www.bjorl.org
Brazilian
Journal
of
OTORHINOLARYNGOLOGY
ORIGINAL
ARTICLE
Preclinical
evaluation
of
Luffa
operculata
Cogn.
and
its
main
active
principle
in
the
treatment
of
bacterial
rhinosinusitis
夽
Leonardo
Silva
a,∗,
Henrique
Olival
Costa
b,
Flávia
Coelho
de
Souza
c,
Elaine
Monteiro
Cardoso
Lopes
d,
Suely
Mitoi
Ykko
Ueda
baSantaCasadeSãoPaulo,SãoPaulo,SP,Brazil
bSantaCasadeSãoPaulo,FaculdadedeCiênciasMédicas,SãoPaulo,SP,Brazil cInstitutodeCiênciasAvanc¸adasemOtorrinolaringologia(ICAO),SãoPaulo,SP,Brazil dUniversidade9deJulho(UNINOVE),SãoPaulo,SP,Brazil
Received7June2016;accepted22November2016 Availableonline26December2016
KEYWORDS Sinusitis; Therapeutics; Luffa; Microbiology; Streptococcus pyogenes Abstract
Introduction:The prevalenceof rhinosinusitis isquite high.Despitethe widespread useof antibioticsforrhinosinusitis,thereareotherformsoftreatment,includingphytotherapy.One ofthemostwidelyusedherbalmedicinesfortreatmentofrhinosinusitisisLuffaoperculata. Objective:Thisstudyaimedtoevaluatetheefficacyoftopicalnasalsolutionoftheaqueous extractofL.operculata,determiningthetoxicitytoitsuseandidentifyingtheactiveprinciples presentedintheaqueousextract.Thesecondaryobjectivewastoevaluatetheactionofactive principlesonbacteriacommonlyinvolvedinacuterhinosinusitis.
Methods:Thestudy wasconducted inexperimentalmodelofsinusitis.Threedifferent con-centrationsofL.operculatawereusedaslocaltreatmentofrhinosinusitis.Theresultswere comparedwiththoseobservedincontrolgroupsthatreceivednasalsalinesolution.Histological examinationoftheliver,kidney,spleen,myocardium,brainandlungsofallanimalsevaluated thetoxicityofL.operculata.Theaqueousextractusedwassubjectedtochromatographic anal-ysisandanactiveprinciplewasisolatedandtestedforinvitroinhibitionofbacterialcolonies usuallyfoundinrhinosinusitis.
Results:IntranasaltreatmentofsinusitiswithL.operculatashowedbetterclinicalevolution thancontrolgroup. Statisticallysignificant difference (p>0.10) between the treatedgroup andthecontrolgroupwasobservedinthehistologicevaluationforinflammatorypattern.The aqueousextractofL.operculatausedpresentedapredominanceof2,3-dicafeoilglicaricacid,
夽 Pleasecitethisarticleas:SilvaL,CostaHO,SouzaFC,LopesEM,UedaSM.PreclinicalevaluationofLuffaoperculataCogn.anditsmain activeprincipleinthetreatmentofbacterialrhinosinusitis.BrazJOtorhinolaryngol.2018;84:82---8.
∗Correspondingauthor.
E-mail:leosilva@uol.com.br(L.Silva).
PeerReviewundertheresponsibilityofAssociac¸ãoBrasileiradeOtorrinolaringologiaeCirurgiaCérvico-Facial. https://doi.org/10.1016/j.bjorl.2016.11.004
1808-8694/©2016Associac¸˜aoBrasileiradeOtorrinolaringologiaeCirurgiaC´ervico-Facial.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).
asubstancenotyetdescribedintheliterature.Therewasasignificantdifferenceinbacterial growthofStreptococcuspyogenesonblood-agarplateswhenundertheinfluenceofboththe aqueousextractandtheactivesubstance.
Conclusion: TopicalnasalsolutionoftheaqueousextractofL.operculataiseffective com-paredtotheapplicationofsalinesolutionfor thetreatmentofbacterialrhinosinusitisinan experimentalmodel.L.operculatadeterminedinvitroinhibitionofgrowthofS.pyogenes.
