Acute Toxicity and Gastroprotection Studies of a New Schiff Base Derived Copper (II) Complex against Ethanol-Induced Acute Gastric Lesions in Rats

1、 AcuteToxicityandGastroprotectionStudiesofaNewSchiffBaseDerivedCopper(II)ComplexagainstEthanol-InducedAcuteGastricLesionsinRatsMaryamHajrezaie1,3.,ShahramGolbabapour1,3.,PouyaHassandarvish1,NuraSuleimanGwaram2,A.HamidA.Hadi2,HapipahMohdAli2,NaziaMajid3,MahmoodAmeenAbdulla1*1DepartmentofMolecularMedi

2、cine,FacultyofMedicine,UniversityofMalaya,KualaLumpur,Malaysia,2DepartmentofChemistry,UniversityofMalaya,KualaLumpur,Malaysia,3InstituteofBiologicalScience,FacultyofScience,UniversityofMalaya,KualaLumpur,MalaysiaAbstractBackground:Copperisanessentialelementinvariousmetabolisms.Theinvestigationwascar

3、riedouttoevaluateacutegastroprotectiveeffectsoftheCopper(II)complexagainstethanol-inducedsuperficialhemorrhagicmucosallesionsinrats.Methodology/PrincipalFindings:Ratsweredividedinto7groups.Groups1and2wereorallyadministeredwithTween20(10%v/v).Group3wasorallyadministeredwith20mg/kgomeprazole(10%Tween2

4、0).Groups47received10,20,40,and80mg/kg of the complex (10% Tween 20), respectively. Tween 20 (10% v/v) was given orally to group 1 and absoluteethanol was given orally to groups 27, respectively. Rats were sacrificed after 1h. Group 2 exhibited severe superficialhemorrhagic mucosal lesions. Gastric

5、wall mucus was significantly preserved by the pre-treatment complex. The resultsshowed a significant increase in glutathione (GSH), superoxide dismutase (SOD), nitric oxide (NO), and Prostaglandin E2(PGE2) activities and a decrease in malondialdehyde (MDA) level. Histology showed marked reduction of

6、 hemorrhagicmucosallesionsingroups47.Immunohistochemicalstainingshowedup-regulationofHsp70anddown-regulationofBaxproteins.PASstainingofgroups47showedintensestainuptakeofgastricmucosa.Theacutetoxicityrevealedthenon-toxicnatureofthecompound.Conclusions/Significance:ThegastroprotectiveeffectoftheCopper

7、(II)complexmaypossiblybeduetopreservationofgastricwallmucus;increaseinPGE2synthesis;GSH,SOD,andNOup-regulationofHsp70protein;decreaseinMDAlevel;anddown-regulationofBaxprotein.Citation:HajrezaieM,GolbabapourS,HassandarvishP,GwaramNS,AHadiAH,etal.(2012)AcuteToxicityandGastroprotectionStudiesofaNewSchi

8、ffBaseDerivedCopper(II)ComplexagainstEthanol-InducedAcuteGastricLesionsinRats.PLoSONE7(12):e51537.doi:10.1371/journal.pone.0051537Editor:DavidD.Roberts,CenterforCancerResearch,NationalCancerInstitute,UnitedStatesofAmericaReceivedJuly11,2012;AcceptedNovember8,2012;PublishedDecember10,2012Copyright: &

9、#223; 2012 Hajrezaie etal. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.Funding:TheauthorswouldliketothanktheUniversityofMalay

10、aforfundingthisresearch,RG373/11HTMandHIR-MOHE(F000009-21001).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.*E-mail:.my.Theseauthorscontributedequallytothisw

11、ork.Introductionreactive oxygen species 3. Lipid-derived free radicals such asconjugated dienes and lipid hydroperoxides cause oxidativereactions. The process of lipid peroxidation is mediated by theinteraction between hydroxyl radicals and the cell membrane,duringwhichthelipid-derivedfreeradicalspr

12、oduce4.Recently,a numerous researches have been inclined to find effectivesynthetic chemical compounds with gastroprotective properties5,6,7.Ulcer is an open sore or lesion, usually found on the skin ormucousmembraneofthebody.Apepticulcerisalesionoccursattheliningofthestomachorduodenum,wherehydrochl

