ContentslistsavailableatSciVerseScienceDirect
Journal
of
Ethnopharmacology
j o u r n al hom ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / j e t h p h a r m
Byrsonima
intermedia
A.
Juss.:
Gastric
and
duodenal
anti-ulcer,
antimicrobial
and
antidiarrheal
effects
in
experimental
rodent
models
Raquel
Cássia
Santos
a,
Hélio
Kushima
a,
Clenilson
Martins
Rodrigues
b,
Miriam
Sannomiya
c,
Lúcia
Regina
Machado
Rocha
a,
Taís
Maria
Bauab
d,
Jorge
Tamashiro
e,
Wagner
Vilegas
c,
Clélia
Akiko
Hiruma-Lima
a,∗aUniv.EstadualPaulista-UNESP–DepartamentodeFisiologia,InstitutodeBiociências,cp510,CEP18618-970,Botucatu,SP,Brazil bUniv.EstadualPaulista-UNESP–DepartamentodeQuímicaOrgânica,InstitutodeQuímica,cp355,CEP14801-970,Araraquara,SP,Brazil cSãoPauloStateUniversity–USP-Leste,EscoladeArtes,CiênciaseHumanidades,RuaArlindoBettio,1000,CEP03828-000,SãoPaulo,SP,Brazil dUniv.EstadualPaulista-UNESP– DepartamentodeCiênciasBiológicas,cp970,CEP14801-970,Araraquara,SP,Brazil
eCampinasStateUniversity–UNICAMP,DepartamentodeBiologiaVegetal,InstitutodeBiologia,RuaMonteiroLobato,255,CEP13083-862,Campinas,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received14August2011 Receivedinrevisedform 18November2011 Accepted2December2011
Available online 13 January 2012
Keywords:
Byrsonimaintermedia Malpighiaceae Pepticulcer Healing Diarrhea
a
b
s
t
r
a
c
t
Ethnopharmacologicalrelevance:AnethnopharmacologicalsurveyindicatedthattheleavesofByrsonima intermediaA.Juss.(Malpighiaceae),amedicinalspeciescommonlyfoundintheBrazilianCerrado,canbe usedagainstgastroduodenaldisorders,suchasgastriculcersanddiarrhea.
Aimofthestudy:TheobjectiveofthisstudywastoevaluatetheeffectsofamethanolicextractofByrsonima intermedia(MBI)leavesongastricandduodenalulcersandtoassesstheantimicrobialandantidiarrheal effectsofthisextract.
Materialandmethods:Theanti-ulcerogeniceffectofMBIwasinvestigatedwithdifferentulcerogenic agentsinrodents(miceandrats),includingnon-steroidalanti-inflammatorydrug(NSAID),HCl/ethanol, pyloricligature,absoluteethanol,cysteamineandischemia–reperfusion.Thegastroprotectiveeffectof MBIwasassessedbyanalysingthevolumeofgastricjuice,pH,totalacidity,mucus,NO,sulfhydryl com-pound,vanilloidreceptor,glutathione(GSH)levels,andmyeloperoxidase(MPO)activityinthegastric andduodenalmucosa.ThegastricandduodenalhealingeffectsofMBIwerealsoevaluatedduring7or 14daysoftreatment.Theantidiarrhealaction(measuredbyintestinalmotilityanddiarrheainducedby castoroil)andanti-bacterialactionofMBIagainstStaphylococcusaureus,EscherichiacoliandHelicobacter pyloriwerealsoevaluatedbymicrodilutionmethods.
Results:ThephytochemicalprofilefromMBIindicatedthepresenceofphenolicacids,flavan-3-ols, oligomericproanthocyanidins,andflavonoids.MBI(500mg/kg,p.o.)significantlyinhibitedtotally gas-tricandduodenallesions(69%)andhealedgastric(49%on14days)andduodenallesions(45%on7and 14days).TheMBIexertgastroprotectiveactionbyparticipationofendogenoussulfhydrylcompounds, vanilloidreceptorsandincreaseinGSHleveltoeffectivegastricandduodenalprotection.MBIalso dis-playedcurative(42%)andpreventive(49%)antidiarrhealeffectsbyinvolvementofopiatereceptorsand alsoantimicrobialeffectsinvitro.
Conclusions:Byrsonimaintermedialeavespresentgastroprotective,healingandantidiarrhealactivities, supportingpreviousclaimsthatitstraditionalusecantreatgastrointestinaldisorders.
© 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Byrsonima intermedia A. Juss. (Malpighiaceae) is a keystone speciesthatplaysacriticalroleinthemaintenanceoftheCerrado Biome structure (Vilas Boas, 2009). This species is popularly called“murici-pequeno”andiscommonly foundintheCerrado
∗Correspondingauthor.Tel.:+5501438116077;fax:+551438153744. E-mailaddress:hiruma@ibb.unesp.br(C.A.Hiruma-Lima).
ofthecityofBotucatu,SãoPaulo,Brazil.Thismedicinalspecies is used in popular medicine in cases of fever, skin infection, stomach ache, diarrhea and dysentery and as a diuretic and anti-asthmatic (Sannomiya et al., 2007; Rinaldo et al., 2010). Rodrigues &Carvalho(2001)have describedthepreparation of this medicinalplant in an infusible form as astringent against diarrheaordysentery(3–4cups/day).Rinaldoetal.(2010)have evaluated the difference between the methanolic extract and theinfusibleformfromthe leavesof Byrsonima intermediaand haveshownthattheextractpresentshigheramountsof flavan-3-ols than the infusible form per gram of leaves. Previous
0378-8741/$–seefrontmatter© 2011 Elsevier Ireland Ltd. All rights reserved.
204 140 (2012) 203–212
investigations regarding the chemical composition of this species have resulted in the isolation of quercetin-3-O--d -galactopyranoside, (+)-catechin, (−)-epicatechin, gallic acid, methyl gallate, quercetin-3-O-␣-l-arabinopiranoside and amentoflavone(Sannomiyaet al.,2007).Althoughsome phyto-chemicalstudiesofthisspeciesexist,nopharmacologicalstudies have been performed to assess the anti-ulcer or antidiarrheal actionofthisspeciesasafolkmedicine.Therefore,theaimofthe presentstudywastoevaluatetheprotectiveandhealingeffectsof methanolicextractfromtheleavesofByrsonimaintermediaagainst gastricandduodenalulcersandtoevaluatetheantidiarrhealeffect ofthismedicinalplantinarodentexperimentalmodel.
