J. Coloproctol. (Rio J.) vol.36 número4

w w w . j c o l . o r g . b r

Journal

of

Coloproctology

Original

Article

Effects

of

hydroalcoholic

extract

of

Ziziphus

jujuba

on

acetic

acid

induced

ulcerative

colitis

in

male

rat

(

Rattus

norvegicus

)

Nader

Tanideh

_,

_Akram

_Jamshidzadeh

_,

_Ali

_Ghanbari

_Saghesloo

_,

Farhad

Rahmanifar

_,

_Maral

_Mokhtari

_,

_Omid

_{Koohi-Hosseinabadi}

_,

Mahmood

Omidi

_,

_Asma

_Najibi

a_{ShirazUniversityofMedicalSciences,StemCellandTransgenicTechnologyResearchCenter,Shiraz,Iran} b_{ShirazUniversityofMedicalSciences,SchoolofMedicine,DepartmentofPharmacology,Shiraz,Iran}

c_{ShirazUniversityofMedicalSciences,SchoolofPharmacy,DepartmentofPharmacologyToxicology,Shiraz,Iran} d_{ShirazUniversity,SchoolofVeterinaryMedicine,DepartmentofAnatomy,Shiraz,Iran}

e_{ShirazUniversityofMedicalSciences,SchoolofMedicine,DepartmentofPathology,Shiraz,Iran} f_{ShirazUniversityofMedicalSciences,CenterofComparativeandExperimentalMedicine,Shiraz,Iran}

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Articlehistory:

Received1March2016 Accepted20April2016 Availableonline27May2016

Keywords: Ziziphusjujuba

Ulcerativecolitis Oxidativestress Histopathology Inflammation

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t

Objective:To investigate the effects of hydroalcoholic extract of Ziziphus jujuba on the histopathological,tissueoxidativestressandinflammationplustoantioxidantpathways ofcolontissueinratwithinducedUlcerativecolitis.

Materialsandmethods:Ulcerativecolitiswasinducedin80ratsthosedividedinto8equal groups.Group1and2werenegativecontrolsreceiving1mL/dayofnormalsalineinenema and oral; group3 and 4as positive control1 and 2received 10mg/kg of intra-colonic asacolandoralmesalazine;groups5and6received20%and40%ofhydroalcoholicextract

ofZ.jujubatrans-rectally;group7and8received1500and3000mg/kgofhydroalcoholic extractofZ.jujubaorally,respectively.After7days,animalswereevaluatedforcolontissue histopathology,levelsofmalondialdehydeandIL-1␤,andactivitiesofsuperoxidedismutase, glutathioneperoxidaseandmyeloperoxidaseincolontissue.

Results:HydroalcoholicextractofZ.jujubainbothformsoftrans-rectalandoral adminis-trationespeciallyinthehigherdosescouldresultintoamorehealingeffectindamaged colonictissue,morereduceglutathioneperoxidaseandIL-1␤level.Also,thesetwodoses (gel40%andoral3000mg/kg)couldmoredecreasethemyeloperoxidaseactivityand stimu-latesuperoxidedismutaseandglutathioneperoxidaseactivities.Also,gel40%intransrectal administrationwasmorepotentthanadministration3000mg/kginoral.

∗ _{Correspondingauthor.}

E-mail:articlepublishers93@gmail.com(A.GhanbariSaghesloo).

http://dx.doi.org/10.1016/j.jcol.2016.04.007

Conclusion: TheresultsofthepresentstudyindicatedthatZ.jujubemaybeconsideredasa treatmentofchoiceforUlcerativecolitisespeciallyingelformandalsoindose-dependent pattern.

©2016PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileirade Coloproctologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Efeitos

do

extrato

hidroalcoólico

de

Ziziphus

jujuba

na

colite

ulcerativa

induzida

pelo

ácido

acético

em

rato

macho

(

Rattus

norvegicus

)

Palavras-chave: Ziziphusjujuba

Coliteulcerativa Estresseoxidativo Histopatologia Inflamac¸ão

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e

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u

m

o

Objetivo: Investigar os efeitos doextrato hidroalcoólico de Ziziphus jujuba noestresse oxidativo emtecidoao nívelhistopatológicoena inflamac¸ão,juntamentecomasvias antioxidantesemtecidodecólonemratoscomcoliteulcerativainduzida.

