Rev. bras. farmacogn. vol.26 número3

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RevistaBrasileiradeFarmacognosia26(2016)375–378

w ww . e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Solanum

paniculatum

root

extract

reduces

diarrhea

in

rats

Jonh

A.B.

Tenório

a

_,

_Dulciana

_S.

_do

_Monte

a

_,

_Thelma

_M.G.

_da

_Silva

a

_,

Teresinha

G. da

Silva

b

_,

_Clécio

_S.

_Ramos

a,∗

a_Department_of_Chemistry,_Universidade_Federal_Rural_de_Pernambuco,_Recife,_PE,_Brazil b_Department_of_Antibiotics,_Universidade_Federal_de_Pernambuco,_Recife,_PE,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received14September2015 Accepted7February2016 Availableonline23March2016

Keywords:

Antidiarrhealactivity Chronicdiarrhea Chlorogenicacid Gastrointestinalmobility Jurubeba

a

b

s

t

r

a

c

t

SolanumpaniculatumL.,Solanaceae,locallyknownas“jurubeba”,iswidelyusedinBrazilforculinary

purposes,andinfolkmedicinetotreatofdiversedisorderincludinggastricdysfunctions.Inthisstudy weinvestigatedtheantidiarrhealactivityofS.paniculatumrootsextractinratsatdifferent concen-trations(125,250and500mg/kg,p.o)usingdifferentexperimentalmodelssuchascastoroil-induced diarrhea,enteropoolingandgastrointestinalmotility,determinedbyinvivoexperimentalmodels.The majorcompoundofrootextractwascharacterizedaschlorogenicacidbasedintheIR,1Dand2DNMR analysis.Alltheextractdosesachievedantidiarrhealpotency,asindicatedbyreducedweightoffecesin castoroil-induceddiarrhea,decreasedintestinalmotilityandsignificantlyinhibitedcastoroil-induced enteropoolingcomparedtothevehiclegroup.Thehighestdose(500mg/kg)producedgreater anti-motilityeffectandbetterreductionofenteropooling,similartothereferencedrugLoperamide(5mg/kg). ExtractfromS.paniculatumL.rootshadantidiarrhealactivity,asshownbythelowerweightofthefeces aswellasdecreaseintheaccumulationofintestinalfluidandslowertransit,justifyingthetraditional useofplantfordiarrhea.

Introduction

ThefamilySolanaceaeA.L.Jussieucomprisesmorethan3000 speciesdistributed in106genera,and theirspecies havea cos-mopolitandistribution.Themosteconomicallyimportantgenus ofthe familyis Solanum,withapproximately1500 species dis-tributedallovertheworld(Nuritetal.,2007).Amongthespecies ofSolanumgenus,SolanumpaniculatumL.istobemorethat high-lightsforitsmanymedicinaluses,widedistributionandespecially forbeingrecognizedasherbalmedicinebytheBrazilian Pharma-copeia,whoserootsandstemsareindicatedinthetreatmentof anemiaandliveranddigestivedisorders.ThespeciesS. panicula-tumL.,knownpopularlyeitherasjurubebaorjurupeba,isused commonlyinBrazilianfolkmedicineforthetreatmentofdiverse disorders,suchasliverandgastricdysfunctionsandhangoveras wellasforculinarypurposes(Mesia-Velaetal.,2002;Botionetal., 2005;Agraetal.,2007;SabirandRocha,2008;Vieiraetal.,2013). Theplantisacomponentofavarietyofpharmaceutical formula-tionsincluding:syrups,infusionsanddecoctions,ethanolextracts,

∗ Correspondingauthor.