© 2016 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). PALAVRAS-CHAVE Sinusite; Terapêutica; Luffa; Microbiologia; Streptococcus pyogenes
Avaliac¸ãopré-clínicadeLuffaoperculataCogn.eseuprincipalprincípioativo notratamentodarinossinusitebacteriana
Resumo
Introduc¸ão: Aprevalênciaderinossinusite(RS)ébastantealta.Apesardousogeneralizadode antibióticosparaRS,existemoutrasformasdetratamento,incluindoafitoterapia.Umadas ervasmedicinaismaisutilizadasnotratamentodaRSéaLuffaoperculata.
Objetivo: Esseestudotevecomoobjetivoavaliaraeficáciadasoluc¸ãotópicanasaldoextrato aquosodeLuffaoperculata,determinandoatoxicidadeaoseuusoeidentificandoosprincípios ativosapresentadosnoextratoaquoso.Oobjetivosecundáriofoiavaliaraac¸ãodosprincípios ativossobreasbactériascomumenteenvolvidasnaRSaguda.
Método: O estudo foi realizado em modelo experimental de sinusite. Utilizaram-se três concentrac¸ões diferentes de Luffa operculatacomo tratamentolocal de RS. Os resultados foramcomparados comosobservadosem grupos decontrolequereceberam soluc¸ão salina nasal.Oexame histológicodofígado,rim,bac¸o,miocárdio,cérebroepulmõesdetodosos animais avaliouatoxicidade deLuffa operculata.Oextratoaquoso utilizadofoisubmetido àanálisecromatográfica eumprincípio ativofoiisolado etestadopara inibic¸ãoinvitrode colôniasbacterianasnormalmenteencontradasemRS.
Resultados: OtratamentointranasaldasinusitecomLuffaoperculatamostroumelhorevoluc¸ão clínica do que o do grupo controle. Foi observada diferenc¸a estatisticamente significante (p> 0.10)entreogrupo tratadoeogrupocontrolenaavaliac¸ãohistológicadopadrão infla-matório.OextratoaquosodeLuffaoperculatautilizadoapresentoupredominânciadoácido 2,3-dicafeoilglicarico,substânciaaindanãodescritanaliteratura.Houveumadiferenc¸a signi-ficativanocrescimentobacterianodeStreptococcuspyogenesemplacasdeágar-sanguequando sobainfluênciatantodoextratoaquosoquantodasubstânciaativa.
Conclusão:A soluc¸ão tópica nasal do extrato aquoso de Luffa operculata é eficaz em comparac¸ão comaaplicac¸ãodesoluc¸ãosalinapara otratamentode RSbacterianaem um modeloexperimental.Luffaoperculatadeterminouainibic¸ãoinvitrodocrescimentode Strep-tococcuspyogenes.
© 2016 Associac¸˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Publicado por Elsevier Editora Ltda. Este ´e um artigo Open Access sob uma licenc¸a CC BY (http:// creativecommons.org/licenses/by/4.0/).