Materiaisemétodos:Induzimoscoliteulcerativaem80ratos,divididosem8gruposiguais. Osgrupos1e2eramcontrolesnegativosquereceberam1mL/diadesalinanormalem enemaeporvia oral;osgrupos3e4eram controlespositivospara1e 2ereceberam 10mg/kgde asacol porvia intracolônicae mesalazinaoral;os grupos5e 6receberam gela20%e40%deextratohidroalcoólicodeZ.jujubaporviatrans-retal;osgrupos7e8 receberam1500e3000mg/kgdeextratohidroalcoólicodeZ.jujubaporviaoral, respectiva-mente.Transcorridos7dias,osanimaisforamavaliadosparahistopatologiadetecidode cólon,níveisdemalonildialdeídoeIL-1␤,eatividadesdesuperóxidodismutase,glutátion peroxidaseemieloperoxidasenotecidocolônico.

Resultados: OusodoextratohidroalcoólicodeZ.jujuba,tantonaformatransretalcomo oral,eemespecialnasdosesmaisaltas,resultouemumefeitodecicatrizac¸ãomaisintensa notecidocolônicolesionado,eemmaiorreduc¸ãonosníveisdeglutátionperoxidase IL-1␤.Alémdisso,essasduasdoses(gela40%e3000mg/kgporviaoral)diminuíramainda maisaatividadedemieloperoxidaseeestimularamasatividadesdesuperóxidodismutase eglutátionperoxidase.Outroachadodoestudofoiqueogela40%poradministrac¸ão trans-retalsemostroumaispotentedoqueaadministrac¸ãooralde3000mg/kg.

Conclusão: OsresultadosdopresenteestudosugeremqueZ.jujubapodeserconsiderado comotratamentodeescolhaparacoliteulcerativa,sobretudoemformadegeletambém emumpadrãoproporcionalàdoseadministrada.

©2016PublicadoporElsevierEditoraLtda.emnomedeSociedadeBrasileirade Coloproctologia.Este ´eumartigoOpenAccesssobumalicenc¸aCCBY-NC-ND(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Inflammatoryboweldiseases(IBDs)includetwoparts, ulcer-ative colitis(UC)and Crohn’sdisease(CD). UCaffected the superficiallayer ofall partsofthe large intestinespecially descendingcolon and rectum.1 _{IncidenceofUC is 1.2–20.3} casesanditsprevalenceis7.6–240casesper100000peoples ineachyear.2_{ThebasisoftheUCisgeneticallysusceptibility} toinflammationandalsoenvironmentaltriggers.3_These envi-ronmentalfactorsincludemicrobiological,4,5_{immunological,}6 smokingandpsychologicalfactors.5_{So,sometreatmentscan} be applicableto overcome these environmental factors.In addition,besidesthecostoftreatment,theimpactofUCon qualityoflifeisstaggering.7_{Therefore,findingeffectiveand} costbenefittreatmentforUCisnecessary.

Beforeeachstudyforfindinggoodtherapeuticagentsfor UC,itisnecessarytosimulatetheconditionssimilartohuman UC inanimal models. There are several modelsfor induc-tionofUCinanimalssuchasuseoftrinitrobenzenesulfonic

acid(TNBS)(7),dextransodiumsulfate,8_{andaceticacid.}9 Rec-tal administration ofacetic acid can mimicthe conditions which occurred inhumanUC10 _{and related UCis a} repro-duciblelaboratoryanimalmodelandisusefulforscreening ofeffectivenessofdrugs.11

Materials

and

methods

Ethicalstatement

TheprotocolofthepresentedstudyisapprovedbytheEthical CommitteeofShirazUniversityofMedicalSciences.Allefforts weremadetopreventtheharmfulhandlingoftheanimals andalsothelowestbutstatisticallysignificantnumberofthe animalswasallocatedineachgroup.