E-mail:clecio.ramos@ufrpe.br(C.S.Ramos).

andelixirs(Júnioretal.,2015).Ethnopharmacologicalstudieshave revealedthatleaves,roots,stemsandfruitsofS.paniculatumare usedtotreatdisordersoftheliverandkidney,anemia,tuberculosis, malariaand hypertension,andinanti-inflammatory,antipyretic and stimulant formulations (Joachimovits, 1954; Ribeiro et al., 1986; Agraet al.,2007).Chemicalstudies revealeda varietyof compoundgroupsinS.paniculatumtissues,suchassteroids, ter-penes, alkaloids (including jurubebina, jubebine and solanine), and saponins (including sojuripidine, isojurubidine, isopanicu-lidine and jurubidine) (Vieira et al., 2010; Ramos and Ramos, 2012).

Diarrheacontinuestobeamajorhealthproblemthroughout theworld,especiallycausingofmalnutritioninchildrenunderfive yearsold.Itisalsoamajorcauseofthehighmorbidityand mor-tality,particularlyamong children. In developing countries, the principalcausesof diarrheais associated withtheenterotoxins thatareproducedandsecretedfrombacterialorganismslike Vib-riocholerae,Salmonella,ShigellaandEscherichiacoli(Walkeretal., 2013).

ConsideringthepharmacologicalpotentialofS.paniculatumand theuseofplantsforthetreatmentofdiarrhea,thisstudy investi-gatedtheactivityofS.paniculatumextractagainstdiarrheainduced byvariousmethodsinrats.

http://dx.doi.org/10.1016/j.bjp.2016.02.003

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376 J.A.B.Tenórioetal./RevistaBrasileiradeFarmacognosia26(2016)375–378

Materialsandmethods

Plantmaterials,extractionandisolation

SolanumpaniculatumL.,Solanaceae,specimenswerecollected inthecityofCamaragibe,Pernambucostate,northeasternBrazil, in January2012. Botanical identificationwas doneby Instituto AgronômicodePernambuco(IPA)and a voucherspecimenwas deposited at the Dárdano de Andrade Lima Herbarium of IPA (88503).Therootsweredriedat40◦_C_by₇₂_h_and_the_dried_material wasmilledtoafinepowderinaMACSALABmill.

Driedroots(46g)werefirstlysubmittedSoxhletextractionwith 200mlofdichloromethanefor2handfollowedbyextractionusing 300ml of ethanol for a period of 4h. The solvent wasfiltered andconcentratedinvacuotoyield4.4g(9.6%)offatty-freecrude extractswithpresenceofprecipitate.Partofextractwaswashed twicewithchloroform,filtratedanddriedtoyielda whitesolid identifiedaschlorogenicacid.

Instrumentationandchromatographymaterials

IRspectraweremeasuredinKBrpelletswithaVarianinfrared spectrometer.1Dand2DNMRspectrawererecordedusingBruker spectrometer NMR (300MHz and 500MHz). HPLC analyses of extractswereperformedinaShimadzuLC10instrumentusinga PhenomenexRP-18 column(250mm×4.7mm,5␮m),eluted in gradientmodestartingwith5%aqueousformicacid/methanol(8:2) for15min,raisingto30%methanolin20min,withdetectionat 325nmandflowrateof1ml/min.Fourmgofthecrudeextractwas suspendedin2mlofmethanolandappliedtoC18cartridges.The cartridgeswereelutedwith4mlofMeOHandtheeluentswere fil-tered(0.45␮mfilter)priortoanalysis.Thestandardofchlorogenic acidwasacquiredoftheSigma–Aldrich(Batch:SLBB6914V).

Experimentalanimals

Malealbinoratsweighingbetween200and250gwereusedfor theexperiments.Theanimalswereobtainedfromtheanimalhouse oftheAntibioticsDepartmentofFederalUniversityofPernambuco, whichisregisteredwiththeBrazilianCollegeofAnimal Experimen-tationunderno.18.AllexperimentswereauthorizedbytheEthical CommitteeforAnimalCare(protocolnumber: 23076.46150/2012-23CCB-UFPE).Theanimalswerekeptinpolypropyleneboxesata temperatureof22±3◦_C_with_a_light-dark_cycle_of₁₂_h_and_received balancedfeedandwateradlibitum.Theanimalsweredeprivedof foodbeforeeachexperiment.

