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Original Research Article

Effect of Pseuduvaria macrophylla in attenuating hyperglycemia mediated oxidative stress and in ﬂ ammatory response in

STZ-nicotinamide induced diabetic rats by upregulating insulin secretion and glucose transporter-1, 2 and 4 proteins expression

Hairin Taha

^a

, Aditya Arya

^b,

*, Ataul Karim Khan

^c

, Nayiar Shahid

^c

, Mohammed Ibrahim Bin Noordin

^d

, Syam Mohan

^e,f

aUniversityTenagaNational,InstituteofEnergyInfrastructure,Kajang,Selangor,Malaysia

bTaylor’sUniversity,SchoolofMedicine,FacultyofHealthandMedicalSciences,DepartmentofPharmacologyandTherapeutics,LakesideCampus,Subang Jaya,47500,Malaysia

cUniversityofMalaya,FacultyofMedicine,DepartmentofPharmacy,KualaLumpur,Malaysia

dMalaysianInstituteofPharmaceuticalsandNutraceuticals,BukitGambir,Gelugor,PulauPinang,Malaysia

eJazanUniversity,MedicalResearchCentre,Jazan,SaudiArabia

fJazanUniversity,SubstanceAbuseResearchCentre,Jazan,SaudiArabia

ARTICLE INFO

Articlehistory:

Received5October2017

Receivedinrevisedform12April2018 Accepted17May2018

Availableonlinexxx

Keywords:

Pseuduvariamacrophylla Type2diabetes GLUT Phenolic Insulin

Glucosetransporter

ABSTRACT

Pseuduvariamacrophylla(Family:Annonaceae)iscommonlyusedasmedicinalplantinMalaysia.Arecent studywiththePseuduvariaspeciesshowedantioxidantandantidiabeticeffects.Thisstudyaimedto ascertain antidiabetic potential of methanolic extract of Pseuduvaria macrophylla bark (PM) using streptozotocin-nicotinamide induced diabetic ratmodels. Various phytochemical and biochemical properties of the plant have been evaluated.The results showedthat the extract haspotentially normalizedtheelevated bloodglucoselevelsbyupregulatingtheinsulinandC-peptidelevelsand alleviatedoxidativestressbyimprovingglutathione(GSH)andreducinglipidperoxidation(LPO)inthe diabeticrats.Inaddition,PMhasdrasticallydownregulatedthelevelsofpro-inflammatorycytokinesand transforminggrowth factorbeta-1 (TGF-b^1).Histopathologicalexaminationofthe pancreasinPM treateddiabeticratsshowedsignificantrecoveryofthepancreaticstructuraldegenerationandthus reflectedtheprotectiveroleofPMagainstperoxidationdamagebyariseininsulinlevelasevidencedby theimmunohistochemistrystudy.TheimprovedexpressionsofGLUT-1,GLUT-2andGLUT-4further confirmedtherestorationofb^-cell^mass^by^PM.Interestingly,thefindingsdemonstratedtheantioxidant, anti-inflammatoryand antihyperglycemic potential of PMwhich may provide future lead forthe managementoftype-2diabetes.

Introduction

Type2diabetesmellitus(T2DM)isacombinationofresistance to insulin action and an inadequate compensatory insulin secretory response (Genuth et al., 2003). In addition, it also compromisedmetabolismofcarbohydrates,proteinsandfats.The releaseoffreeradicalsassociatedwithT2DMconsistofreactive oxygenornitrogenspecies,whichplaycrucialroleindeveloping

diabetes and eventuallycauses damage tothe multipleorgans leadingtodiabeticcomplications(Aryaetal.,2012a).Therefore, interferenceinoxidativestressandinﬂammatoryresponsepath- wayshasbeenhighlightedasanimportantmeansofpreventing diabetesanddiabetesrelatedcomplications(Dandonaetal.,2004).

Synthetichypoglycemicagents arecurrentlyinclinicalpractice, but due to its side effects, scientists have started looking for alternativesfromthenature.Alargeproportionofpeoplearound thegloberelyontraditionalmedicinesespeciallyherbalmedicine and it has been showed that few naturally existing products demonstratesigniﬁcantantidiabeticactivitybystimulating

b

^-cell

andthensecreteinsulinandrecoverinsulinsensitivity(Lombardo

* Correspondingauthor.

