmainbioactives. ©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Rhipicephalus(Boophilus)microplus,Ixodidae,knownasthe cat-tletick,isanectoparasiteendemictothetropicalandsubtropical regionsoftheworld(Rosado-Aguilaretal.,2010;Lopez-Ariasetal., 2014).ThistickhasbecomeincludedinthegenusRhipicephalus aftermolecularandmorphologicalstudiesshowingthe phyloge-neticrelationshipbetweenRhipicephalusandBoophilus(Beatiand Keirans,2001;MurrellandBarker,2003).Thisparasitehasbeena majorproblemforlivestockfarmers,bysettingaveryefficient host-parasiterelationship.Thisfacthascausedahugelossandincreasing spendingonacaricidechemicalstocontroltheinfestation.Sothat controlofthisparasiteisdoneatfarmlevelandtheacaricide prod-uctsarefrequentlyadministratedatamonthlybasisthroughoutthe
∗ Correspondingauthor.
E-mail:carolina.chagas@embrapa.br(A.C.S.Chagas).
year(Bianchietal.,2003).Thecattletickinfestationscausemany economiclossestothelivestockfarmersduetobloodloss, dam-ageintheanimal’sskin,reducedweightgainandtransmissionof pathogenssuchasBabesiabovis,BabesiabigeminaandAnaplasma marginale,whichcausethedisease“TristezaParasitária”,mainlyto thecalves(Grisietal.,2014).
The Haemonchus genus belongs to the Trichostrongylidae family, which present several species that parasite ruminants, including Haemonchus contortus (Rudolphi 1803) Cobb 1898; HaemonchussimilisTravassos1914;Haemonchuslongistipes Rail-lietetHenry1909;and HaemonchusplaceiPlace1893(Jacquiet et al.,1997).H. similis is rarelyfoundin parasitic infections of smallruminantssuchassheepandgoatsbutH.contortusis preva-lentanddominantintermsofintensityofinfection(Achietal., 2003).Therefore,sheepandgoatsshowuphighlysusceptible,with highestablishmentrateofinfectionandlargeexcretionofeggsby females,comparedtootherruminantspecies.Itslifecycleincludes afreelifestage(fromeggtotheinfectivelarvaeL3)andaparasitic
http://dx.doi.org/10.1016/j.bjp.2016.08.005
stage(fromL3ingestedbythehost,totheadultphase)(Jacquiet etal.,1998).NaturalpopulationsofH.contortusinhabittheUnited States,Brazil,Argentina,Australia,Europe(Scotland,Irelandand France)andAfrica(CongoandMauritania)(Jacquietetal.,1997). ThepathogenesiscausedbyH.contortusischaracterizedbyasevere anaemiaduetoblood-suckinghabitoftheworm,leadingtoa seri-ousimpairmentoftheanimal,severeeconomiclossesandeven death(Rodríguezetal.,2015).
The parasite resistance to anthelmintic drugs increasingly affectsanimalssuchascattle,goatsandsheep,andiswidelyspread amongseveralgeneraandphylaofhelminthes(Roseetal.,2015; Paraudetal.,2016).Theresistanceexpressionoccurswhen treat-mentwiththedrugallowsthesurvivalofresistantparasites,which reproduceandcontributewithresistantgenestothenew gen-erations. Thefailure of parasitecontrol providesanincrease of individualsabletosurvivetoanantiparasiticdosethatwouldbe lethaltothemajorityoftheparasitesinasusceptiblepopulation of thesame species (Gearyet al., 1999; Sangster,1999, 2001). Thisresistancecomesmainlyfromtheintensiveuseofsynthetic antiparasiticswithsub-dosagesandapplicationsatshorter inter-valsthannecessary(Alvesetal.,2012;Lopez-Ariasetal.,2014).
