JournalofEthnopharmacology138 (2011) 47–52
ContentslistsavailableatScienceDirect
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
Can
a
Strychnos
species
be
used
as
antiulcer
agent?
Ulcer
healing
action
from
alkaloid
fraction
of
Strychnos
pseudoquina
St.
Hil.
(Loganiaceae)
Flávia
Bonamin
a,
Thiago
Mello
Moraes
a,
Hélio
Kushima
a,
Marcelo
Aparecido
Silva
b,
Ariane
Leite
Rozza
c,
Cláudia
Helena
Pellizzon
c,
Tais
Maria
Bauab
d,
Lucia
Regina
Machado
Rocha
a,
Wagner
Vilegas
b,
Clélia
Akiko
Hiruma-Lima
a,∗aUNESPUnivEstadualPaulista,DepartamentodeFisiologia,InstitutodeBiociências,cp610,CEP18618-000,UNESP,Botucatu,SP,Brazil bUNESPUnivEstadualPaulista,DepartamentodeQuímicaOrgânica,InstitutodeQuímica,cp355,CEP14801-970,UNESP,Araraquara,SP,Brazil cUNESPUnivEstadualPaulista,DepartamentodeMorfologia,InstitutodeBiociências,cp610,CEP18618-000,UNESP,Botucatu,SP,Brazil dUNESPUnivEstadualPaulista,DepartamentodeCiênciasBiológicas,cp970,CEP14801-970,UNESP,Araraquara,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received5July2011
Receivedinrevisedform11August2011 Accepted11August2011
Available online 21 September 2011
Keywords:
StrychnospseudoquinaSt.Hil. Loganiaceae
Ulcerhealing PCNA SOD Angiogenesis
a
b
s
t
r
a
c
t
Ethnopharmacological relevance:Strychnos pseudoquinaSt.Hil.(Loganiaceae) isone Braziliannative medicinalspeciesdescribedinthefirsteditionoftheBrazilianOfficialPharmacopoeiain1929.This medicinalplant,popularlyknownas“quina-quina”,“quina-branca”or“cascaaromaticawasvery com-monlyusedinfolkmedicineinteaformobtainedfromthebarkand/orleavesastonic,antipyretic, antimalarialandmainlyagainstdiseasesoftheliver,spleenandstomach.
Aimofthestudy:PreviousstudyalreadycharacterizedthegastroprotectiveactionofthisspeciesTheaimof thepresentstudyistoelucidatethemechanismofthehealingprocessmediatedbythemethanolicextract (ME)andtheirenrichedalkaloidfraction(EAF)fromStrychnospseudoquinainchronicgastriculceration inducedby5%aceticacidinrats,anexperimentalmodelthataccuratelyreflectshumangastrointestinal disease.
Materialandmethods:TheMEandEAFwasadministeredorallyinasingledose(basedonpreviously studyofdose–responsecurve)for14daysafterchroniculcerationwasinducedinrats.Thehealingeffect ofMEandEAFwasevaluatedbymacroscopicandmorphometricanalyses,immunohistochemicalassay (PCNAandSOD)andanti-Helicobacterpylorieffectwasevaluatedbyinvitroassay.
Results:OurresultsdemonstratedthatEAFsignificantlyreducedborderinternal(42%)andexternal(38%) lesionarea(mm2)bymacroscopicanalyses(P<0.05).AnimalstreatedwithEAFstimulatedsome prolifer-ativefactorsbyincreasingtheheightofepithelialregenerativeareaandtheexpressionofPCNA-positive nuclei.ThenumberofvesselsingastricmucosaofratstreatedwithEAFrevealsanexpressiveincrease (4timesmorethanvehicletreatment)ofvesselsthatstimulatecellsproliferationinthehealingregion. Theseresultssuggestthattherecoveryofvascularizationoftheulceratedareaisinvolvedinthe heal-ingactionofalkaloidfractionofStrychnospseudoquina.TheMIC(minimuminhibitoryconcentration)of 75g/mlfromEAFshowedaneffectiveinvitroanti-Helicobacterpyloriactionofthisfraction.EAFalso wasquiteeffectiveintheprocessofSODreleasethatisanimportantprotectivefactoragainstbacterial agents.TheefficacyofEAFwasaccomplishedsafelywithoutpresentinganyalterationoftoxicological parametersduring14dayoftreatment.