E-mailaddress:[email protected](A.Arya).

https://doi.org/10.1016/j.jab.2018.05.004

xxx–xxx ContentslistsavailableatScienceDirect

Journal of Applied Biomedicine

j o u r n a lh o m e p ag e :w w w . e l s e vi e r . c o m / l o c a t e / j a b

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and Chicco, 2006). Thus, it is essential to investigate the mechanism involved in the antidiabetic activityof the natural plantproductsthathavebeenusedbytraditionalhealersforthe managementofT2DM.

Many plant species are used for the treatment of diabetes attributingtotheantioxidant,anti-inflammatoryandantihyper- glycemic nature. In addition, it has been observed that the presenceofflavonoids,phenolics,alkaloidsandterpenoidsarethe key components responsible for the biological activities (Taha etal.,2014).OnesuchplantisPseuduvariamacrophylla(PM)which belongstotheAnnonaceaefamily and locallyknownas “cagau biasa”inMalaysia.Theantioxidantandantimicrobialpropertiesof thecrudeextractoftheplantwerereportedpreviously(Azizetal., 2016).TheindigenouspeoplesofMalaysia(orangasli)havebeen usingthis plantforthetreatmentofcoughandmanagementof feverbutlittleisknownaboutitsantidiabeticeffectduetolackof documentation.InSouthernThailand,itisconsideredasmedicinal plantknownasKloikhangorPhriknok,andthewoodypartofthe plantis used for treating nauseaand vomiting (Chuakul et al., 2004).Plantsfromthisfamilyandspecieshavebeenreportedto exhibitvariousinterestingpharmacologicalactivitiesduetothe presence of different types of constituents such as alkaloids, flavonoidsandpolyphenols(Tahaetal.,2011).

However, there is not much scientiﬁcevidence of extensive chemical or biological studies on Pseuduvaria macrophylla.

RecentlyTahaetal.(2014)havefoundthatPseuduvariamonticola, a plant from the same genus has antioxidant and antidiabetic potential.Oneofthemechanismsofmedicinalplantsand their phytochemicalsforthe treatmentof diabetesis thecapacity to enhancetheantioxidantsystem(VinayagamandXu,2015).Infact, most of medicinal plants with anti-diabetic property possess antioxidant activity (Hulbert et al., 2005; Nasri et al., 2015;

Raﬁeian-Kopaie and Nasri,2012).SincePseuduvariamacrophylla hasshownanti-oxidantpropertybeforeanditisaplantfromthe same genus which has anti-diabetic potential, it is worth to investigateitsantihyperglycemiceffects. In ourbestknowledge there was no in vivo antihyperglycemic study reported or documented previously on the bark methanolic extract of Pseuduvariamacrophylla.Bykeepingtheaforesaidpointsinview, the current study aims to investigate the antioxidant, anti- inﬂammatory and antihyperglycemic potential of PM on STZ- Nicotinamideinduceddiabeticratsandthe

b

^-cell^protective^effect

oftheextractbydeterminingthepancreaticproteins.

Materialsandmethods Chemicals

Glibenclamide, Streptozotocin (STZ) and Nicotinamide were obtainedfromSigma-Aldrich,USA.

CollectionofPseuduvariamacrophylla

Dried stem bark of Pseuduvaria macrophylla (1kg) were collectedfromKenong ForestPark, Malaysia. Identiﬁcation and authenticationofspecimenwasestablishedbytheDepartmentof Chemistry, Faculty of Science,University of Malaya. A voucher specimen (PM/M2114) had been deposited at the department herbarium,UniversityofMalaya,50,603.

ExtractionofPseuduvariamacrophylla

ThedriedstembarkofPseuduvariamacrophylla(500g) were ﬁnely powdered using blender and extracted with Soxhlet extractorfor48hat43Cwith100%n-hexane.Then,theobtained residuewas extractedagain withmethanoland chloroform. To

collectthe pureextract of Pseuduvariamacrophylla, each crude portionwas driedat 40C byBUCHI-R-215(Rotary Evaporator) with reduced pressure. The extractswere preserved in 20C refrigeratoruntilused.