Castoroilinduceddiarrhea

The method described by Awouters et al. (1978) was fol-lowed.Healthyalbinoratsofeithersex(200–250g)wererandomly selectedand dividedintofivegroupsoffiveanimalseach.They weredeprivedoffoodfor12hpriortothetest,withfreeaccess towater.Group1receivedthevehiclealone(1%Tween80),while thoseingroup2receivedLoperamide(5mg/kg)aspositivecontrol. Animalsingroups3,4and5receivedS.paniculatumrootextracts (125,250,500mg/kg)respectively.Extractadministrationwasby theoralroute.Theanimalswerehousedsinglyincageslinedwith pre-weighedfilterpaper.Onehour afterpretreatmentwiththe extract,theanimalswerethengiven1mlofcastoroilorallyand thetimebetweenoiladministrationandappearanceoffirst diar-rhealdropwasnoted.Thereafter,theywereobservedfor4hfor thepresenceofdiarrheaandtotal weightoffecesexcretedwas obtained.

Gastrointestinalmotilitytest

Malealbinorats(200–250g)wererandomlydividedintofive groupsoffiveratseach.Theyweredeprivedoffoodfor12hbefore thetest,butwereallowedfreeaccesstowater.Group1rats (con-trol)weretreatedwiththevehicle(1%Tween80).Group2rats receivedLoperamide(5mg/kg),whilegroups3,4and5received differentdosesoftheS.paniculatumrootextracts(125,250and 500mg/kg),respectively.Thirtyminutesafterdrugadministration, 1mlofcharcoalmeal(5%)wasadministeredorallytoallanimals and30minlater,alltheratsweresacrificedandtheabdomenwas opened.Thesmallintestine wasdissectedout fromthepylorus tothececumandthetotaldistancetraveledbythecharcoalplug alongthesmallintestinewasestimatedforboththecontrolandthe treatedgroups.Thepercentagedistancetraveledbythecharcoal mealfromthepylorustothececumwasnoted.

Castoroil-inducedenteropooling

Inthismethod,asdescribedbyRobertetal.(1976),ratswere deprivedof foodfor12hbeforetheexperiment. Theratswere dividedintosixgroups offiveeach.Thevehicle(1%Tween80) wasgiventothefirstgroup.Thesecondgroupreceived Lopera-mide(5mg/kg),whilegroups3,4and5receivedgradeddosesof SPRE(125,250and500mg/kg).Thirtyminuteslater,alltherats weretreatedwith1mlofcastoroil.Ashamgroupdidnotreceive anytreatment.After30min,eachratwassacrificedandthewhole lengthoftheintestinefromthepylorustothececumwasdissected andthecontentsweighed.

Statisticalanalysis

Resultswereexpressedasmean±SEM.Thesignificanceof dif-ferencebetweenmeanswasdeterminedusingone-wayanalysis ofvarianceandthestatisticalsignificancewassetatp<0.05.All datawereanalyzedusingGraphPadPrismversion5.0(GraphPad SoftwareInc.,SanDiego,CA,USA).

Results

Chemicalprofile

TheanalysisbyHPLCrootextractofS.paniculatumshoweda majorpeak withretentiontime at 8min(Fig.1).Theethanolic crudeextractoftheS.paniculatumrootswassubmittedto purifica-tionsteps,resultingintheisolationofmajorcompoundasayellow amorphouspowderdeterminedaschlorogenicacid.Structural elu-cidationofchlorogenicacidwasbasedoninterpretationofspectral data,mainlyIR,UV1_H_and13_C_NMR_and_2D-NMR,_including

com-parisonwithvaluesdescribedintheliterature(Agboetal.,2014).

Castoroil-induceddiarrhea

The highest total weight of feces was obtainedin the con-trolgroup (11.17±0.70g), whereas thelowest weight of feces wasrecordedforanimalsthatreceivedLoperamide(3.81±0.13g) (Table1).Alltheextractdosesachievedantidiarrhealpotencyby meansofreducedweightoffecescomparedtothecontrolgroup.