Conclusions:Theexpressivegastrichealingeffectbyincreasingcellularproliferationtogetherwith expres-sionofSODactivityandantibacterialactionagainstHelicobacterpyloriconfirmtheefficacyofthisspecies inhealgastricmucosaandtheseresultsareaimportantcontributiontotheknowledgeofacrudedrug presentsattheBrazilianOfficialPharmacopoeiasince1929.
© 2011 Elsevier Ireland Ltd. All rights reserved.
∗Correspondingauthor.Tel.:+5501438116077;fax:+551438153744. E-mailaddress:hiruma@ibb.unesp.br(C.A.Hiruma-Lima).
1. Introduction
ThegenusStrychnosincludesmorethan200speciesdistributed amongtropicalareasoftheglobe(Philippeetal.,2004).Manyof thesespeciesareknownbytheirmedicinalpropertiesandforthe powerfultoxicitycharacteramongtheirphytocompounds,suchas strychnine,oneofthepoisonousalkaloidsproducedbythespecies
48 138 (2011) 47–52
fromthisgenus(Thongphasuketal.,2003).Amongthemedicinal species,afolkmedicinefromCerradoregionofBrazil,thereisthe speciesnamedStrychnospseudoquinaSt.Hilknownpopularlyas “quina-quina”,“quina-branca”or“cascaaromatica”.Thisplantwas verycommonlyusedinfolkmedicineinteaformobtainedfromthe barkand/orleavesastonic,antipyretic,antimalarialandmainly againstdiseasesoftheliver,spleenandstomach(PottandPott, 1994;Almeidaetal., 1998;Lorenzi andMatos, 2002; Andrade-Netoetal.,2003).ThismedicinalplantwasoneoftheBrazilian nativespeciesdescribedinthefirsteditionoftheBrazilian Offi-cialPharmacopoeiain1929asintermittentfevers,liverandspleen disordersandasdigestive(Brandãoetal.,2008)buteveneighty yearsafterwards fewpharmacologicalstudieshave been devel-opedwiththisspecies.Andrade-Neto etal.(2003)showedthat crudeethanolextractfromthebarkofthisspeciespresentsweak antiplasmodialactivity.Honório-Franc¸aetal.(2008)demonstrated that aqueousextract fromStrychnos pseudoquinabarkprovides hypoglycemicactionbutdidnothealwoundsindiabeticrats.Silva etal.(2005)showedtheprotectiveactionofmethanolicextract (ME)andenrichedalkaloidfraction(EAF)fromleavesofStrychnos
pseudoquinatothegastricmucosaagainstinjuriousagentssuch
asnon-steroidalanti-inflammatorydrugs(NSAID)andanethanol acidifiedsolutioninmice.Thesameauthorsalsoobservedthatanin vivoassayofMEandEAFpresentednotoxiceffectsandno mortal-ityfor14days.However,studiesfromSantosetal.(2006)showed
thatMEfromStrychnospseudoquinawasmutagenicagainststrains
ofSalmonella,buttheEAFpresentedabsenceofmutagenicityinthis
sameassay.
TheconfirmationofgastroprotectiveactionofMEandEAFfrom
StrychnospseudoquinabySilvaetal.(2005)isanimportant
indica-tiveofantiulcerpropertyofthisspecies,corroboratingitsreputed utilityinfolkmedicinethatmainlyobtainedpolarcompoundsas teaform.However,theseactionsdo notimply thatthesesame preparationsalsopresentulcerhealingeffectoninjuredgastric mucosa.Sotheaimofthisstudywastoevaluatethehealingaction
ofMEorEAFofStrychnospseudoquinathroughout14consecutive
daysinthechroniculcermodelinducedbyaceticacidinrats.We alsoevaluatedsometoxicologicalparametersbysubacute treat-mentswithStrychnospseudoquinaandtheanti-Helicobacterpylori actionofthisspecies.