2. Materialsandmethods
2.1. Drugsandchemicals
Analytical grades of the following chemicals were from Sigma Chemical Co. (St. Louis, USA) and used in this study: absolute ethanol, hydrochloric acid, (Sinth, Brazil), lansopra-zole and piroxicam (Hexal, Brazil), N-nitro-l-arginine methyl ester (l-NAME), carbenoxolone, cimetidine, N-ethylmaleimide (NEM), Alcian Blue, cysteamine, castor oil, atropine, lop-eramide, morphine, methanol, and dichloromethane. Gallic acid, (+)-catechin, (−)-epicatechin, methyl gallate and tri-fluoroacetic acid (TFA) were also obtained from Sigma Chemical Co. (St. Louis, USA). 3,4-Di-O-galloylquinic acid, 1,3,5-tri-O-galloylquinic acid, 1,3,4,5-tetra-O-galloylquinic acid, quercetin-3-O--galactopyranoside, quercetin-3-(2′′-O -galloyl)-O--galactopyranoside, quercetin-3-O-␣-arabinopyranoside and quercetin-3-O-(2′′-O-galloyl)-␣-arabinopyranoside were isolated bytheauthors,andtheirstructureswerefullycharacterizedby UV,MS,1H-NMRand13C-NMRdata.Thepurityofeachcompound
was determined to be higher than 98%. HPLC-grade methanol wasobtainedfromTedia (Phillipsburg, USA).HPLC-grade water (18Mcm) wasobtainedusingaDirectQ5 Milli-Qpurification system (Millipore Co., Bedford, USA). Sep-Pak RP18 cartridges (500mg/6mL)forsolidphaseextraction(SPE)wereobtainedfrom WatersCo.(Waters,Milford,USA).IntheSPEstep,theMBI(10mg) was dissolved in 1mL of methanol and applied to a Sep-Pak RP18 cartridge that had been preconditioned with methanol (2×6mL). The cartridge was eluted with methanol (6mL) to removechlorophyll,andtheeffluentwascollectedandevaporated underanitrogenstream.Thesolidobtainedwasre-dissolvedin methanol/water(1:1,v/v),filteredthrougha0.45mnylonfilter membrane (Sigma–Aldrich, St.Louis, USA), and aliquots(20l) weresubmittedtoHPLCanalysis.Allsamplesandreagentswere preparedimmediatelybeforeuse.
2.2. Plantmaterialandextraction
LeavesofByrsonimaintermediaAJuss.werecollectedinJuly 2007fromPratâniacity,SãoPaulo,Brazil.Thisspecieswas identi-fiedbyProf.J.TamashirofromtheInstituteofBiology,UNICAMP, Campinas,SP,Brazil,andavoucherspecimen(Ref.No.24164)was depositedattheIBBherbariumattheUNESP-Botucatu,SP,Brazil. Aftercollection,theplantmaterialwasshade-dried,pulverizedby amechanicalgrinder,passedthrougha40meshsieveandstored atroomtemperatureuntilextraction.Thedriedpowderedplant material(0.59kg)wasextractedexhaustivelywithsuccessionsof methanolatroomtemperature(48h).Theextractwasfilteredand concentratedunderreducedpressureat60◦Cwitharotary evapo-ratortogeneratethemethanolicextractfromByrsonimaintermedia, whichwasnamedMBI(85.2g).Uponlow-pressureevaporationof
thesolventfromtheextract,residueswereobtained(14.5%(w/w) withrespecttodrypowdermaterial).
2.3. ChromatographicanalysesofMBI
TheMBITLCanalyses,whichwereperformedaccordingtothe methodofWagneretal.(1984),showedpositivityforflavonoids and tannins. The crude extract (10.0g) was then suspended in H2O and partitioned withEtOAc.The concentrated EtOAc
frac-tion (4.0g) waschromatographed on a gel permeationcolumn (GPC,SephadexLH-20,Pharmacia)(57cm×3cmH×i.d.),eluted withMeOH,andyielded195fractions(15mL).Thefractionswere analyzedbyThinLayerChromatography(TLC)onsilicagelplates elutedwithamixtureofCHCl3/MeOH/H2O(80:18:2,v/v/v)and
revealedeitherwithNP/PEGreagentorwithanisaldehyde/sulfuric acidsolution(Wagneretal.,1984).FractionsshowingsimilarTLC dataweregrouped,furnishing17majorfractions.Fractions48–52 (319.0mg) were separated over a successive silica gel column (Merck),anditscompoundswereelutedwithCHCl3/MeOH(75:25,
v/v)furnishingquercetin-3-O--d-galactopyranoside1(7.0mg),a mixtureof(+)-catechin2and (−)-epicatechin3(11.0mg). Frac-tions39–42(74.0mg)wereprocessedbysemi-preparativeHPLC using a PhenomenexLuna RP18 (2) column (250mm×10mm; H×i.d.,10m,flowrate:2.0mL/min).Themobilephaseusedwas MeOH/H2O(7:3)and furnishedthefollowing purecompounds:
gallicacid4(6.0mg),methylgallate5(7.2mg)andquercetin-3-O -␣-l-arabinopiranoside6(7.0mg).Thebiflavonoidamentoflavone 7(9.0mg)wasobtainedfromfractions77–94(222mg)by succes-sivechromatographiccolumnsonsilicagel(Merck)eluted with CHCl3/MeOH(90:10,v/v).Theelucidationofthestructureofthe
isolatedcompoundswasdeterminedusingspectraldata,suchas
1H,13C,HSQC,HMQC,COSYandNOESY-NMRspectraelucidation
andEI-MSspectraevaluation.TheNMRspectrawereobtainedon aVarianInova500MHz.
2.4. ChromatographicprofilingandidentificationofMBI
ThechemicalcompositionofMBIwasinvestigatedbyHigh Per-formanceLiquidChromatographycoupledtoaPhotodiodeArray Detector(HPLC-PAD),usingaJasco(Tokyo,Japan)HPLCequipped withaPU-2089quaternary solventpump,a MD-2010 PADand an AS-2055 autosampler. The analytical column maintained at roomtemperature(25◦C),wasaPhenomenexSynergiHydroRP18 (250mm×4.6mm H×i.d.; 4m) with a Phenomenex security guardcolumn(4.0mm×2.0mmH×i.d.).Separation ofphenolic acids,flavonoids,flavan-3-olsandproanthocyanidinswas estab-lishedusingthemobilephaseofwater(eluentA)andmethanol (eluentB), both containing0.1% trifluoroaceticacid (TFA),with the following gradient program: 0–20% B (20min), 20% B iso-cratic (2min), 20–50% B (38min), 50–100% B (5min), 100% B isocratic(5min),returnto0%B(2min),andthecolumnwas re-equilibratedwiththeinitialconditionsfor18minbeforethenext injection.Theflowratewas1.0mL/min,andthetotalruntimewas 90min.EZChromEliteDataSystemsoftware(Chromatec,Idstein, Germany) was used for both detector operationand data pro-cessing.The identificationof thecompounds wasperformedby retentiontimecomparison, UVspectralanalysesand byspiking withcommercialorisolatedstandardsunderthesameconditions.
2.5. Animals
MaleSwissalbinomice(25–35g)andmaleWistaralbinorats (150–250g)fromtheCentral AnimalHouseof theUNESPwere used. The animals were fed a certified Nuvilab® (Nuvital) diet
140 (2012) 203–212 205
The animals were fasted prior to all assays because standard drugsandMBIwerealwaysadministeredorally(gavage)usinga salinesolution(0.9%NaCl,10mL/kg)asthevehicle.Theanimals werehousedincageswithraisedfloorsofwidemeshtoprevent coprophagy.Allexperimentswereperformedinthemorningand followedtherecommendationsoftheCanadianCouncilonAnimal Care(Olfertetal.,1993).TheUNESPInstitutionalAnimalCareand UseCommitteeapprovedalloftheemployedprotocols(CEUAno. 18/05).
2.6. Evaluationofgastroprotectiveactivity
Ethanol-induced ulcer – This experiment was performed as describedbyMorimotoetal.(1991).Maleratsweredistributed into5groups(n=8)andfastedfor24hpriortoreceivinganoral doseofthevehicle(10mL/kg),lansoprazole(30mg/kg)orMBI(250, 500or1000mg/kg bodyweight). After60min,all groupswere treatedorallywith1mLofabsoluteethanoltoinducegastriculcers. Afteranother1h,theanimalsweresacrificed,andtheextentofthe lesionswasmeasuredbyAvSoft®BioviewSpectra,Brazil;thelesion
measurementsareexpressedasmm2.