Introduction
AcuteRSisoneofthecommonestdiagnosesinprimarycare, and its management has significant implications for both publichealthandcosts.1Itisestimatedthatchildrenhave
7---10commoncoldseachyear.Theestimatedfrequencyfor adultsis2---5episodes/year.1About0.5%---2%ofthese
com-moncoldsresultinacutebacterialRS.2Sinusitisaffects1in
7adultsintheUnitedStates;resultinginabout31million individualsdiagnosedeachyear.3
Dataobtainedin2002indicatethatRSaccountfor9%of theprescribedantibioticstochildrenand21%ofprescribed antibioticsforadults,whatmakesitthefifthmostcommon
diseasefor which thistype ofmedication is prescribedin theUSA.4
Despite the widespreaduse of systemicantibiotics for sinusitis,therearemanyotherformsoftreatment, compris-ingseveralmedicationsforsystemicandlocaluse.Systemic corticosteroids and Nonsteroidal Anti-Inflammatory Drug (NSAIDs), antihistamines, systemic and topical deconges-tants,anti-leukotrienesandlocalantiseptics,areemployed fortreatment of RS.5 Herbalmedicineis alsowidely used
bythepopulation,5althoughtherearescarcenocontrolled
experimentsintheliteratureshowingitseffectiveness.2,6,7
Amongtheadvantagesofusingherbalmedicinearethe wide acceptance of herbal and medicinal plants by the
populationduetoculturalfactorsandthebeliefthatbeen ‘‘natural’’,presentfeweradverseeffects.8Thelowcostand
itsabundanceintropicalcountriesareotherfactors.7
OneofthemostwidelyusedphytotherapicstotreatRSin BrazilistheLuffaoperculatausedinpreparationsfornasal use.9
Ina surveyconductedinonepopular marketofBrazil, 86%(13of15)ofplantvendorsrecommendedL.operculata
fortreatmentofRS.10
Chemical analysis of L. operculata shows that it has among its components glycosides, saponins, resin, free sterols,aliphaticesters, quinones,organic acids and phe-nols,anditdoesnotcontaintanninsandflavonoids.Inthe resinarefoundelasterinA,BandDandcucurbitacines isocu-curbitacinB.9
DespitethewidespreaduseofL.operculata,thereare fewstudiesthatproveitstherapeuticvalueforsinusitis.5
Theobjectiveofthisstudyistoevaluatetheefficacyof topicalnasalsolutionoftheaqueousextractofL. opercu-lata,determiningthetoxicitytoitsuseandidentifyingthe activeprinciplespresentedintheaqueousextract.The sec-ondaryobjectivewastoevaluatetheactionofL.operculata
activeprinciplesonbacteriacommonlyinvolvedinacuteRS.
Methods
InductionofRSintheanimalmodel
Thestudywassubmittedtotheethicscommitteeinresearch andapprovedundernumber2011-3.Aveterinarian accom-paniedall proceduresperformed on animals.Institutional guidelinesregardinganimalexperimentationwerefollowed. OnehundredeightyadultwhiteNewZealandrabbits,of bothgenders,weighingapproximately2500gatthe begin-ning ofthe experiment wereused.Throughout the study, animalswereconfinedinindividualcagessuitableforrace andweight.
Theanimalsweredividedinto3groups. Onegroupwas followedfor therapeuticevaluation ofL.operculata. This groupwasfollowedfor3differentperiodsoftime.Another groupwasuntreatedwithL.operculata(controlgroup)and finallyonegroup receivedL.operculata toassess its tox-icity.Therefore, each group hadtwenty animals for each follow-uptime.Thusweevaluated20animalsfor therapeu-ticgroup(n=60),60 for thecontrolgroup and60for the toxicitygroup.
Therabbitsweresubmittedtosurgicalproceduresunder generalanesthesiainordertogeneratea nasal inflamma-toryprocess,similartoacuteinfectiousRS.Initiallyaporous spongeofpolyvinylmeasuring3.0cm×0.5cm×0.3cmwas sterilizedinethyleneoxideandthenintroducedinonenasal cavityofeachanimal.
OnemLofasolutioncomposedof0.8mLoftheanimal bloodand0.2mLof streptococcalandstaphylococcal tox-oid(Toxoidepot®),wasinjected percutaneousin maxillary antrumonthesamesidewerethespongewasintroduced. Thespongesweremaintainedinthenasalcavityofeach ani-malfor aperiodoftendays.Afterthisperiodthesponges wereremoved andthetreatmentperiodbegan.Whenthe spongewasremovedfromthenasalcavityandbeforethe start of the treatment each animal had the secretion of
Figure1 Atomizerusedfordrugapplication.
thenasal cavitycollectedby swab(Cuturet®).Samples of sinussecretionsweresmearedonblood-agarculturemedia andchocolate-agar(ProbacdoBrasil).Theplatesonblood agarandchocolate-agarwereincubatedat35◦±2◦C.Daily readingsoftheplateswereheldupto48h.
Preparationofdrugtreatment---L.operculata solution
Using physiological saline solutionasthe solvent,dilution was prepared containing 0.1g of L. operculata aqueous extractin10mLofsalinesolutiontoachievetheexpected concentration.The solutionwasinserted intoanatomizer (Fig.1),whichproducedajetofthemixtureintoanozzle withresultingformationofmicroparticledaerosol.Eachjet applied0.5mLofsolution.