2. Materialsandmethods
2.1. PlantmaterialandextractionofMEandEAF
Leaves of Strychnos pseudoquina St. Hil. were collected in May2001from PortoNacional city,Tocantins State,Brazil and identified by Prof. E.R. Santos, from theInstitute of Biology at TocantinsUniversity.Aherbariumspecimenvoucher(Nr.3291) wasdepositedattheHerbariumofTocantinsUniversity(HTINS). The leaves were air-dried and powdered in a mill. Leaves of
Strychnos pseudoquina (300g) were extracted in powder form
withmethanolassolventduringsevendays.Solventwas evap-oratedinavacuumtoyield17.7gofextract(ME)(3:10wasthe plant:solventration).Aportion(5.0g)ofthemethanolicextract (MeOH)ofStrychnospseudoquinawassubmittedtocolumn chro-matographyonSephadexLH-20 (100cm×5cm)withMeOH as theeluent.Onehundredfractions(5ml)werecollected,checked byTLCon silicagel plates CHCl3–MeOH–n-PrOH–H2O (5:6:1:4, v/v/v/vlowerphase)and revealedwithDraggendorff, iodoplati-nate or NP/PEG (NaturalProducts/Polyethyleneglycol) reagents. AlkaloidsweredetectedinFr.3–29(denominated“enriched alka-loidfraction—EAF3g)andflavonoidsweredetectedinFr.35–90 (250mg).These exactly the same batchesfrom MeOH and the EAFhave been used in this study were extensively study and
phytochemical profiles of both were described by Silva et al. (2005).
2.2. Animals
MaleWistaralbinorats(150–250g)fromtheCentralAnimal HouseoftheUNESPwereused.Theanimalswerefedacertified Nuvilab®(Nuvital)dietwithfreeaccesstotapwaterunderstandard
conditionsof12hdark-12hlight,humidity(60±1.0%)and temper-ature(21±1◦C).Fasting(24h)wasusedpriortoassaysbecause standard drugs,MEor EAFwere alwaysadministeredbyorally usingasalinesolution(10ml/kg)asvehicle.Animalswerekept incageswithraisedfloorsofwidemeshtopreventcoprophagy. Allexperimentswereperformedinthemorning,andfollowedthe recommendationsoftheCanadianCouncilonAnimalCare(Olfert etal.,1993).TheUNESPInstitutionforAnimalCareapprovedthe employedprotocols.
2.3. Healinginaceticacid-inducedgastriclesion
The experiment was performed according to the method describedbyTakagietal.(1969),withsomemodifications.Three groupsofratsthathad fastedfor24hwereusedinthis exper-iment(n=7–8).Under anesthesia,a laparotomywasperformed onallanimalsthroughamidlineepigastricincision.After expos-ingthestomach,0.05ml (v/v)ofa30%aceticacidsolutionwas injectedintothesubserosallayerintheglandularpartofthe ante-riorwall. The stomachwasbathed withsaline(20◦C) toavoid
adherenceto the external surface of the ulcerated region.The abdomenwasthenclosedandalltheanimalswerefednormally. WeselectedthelowesteffectivedoseofMEorEAF(250mg/kg bodywt.)toevaluatethehealingeffectofStrychnospseudoquina based on datafrom Silva et al. (2005) that evaluated the gas-troprotectiveactionofMEorEAFonacutegastriclesionmodel. Theothergroupwastreatedwith100mg/kgofpurecimetidine (SigmaCo.,USA)orvehicle(10ml/kg)todeterminehealingeffects bysubacutetreatment.Alltreatmentswereadministeredorally onceadayfor14consecutivedaysbeginningonedayaftersurgery. Bodyweightwasrecordeddailythroughouttheexperimentsand themacroscopicanalysesandweightsofvitalorgans(liver, kid-ney,heart,spleenandlung)werecomparedamongthedifferent treatmentsandthegrouptreatedwithvehicletoevaluatethe pos-siblesubacutetoxicity inducedby MEorEAF. Onthedayafter thelastdrugadministration,theratswerekilledandtheir stom-achsweregentlyremoved.Thegastriclesionswereevaluatedby examiningtheinnergastricsurfacewithadissectingmagnifying glass.Themacroscopiculcerareaoftheinternalborderand exter-nalborder(mm2)weredeterminedasdescribedbyTakagietal.