NSAID-inducedgastriculcersinmice–Inthismodel(Puscasetal., 1996),gastriclesionswereinducedwithpiroxicam(30mg/kg,s.c.) administeredtomaleSwissmice(n=8)aftera24hfast.MBI(250, 500or1000mg/kgbodyweight),cimetidine(100mg/kg)orvehicle wasorallyadministered30minbeforetheinductionofthegastric lesions.Theanimalsweresacrificed4haftertreatmentwiththe ulcerogenicagent.Thestomachswereremoved,andgastriclesions weremeasuredasdescribedabove.
HCl/ethanol-inducedulcer–Theexperimentswereperformed asdescribed byMizuiandDoteuchi(1983),withmodifications. Male Swiss mice (n=5) were distributed into groups of 5 ani-mals that were fastedfor 24h prior to receiving an oral dose ofthe vehicle(10mL/kg), lansoprazole(30mg/kg)or MBI(250, 500or1000mg/kg bodyweight). After50min,all groupswere orally treated with 0.2mL of a 0.3M HCl/60% ethanol solution (HCl/ethanol)forgastriculcerinduction.Theanimalswere sacri-ficed1haftertheadministrationofHCl/ethanol,andthegastric damagewasmeasuredasdescribedabove.
Shayulcer–Allmalemice(n=8–9)wererandomlydividedinto threegroupsandfastedfor18hwithfreeaccesstowater.Thirty minutesafteroraladministrationofMBI(500mg/kg,whichisthe lowestdosethat presentssignificantresultsin allacute exper-iments),cimetidine (100mg/kg)asa positive controlorvehicle (10mL/kg),pylorusligaturewasperformedasdescribedbyShay (1945).Fourhourslater,theanimalsweresacrificed,theabdomen wasopened,andanotherligaturewasplacedaroundthe oesopha-gusclosetothediaphragm.Thestomachwasremovedandlesion wasmeasured(mm2),andthegastricjuicevolume(mL),hydrogen
ionconcentration(mEq/mL/4h)andpH(unit)wererecorded. Determinationofgastricsecretion– Theassaywasperformed according to themethod of Shay (1945) witha few modifica-tions. All groups of male mice (n=9–10) were fasted for 18h withfreeaccesstowater.Immediatelyafterpylorusligature,MBI (500mg/kg),cimetidine(100mg/kg)aspositivecontrolor vehi-cle(10mL/kg)wasadministeredbyanintraduodenalroute.The animalsweresacrificed4hlater,theabdomenwasopened,and anotherligaturewasplacedaroundtheoesophagusclosetothe diaphragm.Thestomachswereremoved,thegastriclesionswere measured(mm2)andthegastriccontentwascollectedto
deter-minethetotal amountofgastricjuice acid(mL)and pHvalues (unit).Distilledwaterwasadded,andtheresultantsolutionwas centrifugedat3000×gfor10min.Totalacidinthegastric secre-tionvolumewasdeterminedinthesupernatantbytitrationtopH 7.0with0.01NNaOH(mEq/mL/4h).
Assessmentofmucusadheredtothegastricwall–Theprocedure wasperformedaccordingtothemethodsofRafatullahetal.(1990). Afterfastingfor18h,alongitudinalincisionwasmadebelowthe xiphoidapophysisforthepylorusligaturein anaesthetizedrats (n=6–7).Vehicle(10mL/kg),carbenoxolone(200mg/kg)orMBI (500mg/kg)wasadministered(p.o.)for1hbeforetheligature.Four hourslater,theanimalsweresacrificed,andtheglandularportion ofthestomachwasseparated,weighedandimmersedina solu-tionofAlcianBluetoquantifythemucus.Foreach sample,the absorbanceat598nmwasmeasuredinaspectrophotometer,and theresultsareexpressedasgofAlcianblue/goftissue.
Ethanol-inducedgastriclesionsinl-NAME-,NEM-,andRuthenium
Red-pretreatedrats–Theprocedurewasperformedaccordingtothe methodsofPongpiriyadachaetal.(2003),Matsudaetal.(1999)and Morimotoetal.(1991).
GastricmucosallesionswereinducedinmaleWistarrats.To investigate the involvement of SHs in the protective effect of MBI(500mg/kg),NEM(anSH-blocker,10mg/kg)orvehiclewas injected30minbeforetheadministrationofMBI.Toinvestigate thepossibleinvolvementofendogenousNOintheprotectiveeffect of MBI,l-NAME (aninhibitorof NOS,70mg/kg)or vehiclewas administered30minbeforetheadministrationofMBI.To inves-tigatethepossibleinvolvement of thevanilloidreceptor in the protectiveeffectofMBI,RutheniumRed(RR,6mg/kg),whichhas beenreportedtobeanantagonistagainstcapsaicin,orvehiclewas injected30minbeforetheadministrationofMBI.After60min,all groupswereorallytreatedwith1mLofabsoluteethanolfor gas-triculcerinduction.Theanimalsweresacrificed1hafterethanol administration,thestomachswereexcised,andthegastriclesions weremeasuredasdescribedabove.
Determinationoftotalglutathione(GSH)ingastricmucosain NEM-pretreated ratsandRR-pretreatedrats – TheGSHpresent in the samples wasquantified by the methoddescribed by Anderson (1985).Stomachsobtainedfromtheinvestigationoftheroleof sulfhydrylcompoundsandtheinvolvementofthevanilloid recep-torinthegastricprotectionexperimentwerecutintoslicesand placed inanEppendorfcontaining5% TCAat a1:20 (w/v) pro-portion.Thesampleswerehomogenizedin5%TCAusingaPotter cellmacerator(Marconi®,Brazil).Aftertwocentrifugationsteps
(2000×gand9000×gfor5and10min,respectively)at4◦C,the supernatantswereremovedtoquantifytheglutathionethrough reactionwithDTNB(5,5′-dithiobis(2-nitrobenzoicacid).Theresults wereobtainedasnmol/gofwettissue.
Acutegastricmucosallesionsinducedbyischemia–reperfusionin rats–Ischemia–reperfusion(I/R)erosionswereproducedinthree groupsofrats(n=8–10)bythemethodoriginallyproposedbyUeda etal.(1989).Theratsweredeprivedoffoodbutallowedaccessto tapwateradlibitumfor18hbeforetheexperiments.Briefly,under anaesthesia(0.8mg/kgofketaminehydrochlorideand0.4mg/kg xilazinehydrochloride),theceliacarterywasclampedwithasmall clampfor30min.Thentheclampwasremoved,andreperfusion occurredfor60min.Vehicle(10mL/kg),lansoprazole(30mg/kg) orMBI(500mg/kg)wasadministeredorally60minpriortothe experiments.Theuntreatedgroupcomprisedanimalsthat were submittedtotheabdominalincision,butnottoI/R.After60min of reperfusion,the animalsweresacrificed, and theirstomachs wereexcised.ThegastricmucosalinjurywasmeasuredbyAvSoft®
BioviewSpectra,Braziland expressedasmm2.Thenthegastric
mucosawasstoredat−80◦CforbiochemicalmeasurementsofGSH levelsandMPOactivity.
206 140 (2012) 203–212
subsequentlydilutedtoafinal1:20(w/v)ratioandhomogenized for1minwithanautomaticHeidolphhomogenizer,sonicatedfor 10sandsubjectedtothreefreeze–thawcycles.Thehomogenates werethencentrifugedat7000×gat4◦Cfor10min,andthe super-natantswereassayedforMPOactivity.Theresultsareexpressedas MPOunitspergram(U/g)ofwettissue.ThemeasurementofGSH contenthasalreadybeendescribed.