Controlstrategiesforparasitesthatminimizetheuseof syn-theticantiparasiticsareofincreasingimportance,aswellasthe trendtowardsnon-chemical(ecological,organic,green)farming oflivestock(Learmountetal.,2016).Secondarymetabolitesfrom plants arethesubjectof researchersin thescreeningfor prod-uctsthat are less harmful to theenvironment, therefore more sustainable,moreselective andwithpotenteffect ona specific target.Hesperozygismyrtoides(A.St.-Hil.exBenth.)Eplingbelongs totheLamiaceaefamilyandispopularlyknownas“poejo” (pen-nyroyal).ThisplantisendemictosoutheasternBrazil,growingin theCerradoandAtlanticForestbiomes,ataltitudeabove1800m. It possesses a mint aroma that has been associated with its essentialoilrichinmonoterpeneketones.Themainchemical com-poundsin theessentialoilfromtheaerialparts ofH.myrtoides arepulegone(19.8–57.3%),isomenthone(14.3–47.7%),limonene (2.1–22.7%) and isomenthyl acetate (0.3–14.3%) (Martini et al., 2011;Castilhoetal.,2016).
The essential oils and terpenoids from many plants rich in monoterpeneketones,suchasmenthone,isomenthoneand pule-gone,havebeenextensivelytestedagainstsomeparasites,showing acaricidalandanthelminticactivity(Faceyetal.,2005;Taketal., 2006; Rosado-Aguilaret al., 2010; Amer et al., 2011; Jeon and Lee,2011;KamarajandRahuman,2011;Martinez-Velazquezetal., 2011;Carvalhoetal.,2012;Chagasetal.,2012;Molefeetal.,2012; Ferreiraetal.,2013;Kocetal.,2013).Therefore,thepresentstudy aimedtoinvestigatetheinvitroacaricideandanthelmintic activi-tiesoftheessentialoilfromH.myrtoidesaerialparts.
Materialandmethods
Plantoriginandextraction
AerialpartsofH.myrtoides(A.St.-Hil.exBenth.)Epling, Lami-aceae,werecollectedinJuly2012inthe“Campodospoejos”(GPS coordinates:22◦231.83′S/44◦3830.20′W,Aiuruoca,MG,Brazil). Plant identificationwasperformedbyDr. RosanaC. Lopes,and voucherspecimensweredepositedintheDepartmentofBotany, FederalUniversity of Riode Janeiro,Rio de Janeiro,Brazil(RFA 39448).Samplesof200goffreshaerialpartsweresubjectedto hydrodistillationinaClevenger-typeapparatusfor2h.The essen-tialoilwasseparatedfromthehydrolatebycentrifugationanduse ofmanualpipette,andthenstoredunderrefrigerationinsealed amberflask.Theyieldwascalculatedasmlofoilg−1offreshweight oftheplantmaterial.
Chemicalanalyzes
ChemicalanalysesoftheH.myrtoidesessentialoilwere per-formedatEmbrapaAgroindústriadeAlimentos.Theidentification of the essential oil components was carried out by gas chro-matographyanalyses, performedwithin2–3daysafteressential oilextraction,using anAgilent6890Ngaschromatograph(Palo Alto,CA, USA)equipped witha flame ionization detector(FID) and HP-5 (5% phenyl – 95% dimethylpolysiloxane) fused sil-ica capillary column (30m×0.32mm×0.25m). The injector temperaturewaskeptat250◦C and theovertemperature pro-grammed from 60 to 250◦C at 3◦Cmin−1. Detector (FID) was operatedat280◦C. Onemicroliterofa 1%solutionoftheoilin dichloromethanewasinjectedinsplitmode(1:50).The percent-agesofeachcomponentwerereportedasrawpercentageswithout standardization. GC-MS analyseswere performedin anAgilent 5973NmassselectivedetectorcoupledtoanAgilent6890gas chro-matograph(PaloAlto,CA),fittedwithaHP-5MScapillarycolumn (30m×0.25mm×0.25m),operatinginelectronicionization(EI) modeat70eV.Transferlinewasmaintainedat260◦C,whilemass analyzer and ion source temperature were held at 150◦C and 230◦C,respectively.Helium(1mlmin−1)wasusedascarriergas. Oventemperatureprogramme,injectortemperatureandsplitrate werethesameasstatedforGCanalyses.Astandardsolutionof n-alkanes(C7–C26),injectedinthesamecolumnandconditionsas above,wasusedtoobtainthelinearretentionindices.Individual volatilecomponentswereidentifiedbycomparisonoftheirmass spectra(MS)andlinearretentionindexes(LRI)withthosereported inliterature(Adams,2007),aswellastotheWileyRegistryofMass SpectralData,6thEdition(1994).