(1969).
2.3.1. Histologicalmethods
The stomach lesionsinduced by acetic acid in rats submit-tedtothedifferenttreatmentswerelocated,sectioned,andfixed inALFACsolution(70%ethanol,4%formaldehydeand5%glacial acetic acid) for 24h at 4◦C. Then the samples were routinely
processedforembeddinginparaplast,andcutinto7m-thick sec-tionsthatwerestrainedwithperiodicacid–Schiff(PAS) (Vacca, 1985)andhematoxylin-eosin(HE)(Behmeretal.,1976).Paraffin slides wereprocessedfor HEstrainingand imunohistochemical reactionin bloodvessels. Forboth analyses, weused atleast6 fieldsforeachgroupandtheresultsweresubmittedtostatistical analyses.
2.3.2. Morphometricanalyses
138 (2011) 47–52 49
Table1
Regenerationmeasuresofthegastriclesionborders(epithelialheight)andthenewbloodvesselsintheregenerationareasunderthemucousmembraneinhistological analysisofthestomachtreatedwithEAFofStrychnospseudoquinaunderthemodelofulcerinductionbyaceticacid.
Treatments(p.o.) Doses(mg/kg) Epithelialheight(m) Numberofnewbloodvessels(m2)
Saline – 1462.00±25.27 178.01±20.64
Cimetidine 100 1489.50±17.56 309.17±30.33*
EAF 250 1611.70±28.14** 633.95±33.97**
Resultsaremean±S.E.M.Statisticalanalysisamongthegroups:ANOVAfollowingbyDunnett’stest. *P<0.05.
**P<0.01.
Fig.1. Influenceofulcerhealingactivityofmethanolicextract(ME=250mg/kg) andalkaloidsfraction(EAF=250mg/kg)oftheStrychnospseudoquinainacetic acid-inducedgastriclesion.Datafromblackbarsrepresentsinternalborderandgraybars representsexternalborderfromlesion.
England),wheretheheightofregeneratedmucosawasmeasured byemployingavariationofthemethodusedbyIshiharaandIto (2002).
2.3.3. Immunohistochemicalanalyses
Theantibodiesutilized(NovoCastraNCL-PCNA)included anti-PCNA(celldivisionmarker,toevaluateregenerationpotential)and anti-SOD(superoxidedismutase,toevaluateantioxidantactivity on the superoxide radicals). Slices were deparaffinized, dehy-dratedandimmunostrainedwithperoxidasebytheavidin–biotin method.Hightemperatureantigenunmaskingtechniquein0.01M citrate buffer, pH6, in a microwave oven was performed two timesfor5mintotal. Blockingofnonspecificreactionwas com-pleted with1% normal goat serum and 3% not-fat milk. After risingin phosphatebufferedsaline(0.01mol/LPBS pH7.4),the sectionswereincubatedinsecondaryantiserum.Theywerethen washedinPBSandincubatedinABC(avidineandbiotinecomplex) reagents (ABC kit—Vector) and incubated in peroxidase reac-tion(3,3′-diaminobenzidinetetrahydrochoridecontaining 0.01%
H2O2 in PBS buffer. After being immunostrained, the sections werelightlycounterstainedwithMayer’shematoxylin.Theslides wereobservedunderaLeicalightmicroscope.Forpositive con-trolwe used the stem cell region in stomach mucosa and for negative control the primary and secondary antibodies were omitted.