13、oricacidand pepsin are present. In the past, it was thought that lifestylefactors, such as stress and diet caused ulcers. Later, researchersfoundthatstomachacids(i.e.,hydrochloricacid)andpepsintookpart in ulcer formation. Many researches have shown that mostulcers (80% of gastric ulcers and 90% of d

14、uodenal ulcers)contribute significantly to bacterium Helicobacter pylori . Amongthe three factors (lifestyle, hydrochloric acid and pepsin, and H.Pylori) important in ulcer development, H. pylori is the primarycause of the gastric ulcer 1,2. Stress is an important etiologicalfactor in the incidence

15、of gastric ulcer. Free radicals, in stress-involved gastrointestinal injuries, may inactivate synthetize ofmucosal prostaglandin (by accumulating H2O2, an inhibitor insynthesisoftheprostaglandin,whichpropitiatesthegenerationofCopperisanessentialtraceelementinhumanmetabolism,butdoesnotexistinionicfor

16、minbiologicalsystems.Theamountofcopper in the body is measured in complexes with organiccompounds. The process of chelating metals smuggles them tobypass the intestinal wall and to enter into the mainstream ofnutrient flow and usage in the body. Copper complexes such ascopperaspirinateandcoppertrypt

17、ophanate,remarkablyincreasehealing rate of ulcers and wounds 8. The Copper complexesshorten the healing period of gastric ulcers for five days andpromote the wound healing process at the same time retainingPLOSONE | 1December2012 | Volume 7 | Issue 12 | e51537 GastroprotectionStudieso

18、fCopper(II)ComplexElmer FT-IR spectrometer. Elemental analysis (C, H, N) wereperformed using a Flash EA 1112 Series elemental analyzer inUniversityofTechnologyMalaysia.SynthesisoftheCopperComplex2-hydroxyacetophenone (0.21g, 1.65mM) was weighed accu-ratelyandwasdissolvedin25mLofabsoluteethanoldrippi

19、ngtoan ethanolic solution and N,N9dimethylethyldiamine (0.14g,1.65mM)atroomtemperatureandwasrefluxedfor3h.Aclearyellowoilyproductwasformedaftertheevaporationprocess.Theproduct was isolated in liquid form by adding a few drops ofdiethyl ether/n-hexane and the purity was confirmed by TLC.Stoichiometri

20、c amount of the synthesized schiff base ligand(1.65mM)wasdissolvedin25mLabsoluteethanol,anequimolarquantityofCopper(I)bromide(0.21g,1.65mM)inaminimumamountofethanolwasadded.Ablueprecipitatewasformed.Theprecipitatewasfiltered,washedwithethanolanddriedinvacuumdesiccators. Recrystallization was perform

21、ed in ethanol. Thecrystal structure of the compound X-ray 13 is as shown in theproposed structure in Figure 1. Elemental analysis and spectralcharacterizationfortheligandanditsmetalcomplexarepresentedintheTable1.Figure1.ChemicalstructureofCu(BrHAP)2.doi:10.1371/journal.pone.0051537.g001anti-inflamma

22、tory activity 8 while non-steroidal anti-inflamma-tory drugs (e.g. ibuprofen and enefenamic acid) suppress woundhealing process.In recent years, copper coordination compoundswithschiff-baseligandshavebeenextensivelystudied.Itseemsthattheir reactivity with dioxygen or its reduced derivativesistheir-u

23、niquebiologicalproperty9.Someofschiffbasederivedcoppercompounds causesevere cytotoxicity, through the generation ofreactiveoxygenspecieswhichcandamagedifferentbiomolecules10.However,theymaybeconsideredasefficientcatalystfortheoxidation of different substrates 11. Many of these complexeshavebeeninves

24、tigatedfortheireffectivescavengersofsuperoxideradicals 12, acting as antioxidants.This study introduced theCopper(II)complexderivedfromN,Ndimethylethylenediamineand2-hydroxyacetophenoneschiffbaseligand,itssynthesisanditschemicalcharacterization.Also,thestudyevaluatedacutetoxicityand gastroprotective