Invitroassays
Adultimmersiontest(AIT)
TheinvitrotestswerecarriedoutintheAnimalHealth Lab-oratoryofEmbrapaPecuáriaSudeste.EngorgedfemalesofR.(B.) microplus were collected from cattle kept at the experimental farm.Accordingtoaresistanttestperformedin2016,theseticks areresistanttopyrethroids,organophosphatesandamidines.The specimenswereselectedaccordingtotheirintegrity,motilityand maximumengorgement.Thetickswerethenweighedand sepa-ratedintogroupsoftenwithhomogeneousweights,withthree repetitions for each concentration including the control group (immersedindistilledwateralone)andblank(2%tween80).
Theengorgedfemaleswereexposed totheoilin concentra-tionsof25.0;12.5;6.25;3.13;1.56;0.78and0.39mg/ml.Females wereimmersedinthetreatmentsolutionsfor5min,afterwhich weredriedonabsorbentpaperandplacedinsterilePetridishes andincubated(27±1◦C,RH>80%)tocompletethelifecycle.At theendofoviposition,theeggswereweightedandtransferredto adaptedplasticsyringes,identifiedandsealedwithcotton.Theeggs wereputbackintotheincubator(Tecnal,TE-391model)underthe sameconditionsforlarvalhatching.Thedataobtainedwereused todeterminethepercentageofreductiononeggovipositionand larvalhatching,aswellastocalculatethereproductiveefficiency index(REI)and theefficacyoftheessentialoil(E),accordingto Drummondetal.(1973).
Larvalpackettest(LPT)
6.25mg/ml.Theexperimentwasconductedintriplicateforeach dilution,withcontrolgroup(distilledwater)andblank(2%Tween 80).Thepacketswereincubatedundertheconditionpreviously described.Thenumbersofdeadand livinglarvaewerecounted after24hincubated(27±1◦C,RH>80%)withtheaidofavacuum pumpwithanadaptedpipettetip.Larvaethatwerecompletely immotilewereconsidereddead.
Egghatchtest(EHT)
ThetestswereperformedusinganH.contortusisolate resis-tanttobenzimidazole,macrocycliclactone,andimidazothiazole (Embrapa,2010;Chagasetal.,2013).Faeceswerecollecteddirectly fromtherectumofthesheepdonorandtheeggsrecoveredwith theuseofsequentialsievesaccordingtoColesetal.(1992).Briefly, faecesweremixedwithdistilledwaterandfilteredthrough100,56, 30and25mmeshsieves.Inthelastsieve,theeggswereretained andwashedwithdistilledwaterandcentrifugedinFalcontubes (50ml)at3000rpm/5minfilledwithwater.Thenthesupernatant wasremovedandaNaClsaturatedsolutionwasadded.This solu-tionwasonceagaincentrifugedunderthesameconditionsandthe supernatantwaswashedthrougha25msieve.Theeggswere col-lectedandquantifiedunderaninvertedmicroscope(Zeiss,Axiovert 40CFLmodel).Approximately100eggswerethenplacedineach wellof24-wellplates,alongwiththetreatments,reachingatotal volumeof500l.Alltestsweredoneinsixreplicates,including controlgroup(distilledwater)andblank(2%Tween80).Thenthe plateswereincubated(27±1◦C,RH>80%)for24hwhenlarvaeand eggswerecountedintheinvertedmicroscopetocalculatehatching inhibitionpercentage(Chagasetal.,2011).