2.7. Evaluationofhealingofgastriculcersinducedbyaceticacid inrats
The experiments were performed according to the method describedbyOkabeetal.(1971)modifiedbyKontureketal.(1988). SixgroupsofmaleWistarratsthatwerefastedfor18hwereused inthisexperiment (n=8).Under anaesthesia,alaparotomywas performedonallanimalsthrough amidline epigastricincision. Afterexposingthestomach,0.05mL(v/v)of80%aceticacid solu-tionwasinjectedintothesubserosallayerintheglandularpartof theanteriorwall.Thestomachwasbathedwithsaline(20◦C)to avoidadherencetotheexternalsurfaceoftheulceratedregion. Theabdomenwasthenclosed, andallof theanimalswerefed normally.Alltreatments,includingMBI(500mg/kgbodyweight), lansoprazole(30mg/kg)orvehicle(10mL/kg),wereadministered orallyonceadayfor7or14consecutivedaysbeginningoneday aftersurgery. Onthedayafterthelast drugadministration,the ratsweresacrificed andtheirstomachswereremoved.The gas-triclesionswereevaluatedbyexaminingtheinnergastricsurface withadissectingmagnifyingglass.Themacroscopiculcerareawas recordedusingAvSoft®BioviewSpectra,Brazilandexpressedas
mm2.
2.8. EvaluationofthepreventiveeffectofMBIonduodenalulcers inrats
CysteamineduodenalulcerinratsanddeterminationofGSHand MPOactivities–MaleWistaralbinorats,weighing150–250gwere used,andfoodandwaterwereavailableadlibitumthroughoutthe assay.Duodenalulcerswereinducedbytwooraladministrations ofcysteaminehydrochloride(400mg/kg)insalinesolutionatan intervalof4h.MBI(500mg/kg),lansoprazole(30mg/kg),or vehi-cle(10mL/kg)wereadministered30minbeforethefirstdoseof cysteamine.Oneuntreatedgroupwasusedtoevaluatethe nor-malparametersoftheduodenummucosa.Alloftheanimalswere sacrificed48hafterthefirstdoseofcysteamine.Eachduodenum wascutopenalongtheantimesetericsideandrinsedwithsaline. Theduodenalscorewasdeterminedusingthefollowing4-point scale:0=nolesion;1=superficialmucosalerosion;2=deepulcer ortransmuralulcer;and3=perforatedorpenetratedulcer(Szabo, 1978).
2.9. Evaluationofthehealingofduodenalulcersinducedby aceticacidinrats
The experiments were performed according to the method describedbyOkabeetal.(1971)modifiedbyKontureketal.(1988). SixgroupsofmaleWistarrats(200–250g)weredeprivedoffood for18h, buthad freeaccesstowater.Anincisionwasmadein theabdomenofanimalsunderanaesthesia,anda plasticmould (4.2mmin diameter)wasplaced tightly ontheserosal surface oftheduodenalwall,approximately5mmdistaltothepylorus. Aceticacid(80%,70l)waspouredintothemouldandallowedto remainfor10s.Afterremovalofallaceticacid,theabdomenwas closed,andtheanimalswerefednormally.Alltreatmentswere administeredorallyonceadayfor7or14consecutivedays begin-ningonedayaftersurgery.Toassesstheduodenalhealingeffects, theselecteddoseofMBI(500mg/kgbodyweight),lansoprazole
(30mg/kg)orvehicle(10mL/kg)wasadministeredonceadayto3 groupsfor7daysandtoanother3groupsfor14days.Ontheday afterthelastdrugadministration,theratsweresacrificed,andtheir duodenawereremoved.Theduodenallesionswereevaluatedby examiningtheinnerduodenalsurfacewithadissecting magnify-ingglass.Themacroscopicduodenalulcerareawasrecordedand expressedasmm2.
2.10. Antidiarrhealactivity
Gastrointestinalmotility (charcoalmeal) inmice– MaleSwiss mice(n=7)wereweighedanddeprivedoffoodfor6h,buthad freeaccesstowater. Attimezero,theanimalsreceivedvehicle (saline)asthenegativecontrol,5mg/kgofatropineasthepositive control,orMBIatdosesof250,500or1000mg/kgbyoralgavage. Thirtyminafterthetreatments,allgroupsreceived10activated charcoal((01,mL/10g),orally)and weresacrificed 30minlater. Theresultswereexpressedaspercentageofthetotallengthofthe smallintestine.Thedistancetravelledbythecharcoalrelativetothe totallengthofthesmallintestinewascalculatedforeachmouse toexpressthepercentageofdistancetravelledandconvertedby arcosine(StickneyandNorthup,1959,withmodifications).
Intestinalfluidaccumulation–FollowingthemethodofRobert et al. (1976),male Swiss micewere dividedinto six groups of sevenoreightanimalspergroupandhalfofthemwerepretreated with saline and another one with naloxone – opioid antago-nist (30mg/kg, i.p.).Fifteen minutes later, groups were treated withvehicle (10mL/kg,p.o.), morphine (10mg/kg, i.p.), or MBI (500mg/kg,p.o.)andonehourlater,themicereceivedcastoroil (2mL/animal)orally.Theanimalsweresacrificed1hlater,andthe smallintestineswereremovedafterligationatthepyloricendand ileocaecaljunctionandweighed.Thecontentsoftheintestinewere thenexpelledintoagraduatedtube,andthevolumewasmeasured. Theintestineswerere-weighed,andthedifferencesbetweenthe fullandemptyintestineswerecalculated.
Diarrhea induced by castor oil: evaluation of preventive effect of MBI – Three groups of male Swiss mice (n=7) were orally treated with vehicle (10mL/kg), MBI (500mg/kg body weight) orloperamide(5mg/kg)30minbeforecastoroil(0.2mL/animal) administration.Immediatelyafteringestingcastoroil,eachanimal waskeptinanindividualcage,whosefloorwaslinedwithblotting paper,andobservedfor5h.Thefollowingparameterswere moni-tored:timetoinitialevacuation(min),evacuationclassification–1 (normalstool),2(semi-solidstool),and3(waterystool)and evac-uationindex(EI),expressedaccordingtotheformula:EI=1×(no stool1)+2×(nostool2)+3×(nostool3)(Awounters,1978, mod-ifiedbyMukherjeeetal.,1998).
Diarrheainducedbycastoroil:evaluationofcurativeeffectofMBI– ThreegroupsofmaleSwissmice(n=8–9)wereorallytreatedwith 0.2mL/animalwithcastoroil(p.o.).Then15minafter administra-tionofthecatharticagent,thegroupsweretreatedwithvehicle (10mL/kg),MBI(500mg/kgbodyweight)orloperamide(5mg/kg). Immediatelyafteringestingthedifferenttreatments,eachanimal waskeptinanindividualcagewhosefloorwaslinedwith blot-tingpaperandobservedfor4h.Thesameparametersasdescribed aboveforthepreventiveevaluation(Awounters,1978;Mukherjee etal.,1998)weremonitored.