Gastrointestinalmotilitytest

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J.A.B.Tenórioetal./RevistaBrasileiradeFarmacognosia26(2016)375–378 377

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 Min

0 100 200 300 400 500 600 700 800 900

mAU

O O HO

HO

OH

OH OH O

Chlorogenic acid

Fig.1.ChemicalprofileobtainedbyHPLCofcrudeextractofSolanum panicula-tumroots.(C18column250mm×4.7mm,5␮m,elutedwith5%aqueousformic

acid/methanol8:2for15min,raisingto30%methanolin20min; =325nm;flow 1ml/min).

Table1

EffectoftheSolanumpaniculatumrootsextractoncastoroil-induceddiarrheain rats.

Group Dose(mg/kg) Totalweightof feces(g)

Inhibition(%)

Vehicle – 11.17±0.70 –

Loperamide 5 3.81±0.13a _65.9

125 6.54±0.16a _41.5

Extract 250 6.26±0.28a _44.0

500 5.59±0.10a _50.0

Valuesareexpressedasmean±SEM(n=5).

a_p_<_0.01_with_respect_to_the_{vehicle-treated}_group,_using_one-way_ANOVA

fol-lowedTurkey’stest.

Table2

EffectoftheSolanumpaniculatumrootsextractongastrointestinalmotilitytest.

Group Dose(mg/kg) Distancetraveledby charcoal(%)

Inhibition(%)

Vehicle – 73.88±1.34 –

Loperamide 5 52.45±2.28b _47.5

125 64.62±1.87a _35.4

Extract 250 61.92±1.80a _38.0

500 55.86±3.40b _44.1

b_p_<_0.01_with_respect_to_the_{vehicle-treated}_group,_using_one-way_ANOVA

The highest dose of SPRE (500mg/kg) produced greater anti-motilityeffect,similartoLoperamide(5mg/kg).

Castoroil-inducedenteropooling

As shown in Table 3, the intestinal weight content was much higher in the control group (3.277±0.18g) compared withshamgroup(2.040±0.03g).Consistently,5mg/kgof Loper-amide reduced the intestinal content significantly compared with the control group (p<0.01). Treatments with SPRE (125, 250and500mg/kg)decreasedtheintestinalweightcontentsto 2.788±0.08g(p<0.05),2.657±0.08g(p<0.05)and2.402±0.14g (p<0.01),respectively.

Table3

EffectoftheSolanumpaniculatumrootsextractoncastoroil-inducedenteropooling.

Group Dose(mg/kg) Weightofintestinal content(g)

Inhibition(%)

Sham 2.040±0.03 –

Vehicle 3.277±0.18 –

Loperamide 5 2.392±0.05b _27.0

125 2.788±0.08a _14.9

Extract 250 2.657±0.08a _18.9

500 2.402±0.14b _26.7

b_p_<_0.01_with_respect_to_the_{vehicle-treated}_group,_using_one-way_ANOVA

Discussion

Diarrheaisoneofthemaincausesofinfantmortalityin devel-opingcountries,causingabout5to8milliondeathsayear,mainly among children under five years of age. Medicinalplants have beenusedtraditionally,includingS.paniculatum,asantidiarrheal withoutanyscientificbase(Konatéetal.,2015).Diarrheainvolves increasedgastrointestinalmobility andsecretionand decreased absorptionofwaterandelectrolytes(Wangetal.,2015).

Thegastrointestinalmodelusingactivatedcharcoalasmarker hasbeenusedforover60yearsasasimpleandeffectivetoassess theeffectsoflaxatives.Thismethodindicatesthemaximum dis-tancetraveledbythemarkerinthesmallintestineinagiventime intervalafteritsadministration(Silvaetal.,2012).Accordingtothe model,theS.paniculatumextractsatthedifferentconcentrations evaluatedsignificantlyreducedtheintestinalmotility,witha dose-dependentpatternwhencomparedtothecontrolgroup(Table2). ThemotilityintheanimalsthatreceivedSPREattheconcentration of500mg/kgwasabout44%slower,demonstratingthattheextract hasapositiveeffectinreducingintestinaltransit.