PhytochemicalcharacterizationofPseuduvariamacrophylla ThephytochemicalconstituentsofPMwereanalyzedbyLiquid Chromatography Mass Spectrometry (LCMS-QTOF, AB Sciex TripleTOF 4600)equippedwithHPLC(SparkHolland Symbiosis PICO)systemwithaC18Kinetexreversephasecolumn(2.6

m

^m,

1003.0mm,Phenomenex,USA).Thesolventsystemconsistedof amixtureof0.1%formicacidinwater(SolventA)andacetonitrile with0.1%formicacid(SolventB),whichwasusedasmobilephase byelutingataﬂowrateof0.2ml/minwithaninjectionvolumeof 10

m

^l,^from^10%^to^100%^gradient^system.^The^complete^scans^of^the

massspectrawererecordedfrom50m/zto1000m/zanddatawas processedbyAnalystSoftware.Thewavelengthsusedwere210nm and350nm.1mg/mlofPMmethanolextractwasfractionated.15 fractionswerecollectedandpooledtogetherbasedontheirTLC pattern. Thepresenceof alkaloidswas detected byexposureto Dragendorff’sreagent.Thestructuresoftheisolatedcompounds wereidentiﬁedbycombinationof MSspectroscopictechniques, UV,¹H-NMR,¹³C-NMR(JEOLECA500,Japan).ForNMRanalysis, approximately5mgofdriedisolatedcompoundwasdissolvedina 2mldeuteratedsolvent(deuteratedmethanolorchloroform)and pipettedintoNMRtube(5mmindiameter).ThentheNMRtube was inserted into a probe and analyzed for 5min for nuclear magneticﬁeld.

Experimentalanimalsanddosing

Sprague Dawley rats (250–280g) were obtained from the Experimental Animal House, Faculty of Medicine, Universityof Malaya.Theratsweremaintainedinwire-bottompolypropylene cages underpathogen-free and light controlled(12hlight/dark cycle)roomata temperature(252C) with35–60%humidity andadministeredwithstandardratpelletdietanddistilledwater.

Alltheexperimentswereperformedaccordingtotheinstitutional ethicalguidelinesafterobtaininganethicalapproval(2014-07-01/

PHARM/R/NS). Animals were divided in to 5 groups (n=6) as follows;group-1administeredonlywithdistilledwater(Normal control, NC); group 2 were treated with STZ-Nicotinamide to induce diabetes (Diabetic Control, DC); group 3 were kept as diabeticratswithSTZ-Nicotinamidetreatmentand200mg/kgPM (PMa);group4werekeptasdiabeticratswithSTZ-Nicotinamide treatmentand400mg/kg PM(PMb);andgroup5werekeptas diabeticratswithSTZ-NicotinamidetreatmentandGlibenclamide (2.5mg/kg)(PositiveControl,PC).

The treated rat groups were orally administered with PM (200mg/kg and 400mg/kg) and Glibenclamide(2.5mg/kg) dis- solvedindistilledwater,onceadaybyintra-gastrictube.After24h oflasttreatmentdose,alltheexperimentalratsweresacriﬁced.

Toxicitystudy

InlinewiththeOECD(OrganisationforEconomicCo-Operation and Development) guidelines, the acute toxicity tests were performed onPM treated groups (PMaand PMb) withnormal rats.Afterovernightfasting,theratswereorallyadministeredwith three different doses of PM (500, 1000 and 3000mg/kg body weight) and the normal control group was administered with distilled water alone. Next 2h, all the rats were continually observed for any abnormalities in behavioral, anatomical and neurologicalfeatures.Thismonitoringwasfurthercontinueduntil next24and72h(Turner,2013).

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InductionofT2DMbystreptozotocin-nicotinamide

ForthepurposeofinducingT2DM,thenormalratswerefasted overnight(15h)andthenasingledoseofnicotinamide210mg/kg body weight (bw) was injected intraperitoneally. After 15min, 1ml/kgbwoffreshlypreparedSTZ(55mg/kgbw)incitratebuffer (0.1M,pH4.5)wasadministeredintraperitoneally.Type2diabetes wasconﬁrmedinratsafter72hofSTZ-Nicotinamideinjectionby measuring the blood glucose levels (Considered as T2DM>16.7mmol/l)(Aryaetal.,2012b).