2.11. Antibacterialsusceptibilitytest
140 (2012) 203–212 207
platesand incubatedat37◦Cfor 24h, andH.pyloriwas inocu-latedonMueller–Hintonagarplatescontaining5%sheepbloodand incubatedat36◦Cfor3daysina10%CO
2atmosphere.Inoculates
werepreparedinthesamemediumatadensityadjustedtoa0.5 McFarlandturbiditystandard[108colony-formingunits(CFU)/mL]
forE.coliandS.aureusand2.0McFarlandturbiditystandardforH. pylori.Theworkingsuspensionforeachmicroorganismwasdiluted 1:10,anda100lvolumewasaddedtoeachwellofamicroplate. A100lvolumeofMueller–Hintonbrothorthesamevolumeof Mueller–Hintonbrothsupplementedwith10%foetalbovineserum wasaddedtoeachwellofthemicroplatesofE.coliandS.aureus,and H.pylori,respectively.Theconcentrationsforeachextract,prepared in2%DMSO,rangingfrom0.5to1.000g/Lwereobtainedwhen a100lvolumeoftheextractwastransferredtothefirstwellof eachrow,andserial2-folddilutionswereperformed.Amoxicillin, metronidazole,andtetracyclinewereusedasreference antimicro-bialcompounds.TheMICswererecordedafterincubationofthe microplatesat37◦Cfor24h(E.coliandS.aureus)or36◦Cfor3 daysina10%CO2atmosphere(H.pylori),andthegrowthwas
visu-allyexamined.Theabsorbancewasdeterminedusinganautomatic microplatereaderadjustedto600nm.Thelowestconcentrationof thetest extractthatultimatelycausedaninhibitionofbacterial growthofmorethan90%wastakenastheMIC.
2.12. Statisticalanalysis
Theresultsareexpressedasthemean±S.E.M.Statistical signif-icancewasdeterminedbyone-wayanalysisofvariancefollowed by Dunnett’stest, or noparametric resultswasdetermined by Kruskal–WallisfollowedbyDunn’stest;levelsofP<0.05were con-sideredtobestatisticallysignificant.
3. Resultsanddiscussion
The HPLC-PAD phytochemical characterization showed that MBI presented several peaks that eluted in the retention time (Rt) range of 5–65min (Fig. 1). The metabolite classes
were identified with PAD scanning from 200 to 600nm. In the chromatogram in Fig. 1, the constituents of MBI can be divided into the following three main classes: (I) phenolic acids and catechin derivatives, (II) oligomeric proanthocyani-dins and (III) flavonoids. Based on this information and after co-injectionexperiments,elevenpeakswereunambiguously iden-tified as being associated with gallic acid (1, Rt=15.1min),
3,4-di-O-galloylquinic acid (2, Rt=23.9min), methyl gallate
(3, Rt=28.6min), (+)-catechin (4, Rt=29.9min), (−)-epicatechin
Fig. 1.Analytical HPLC-PAD chromatogram recorded at 275 nm of the compoundsidentifiedontheMBI.Peakidentification:(1)gallicacid,(2) 3,4-di-O-galloylquinic acid, (3) methyl gallate, (4) catechin, (5) epicatechin, (6) 1,3,5-tri-O-galloylquinicacid,(7)1,3,4,5-tetra-O-galloylquinicacid,(8) quercetin-3-O--galactopyranoside, (9) quercetin-3-(2′′-O-galloyl)-O--galactopyranoside, (10)quercetin-3-O-␣-arabinopyranosideand(11)quercetin-3-O-(2′′-O-galloyl)-␣ -arabinopyranoside.ForchromatographicconditionsseetheMaterialsandMethods section.
(5, Rt=38.1min), 1,3,5-tri-O-galloylquinic acid (6, Rt=39.5),
1,3,4,5-tetra-O-galloylquinic acid (7, Rt=41.7min),
quercetin-3-O--d-galactopyranoside (8, Rt=56.8min), quercetin-3-(2′′-O -galloyl)-O--d-galactopyranoside (9, Rt=57.9min),
quercetin-3-O-␣-l-arabinopyranoside (10, Rt=59.4min) and quercetin-3-O -(2′′-O-galloyl)-␣-l-arabinopyranoside(11,Rt=61.2min).In addi-tion to the monomeric flavan-3-ols found in MBI, oligomeric proanthocyanidinsweresuggestedasthebroadunresolvedpeak thatelutedintheRt rangeof35–60min.Thisresultisin
accor-dancewiththoseobtainedbyRinaldoetal.(2010),whocompared themethanolicextractandinfusibleformfromtheleavesof Byr-sonimaintermedia.Theextractionwithmethanolinsteadofwater didnotshowanyselectivityintheextractionprocess;however, theconcentrationof(+)-catechinand(−)-epicathechinwaslower intheinfusibleform.
Asapartofthepharmacologicalstudyofthegastroprotective effectsofMBI,theextractwasevaluatedinexperimentalmodelsof gastriculcersinducedbymultipledamagingagents(Table1). Gas-tricmucosalinjuryfrequentlyoccurswhennoxiousfactors,such asNSAID,increasedacidsecretion,thepresenceof thebacteria Helicobacterpylori,ingestionofalcoholormucosalischemia over-whelmthemucosaldefencefactors(Laineetal.,2008).Amongthe threedifferentdosesofMBIexaminedinthisstudy,the gastropro-tectiveeffectwasfoundtobegreatestatthedoseof500mg/kgin allexperimentalmodels.Thegastricdamageinthegrouptreated
Table1
EffectsofdifferentdosesfrommethanolicextractofByrsonimaintermedia(MBI)onmodelsofgastriclesionsinducedinrodents.
Methods(animal) Treatments(p.o.) Dose(mg/kg) N Gastriclesion(mm2) Inhibition(%)
Absoluteethanol(rats) Vehicle – 8 150.5±17.1 –
Lansoprazole 30 8 65.6±16.7** 56
MBI 250 8 68.4±13.9** 55
500 8 4.2±1.8** 97
1000 8 0** 100
NSAID(mice) Vehicle – 8 27.2±4.3 –
Cimetidine 100 8 11.8±2.6** 57
MBI 250 8 21.3±3.6 –
500 8 12.4±1.5** 54
1000 8 9.8±2.6** 64
HCl/ethanol(mice) Vehicle – 5 91.4±7.7 –
Lansoprazole 30 5 34.8±6.6** 62
MBI 250 5 61.4±3.2** 33
500 5 45.4±6.6** 50
1000 5 20.6±3.4** 78
208 140 (2012) 203–212
Table2
EffectsofcimetidineandmethanolicextractofByrsonimaintermedia(MBI)administeredorallyorintraduodenallyongastricjuiceparametersinpylorusligature-induced gastriclesionsinmice.
Treatments Route Dose(mg/kg) N Gastriclesion(mm2) Inhibition(%) pH(unit) Volume(mL) [H+](mEq/mL/4h)
Vehicle p.o. – 8 16.7±2.5 – 3.5±0.3 0.9±0.1 10.3±1.0
Cimetidine 100 8 12.0±1.7* 28 5.3±0.5** 1.0±0.1 6.2±1.0*
MBI 500 9 7.5±1.1** 55 3.3±0.3 1.5±0.2* 7.3±1.2
Vehicle i.d. – 10 19.1±2.8 – 3.0±0.3 1.1±0.1 10.2±0.7
Cimetidine 100 10 7.6±1.3** 60 4.9±0.5* 0.8±0.1 6.1±1.3*
MBI 500 9 6.6±1.3** 66 3.7±0.5 1.5±0.1* 8.8±1.0
Resultsaremean±S.E.M.ANOVAfollowedbyDunnett’stest:*P<0.05,**P<0.01
withMBIwassignificantlyreducedby50%(HCl/ethanolmodel),
54% (NSAID model) and 97% (absolute ethanol model) relative
to the control groups treated with vehicle. The lower dose of
MBI(250mg/kg)didnotexertacompletegastroprotectiveaction
againstNSAIDs, and a statisticallysignificantdifference existed
betweenthedoseof250and500mg/kg(P<0.05).