Castoroilinducesdiarrheabycausinganincreaseinthe secre-tionoffluidsandelectrolytesintheintestinallumenthroughthe mucosa.Thisinturncausesa build-upof fluidandan aqueous lumencontentthatpassesquicklythroughthegut(Wangetal., 2015).Thereleaseofricinoleicacidproduceschangesinthe trans-portofwaterandelectrolytes,diminishingtheabsorptionofNa+

andK+_,_in_turn_reducing_the_activity_of_Na+_,_K+_-ATPase_in_the_small

intestine and colon,resulting ina hypersecretoryresponse and fasterintestinaltransit(Dashetal.,2014).Castoroilincreasesthe productionorreleaseofprostaglandins(Saitoetal.,2002), chang-ingthepermeabilityandcausinginjuriestotheintestinalmucosa. Italsostimulatesbiosynthesisofplateletaggregationfactor(Izzo etal.,1998),whichcanresultininflammationofthemucosa.

TheextractfromS.paniculatumrootsinthecastoroil-induced diarrheamodelreducedthediarrheasignificantly,atallthetested doses,whencomparedtothecontrolgroupthatonlyreceivedthe vehicle(Table1).Theextractcausedadiarrheareductionofabout 50%,besidesmorefeceswithsolidconsistencyandreduced evac-uationfrequencyinrelationtothegroupcontrol.Anotherresult observedinthisworkwasthereductionoftheaccumulationof intestinalfluidinduced bycastoroil. TheS.paniculatumextract reducedtheaccumulationofintestinalfluidatalltheevaluated doses(125,250and500mg/kg),withinhibitionratesof14.9,18.9 and26.7%,respectively,showinga dose-dependentrelationship. Thedoseof500mg/kgproducedthebestresults,withsimilar inhi-bitionratetoLoperamide(Table2).

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inhibiteddiarrhealdroppingsby84.81%,resultsimilarto antidiar-rheal compounds Loperamide (72.65%) and diphenoxylate HCl (84.81%)(Tekeetal.,2010).Thediterpenoid19-deoxyicetexone iso-latedfromSalviaballotifloraatdosesof12.5and25mg/kgreduced diarrheafor61.8and89.6%,respectively (Pérez-Gutiérrezetal., 2013).A previousstudy,theethanolicextractfrom S. panicula-tumaerialpartsshowedantidiarrhealactivityatdoseof125,250, 500,and750mg/kg,andthechlorogenicacidalsowasidentified in theaerial parts of plant(Clementino-Netoet al., 2016).The reducedaccumulationofintestinalfluidpromotedbytheS. pan-iculatumextractfromrootsmaybeassociatedwiththepresence ofchlorogenicacid,aknownanti-inflammatorycompound,since theintestinal hypersecretion of fluid involves aninflammatory response(Izzoetal.,1998;Guoetal.,2015).

Conclusion

OveralltheextractofS.paniculatumL.rootshadantidiarrheal activity,as shown bythe lower weightof thefeces as wellas decreaseintheaccumulationofintestinalfluidandslowertransit, justifyingthetraditionaluseofplantfordiarrhea.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authors’contributions

JABT,DSMandTGSdevelopedtheanimalmodel.JABTandTMGS wereresponsibleforthecollectionofplantsampleandthe chem-icalstudies.CSRandJABTdevelopedtheanalyticalmethodology (HPLC).CSRandTGSdesignedthestudyandsupervisedthe labo-ratorywork.CSRandJABTwrotethemanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

JABTthanksCAPESforprovidingascholarship.Theauthorsare indebtedtotheCentrodeApoioaPesquisa(CENAPESQ),UFRPE,for thelaboratoryfacilities.

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