Larvaldevelopmenttest(LDT)
Approximately100eggsobtainedaccordingtothetechnique describedabove,wereplacedineachwell.Eachonealsoreceived 50lof Escherichiacoli lyophilized in suspension(ATCC 9637), 10lofamphotericin Band 20lof nutrientmedium (Hubert andKerboeuf,1992).Distilledwaterwasaddedtoreachatotal volumeof250l.Thematerialwashomogenizedandincubated (27◦C±1◦CandRH>80%)for24h.Afterthisperiod250lofthe solutionswereaddedtoeachwell.Alltestsweredoneinsix repli-cates,includingthecontrolgroup(distilledwater)andthesolvent blank(0.5%DMSO).Plates wereincubatedfor6 days,when L1, L2andL3werecountedtocalculatelarvaldevelopmentinhibition percentage(Chagasetal.,2011).
Dataanalyses
IntheAITthereproductiveefficiencyindex(REI)andtheefficacy oftheessentialoil(E)werecalculatedaccordingtotheformulas proposedbyDrummondetal.(1973):
REI=eggmassweight×%egghatching/engorgedfemalesweight
×20,000∗
*Constantthatindicatesthenumberofeggspresentin1g
E=(REIcontrol−REItreated)/REIcontrol×100
IntheEHTtheHatchingInhibitionPercentagewascalculated accordingtotheformula:[(numberofeggs/numberofeggs+larvae in the well)]×100. In theLDT theLarval Development Inhibi-tionPercentage=[(numberofL1+L2/numberofL1+L2+L3 inthe well)]×100.TheLC50andLC90weredeterminedbystatistical anal-ysisusingtheProbitprocedureoftheSASprogram(SAS2002/2010) andthedataonconcentrationswerecomparedwithineach treat-mentwiththeTukeytest(p<0.05).
Resultsanddiscussion
Chemicalanalyzes
TheessentialoilofH.myrtoides,obtainedfromplantscollected inJuly2012with0.74%yield,wasusedinfourdifferentinvitrotests todeterminetheirantiparasiticactivity.Inpreviousworksfrom ourgroupwehavedeterminedthattheessentialoilyieldsofthis plantvariesconsiderably(0.74–2.99%),dependinguponthe collec-tionsite,altitude,andtimeofyear(Martinietal.,2011;Castilho etal.,2016).Theanalysisoftheoilallowedtheidentificationof 99.2%compounds(Table1)andthemainoneswereasfollows: iso-menthone(47.7%),pulegone(21.4%),limonene(7.7%),isomenthyl acetate(6.8%)andneoisomenthol(3.9%),respectively,asdepicted inthechromatogram(Fig.1).Themainsubstancesidentifiedinthe presentstudyarealsothemajorcompoundsintheH.myrtoidesoil reviewedbyMartinietal.(2011),featuringwellthisplantspecies.
Acaricidalactivity
Laboratory tests were conducted to determine the efficacy oftheH.myrtoidesessential oilonengorgedfemale andlarvae ofR.(B.)microplus.Intheadultimmersiontest(AIT)theactionof theoilwasevaluatedbymeasuringtheinhibitionofegg hatch-ing, reproductive efficiency index and efficacy of the oil (E),
Table1
Chemicalcompositionandrelativepercentagesoftheidentifiedcompoundsinthe essentialoilofHesperozygismyrtoides.