25、 activity of the complex against absoluteethanol-inducedacutehemorrhagicmucosallesionsinrats.ChemicalsIn this study, omeprazole (obtained from UMMC Pharmacy)wasusedasthereferenceanti-ulcermedicine.Themedicinewasdissolved in 10% Tween 20 (Merck Schuchardt OHG, 85662Hohenbrunn, Germany).Adilutionof(10

26、%v/v)Tween20wasusedasthevehiclefordosingalloftherats(5mL/kg)andwereadministered orally to the rats in a dosage of 20mg/kg bodyweight(5mL/kg)accordingtotherecommendationofMahmoodetal.14.ExperimentalAnimalsAcute toxicity study. Healthy male and female Sprague-Dawley rats (68 weeks old) were obtained f

27、rom the AnimalHouse,FacultyofMedicine,UniversityofMalaya,KualaLumpur(Ethics No. PM/27/07/2010/MAA (R). The rats were weighedbetween150180g.Theanimalsweregivenstandardratpelletsand tap water ad libitum. The acute toxicity study was used todetermine a safe dose for the Copper (II) complex. 36 rats (18

28、males and 18 females) were randomly assigned into 3 groupslabeledasvehicle(10%Tween20),and100mg/kgand2000mg/kgoftheCopper(II)complex(10%Tween20)15.TheanimalsMaterialsandMethodsExperimentalSectionAllofthechemicalsusedinthisstudywereobtainedfromFlukaand Aldrich and used as received without further pur

29、ification.Schiffbases weresynthesized bycondensationreaction. Thetwoethanolic solutions were stirred for 2h or 5h after dissolving it.TheethanolwasthenevaporatedusingRotaevaporator.InfraredspectrawereobtainedusingKBrdiscs(4000400cm21)onPerkinTable1.Elementalanalysisandspectralcharacterizationforthel

30、igandanditsmetalcomplex.LigandElementalAnalysisAnalyticalCalculated:C,69.87;H,8.80;N,13.58.Found:C,68.48;H,8.98;N,14.98.IR(KBrdisccm21)u(ArOH),3436sb;u(CH),2836s;u(C=N),1628s;u(CC),1438s;u(CN),1166s;279nm(3091.79,p-p*);303nm(3152.21,CT)UV-Vis(DMSO),lmax(e,Mol21cm21):1H-NMR(DMSO-d6)2.23(s,3H,CH3),2.2

31、8(6H,2CH3),2.49(t,2H,2CH2),6.787.02(4H,ArH),10.7(s,1H,phenolic)13C-NMR(DMSO-d6)16.72(CH3),45.54(2CH3),45.86,57.53(2CH2),167.91(C=N)ArC:125.15(CH),128.48(CH),141.33(CH),1148.29(CH),149.82(C)ComplexElementalAnalysisAnalyticalCalculated:C,40.29;H,5.07;N,7.83Found:C,40.88;H,5.20;N,7.23.IR(ATRcm21)2843.5

32、3m,2800.66mn(C-H),1102.37mn(C-N),1474.35m,1436.35mn(C-C),1605.47m,1593.89n(C=N),518.09mn(M-N),473.95n(M-O).UV-Vis(DMSO)296(pRp*);362(nRp*);376(LMCT);610(dRd*).doi:10.1371/journal.pone.0051537.t001PLOSONE | 2December2012 | Volume 7 | Issue 12 | e51537 GastroprotectionStudiesofCopper(II

33、)ComplexFigure2.Histologicalsectionsinacutetoxicitytest(H&Estaining,20x).Histologicalsectionsofliver(firstrow)andkidney(secondrow)inacutetoxicitytest.Ratstreatedwith5mL/kgvehicle(10%Tween20)(AandD).Ratstreatedwith500mg/kg(5mL/kg)theCopper(II)complex(BandE).Ratstreatedwith2000g/kg(5mL/kg)theCoppe

34、r(II)complex(CandF).Thereisnosignificantdifferencesinstructuresofliverandkidneybetweentreatedandcontrolgroups.doi:10.1371/journal.pone.0051537.g002werefastedovernightpriortothedosing.Foodwaswithheldforanother3to4hafterdosing.Theanimalswereunderobservationfor0.5,2,4,8,24and48hafteradministrationtomon