Bloodsamplescollection

After 45 days of the treatment period, all the rats were anesthetized by Ketamine 50mg/kg and Xylazine 5mg/kg combinationandcollected3mlofbloodsamplesfromtheinner canthus of therat’seye. Serum preparationwas carried out at ambienttemperature byallowingthe blood samples toclot in vacutainertubefor15–30min.Then,thesampleswerecentrifuged inarefrigeratedcentrifugeat4000rpmfor10mintocollectthe serum,whichwasstoredat 80Cforfurtheruse.

Biochemicalassessment

Thebody weightandfastingbloodsugarofalltheratswere measuredon7,21and 45thdays.Theglucometer (Accu-Check NanoPerforma)wasusedtodeterminethebloodglucoseleveland ELISAAssay (589501, Cayman, USA& RatELISA Kit K4757, Bio VisionCorporation,USA)wasperformedfollowingmanufacturer's protocoltoquantifytheSerumInsulinandC-peptidelevelsineach groupofrats.

Antioxidantparameters

Serumglutathione(GSH)andlipidperoxidation(LPO)levelsof diabetic rats were measured using GSH and LPO assay kits accordingtothemanufacturerinstruction(CaymanChemical,USA, 703002,705,003).

Transforminggrowthfactor-beta1(TGF-

b

¹⁾^assessment

Atﬁrst, the collectedserum samples fromexperimental rat models were frozen at 20C before starting the analysis of determining TGF-

b

¹ ^using ^ELISA ^kit ^(TGF-

b

¹ ^Rat ^ELISA ^Kit,

ab119558)followingmanufacturer’sprotocol.Activationofeach 50

m

^lôf^the^sample^wasînitiated^byîncubatingît^with¹

m

^l^of^1.0^N

HClatroomtemperaturefor15minandthenneutralizationwas doneby1

m

^l^of¹^N^NaOH^to^further^activate^the^serum^TGF-

b

¹

untilit reaches theimmunoreactive form(Rajaveletal., 2012).

SerumTGF-

b

¹îsêxpressedâs^totalâmountêxcretedⁱⁿ^pg/ml.

Pro-inﬂammatorycytokinesassessment

Thelevelsofpro-inﬂammatorycytokines(TNF-

a

^,^IL-1

b

^and^IL-

6)weremeasuredinserumusingRatTNF-

a

^ELISA^Kit^(Item^no:

ab46070),RatIL-1

b

ÊLISA^Kit^(Item^no:âb100767)ând^RatÎL-6

ELISA Kit (Item no: ab100772) according to the manufacturer protocol(Abcam,USA).

Histology

Attheendoftheinvivoexperiments,theanimalswerescariﬁed andrat’spancreaswaswashedproperlywithnormalsaline.The tissuessampleswerethenpreservedforaweekin10%formalinat room temperature. Later the samples were sliced to 5

m

^m

diameters in size, and the tissue sections were stained with

hematoxylinandeosin(H&E)formicroscopicexaminationsafter gradualrehydrationinaseriesofgradedalcohols.

Immunohistochemistry

Pancreas wereﬁxedin formaldehyde,embeddedinparafﬁn, and sectioned(5

m

^mthickness).Thedewaxedsectionswerere- hydrated with tris-buffered saline (TBS) in a 3% hydrogen peroxide–methanol solution for 30min to block endogenous peroxidase activity, followed by 30min incubation at 90C.

Immunolabelingofinsulinwasperformedwitha1:500dilution ofgoat-polyclonal-Insulin-A(sc-7839;SantaCruz,)antibodyand slides wereincubatedfor 2hat 37C and subsequently rinsed three times withTBS for 3min per wash. Further, slides were incubatedwithperoxidase-conjugatedproteinA(1:100)for1hat roomtemperature(25C) andwashed againwith3.3-diamino- benzidinetetrahydrochloride(DABChromogenKit,BiocareMedi- cal,Concord,USA)forimaging.Next,slideswerecounterstained withMayer’shematoxylinand mounted.Imageswereobtained usingalightmicroscopepluscamera(Eclipse50i,Nikon,Tokyo, Japan)atamagniﬁcationof40.