Fruitandextraction

Z.jujubafruitswerepurchasedfrom localmarketand after genusandspeciesconfirmationbyBotanistaffiliatedto Agri-cultureSchoolofShirazUniversity,werefinelygroundedby mixer.Thehydroalcoholicextractionwasperformed accord-ing to previous report.15 _{The antioxidant content of this} extractwasevaluatedusingferricreducingantioxidantpower (FRAP)testasdescribedpreviously.16

Animals

Inthisstudy,80 maleSprague Dawleyratswithmean and SDofweighof200±20gwerepurchasedfromtheCenterof Comparativeand Experimental Medicine,Shiraz University of Medical Sciences. They kept under conventional condi-tionsinclude22±1◦_{C,55±5%relativehumidity,and12/12h} light/darkcyclesbeforethe onsetofthestudy for acclima-tionandduringthestudyperiod.Allratswerefastedfor24h andthenUCwasinducedbyrectaladministrationof1mLof 3%aceticacid.Animalswererandomlyallocatedinto8equal separatedgroupsandreceiveddifferenttreatmentafter24h, basedontheTable1.

Attheendofthe7thday,allratsweresacrificedby cervi-caldislocationandsamplesfromcolontissuewereobtained

Table1–Experimentalsetupandtreatmentswhich usedinthisstudy.

Groupno. Abbreviation Treatment

1 Negativecontrol1(NC1) 1mLnormalsaline, enema

2 Negativecontrol2(NC2) 1mLnormalsaline, oral

3 Positivecontrol1(PC1) Asacol10mg/kg, enema

4 Positivecontrol2(PC2) Mesalazine10mg/kg, oral

5 Gel20% 1mLofgel20%of

ZJHE,enema

6 Gel40% 1mLofgel40%of

ZJHE,enema

7 Oral1500 1mLofsolutionof

ZJHEasdose 1500mg/kg,oral

8 Oral3000 1mLofsolutionof

ZJHEasdose 3000mg/kg,oral

ZJHE,Z.jujubahydroalcoholicextract.

andfixedin10%bufferedformalin.Thetissueprocessingand histopathologicalslidepreparationwereperformedaccording to previous procedures.17–19 _{The histopathological sections} wereevaluatedforseverityandextentofinflammation,crypt damage,percentofinvolvementandregeneration.

Measurementofmalondialdehyde(MDA)level

Briefly,500mgoftissuewashomogenizedin5mLof1.15% coldKCl.Then,3mLof1%phosphoricacidand1mLof0.6% tiobarbitoric acid were added to500␮lofhomogenate and shakewell.Afterindirectheatingat100◦_{Cfor45min,the} cen-trifugationwasperformedat10000rpmfor10minand the absorbanceofthesupernatantwasmeasuredin532nmusing UV-visiblespectrophotometer.

Myeloperoxidase(MPO)activity

TwomLofphosphatebuffercontain0.5%hexadecyltrimethyl ammoniumbromide(HTAB)wasaddedto100mgofcolon tis-sueandhomogenizedoniceforsixtimesof45s.Then,10sof sonicationandfreezebyliquidnitrogenwereappliedforthree times.Centrifugationat3000rpmand4◦_Cfor30minwas per-formedandsupernatantwasharvested.2.9mLofphosphate buffer containo-Dianisidineand0.005%hydrogen peroxide wasaddedto0.1mLofsupernatantandafter5min,0.1mLof 1.2MHClwasaddedtotubetoorangecolorwasappeared. Theabsorbanceofthesampleswasmeasuredat460nmby UV–visible spectrophotometer and theactivity ofMPO was calculatedusingastandardcurve.

Superoxidedismutase(SOD)andglutathioneperoxidase (GPx)activities

The activities of these two antioxidative enzymes were assessedusingcommercialkits(Biorexfars,Iran).