Compoundsa LRI
calc.b LRIlit.c %
␣-Pinene 932 932 0.4
Sabinene 972 969 0.3
-Pinene 976 974 0.5
Myrcene 990 988 1.0
p-Cymene 1024 1022 0.1
Limonene 1027 1024 7.7
1,8-Cineole 1030 1026 0.8
cis-Ocimene 1036 1032 0.2
trans-Ocimene 1046 1044 1.3
Linalool 1103 1095 0.4
Octen-3-ylacetate 1113 1110 0.2
Menthone 1153 1148 0.5
Isomenthone 1165 1158 47.7
Isopulegone 1176 d 0.4
Terpinen-4-ol 1178 1174 0.1
Neoisomenthol 1185 1184 3.9
␣-Terpineol 1193 1186 0.1
Pulegone 1240 1233 21.4
2-Phenylethylacetate 1259 1254 0.1 Isomenthylacetate 1308 1304 6.8 Neoiso-pulegylacetate 1312 1312 0.3
␣-Terpinylacetate 1349 1346 0.3
␣-Copaene 1373 1374 0.1
-Bourbonene 1381 1387 0.1
-Caryophyllene 1415 1417 1.8
n.i.(MW:166) 1421 n.i. 1.4
␣-Humulene 1449 1452 0.2
GermacreneD 1477 1480 0.7
Bicyclogermacrene 1492 1500 0.5
Spathulenol 1576 1577 0.2
Caryophylleneoxide 1578 1582 0.2
Monoterpenes 87.1
Sesquiterpenes 3.8
Totalofidentifiedcompounds 99.2
aComponentswerereportedaccordingtheirelutionorderonSE-52.
bLinearretentionindexbasedonaseriesofn-hydrocarbonsreportedaccording
totheirelutionorderonHP-5MS.AdaptedfromCastilhoetal.(2016).
c Identificationconfirmedbycomparingmassspectraandretentiontimeswith
thoseobtainedfromtheliterature(Wiley,1994;Adams,2007).
d Tentativelyidentifiedbycomparingmassspectra;t,traces(<0.1%).
Table2
Averageofthefemales’weight(FW),eggmassweight(EW),percentageofegghatching(%H),reproductiveefficiencyindex(REI),andefficacyoftheessentialoil(E)in engorgedfemalesofRhipicephalus(Boophilus)microplus,testedbymeansofemersionindifferentconcentrations(mg/ml)ofHesperozygismyrtoidesoil.
Concentrations FW(g) EW(g) %H REI E(%)
25.0 2.05±0.02A 0.85±0.06A 76.67±3.33A 63.90±5.86A 24.97
12.5 2.06±0.00AB 0.84±0.07A 83.33±3.33AB 68.15±7.86A 19.98
6.25 2.03±0.00A 0.90±0.04A 80.00±0.00AB 70.79±3.36A 16.89
3.12 2.07±0.01AB 0.88
±0.03A 86.67
±3.33AB 74.03
±3.37A 13.08
1.56 2.07±0.01AB 0.92
±0.03A 85.00
±2.89AB 75.36
±2.84A 11.52
0.78 2.06±0.01AB 0.89
±0.06A 90.00
±0.00B 77.83
±5.49A 8.62
0.39 2.11±0.03B 0.95
±0.05A 86.67
±3.33AB 78.16
±6.46A 8.23
Control 2.05±0.01A 0.97±0.08A 90.00±0.00B 85.17±7.02A 0.0
Differentlettersinthecolumnindicatestatisticallysignificantdifference(p<0.05).
pA 180
160
140
120
100
80
60
40
20
0 10
a
b d
e
c
2266
4660 5592 7011
7125
9975 11 491 12 443
12 709
11 935
14 878
15 664 17 830
17 639
19 309 20 594 21 542 23 004 24 961 28 206
20 30
Fig.1. Representativechromatogram(GC-FID) oftheessential oilfrom fresh aerialpartsofHesperozygismyrtoides.alimonene;bisomenthone;cneoisomenthol; dpulegone;eisomenthylacetate.
demonstratinglow hatchinginhibitionandloweffecton repro-ductiveefficiencyandtherefore,lowefficacy(Table2).
Todate,nostudyreportingtheacaricideactivityofH.myrtoides essentialoilonthecattletickR.(B.)microplushasbeendescribedin theliterature.ThereisonlyonestudyofRibeiroetal.(2010)with thespeciesHesperozygisringens,inwhichitsacaricideactivitywas evaluatedagainsttheR.(B.)microplusengorgedfemales,by eval-uatingtheeggproduction.Itseemsthatitdidnotcausefemales’ mortality,buttheauthorsobservedthattheoilatconcentrations of50and25l/ml,inhibittheegglayingofthefemalesin76.4 and48.0%,respectively.Inaddition,theegghatchingwasinhibited in95%and30%inthesameconcentrations.Theisolatedpulegone wasalsotestedanditseffectwassimilartothatoftheessentialoil (Ribeiroetal.,2010).Inthepresentstudy,thegreateracaricide effi-cacywas24.97%onengorgedfemalesandtherewassomeaction onthelarvalhatchingofthefemalestreatedwiththeoil.