Treatment
AfterRSinductionperiod,animalsinthetherapeuticstudy groupreceivedtreatmentwithnasalapplicationofL. oper-culataaqueousextractdilutedto1%insalinesolution.The animal head was hold vertically and the atomizer nozzle wasinsertedintotherightnostril.Theatomizernozzlewas pressedonce.Thenthedevicewascleanedandtheprocess wasrepeatedintheleftnostril.Theanimalsreceivedaspray ofthesolution,threetimesdailythroughoutthetreatment period.Thecontrolgroupreceivedtreatmentwithsalinein thesameformandamountofthestudygroupforaperiodof 30days.Afterfivedaysoftreatment,20animalsfromeach group were sacrificed. The same procedure wasrepeated after15daysandafter30days.
Histologicalevaluationofsinusmucosa
Immediately after sacrifice maxillary sinus lining mucosa sampleswerecollected.Thehistologicparametersobserved were inflammatory cells infiltration (infiltrate mild, mod-erate or severe), neovascularization (present or absent) andconnective-fibrousproliferation(absentinisolated out-breaks or diffuse proliferation). Allslides wereevaluated by two different pathologists, blinded to the treatment protocol.
Toxicitystudy
Sixtyrabbitsweredividedinto3groupsreceivingL. oper-culata solution at therapeutic concentration for a period of 30days. After receiving the drug, the animals had blood samples, liver, kidney, brain and lung collected to histopathologicalevaluation.
Statisticalanalysis
Thevaluesofmucosalhistologywerealsodescribed accord-ingtogroupsandtimeswiththeuseofabsoluteandrelative frequencies. They were compared using the nonparamet-ricWilcoxontestfortheinfiltrationofinflammatorycells, connective, vascular and fibrous proliferation variables. Comparisonswereperformedtoinvestigatethedifferences betweengroupsorfollow-uptimes.Alltestswereperformed at10%significancelevel.
Results
Of the 180 animalsstarted the experiment 8 died before thesacrificetime.Threeoftheseanimalsbelongedtostudy group, 2to controlgroup and1 totoxicity group.Among these8,3 diedduring theinduction RSperiod.The other animalsdiedsoonafterthestartofthetreatmentperiod.
Oneanimal that belongedtostudy groupdied sixdays aftertheonsetofadministrationofL.operculatanasal solu-tion,1belongedtothecontrolgroupanddiedbetween2and 6daysaftertheonsetofnasaladministrationofphysiologic solution.Ofthe8animals,7diedfromgastro-enterocolitis and1duetopneumonia.Alltheanimalsat theendofRS inductionperiodhadpurulentrhinorrheaatthesidewhere thespongewasplacedandnoneofthemhadcontralateral rhinorrhea.
In order to identify possible histological changes that couldberelatedtothecontinuoususeofthetestsubstance wehistologicallyevaluatedbyhematoxylineosinthe follow-ingorgans:brain,heart,lung,kidneyandliver.Therewere noabnormalitiesthatcouldberelatedtodruguse. Sinussecretionculture
Sinus secretions were takenfrom the inside of each rab-bit maxillary sinus with swab (Cuturet®) after sacrifice. Thesecretionscollectedwereharvestedinbloodagarand chocolate-agar.Thebacteriafoundaftertheprocedureare describedinTable1.
Histologicalfindings
Histological evaluation of sinus mucosa showed various degreesof inflammation,characterized fromintense infil-trationofinflammatorycells(Fig.2),epithelialalterations (Fig. 3), neovascularization, glandular destruction and connective-fibrousproliferationtomucosapractically nor-mal(Fig.4).Suchvariationswerepresentinbothtreatment groups(Table2).
These data show a statistically significant difference between groups and treatment times in the overall
Table1 Descriptionofbacteriaidentifiedinthecultures ofmaterialcollectedinthenostrilsofanimalsatthestart ofthetreatment,controlandstudygroups.