The next step of this study was the evaluation the effect
of MBI on gastric juice parameters to evaluate the possible
anti-secretory action of the extract. The administration of this
extractshowed(Table2)thatoraltreatmentwithMBIdecreased
thegastriclesion(55%) and increasedthegastricvolume (67%) withoutchangingthepHorH+concentrationrelativetothecontrol
grouptreated withvehicle.Thesystemiceffectof gastroprotec-tionbyMBIwasalsoobservedbyadministrationofthisextract throughanintraduodenalroutethatinducedthesamealterations inthe gastricmucosa and gastricjuice. Both resultsinTable 2 excludedapossibleanti-secretoryactionofMBI,likecimetidine, andshowedthatMBIdidnotpresentacoatingeffectongastric mucosa.Therefore,thegastroprotectiveactivityofMBIcouldbe relatedtoastrengtheningofthedefencemechanismsofgastric mucosa,suchasmucus(Laineetal.,2008).Thecopious quanti-tiesofmucusfromthegastricmucosaarethefirstlineofmucosal defenceagainstnoxiousagentsinthelumen(Abdel-Salametal., 2001).TheeffectofMBIonincreasingthegastricmucus produc-tionwasevaluatedinpylorusligaturerats.Inthegrouptreated onlywithcarbenoxolone(positivecontrol),theamountofmucus adheredtothegastric mucosa(12.4±1.2g/gof tissue)inrats increasedsignificantly(P<0.05)inrelationtocontrolgroup.The grouptreatedwithMBImaintainedthegastricmucusamountat thesamelevelasthecontrolgrouptreatedwithvehicle(8.6±1.2 and8.5±0.7g/goftissue,respectively).Thisresultalsodiscards theeffectofMBIinincreasingtheamountofmucusasa gastro-protectivedefencemechanism.Inadditiontomucus,otherdefence mechanismswerealsoinvestigated,suchasendogenousNO(nitric oxide),NP-sulfhydrylcompoundsandtheTRPV1receptor (tran-sient receptor potentialvanilloid type 1),which are important factorsthatmaintainmucosalintegrity (Szabo andNagy, 1992; Holzer,2006).
Mucosalbloodflowisessentialforthedeliveryofoxygenand nutrients,removaloftoxicsubstancesandnormallybloodflowin thegastricmucosaisincreasesinresponsetoacidsecretion( Abdel-Salametal.,2001).GastricbloodflowismediatedinpartbyNO, howeversensorynervesarealsoinvolvedintheregulationof gas-tricbloodflow(Pawliketal.,1998;Mártinetal.,2001;Jaworek etal.,2002).Thegastricmucosaisdenselyinnervatedby capsaicin-sensitiveafferentneuronscontainingvasodilatorpeptides,suchas therelatedpeptidecalcitoningene(CGRP)(Sterninietal.,1987). Capsaicinaffectsthesenervesbytwodifferentways;alow-doses stimulationofsensorynerves,accompaniedbythereleaseofCGRP, whilehighdosesofcapsaicinleadtoablationorfunctional inac-tivationofsensorynerves(Warzechaetal.,2000).Thereleaseof CGRPbycapsaicin-sensitivenerveshavealsobeeninvolvedinthe maintenanceof mucosalintegritybecausetheablationof func-tionalneuronsresultsadecreaseinreactivitytoCGRPinthemucosa
and leadsto a worsening of gastriclesions inducedby various ulcerogeniccompounds(Brzozowskietal.,1996;Kwiecie ´netal., 2002).Forexample,ethanolprovidesnotonlyharmfuleffectby decreasingthebarrierfunctionofmucus,butalsoactivatesTRPV1 receptorsinsensoryneuronsofthegastricmucosabystimulating thereleaseofsubstancePinthestomach,whichinturnactivates NK1receptorsofgastricepithelialcellspromotingtheincreaseof ROS(oxidativestress),thatcausehemorrhagiclesionsinthe stom-achwallduetolipidperoxidation(Gazzierietal.,2007).
Ourresultsshowedthatpretreatmentofanimalswithl-NAME (aNO-synthaseinhibitor)didnotchangethegastroprotectiveeffect ofMBI(P>0.05)comparedtothesaline-pretreatmentMBIgroup (inhibition 98% vs. 99%).Therefore, our resultsalso excludean involvementofendogenous NOinthegastroprotectiveeffectof MBI(datanot shown).However,thegroupofanimalsthat was pretreatedwithRutheniumRed(whichblockstheTRPVreceptor) andtreatedwithextractdisplayedasignificantincreaseingastric lesionsbyapproximately10-fold(37.7±12.5mm2)comparedto
thegrouptreatedwithMBI(3.1±2.9mm2).Theprotectiveeffect
ofMBIwasalsoeliminatedwhenthegroupwastreatedwitha sulfhydrylinhibitor(NEM)(P<0.001).Bothoftheseresults,which areshowninTable3,suggestthattheactivityofMBImayinvolve theTRPVreceptorandthepresenceofSHcompoundsinthe gas-tricmucosa.ThisgastroprotectiveeffectofMBIdemonstratedin Table3wasalsoevidencedbyasignificantincreaseinthelevels oftotalglutathione(GSH).GSHactsasanimportantantioxidantin thegastricmucosa,anditspresenceisimportantforthe mainte-nanceofmucosalintegrity(SzaboandNagy,1992).Thelevelsof GSHinthegastricmucosatendtodecreaseafteradministrationof absoluteethanol,butourresultsshowedthatpretreatmentwith MBIwasabletoinhibitthisdecreaseinGSH(P<0.05).
Ischemiaalsohasdeleteriouseffectsongastricmucosaandis oneof thestress-inducing gastricmucosalinjuries (Laine etal., 2008).Therestorationofbloodflow(reperfusion)afteraperiod ofischemiainitiatesacascadeofchanges,includingthereleaseof localofreactiveoxygenspecies(ROS)andanincreaseinthe adhe-sionofneutrophilstoendothelialcells,whichcausesdamageto theintegrityofthemucosallining;thisphenomenonisknownas ischemia–reperfusion(I/R)(Abdallahetal.,2009).
140 (2012) 203–212 209
Table3
EffectofmethanolicextractofByrsonimaintermedia(MBI)ongastriclesionsandtotalglutathionelevel(GSH)inrats(n=7–8)pretreatedwithNEM(SHblocker)orRuthenium Red(RR–TRPVblocker)andtreatedwithabsoluteethanol.
Pretreatment(i.p.) Treatment(p.o.) Dose(mg/kg) Ulcerativelesion(mm2) GastriclesionInhibition(%) GSH(nmol/g)
Untreated 1070.2±92.3
Saline+ Vehicle – 212.8±49.8 – 607.7±46.7&
Saline+ Carbenoxolone 100 0.1±0.1** 100 895.2
±48.0**
Saline+ MBI 500 4.35±4.4** 98 768.1±27.8*
NEM+ Vehicle – 360.3±91.9# – 411.2
±40.1&
NEM+ Carbenoxolone 100 240.2±101.4*,# 33 661.6
±73.2&
NEM+ MBI 500 147.3±42.2**,## 59 486.7±51.1&
Saline+ Vehicle – 131.6±42.4 – 711.4±43.2&
Saline+ Capsaicin 4 1.6±0.9** 99 1077.4
±52.2**
Saline+ MBI 500 3.0±2.9** 68 991.8±58.1*
RR+ Vehicle – 151.5±12.4 – 647.8±61.8&
RR+ Capsaicin 4 103.8±44.7## 31 967.3
±98.3*
RR+ MBI 500 37.7±12.5**,# 75 793.3
±34.7&
Dataarepresentedasmean±S.E.M.ANOVAfollowedbyDunnett’stest.Ulcerativelesion:*P<0.05,**P<0.01representsdifferenceinrelationtocontrolgrouptreated withvehicle;#P<0.05,##P<0.001representsdifferenceinrelationtopre-treatmentssaline+treatmentandRR+treatmentorsaline+treatmentandRR+treatment.Total Glutathione&representsdifferenceinrelationtountreatedgroupand*P<0.05,**P<0.01representsstatisticaldifferenceinrelationtocontrolgrouppre-treatedwith saline+vehicle,NEM+vehicleorRR+vehicle.