The LPT evaluatesthe mortalityof the R. (B.) microplus lar-vaeaftercontactwiththeH.myrtoidesessentialoil,wherelarvae withoutmovementwereconsidereddead.Itcanbeobservedin the present study that up fromthe concentration of 25mg/ml theH.myrtoidesoileliminated100%ofthelarvae(Table3).This concentrationmaybeconsideredtoohigh,becauseaccordingto
Table3
PercentageofmortalityofRhipicephalus(Boophilus)micropluslarvae(±standard deviation)submittedtotheLarvalPacketTest(LPT)causedbyHesperozygis myr-toidesessentialoilindifferentconcentrations.
mg/ml %
100.0 100±0.0E
50.0 100±0.0E
25.0 100±0.0E
12.5 57.25±0.38D
6.25 13.79±0.11C
3.12 5.60±0.40B
Control 0.0±0.0A
Differentlettersinthecolumnindicatestatisticallysignificantdifference(p<0.05).
Ribeiroetal.(2010),theessentialoilofH.ringensandtheisolated pulegone,inconcentrationsof20.0–0.625l/mlwerelethaltothe totalityofthelarvae.Theauthorssuggestthatthemonoterpene pulegoneisresponsiblefor thepowerfulacaricide activityof H. ringensoiltofemalesandlarvaetickofR.(B.)microplus.
TheLC50andLC90hereobtainedwere13.50and21.80mg/ml, respectively,anditcanbebestviewedinTable4.ThesehighLCs suggestthattheH.myrtoidesoilisnotapotentacaricide.Itis possi-blethatthisresulthascorrelationwiththepulegoneleveldetected intheoil(21.4%)comparedtothatonH.ringensoil(86.0%)(Ribeiro etal.,2010).AccordingtothediscussionofRibeiroetal.(2010), attemptstoevaluatepenetrationandcutaneousabsorptionofthe oilcomponentsareneeded,mainlyconsideringtheconsumption ofmeatanddairyproductsfromcattle.Pulegonehasbeen asso-ciatedwithseverehepatotoxicityanddeathindogsandmouse, aswellasmenthofuran,itsmetaboliteproducedbyoxidationvia CytochromeP450(Sudekumetal.,1992;Sztajnkryceretal.,2003). The acaricidalactivityof many pureterpenes againstR. (B.) microplus has been shown in previous studies, due mainly to oxygenatedcompounds,suchas1,8-cineole,citronellol,linalool, isopinocamphone,camphor(Pratesetal.,1998),carvacrol (Cruz etal.,2013),thymol(Monteiroetal.,2010;Scoraliketal.,2012;Cruz etal.,2013;Araújoetal.,2015),cadina-4,10(15)-dien-3-one(Porter etal.,1995),eugenol(Valenteetal.,2014),carvone(Peixotoetal., 2015),nerolidol(Lageetal.,2013)andnon-oxygenatedcompound, likelimonene(Ferrarinietal.,2008;Peixotoetal.,2015).
SeveralspeciesoftheLamiaceaefamilyhadtheiracaricide activ-itytested,especiallythosefromthegenusCunila.Speciesofthe genus are also known in Brazil as Poejo, because of its minty aroma(Apeletal.,2009).TheessentialoilfromCunila angustifo-lia,CunilaincanaandCunilaspicatacaused100%ofmortalitytothe R.(B.)micropluslarvaeat5l/ml,evaluatedintheLarval Immer-siontest.Intheseoils,themaincompoundswerethemonoterpenes sabinene(32.1%);␣and-pinene(26.7and27.5%);andlimonene, menthonfuranandborneol(12.0,34.8and19.7%,respectively).In theoilsfromthesespecies,nopulegonewasdetected.