35、itoranyonsetofclinicalortoxicologicalsymptoms.Mortality,ifany,wasrecordedoveraperiodof2weeks.Theanimalsweresacrificedonday15.Histology,hematologicalandserumbiochemicalparam-eters were determined according to the OECD 15. This studywas approved by the Ethics Committee for animal experimenta-tion, Fac

36、ulty of Medicine, University of Malaya, Malaysia EthicNo.PM/07/05/2011/MAA(R).Throughoutexperiments,alloftheanimalsreceivedhumanecareaccordingtotheGuidefortheCareandUseofLaboratoryAnimalspreparedbytheNationalAcademyofSciences.orallyadministeredwiththeCopper(II)complexdissolvedin10%Tween20(5mL/kg)atd

37、ifferentdosagesof10,20,40and80mg/kg (Groups 4, 5, 6 and 7, respectively). Then,1h after this pre-treatment,group1wasorallyadministeredwith5mL/kg10%ofTween 20.Groups 27 were received absolute ethanol orally(5mL/kg). The rats wereeuthanized 60min later (based onthepreviously used method 16) with an ov

38、erdose of xylazine andketamineanesthesiaandtheirstomachswereimmediatelyexcised.Macroscopicappearance of acute gastric mucosalLesionsofthegastricmucosaappearaselongatedbandslesion.ofsuperficialhemorrhagicmucosallesionsparalleltothelongaxisofthestomach.Gastricmucosaofeachratwasthusexaminedforthedamage

39、s. Thelength andwidthofthehemorrhagicmucosallesions (mm) were measured with a planimeter (10 6 10mm2=ulcer area) under a dissecting microscope (1.8x). Thehemorrhagicmucosallesionsareawasmeasuredbycountingthenumberofsmallsquares(2mm62mm)coveringthelengthandthewidthofeachlesionband.Thesumoftheareasoft

40、helesionsforeachstomachwasappliedinacalculationoftheUAwherethesum of small squares 64 61.8=UA (mm2) were calculatedGastriculcerstudy. SpragueDawleyhealthyadultmaleratswere obtained from Experimental Animal House, Faculty ofMedicine, University of Malaya; Ethic No. PM/28/09/2011/MAA (R). The rats (we

41、ighed between 225250g) were dividedrandomlyinto7groupsconsistingof6ratseachgroup.Eachratwascagedindividuallywithwide-meshwiredbottomstopreventcoprophagiaduringtheexperiment.Theanimalsweremaintainedonastandardpelletdietandtapwateradlibitum.according to the recommendation of Zahra etal. 17. Theinhibit

42、ionpercentage(I%)wascalculatedbythefollowingformulaaccordingtotherecommendationofAbdullaetal.18.Gastriculcer-inducedbyethanol. Priortotheexperiment,theratswerefastedfor24h,accordingtoarecommendedmethod14. They had free access to drinking water till 2h before theexperiment.Gastriculcerwasinducedbyoro

43、gastricintubationofabsolute ethanol (5mL/kg) according to the method recom-mendedbyAbdullaetal.14.Group1andgroup2wereorallyadministeredwith10%Tween20(5mL/kg).Group3receivedanoraldoseof20mg/kgomeprazolein10%Tween20(5mL/kg),as the reference control group. The experimental groups wereðI%Þ

44、9;ðUAcontrolUAtreatedÞ=UAcontrol|100%:Gastric wall mucus determination. The glandular seg-ments of the stomach were removed, weighed and assessed todetermine gastric wall mucus in rats 19. Each segment wasPLOSONE | 3December2012 | Volume 7 | Issue 12 | e51537 Gastroprotectio

45、nStudiesofCopper(II)ComplexFigure3.Macroscopicalappearanceofthegastricmucosainrats.Thenegativecontrolgroup(A)hasnoinjuryofgastricmucosa.Theulcercontrolgroup(B)showsseverinjuriesinthegastricmucosa.Absoluteethanolproducedextensivevisiblehemorrhagicnecrosisofgastricmucosa.Thereferencecontrolgroup(omepr

46、azole,20mg/kg)(C)showsmildinjuriesinthegastricmucosacomparingtotheinjuriesseeningroup2.Ratsreceived10mg/kgofthecomplex(D)hasmoderateinjuriesinthegastricmucosa.Theextractreducestheformationofgastriclesionsinducedbyabsoluteethanol.Ratsreceived20mg/kgofthecomplex(E)showsmildinjuriesinthegastricmucosa.R

47、atsreceived40mg/kgofthecomplex(F)shows mild injuriesin the gastric mucosa.Rats received 80mg/kgof the complex (G) does not show any injuries of the gastric mucosa insteadflatteningofgastricmucosaisvisible(whitearrow).Blackarrowspointtothesuperficialhemorrhagicmucosallesions.doi:10.1371/journal.pone.