Interleukin(IL)-1ˇ

ThecolontissuecontentofIL-1␤determinedbycommercial quantitiesenzymelinkedimmunosorbent assay(ELISA)kit (Biosource,USA)accordingtothemanufacturerinstruction.

Statisticalanalysis

DatawereexpressedasmeanandSDandanalyzedusingSPSS version21.Onewayanalysisofvariance(ANOVA)withusing TukeyasPosthoctestwereusedtofindstatisticalsignificant differences (p<0.05). GraphPad 6wasusedfor drawingthe charts.

Results

a a

b b

b

Groups Inflammation severity

Deg

ree

b b

b

NC1 NC2 PC1 PC2

Gel 20% Gel 40% Oral 1500Oral 3000

4

3

2

1

0

Groups

a a

b b

b b

b b Crypt damage

Deg

ree

NC1 NC2 PC1 PC2

Gel 20% Gel 40% Oral 1500Oral 3000

5

4

3

2

1

0

a _a

b b

b

Groups Extent of inflammation

Deg

ree

b b

b

NC1 NC2 PC1 PC2

Gel 20% Gel 40% Oral 1500Oral 3000

4

3

2

1

0

Groups

a a

b b

b

b b

b Percent of involvment

Deg

ree

NC1 NC2 PC1 PC2

Gel 20% Gel 40% Oral 1500Oral 3000

4

3

2

1

0

Groups

a a

b b

b b

b

b Regeneration

Deg

ree

NC1 NC2 PC1 PC2

Gel 20% Gel 40% Oral 1500Oral 3000

4

3

2

1

0

Fig.1–Comparisonofhistopathologicalindicesofdamageandregenerationbetweendifferentgroups.Significant statisticaldifferencesbetweengroupsineachindexareindicatedbydifferentsuperscriptletter(p<0.05).

all pathological indices include inflammation severity and extent,cryptdamage,percentageofinvolvementand regen-eration(p>0.05)(Fig.1).

ComparisonofMDA level as oxidativestress index and activities of two antioxidative enzymes, SOD and GPx, in colontissuearepresentedinFig.2.Asshown,thecolon tis-sue MDA concentrationin NC1 and NC2 were significantly higher than all other groups (p<0.05) but not statistically significanttogether(p>0.05).ThetissueMDAconcentration

3.0

A

B

C

2.0

1.5

1.0

0.5

0.0

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

1.5

1.2

0.9

0.6

0.3

0.0

a b

c c

d e

f g f

d e

e d c b a a

c

f

d

c b

a a

Groups

Concentr

ation (

µ

mol/L)

Activity (IU/L)

Activity (IU/L)

NC1 NC2 PC1 PC2

Gel 20% Gel 40% Oral 1500Oral 3000

Groups

NC1 NC2 PC1 PC2

Gel 20% Gel 40% Oral 1500Oral 3000

Groups

NC1 NC2 PC1 PC2

Gel 20% Gel 40% Oral 1500Oral 3000

Fig.2–ComparisonofmeanandSDofA,malondialdehyde level;B,glutathioneperoxidaseactivityandC,

superoxidasedismutaseactivityincolontissuesof differentexperimentalgroups.Significantstatistical differencesbetweengroupsineachindexareindicatedby differentsuperscriptletter(p<0.05).

Thisincludedhigher activitiesofenzymes inall groups in comparisontobothNCgroups,highestandlowestactivities ofbothenzymes between treatmentgroup ingel 40% and PC2,respectively, and highestactivities ofbothenzyme in responsetooralmesalazineincomparisontoenemaasacol. OtherdifferencesandtheirsignificancesareshowninFig.2B andC.

ComparisonofinflammatorybiomarkersofIL-1␤andMPO activity in colon tissue of different groups are presented in Fig. 3. As shown, these two inflammatory indices were

decreasedinresponsetotreatmentsincomparisontobothNC groups(p<0.05).Themostdeclineswerebelongedtothegel 40%group(210.00±22.47pg/mLforIL-1␤and1.19±0.05IU/L forMPO)followedbyoral3000group(318.20±31.49pg/mLfor IL-1␤and1.37±0.03IU/LforMPO).Othervaluesandtheir sig-nificancescanbeseeninFig.3AandB.