Microorganism Blood agar Chocolate agar Acinetobacterlwoffii 16 3 Acinetobacterbaumanii 1 ---Alcaligenessp. 41 24 Bacillussp. 7 20 Bacillussubtillis 3 2 Micrococcussp. --- 1
Negativeafter48hincubation 8 7
Escherichiacoli 1
---Pseudomonassp. 7 1
Sphingomonassp. 1 1
Staphylococcusaureus 23 7
Streptoccocuspyogenes 8 45
Staphylococcuscoagulasenegative 3 3
Streptococcusviridans 1 2
Pseudomonasaeruginosa --- 2
Figure2 Mucosaofthemaxillarysinusshowingintense lym-phocytic infiltrate (narrow arrow) and glandular destruction (widearrow)---opticalmicroscopy,HEstaining,200×.
Figure3 Mucosaofthemaxillarysinusshowingareasof ero-sionintheepithelium(arrows)andinflammatorycells.Optical microscopy,HEstaining,100×.
Figure 4 Mucosa of the maxillary sinus showing normal epithelium,withoutinflammatoryprocess.Opticalmicroscopy, HEstaining,100×.
Table 2 Results of data comparisons between parameters---acute inflammation, neovascularization andfibrousconnectiveproliferationfordifferenttreatment followup. Acuteinflammation p Time5 0.186299 Time15 0.052137 Time30 0.08863 General 0.08863 Neovascularization Time5 1 Time15 0.567169 Time30 0.002849 General 0.002849
Fibrousconnectiveproliferation
Time5 1
Time15 1
Time30 0.025582
General 0.025582
assessment of the threedifferent criteria used for histo-logicalanalysis.
Histological studies showed no toxicity in the studied organsthatcouldberelatedtotheuseofL.operculata. Chemicaldefinition
Phytochemical assessment of L. operculata was done throughliquidchromatographywithreversedphasecolumn. Elution was carried out in the gradient mode at a flow rateof1.0mL/minandUVdetectionat254nm.Themajor substancewaschosenasphytochemicalmarker.The quan-titativeanalysiswasperformedusingtheexternalstandard method,andthemarker,dulypurifiedandidentified,used asstandard.
Purificationofphytochemicalmarker
Aqueous crude extract was fractionated by solid phase extractiononreversephase(EFS-C18),usingC18silicagel asadsorbent. Ten fractions were obtained and then they wereelutedwithwaterinitiallyandsubsequentlymixtures
of water/MeOH to 100% MeOH. Final purification of the compoundwascarriedoutbyHPLC.
Structuralelucidationofthemarker
The NMR spectra were obtained on spectrometer 500 (11.7T),at500MHzand125MHzfor13C,withsamples dis-solvedinD2O.High-resolutionmassspectrawereobtained and the spectra were obtained in positive and negative modes.
The purifiedsubstance obtainedin the formof an off-whitesolid,wasidentifiedas2,3-dicafeoilglicaricacid.This compoundwasnotdescribedpreviouslyinliterature.
The spectrum of high-resolution mass in the positive mode presented cations at m/z 543.1177 [M+Na]+(calculated543.1109)and negativemode showed the ion at m/z 519.1221 [MH] --- (calculated 519.1144) indicating molecular formula C24H24O13. Furthermore, one can observe loss of two caffeoyl units (162) for MS2 in both thepositive modeand inthe negativemode.The chromatographicpurityof thispatternwasdeterminedto beequalto86.97%.
Invitroevaluation
Staphylococcus aureusATCC25923---methicillin-susceptible (MSSA)andStaphylococcusaureusATCC 43300---methicillin-resistant (MRSA) were used as bacterial subjects and cultivatedonMuller-Hiltonplates.After24hincubation it wasobserved bacterial growthonall samples.The plates werecultivatedwithandwithoutthepresenceofL. opercu-latainaqueousextract1%and0.5%and2,3-dicafeoilglicaric acid 1%. The higher the concentration of the drug, the greaterwastheinhibition ofbacterialgrowthonthe sam-ples. When isolated, active principle proved to be more effective than the samples with L. operculata extractat thesameconcentrations.Theactiveprinciple1%wasmore effective than the1% aqueous extract,which inturn was more efficientthan 0.5% aqueous extract.All plateswith
Streptococcus pyogenes ATCC 19615 had their bacterial growthcompromisedbytheactionofL.operculata in dif-ferentconcentrations and nodifferencewasobservedfor
StaphylococccusaureusATCC25923---Methicillin-Susceptible (MSSA)andStaphylococcusaureusATCC 43300---Methicillin-Resistant(MRSA).