48、0051537.g003transferred immediately to a 1% alcian blue solution (in sucrosesolution, buffered with sodium acetate at pH5).Rinsing withsucrosesolution,theexcessdyewasremoved.Thedyecomplexedwiththegastricwallmucuswasextractedbymagnesiumchloridesolution.A4mLaliquotofblueextractwasthenshakenwithanequal

49、 volume of diethyl ether. The resulting emulsion wascentrifuged then the absorbance of the aqueous layer wasmeasured at 580nm. The quantity of alcian blue extracted pergramofglandulartissuewasthencalculated.Preparation of stomach homogenate. The gastric tissuehomogenate from each rat was prepared fo

50、r PGE2 and MDAassays.Theentireexperimentwasperformedat4uC.Gastrictissuewascutinto3smallpieces(approximately200mgforeach),andtheexactweightofeachpiecewasrecorded20.Thetissueswerehomogenizedinateflonhomogenizer(Polytron,HeidolphRZR1,Germany)usingtheappropriatebuffer.Theamountofbufferusedwas dependent

51、on the weight of the tissue used. After centrifu-gationat4,500rpmfor15minat4uC,thesupernatantwasusedforthePGE2andMDAassays.Antioxidantactivitiesofstomachhomogenate. TheGSHlevels of the gastric tissues were measured using gastric tissuesupernatant with the GSH kit (Cayman Chemical Co., Mich,USA), acc

52、ording to the manufacturers instructions. The SODactivities in the gastric tissues were determined, according to themanufacturers protocol, using the SOD assay kit (CaymanChemical Co., Mich, USA). Using a commercial kit (CaymanChemicalCo.,Mich,USA),theNOlevelsofgastrictissuesweremeasuredaccordingtot

53、hemanufacturersinstructions.Figure4.Effectsofthecomplexongastriculcerarea,inhibitionpercentageandalcianbluebindingcapacity.Alcianbluebindingcapacity is defined as Gastric wall mucus (GWM). Groups 1 to 3representthenegativecontrolgroup,theulcercontrolgroupandthereference control group (omeprazole, 20

54、mg/kg), respectively. Theexperimentalgroupsreceived10,20,40and80mg/kgofthecomplexare presentedas groups 47, respectively.All valuesare expressed asmean6standarderrormean.Meandifferenceissignificant,inGWM,atthe p,0.05 level (one-way between groups ANOVA with post-hocanalysis).*significantwhencompared

55、withthegroup2.#significantwhen compared with the group 3. Inhibition of gastric lesions (%) isindicatedinbracketsabovethecolumns.EnzymaticActivitiesofStomachHomogenateMeasurement(MDA).ofmembranelipidsThe rate of lipoperoxidation in the gastric mucousperoxidationmembraneisvaluedthroughthemeasuremento

56、fMDAusingtheTBARS test. The stomachs were washed with normal saline tominimizeanyinterferenceofhemoglobinwithfreeradicalsandtoremove blood adhered to the mucous membrane. The stomachswerehomogenizedwithpotassiumphosphatebuffer(10%(w/v).Adoi:10.1371/journal.pone.0051537.g004PLOSONE | 4

57、December2012 | Volume 7 | Issue 12 | e51537 GastroprotectionStudiesofCopper(II)ComplexFigure5.Effectofthecomplexonglutathione(GSH),superoxidedismutase(SOD)andnitricoxide(NO)(A,BandC).Groups1to3represent the negative control group, the ulcer control group and the reference control group (omeprazole, 20mg/kg), respectively. Theexperimentalgroupsreceived10,20,40and80mg/kgofthecomplexarepresentedasgroups47,respectively.Allvaluesareexpressedasmean6standarderrormean.Meandifferenceissignifi