Fig.2. GastroprotectiveactivityofmethanolicextractofByrsonimaintermedia (MBI)onleveloftotalglutathioneandmieloperoxidaseonmodelofgastricinjury inducedbyischemia/reperfusioninrats(n=8–10).Thebarsrepresentmean±S.E.M. Thenumbersindicatethepercentageprotectioninrelationtothecontrolgroup treatedwith vehicle.++P<0.01fortotal gluthatione; F(3,21)=9,70 ##P<0.01for myeloperoxidase,ANOVAfollowedbyDunnett’stest;**P<0.01.
anti-inflammatoryeffectofthisspecies.Thephytochemical
com-positionofMBI(mainlyflavonoids)is probablyinvolvedin this
resultbecauseastudybyRaoandVijayakumar(2008)hasalready
describedtheeffectof(+)-catechin,themainflavan-3-olpresent
inMBI,asanimportantgastroprotectivecompoundagainstgastric mucosalinjuryinducedbyischemia–reperfusioninrats.
InadditiontotheprotectiveeffectofMBIingastric experimen-talmodelsinvivo,wealsodeterminedtheprotectiveeffectsofMBI againstduodenallesionsinducedbycysteamineinrats.Cysteamine hydrochlorideinhibitsalkalinemucussecretionfromtheBrunner’s glandintheproximalduodenumandstimulatestherateof gas-tricacidsecretion(Schwedesetal.,1977).AccordingtoKhomenko etal.(2009)animalstreatedwithcysteaminealsodisplaya sig-nificantincreaseinduodenallesionsduetothegenerationoffree radicals.Fig.3showsthatduodenalinjuriescausedbycysteamine significantlydecreasedupon treatmentwithMBIrelativetothe grouptreatedwithvehicle.TheduodenalprotectiveeffectofMBI (69%)wasalsoaccompaniedbyasignificantdecreaseinMPO,a markerofneutrophilactivity.Neutrophilsarethemajor inflam-matorycelltypethatinfiltratestheinjuredgastroduodenalmucosa. OurresultsindicatethatMBIcounteractsthisinfiltrationtoprotect theduodenalmucosaagainstulcers.
Basedonalloftheresultsregardingthegastroprotectiveeffectof MBI,theprotectiveeffectofthisextractagainstgastricand duode-nalinjuriesinducedbydifferentulcerogenicagentswasconfirmed. However,foranynewanti-ulcerdrug,itisdesirableforthis pre-ventiveeffecttoalsobeaccompaniedbyulcer-healingeffectsin bothofthesetissues.
Fig.3. EffectofmethanolicextractofByrsonimaintermedia(MBI)onleveloftotalglutathioneandmieloperoxidaseonmodelofduodenalinjuryinducedbycysteaminein rats(n=8–10).Thebarsrepresentmean±S.E.M.Thenumbersindicatethepercentageprotectioninrelationtothecontrolgrouptreatedwithvehicle.ANOVAfollowedby Krusal/Wallis’stest;*P<0.05,**P<0.01.++P<0.01fortotalglutathione;F
210 140 (2012) 203–212
Fig.4.GastrichealingeffectofmethanolicextractofByrsonimaintermedia(MBI)by treatmentduring7or14daysafterinjuryinducedbyaceticacidinrats(n=8). Thebarsrepresentmean±S.E.M..Thenumbersindicatethepercentage protec-tioninrelationtothecontrolgrouptreatedwithvehicle.ANOVAfollowedby Dunnett’s;*P<0.05,**P<0.01.
OkabeandAmagase(2005)haveelegantlydescribedthebenefit oftheappearanceofaceticacidulcermodelsfor pathophysiolog-icalandpharmacologicalstudiesofpepticulcers.Newdrugsthat preventulcerrelapseandenhanceulcerhealingcouldpotentially bedevelopedusingthisestablishedmethod.Thusthisstudyalso determinedtheeffectofMBIduring7or14consecutivedayson thehealingofgastric(Fig.4)andduodenalulcers(Fig.5)induced byaceticacidinrats.
Duetoseverityofthelesionsinducedbyaceticacidinthegastric mucosaofrats,neitherlansoprazolenorMBIwereabletoheal gas-triclesionswithin7consecutivedays(Fig.4).However,theresults inFig.4alsoshowthattreatmentwithMBI(500mg/kg)orwith lan-soprazolefor14consecutivedayswereabletohealgastriculcers bydecreasingthelesionarea(49%and59%,respectively)in rela-tiontothecontrolgrouptreatedwithvehicle.Fig.5showsthatMBI wasmoreeffectiveinhealingduodenalulcersthangastriculcers inrats.MBIwasabletohealduodenalulcerswithin7(44%)and14 consecutivedaysoftreatment(45%).Takentogether,theseresults reinforcethemarkedeffectofMBIininhibitinggastricand duo-denallesionsandinpromotingthehealingofa chronictype of gastricandduodenalulcerinrats.Otherimportantdataobtained inthisassayindicatedthatduringtheconsecutivetreatmentsof animalswithMBI(7or14days),noanimalsdiedorshowedsigns oftoxicityfromthisextract,suchasweightloss.However,more specificstudiesregardingpotentialinvivotoxicityofMBImustbe
Fig.5.DuodenalhealingeffectofmethanolicextractofByrsonimaintermedia(MBI) bytreatmentduring7or14daysafterinjuryinducedbyaceticacidinrats(n=8). Thebarsrepresentmean±S.E.M.Thenumbersindicatethepercentage protec-tioninrelationtothecontrolgrouptreatedwithvehicle.ANOVAfollowedby Dunnett’s;*P<0.05,**P<0.01.
Table4
EffectofmethanolicextractofByrsonimaintermedia(MBI)onintestinalpropulsion inducedbyactivatedcharcoalinmice.
Treatment(p.o.) Dose(mg/kg) N Distancemovedby charcoal(arcosine)
Vehicle – 7 56.1±2.6
Atropine 5 7 35.8±3.8**
MBI 250 7 62.6±5.0
500 7 51.4±1.9
1000 7 52.4±2.0
Dataarepresentedasmean±S.E.M.ANOVAfollowedbyDunnett’stest. **P<0.01.
performed,basedondataobtainedfromSannomiyaetal.(2007), whichdescribestheabsenceof invivo mutagenicaction ofthis extractbutthepresenceofamutageniceffectinvitro.
Additionallytoprovingtheanti-ulceractivityofMBI,thisstudy also aimed to evaluate the claimed antidiarrheal effect of the traditionalindicationofthisspecies.Therelevanceofthis thera-peuticeffectisdemonstratedbyintestinalinfectionbeingthemost commoncauseofdiarrheaintheworldandbeingresponsiblefor thedeathsofapproximately4millionindividualseachyear(WHO, 1996).