2.4. Anti-Helicobacterpyloriactivity
TheMEandEAFweretestedtodetectanti-Helicobacterpylori activity (Hachem et al., 1996).The strain of Helicobacter pylori (ATCC43504)wasisolatedfrompatientswithduodenalulcer dis-ease.FrozenHelicobacterpyloriisolatewasthawedandgrownon 5%sheepbloodagarplatesfor3–4daysat37◦Cin10%CO2and
98%humidity.Eachplatewasswabbedwithasterilecotton-tipped applicatorandthecellsweresuspendedinsterilesalinetoobtain turbidityequivalenttoa2.0McFarlandstandard.Muller–Hinton brothcontaining10%horseserumwasaddedtoallwellsofa 96-wellmicrotiterplate(Corning,USA).Eachwellwasincubatedwith
Helicobacterpyloriatafinalconcentrationof∼1×105CFU/ml.The
plates wereincubatedfor5days ina microaerobicatmosphere at37◦C.Followingincubation,theplateswereexaminedvisually
andspectrophotometricallyandthelowestconcentrationshowing completeinhibitionofgrowthwasrecordedastheMIC(minimum inhibitoryconcentration).Staphylococcusaureus(ATCC25923)and
Escherichiacoli(ATCC25922)wereusedascontrolorganismsfor
clarithromycinandampicillin,respectively.Theresultswere con-sideredvalidonlywhentheMICvaluesforthecontrolorganisms werewithintherangesestablishedbytheNationalCommitteefor ClinicalLaboratoryStandards(NCCLS).
2.5. Statisticalanalysis
Resultswereexpressedasmean±S.E.M.Statisticalsignificance wasdeterminedbyone-wayanalysisofvariancefollowedby Dun-nett’sorTukey’stest,withthesignificancelevelatP<0.05.
3. Resultsanddiscussion
Sincethegastriculcermodelinducedbyaceticacidwas estab-lishedmorethan40yearsago(Takagietal.,1969)thisassayhas beenusedforthestudyofunderlyingmechanismsofulcer cicatri-sationbysubacuteandchronictreatmentofanti-ulcerdrugsthat acceleratedthelesion-healingeffect(Pawliketal.,2002).Basedon datafromOkabeandAgamase(2005),thismodelwastheonethat mostcloselyapproximateshumanpepticulcerdisease,becausethe damagecausedbytheaceticacidpenetratesingastricmucosanot
50 138 (2011) 47–52
onlyinthemucousmembraneandsubmucouslayerbutalsothe muscularlayer.
Fig.1showstheresultofthemacroscopicanalysisofthe gas-tric lesionsinduced by aceticacid in ratstreated withvehicle, cimetidine, MEor EAF fromStrychnos pseudoquina. Our results shownthatalthoughMEfromStrychnospseudoquinainduced sig-nificantgastroprotectiveeffectagainstinjuriescaused byNSAID andethanol/HClinmice(Silvaetal.,2005),thetreatmentofrats for14consecutivedayswiththisextractdidnotrevealsignificant healingactionbyreducinginternalorexternalborderarea,when comparedmacroscopicallywithvehicle-treatedanimals(P>0.05). However,thegastriclesionsoftheanimalstreatedwithEAF,at thesamedose,inducedsignificantreduction(P<0.05)ininternal (42%)andexternal(38%)border.Thismacroscopicresultwasalso provenbyhistologicalanalyses(Table1)in whichtheheightof theregenerationarea(m)wasmeasured.Theanimalstreatwith
EAFfromStrychnospseudoquinasignificantlyincreasedepithelial
heightinregenerativeareaofgastricmucosawhencomparedwith vehicle-treatedones.TheseresultssuggestedEAFstimulatedsome proliferativefactorsingastricmucosathatwereimportantfactors toregenerationofinjuredgastricmucosa.
WallaceandDevchand(2005)reportedthatreconstructingthe entirestructuralarchitectureofgastricmucosadamagecanrequire severalweeksandinvolvestheformationofgranulationtissueat thebaseoftheulcers,formationofnewvessels(angiogenesis),and re-establishmentofthewholeglandulararchitecture.The qual-ityofmucosalstructuralrestorationmaybethemostimportant factorindeterminingfutureulcerrecurrence(Tarnawski,2005). H&Estrainingofhistologicalcuts(Fig.2)fromEAF-treatedanimals revealswellstructuredmucousglandsinthehighlyregenerated areathatprobablyprovokedtheirelongationandalignment.The architecturalreorganizationofregeneratedareainEAF-treated ani-mals (Fig. 2)facilitates thepassage of themucus and of other substancestowardsthesurfaceofthegastricmucousmembrane.