Ontheotherhand,theessentialoilsfromCunilaincisaandCunila microcephalaat10l/mlkilled18and5%ofthelarvae,respectively, i.e.,presentedlowlarvicidalactivity.Themaincompoundsfound intheseoilswerethemonoterpenes1,8-cineole(55.4%)and men-thofuran(72.7%),respectively(Apeletal.,2009).Theessentialoilof Hyptisverticillata,alsobelongingtothefamilyLamiaceae,wasquite
Table4
CL50andCL90(±95%confidencelimits)obtainedintheLarvalPacketTest(LPT)
withRhipicephalus(Boophilus)micropluslarvae,EggHatchTest(EHT)andLarval DevelopmentTest(LDT)withH.contortussubmittedtotheHesperozygismyrtoides
essentialoil.
Invitrotest LC50(mg/ml) LC90(mg/ml)
LPT 13.50(12.05–15.23) 21.80(18.76–27.43)
EHT 0.249(0.238–0.261) 0.797(0.743–0.860)
moreeffectivefortickfemalesinrelationtothepresentstudy.The oilat4mg/mlcausedmortalityofR.(B).microplusadultfemalesin 96h(45%),interruptionofoviposition(87%)andoftheegg hatch-ingofthistick(90%)(Faceyetal.,2005).However,it’schemical compositionisquitedifferentfromthosemonoterpenerichoilsof HesperozygisandCunilaspecies,beingrichinsesquiterpenes,with cadina-4,10(15)-dien-3-one(15.1%)and aromadendr-1(10)-en-9-one(30.7%),asmajorsesquiterpene-ketones,followedbytheminor compoundsviridiflorol(4.3%)andspathulenol(2.2%).
Anthelminticactivity
The EHT assessed the percentage of hatching inhibition on H.contortuseggs,afterbeingtreatedwiththeoftheessentialoil solutionatconcentrationsfrom25.0to0.012mg/ml.Inthistest,up fromtheconcentrationof3.12mg/ml,100%ofegghatching inhi-bitionwasdetected(Table5).TheLC50andLC90resultsintheEHT were0.249and0.797mg/ml,respectively(Table4).
Concentrationsfrom0.40 to0.0125mg/ml wereusedin the LDT and the oil demonstrated better efficacy at this parasite stagebecause100% ofthe larvaldevelopmentwasinhibited at 0.40mg/ml(Table6).TheLC50andLC90 resultswerealsolower thanin theEHT, wherethe values obtainedwereof0.072 and 0.167mg/ml,respectively(Table4,Fig.2).
TheH.myrtoidesessentialoilevaluatedinthepresentstudyis richinmonoterpenes,whichareaclassofsecondarymetabolites withavastarrayofbiologicalactivities,includingantimicrobial, acaricideandanthelmintic(Katikietal.,2011;Chagasetal.,2012; Chagasetal.,2014).Inthis oil,36 constituentswereidentified, beingthemajoronesisomenthone,pulegone,limonene, isomen-thylacetateandneoisomenthol.Thesesecondarymetabolitesare knowntopresentabroadaction againstvariousorganisms and synergismthatcanbeeffectivefordifferentparasites,since essen-tialoilsareacomplexmixtureofsubstancesthatcaninteractwith
Table5
AveragepercentageofhatchinginhibitionofHaemonchuscontortuseggs(±standard deviation)submittedtodifferentconcentrations(mg/ml)ofHesperozygismyrtoides
essentialoil,evaluatedintheEggHatchTest(EHT).
mg/ml %
5.0 100±0.0G
12.5 100±0.0G
6.25 100±0.0G
3.12 100±0.0G
1.56 99.3±1.0G
0.78 92.0±1.9F
0.39 59.3±3.1E
0.19 39.4±4.5D
0.098 16.9±1.6C
0.049 4.5±1.8B
0.024 1.7±0.69AB
0.012 0.74±0.87A
Control 1.4±1.3AB
Differentlettersinthecolumnindicatestatisticallysignificantdifference(p<0.05).