WestartedtoevaluatethispotentialeffectofMBIonnormal intestinalpropulsionofmicewithdifferentdosesoftheextract (Table4).IntheMBI-treatedgroupofmice,alldosesofextractdid notchangethenormalintestinalpropulsivemovementrelativeto theatropinegroup;atropineproducedagreateranti-motilityeffect (P<0.05).ToevaluatetheantidiarrhealeffectofMBI,wechallenged thisextractagainstthecatharticagent,castoroil.Oral administra-tionofcastoroilproducedamarkedandsignificantincreaseinthe intestinalfluidvolumeofcastoroil-treatedmice(Table5).The stan-darddrug(morphine)andMBI(500mg/kg)producedasignificantly inhibitoryeffectonthecastoroil-inducedfluidaccumulation.These findingsuggestthatthisextractproducesdiarrheareliefthrough itsanti-enteropoolingeffect.
We evaluated the antidiarrheal activity of this extract by pretreament with naloxone (competitive opioid antagonist) to determinetheinvolvementofopioidreceptorsattheMBIeffectson managementofdiarrhea.Ourresultsshownthatpretreamentwith naloxonewasabletoreversedtheantidiarrhealeffectofMBI con-firmingtheactionMBImayoccurbyopioidreceptors.Theseresults arealsoinconcordancewithobtainedbyOrlandietal.(2011)that foundexpressiveantinociceptiveactivityofByrsonimaintermedia extract.Inthisstudy,theauthorssuggestthatatleastpartofthe anti-hyperalgesiceffectwasduetoinvolvementofopioidsystem.
Table5
EffectsofmethanolicextractofByrsonimaintermedia(MBI)oncastoroil-induced enteropooling(fluidaccumulation)inmice.
Pretreatment
(i.p.)
Treatment
(p.o.)
Dose
(mg/kg)
Intestinal fluid N
(g)
Inhibition
(%)
- 1.5 ± 0.1
7 - Vehicle Saline +
48 0.8 ± 0.1 ** 7
10 Morphine Saline +
36 0.9 ± 0.1 *
7 500 MBI
Saline +
Naloxone + Vehicle - 8 1.4 ± 0.1
-Naloxone + Morphine 10 8 1.4 ± 0.1 ## 0
Naloxone + MBI 500 8 1.6 ± 0.1 ## -14
140 (2012) 203–212 211
Table6
PreventiveandcurativeeffectsofmethanolicextractofByrsonimaintermedia(MBI)onantidiarrhealinducedbycastoroilinmice.
Effects Treatment(p.o.) Dose(mg/kg) N Timetoinitial evacuation(min)
Evacuationclassification Evacuationindex(EI) Inhibiton(%)a
Normal Semi-solid Liquid
Preventiveeffect Vehicle – 7 74.1±16.3 1.7±0.3 1.3±0.4 3.9±0.5 15.9±0.9 –
Loperamide 5 7 220.8±19.2** 0.0±0.0** 0.0±0.0 0.5±0.5** 1.5±1.3** 91
MBI 500 7 167.0±30.8* 2.4±0.2 1.6±0.4 0.9±0.6* 8.1±1.9* 49
Curativeeffect Vehicle – 8 71.1±5.7 1.5±0.3 0.9±0.3 5.0±0.4 18.3±1.2 –
Loperamide 5 9 187.0±28.7** 0.7
±0.2 0.6±0.2 0.7±0.2*** 3.8
±0.9*** 79
MBI 500 9 103.4±18.8 1.8±0.4 1.1±0.2 2.2±0.4* 10.7±0.7* 42
Dataarepresentedasmean±S.E.M.ANOVAfollowedbyDunnett’stest;*P<0.05,**P<0.01.ForclassificationofevacuationsandcalculationofIE,Kruskal-Wallisfollowed byDunn*P<0.05,**P<0.01.#Inhibitioninrelationtoevacuationindex.
InadditiontothepreventiveeffectofMBIondiarrheainduced
bycastoroil,wealsoevaluatedthecurativeeffectofthisextract
to reverse existing diarrhea, simulating a more realistic
situa-tioninthetherapeuticuseofthemedicinalplant.Thediarrhea
inducingbycastoroilisknowntobeduetoricinoleicacidthat
presentsalaxativeaction,causelocalirritation,affectsintestinal
motility,increasesgastrointestinalmucosalpermeability,induced
damageofintestinalmucosaandalsohaspro-inflammatoryaction
byincreasedprostaglandinslevelsandactivatedplatelet
activat-ingfactors(Izzoetal.,1998;Vieiraetal.,2001;Saitoetal.,2002).
Allratsinthecontrolgroup(treatedwithvehicle)aftercastoroil administrationproducedcopiousdiarrhea(Table6).However, pre-treatmentwithMBI(500mg/kg)inducedasignificantdelayinthe onsetof thecopiousdiarrhea (49%).The decreasein the sever-ityofthediarrhea(evacuationindex)wasalmostthesamewith pre-treatmentwithMBI(preventiveeffect)orpost-treatmentwith thisextract(curativeeffect).TreatmentwithMBIafterorbefore thecatharticagent(castoroil)wasabletoproducea significant decreaseinliquidevacuation,similartoloperamide,butonlyMBI showedpreventiveeffectsbyincreasingthetimetoinitial evacua-tion(P<0.05).
Together,alloftheseresultsshowthatMBIwasabletoboth preventand reversediarrhea inducedbythecatharticagentby decreasingtheliquidfaecesandintestinalfluidwithoutchanging theintestinalmotility.Ourphytochemicalstudydemonstratedthe presenceofoligomericproanthocyanidinsinthisextractandthese compoundshavebeendemonstratedantidiarrhealactivity(Fischer etal.,2004).Butthepresenceofcompoundssuchasflavonoidsin MBIwithdemonstratedanti-inflammatoryeffectsalsocontributes toantidiarrhealactionagainstthecatharticagent.
The antidiarrheal effects of MBI were disabled because the mostcommonintestinalinfectionsthatinduceacutewatery diar-rheainapproximately80%ofpeoplearecausedbyEscherichiacoli and Staphylococcus aureus (Hunter et al., 2010). In this study, wealsoevaluatedtheinvitroantibacterialactivityofMBI.MBI was found to present antibacterial activity against Helicobac-ter pylori(MIC=0.125mg/mL).But wealso evaluatedtheeffect of MBI which the most important pathogenic agents, such as Staphylococcus aureus (MIC=0.250mg/mL) and Escherichia coli (MIC=0.500mg/mL).
4. Conclusions
The present study clearly demonstrates the gastroprotec-tive,ulcer-healing, antibacterial and antidiarrheal action of the methanolic extract from Byrsonima intermedia leaves. The pro-tectiveaction ofthe extract against various ulcerogenic agents is related tothe strengthening of thegastric protective barrier throughtheactionofendogenoussulfhydrylcompounds,increases intheglutathionelevelandactivationofvanilloidreceptors.The protectiveeffectoftheextractisalsomediatedbyareductionofthe
myeloperoxidaselevelsingastricandduodenalmucosa.The ulcer-healingactionagainstgastric(14days)butmostlyduodenalulcers (7and14days)demonstratedthepowerfulhealingeffectofthis extract.Thisworkalsoconfirmedthattheanti-diarrhealactionof thisextractisexecutedbydecreasingdiarrhealfaecesand intesti-nalfluidformationandsuggesttheinvolvementofopiatereceptors. Theseeffectsoftheextractareaccompaniedbyanantimicrobial actionagainstthemostcommonbacteriathatcauseulcers.
Conflictsofinterest
Thereisnotconflictofinterest.
Acknowledgments
This work was supported by the Biota-FAPESP project (Fundac¸ãodeAmparoaPesquisadoEstadodeSãoPaulo),CNPq (Conselho Nacional de Pesquisa) and CAPES (Coordenac¸ão de Aperfeic¸oamentoPessoaldeNívelSuperior).
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