Tarnawskietal.(1991)indicatedthatsomegastricmucosashowed re-epithelializationofthemucosalsurfacebutthatsubepithelial mucosadisplayedprominentabnormalities.Theseabnormalities couldinterferewithoxygenation,nutrient supply,and mucosal resistanceanddefense;therefore,theycouldbeabasisforulcer recurrence.
Cellularproliferation playsanimportant role inmaintaining theintegrity ofthegastrointestinalsystem.Cell divisionin gas-trointestinaltreatmentis limited toa discreet anatomical area, theproliferativecompartment(OkamotoandWatanabe,2004).To investigatetheeffectofEAFoncellproliferationinthegastriculcer area, this parameterwas determined by immunohistochemical assay(Fig.3).Inthevehiclegroup,theimmunoreactionsforPCNA (expressed throughout thecell cyclewhereas itsconcentration increasedfurtherintheS-phase)wereobservedasdark accumu-lationsofDAB(diaminobenzidine)reactionproductsinthenuclei ofthegastricmucosalayer.WeobservedthattheEAF-ulcerated groupstimulatedthecellproliferationintheregeneration area, mainlyatthebaseofthemucousglands(Fig.3).SoPCNA-positive nucleifromEAF-groupwasincontestablymoreintensethaninthe vehicle-treatedorthecimetidinepositivecontrolgroup.
Previousstudiesalsosuggestedthatthequalityofepithelial sur-facereconstitutionwasguaranteedbyangiogenesis(formationof anewvascularnetwork)thatensuresanadequatesupplyof oxy-genandnutrientstothehealingmucosa(Tarnawskietal.,2001; Tarnawski,2005).Wequantifiedvessels ingastricmucosa sub-mittedtodifferenttreatments(Table1).Thenumberofvesselsin ratstreatedwithcimetidinewasdoublethatinthosetreatedonly withvehicle.Butthenumberofvesselsingastricmucosaofrats treatedwithEAFfromStrychnos pseudoquinarevealsan expres-siveincrease(4timesmorethanvehicletreatment)ofvessels.This
resultindicatesthatEAFtreatmentinducedhealingactioningastric Table
138 (2011) 47–52 51
Fig.3.ColorationimmunohistochemistryforPCNAinstomachsofratstreatedwithsaline,cimetidine(100mg/kg)andStrychnospseudoquina(EAF).(→)Theareaof
reverse-organizationofthemucousmembrane(EAF)iswithalotofcellspositivePCNA(browncolor,suitableforthearrow).Microscopymagnification20×.(Forinterpretationof
thereferencestocolorinthisfigurelegend,thereaderisreferredtothewebversionofthearticle.)
mucosabyproliferatingvesselsingastricmucosathatstimulated cellproliferationinthehealingregion.
Leiteetal.(2004)observedthatreplenishmentofSOD (super-oxidedismutase)ledtodecreaseinconstrictiveremodelingand enhanced angiogenesis. SOD is the most important enzymes responsibleforeliminatingfreeradicalsfromthecell,giventhat ittransformsO2•−radicalsintoH2O2andprotectsthecellsfrom damagethroughtheremovalofthoseradicals.TheSODactivity levelrepresentstheintracellularantioxidantabilityofthecell.The freeradicalsaffectthestructureofthecellmembraneaswellas themembranesofvariousorganellesincludingthemitochondria, lysosomesandtheendoplasmicreticulum(Wangetal.,2005).