Table6
AveragepercentageofdevelopmentinhibitionofHaemonchuscontortuslarvae (±standarddeviation)submittedtodifferentconcentrations(mg/ml)of Hesperozy-gismyrtoidesessentialoil,evaluatedintheLarvalDevelopmentTest(LDT).
mg/ml %
0.40 100.0±0.0F
0.20 94.1±1.9E
0.10 67.4±4.6D
0.05 26.6±1.9C
0.02 6.2±1.1B
0.01 2.0±0.4A
Control 2.0±0.7A
Differentlettersinthecolumnindicatestatisticallysignificantdifference(p<0.05).
0.1 0.08
0.06 0.04
0.02 0
0 10 20 30 40 50 60 70 80 90 100
1 0.8
0.6 0.4
0.2 0
LDT concentrations (mg/ml)
% i
n
h
ib
iti
on
EHT concentrations (mg/ml)
EHT LDT
Fig.2.Inhibitionoftheegghatchingandlarvaldevelopment(LC50andLC90)ofH.
contortusofsheepsubmittedtotheHesperozygismyrtoidesessentialoil concentra-tions.
multipletargetsin variousstages oftheirdevelopment (Marie-Magdeleineetal.,2009).
Ingeneral,theactionofessentialoilsistheresultofcombined effectsthatactivateorinactivatestructures.Theypossiblyactby breakingordisruptingmembranesbyactiononthelipophilic com-pounds, causing lossof severalenzymesand nutrientsthrough thecellmembrane(Cowan,1999;Coxetal.,2000).Accordingto Sikkemaetal.(1993,1995),thecellmembranecanbesignificantly differentbetweenoneorganismandanother,affecting permeabil-ityandhencetheresponseofthesameinhibitorycompounds.Thus, thedifferencesbetweentheperformancesoftheessentialoilon theparasitescanbeattributedtointeractionsbetweenthese com-poundsandparasiticstructures,beingmoreefficientinH.contortus. Basedontheresultsobtained,theessentialoilfromaerialparts ofH.myrtoidespresentedinvitroanthelminticpropertyagainstthe gastrointestinalnematodeH.contortus.Thosetestsarethemost usedtodetectinvitroanthelminticactivityandresistancein nema-todesofsmallruminants.Theyareinterpretedbasedonthelethal concentrations,becausehighLC50suggestthepresenceof para-siticresistance(Coles,2005;Váradyetal.,2009).Accordingtoour knowledge,thisisthefirstreportoftheanthelminticactivityofthe H.myrtoidesessentialoilonthisimportantparasite.
Conclusion
Insum,fromthesignificantresultsobtainedinthiswork, espe-cially against the parasite H. contortus, the possibility of using H.myrtoides essential oilto control gastrointestinalnematodes encouragesfurtherresearchfocusingonbioactiveconstituentsfor veterinaryuseinsmallruminantsandcattle.Suggestionofa pos-sibleprotection of ruminantsagainst parasites requires careful studiesandconsiderationduetointakeofmeatandmilkfromthose animals.
Ethicaldisclosures
Protectionofhumanandanimalsubjects. Theauthorsdeclare thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).
Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.
Authors’contributions
CVVCextractedtheessentialoilanddraftedthepaper.RRF,YAG performedthebioassayworkandHRBperformedGCanalyses.NSB, SGLparticipatedintheresults,discussionandwritingthepaper. ACSCorganizeddata,setstatisticalanalysesandthemanuscript finalwriting.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
We are grateful to Dr. Rosana C. Lopes from the Federal UniversityofRiodeJaneiroforthetaxonomicidentificationofplant material,toEmpresaBrasileiradePesquisaAgropecuária,project 03.11.01.023.00.00,CAPES(CAPESMESCUBA952/2013),CNPq,and FAPERJforfundingthisstudy.
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