Discussion

In the present study antioxidative, anti-inflammatory and regenerative effects of hydroalcoholic extract of Z. jujuba

in 4 doses of 20% and 40% in gel form and enema route and 1500mg/kg and3000mg/kginoralrouteinacetic acid induced UCinrat modelwere investigated.Overall, stimu-latory effects onthe activitiesof SODand GPx, decreasing effectsonIL-1␤ andMDAlevelplusMPOactivity and heal-ingandregenerativeeffectsinhistopathologicalfeatureswere detected in response to use ofplant extract. Also, enema route is better than oral administration in all effects and dose-dependent response was detected in both routes of administration.

Inpreviousstudies,differentplantsandderivativeswere explored for treatment of UC. It has been reported that hydroalcoholic extract of Teucrium polium could increase healthycellsinthecolontissue,decreasetheinflammation severityandresolvetheinflammationofcolontissue.20_Effects ofdifferentdosesandroutesofadministrationof hydroalco-holicextract oflicoricefor7dayintreatmentofUC inrat wereinvestigated.Itisfoundthatthesetreatmentdecreased theintestinalepitheliumdamages,TNF-␣,IL-6andNOand increasedSOD activity indose-responsepattern.21 Improv-ing ofpathological conditions ofcolon, increase ofweight anddeclineinMDAlevelwereseeninratsufferedfromUC in response to strawberry extract indose-response type.22 Similar findings are reported by our group and other sci-entists forBerberis vulgaris,23 _{Melilotus officinalis,}9 _Hypericum perforatum,24,25_{Calendulaofficinalis}26_{andPistachiaatlantica}27_in linewithfindings ofthis study.However,the reportsabout thebeneficialeffectsofusingofZ.jujubaininflammatory dis-easesarescarce.HydroalcoholicextractofZ.jujubecanreverse oxidativestressinducedbypentylenetetrazole(PTZ)and elec-troshockinexperimentalmodelsofepilepsyinrats.28_Also,the beneficialeffectshydroalcoholicextractofZ.jujubaas alterna-tivetreatmentinOMasanotherinflammatorydiseaseswas reportedbyourgrouppreviously.14

1000

A

B

a b

c c

d

e f

g

a _a

b c _d

e bc

f 4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0 900

800

700

600

500

400

300

200

Groups Groups

Concentr

ation (pg/mL)

Activity (IU/L)

100

0

NC1 NC2 PC1 PC2

Gel 20% Gel 40% Oral 1500Oral 3000

NC1 NC2 PC1 PC2

Gel 20% Gel 40% Oral 1500Oral 3000

Fig.3–Changesofinflammatoryindicesincolontissuesofratsindifferentgroups.A,IL-1␤level;B,myeloperoxidase activity.Significantstatisticaldifferencesbetweengroupsineachindexareindicatedbydifferentsuperscriptletter(p<0.05).

Conclusion

Inflammatory diseases such as UC are currently treated with steroidal and non-steroidal anti-inflammatory drugs (NSAIDs),butourfindingssuggesthydroalcoholicextractof

Z.jujubaasanewtherapeuticagentforUC.Thiscanbe con-cludedfromstimulationofhealingprocessandinhibitionof theinflammatoryandoxidativepathways.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Authorswishtothankallstaffs ofDepartmentof Pharma-cologyToxicology,SchoolofPharmacy,ShirazUniversity of MedicalSciences,Shiraz,Iranfortheircooperationin exper-imentalprocedure and analysis. Thisarticle wasextracted fromMScThesisofMr.Ali GhanbariSaghesloowith super-vision ofDr. NaderTanideh and Dr. AkramJamshidzadeh. TheauthorsalsothanksTheArticlepublishersgroup(www. articlepublishers.ir)forkindlyhelpinarticlerevision.

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