TheparticipationofSODintheEAFactionmechanismwasalso evaluated,duetoitsimportanceasanantioxidantand elimina-toroffreeradicalsfromthecells(Wangetal.,2005;Fanetal., 2005).The result of this study evidenced a greater number of strainedcells(inbrown)fromtheEAF-treatedgroupthaningroups treatedwithvehicle(Fig.4).WealsoobservedthatSODstrained cellswerenotpresentincimetidine-treatedgroup.Kontureketal. (2000)observedthatSODacceleratesthehealingofischemiaand reperfusionlesionsduetosuppressionofoxygenfreeradicalsand improvementofgastricmicrocirculation.Basedonourresultwe concludedthattheEAFisquiteeffectiveintheprocessofreleasing SOD,eliminatingfreeradicalsand,thus,healinggastriclesions.
Somenewantiulcerdrugs,suchasrebamipide,wereeffective in inducing ulcerhealing but failed in Helicobacter pylori erad-ication therapy (Terano et al., 2007). As it is highly desirable toidentifyonedrugthatproducesgastroprotectiveaction,heals injuredgastricmucosaandalsoactsagainstHelicobacterpylori,we evaluatedtheanti-Helicobacterpylori effectofEAFfrom
Strych-nos pseudoquina against a strain of Helicobacter pylori isolated
frompatientswithduodenalulcerdisease.Recently,Castillo-Juárez etal.(2009)evaluatedplantsusedintraditionalmedicineagainst
Helicobacter pylori while considering an MIC value lower than
125g/ml,toconstitutestrongantibacterialaction.Weobtained averyinterestingMICfromEAFof75g/ml,afindingthat indi-catesahighlysatisfactoryanti-Helicobacterpyloriactionfromthis fraction.
OvertwentyyearsagoGarner(1986)predictedthatthefuture of antiulcer drug research must address the multifactorial eti-ologies of gastric ulcer as well as attempt to cure the disease rather thansimplyinduceantisecretoryaction. Buttheefficacy of the new drug must alsobe accompanied by a clear indica-tion for safe human use. Silva et al. (2005)observed that EAF
and MEfrom Strychnos pseudoquinadidnot present acute
tox-icity given that no mortality was observed up to 5g/kg (p.o.). Animals submitted to different treatments (EAF, ME, cimeti-dine andvehicle) in gastriculcer modelinduced byacetic acid supply some important toxicity parameters such as evolution of body weight during 14 days(data not show), mortality, and weight of the vital organs (Table 2). The 14-day evolution in body weight alteration under EAF did not differ significantly fromthevehiclegroupwhiletheaverageweightsofvitalorgans and visceral conditions were normal and comparable to those of the control group(P>0.05).The finding of one death regis-teredineachofthesaline,cimetidineandEAFgroups,doesnot necessarily represent a toxicity signbut rather a consequence of the surgicalprocedure of this model. Table 2 also presents resultsobtainedbybiochemicalanalysesofserum,organizedby treatments, among which we observed no alteration of these parameters.
Santosetal.(2006)describedthatmutageniceffectof methano-lic extract from Strychnos pseudoquina present in Ames and micronucleus tests. But they also recommend that EAF from
Strychnos pseudoquinashould be explored as a possible source
ofnewantiulcerogenicphytotherapeuticpreparationbecausethe absencesofmutagenicitybutnewtoxicologicalstudiesare neces-sarytoensureitssafetyuseinhuman.
52 138 (2011) 47–52
Phytochemicalstudiesof EAF, inagreementwithSilvaet al. (2005),yieldednordiidrofluorcurarine,analkaloidindole,in addi-tiontorutinandkaempferol 3-O--rutenoside.Thestructure of indolicalkaloid3-hydroxy-enolateresemblesthatofomeprazole, awellknownantiulceragent.However,thisalkaloidmaypossess areversiblebondbetweensulfhydrylgroupsanddoesnotpresent adversesideeffectsfromomeprazolerelatedtocarcinoma.
4. Conclusions
Basedontheresultsofthepresentworkweconcludedthatthe
EAFfromStrychnospseudoquinapresentsexpressivehealingeffect
ongastricmucosathroughtheincreaseofangiogenesis,cell pro-liferation,antioxidantactivitythroughexpressionofSODactivity andantibacterialactionagainstHelicobacterpylori.Theabsenceof toxicitythroughoutthe14consecutivedaysoftreatmentensures itssafetyforusebythepopulation.
Acknowledgements
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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