Discussion
Differentstudieshave highlightedthe importanceoflocal treatment of sinus disease.11,12 L. operculata is used to
treatinflammatorydiseasesoftheupperairwayin homeo-pathicandallopathicmedicationsproducedinEurope,North America and Brazil.13,14 Despite the widespread use of L.
operculata thereare few studies thatattest toits thera-peuticvalue.15
TheexactmechanismofactionofL.operculattaisstill unclear.AccordingtoMatosandGottlieb(1967),16theactive
ingredient isocucurbitacin B presents biological activities withdecongestantsactionsaswell aslaxative,hemolytic, embryo toxic andabortion inductiveproprieties. Thus, in
viewofreportsconfirmingthetoxicityofcucurbitcinesand itisassumedthattheisocucurbitacinBisthetoxicprinciple ofL.operculata.16
Aftertheestablishmentofeffectiveexperimentalmodels and reproducible rhino sinusitis in rabbits, many authors haveusedthistooltostudyandcomparevariousformsof treatmentfornasalinfection.17---19Someauthorsevenrated
theherbaleffectoflocalapplicationinthesesituations.20
Some studies have already evaluated the histological inflammatorypatternofsinusitisinanimalmodels.21,22
Some ofthesestudies haveusedexperimental sinusitis modelstocomparedifferenttreatments.Someauthorsuse thehistologicalevaluation ofthenasalor sinusmucosaas inflammationintensityparameter.Inmostcases,this anal-ysisisperformedqualitatively.22However,inotherstudies,
thisanalysisisalsodonebysemi-quantitativetechnique.21
Therefore,theyareoftenassessedfactorssuchas infiltra-tionofinflammatorycells,epithelialulceration,cilliaryloss, edemaandconnective-fibrousproliferation.23
Inagreementwithourstudy,otherauthorspointoutthat thebacterial sinusitisinductiontechnique iseffective19---23
andtheetiologicalagentsofinfectionisrelatedtothe infec-tionmodelused.24
The antimicrobial effect and secretive induction are probablythemainactivitiesofL.operculata.Theseactions areknowntobeimportantfortreatingdifferentrespiratory infectionssuchasRS.23
NasaltopicaladministrationLuffaextractsolution oper-culata1%hasshownsuperiorefficacytothesalinefor the treatmentofbacterialrhinosinusitisinexperimentalrabbit model,takingintoaccount,histologicalandsinussecretions cultureparameters.
The positive results observed in the in vivo test com-bined withirritant effectontherespiratory tractalready describedinliteratureasasideeffectofLuffa,stimulated ustoidentifyactiveingredientsinLuffaextractandsubmit themtoantibacterialactivitytests.
The purifiedsubstance obtained wasidentified as 2,3-dicafeoilglicaric acid. This compound was not described previouslyinliterature.Thesubstanceprovedtobe effec-tivein vitro testsinhibitingthe growthof bacteriaof the species Streptococcus pyogenes. Since the treatment of rhinosinusitiswithtopicalaqueousextractofL.operculata
hassidesymptoms duetothepresence ofsaponinswhich causesomeirritationtotheairways,theuseoftheactive principleinthe absencethereofmayprovetobeofgreat valuein thetreatment ofRSin humans,bringingthe pos-sibility of a newdrug for topicaluse withgreatpractical utilityandcommercialviability.
Conclusion
TopicalnasalsolutionoftheaqueousextractofL.operculata
iseffective comparedtotheapplication ofsalinesolution forthetreatmentofbacterialRSinanexperimentalmodel.
L.operculatadeterminedinvitroinhibitionofgrowthofS. pyogenes.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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