GLUT-1,-2and-4analysisbyenzymelinkedimmunosorbent(ELISA) assay

ELISAkit for Glucose Transporter1 (GLUT1), Glucose Trans- porter 2 (GLUT2) and Glucose Transporter 4 (GLUT4) were obtained from USCNLIFE Science Inc, (Wuhan, China). Rat’s pancreaticsamplewererinsedinice-cold PBStoremoveexcess bloodthoroughlyandweighedbeforehomogenization.Mincedthe tissuestosmallpiecesandhomogenizedthemin5–10mlofPBS witha glass homogenizeronice. Theresulting suspensionwas sonicated with an ultrasonic cell disrupter. After that, the homogenates were centrifuged for 5min at 5000g. The supernatant was collected and the protein was measured and usedfortheassayaccordingtotheprotocolofmanufacturer.The concentrations of target proteins were determined at 450nm immediately in a microplate reader. The optical density of treatmentwascomparedwiththeopticaldensityofcontroland the valuewas expressed as percentage differencecompared to control.

Statisticalanalysis

All Values were reported as meanSD. The Statistical signiﬁcance of differences betweengroups was assessed using one-wayANOVAfollowedbyposthocTukey’smultiplecompari- sontests.AValueofP<0.05wasconsideredsigniﬁcant.

Results

Pseuduvariamacrophyllaextractyield

ExtractyieldofPseuduvariamacrophyllastembarkwas2gfor hexane(PH),3.11gforchloroform(PC)and152.35gformethanol (PM).Accordingtopreliminaryscreeningofallextracts, highest percentageyieldofPseuduvariamacrophylla(methanolicextract) showedhypoglycemiceffectsondifferentbiochemicalassays(data notshown).Therefore,methanolicextractwaschosenforfurther studies.

LCMS-QTOFandNMRanalysis

The characterisation ofthe chemical compoundsin PMwas achievedthroughLCMS-QTOFandNMR.Inthisexperiment,ﬁve majorcompoundsweredetectedfromPM(Fig.1A).Phytochemical

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analysisin positive ion mode by LCMS-QTOF has revealedthe presenceoftwooxoaporphinealkaloidssuchasliriodenine1([M+ H]⁺ionatm/z276),O-methylmoschatoline2([M+H]⁺ionatm/z 322);andtwobenzopyranderivativessuchaspolycerasoidol4([M +H]⁺ionatm/z359and[M+H] ionatm/z357)andpolycerasoidin 5([M+H]⁺ionatm/z373and[M+H]–ionatm/z371)basedon

mass fragmentations pattern by comparison with reportedMS data(Fig.1B).A secondrun in negativemodeshowsthethree majorpeakstentativelyidentiﬁedascaffeicacid3([M+H]–ionat m/z179),polycerasoidol4andpolycerasoidin5(Fig.1C).Allthe structuresofisolatedcompoundswereconﬁrmedby¹Hand¹³C NMRexperimentandbycomparisonwiththoseofauthenticdata.

Fig.1.MajorcompoundsofPseuduvariamacrophyllabarkswereidentified.(A)Molecularstructuresoffivemajorcompoundsthatwereidentifiedandisolated;(B)LCMS- QTOFchromatogramsshowingfourmajoridentifiedcompoundsdetectedinpositiveionmode,liriodenine1,O-methylmoschatoline2,polycerasoidol4andpolycerasoidin5;

(C)LCMS-QTOFchromatogramsofmajorpeaksdetectedinnegativemodeshowingcaffeicacid3,polycerasoidin5andpolycerasoidol4.

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Acutetoxicitystudy

Non-toxicnatureofPMwasrepresentedbyacutetoxicitystudy, asnomortalitywasobservedatminimum,mediumandmaximum dosesafter24and 72hoftreatment. Therewerenosigniﬁcant changesin the body weightand foodconsumption against the normalcontrolgroup.

EffectofPMonbodyweight

Fig. 2A represents the body weight of the treated groups comparedtothatofthediabeticcontrolgroup(P<0.05).There was a significant decrease in the body weight of DC group comparedtoNCgroup(P<0.05).Duringthefirsttwoweeks,there werenotmuchchangesinbodyweightagainstDCgroupandthe changestartedtoshowonweek3.However,fromweek4,thePMa andPMbtreateddiabeticratgroupsshowedsignificantincreasein bodyweightcomparedtoDCgroup(P<0.05).

EffectofPMonbloodglucoselevels

ThepotentialofPMin regulatingtheblood glucoselevelsof normalanddiabeticratsisdemonstratedinFig.2B.Duringtheﬁrst week of experiment, the blood glucose level was considerably increased(P<0.05)inDCgroup;whereas,fromthesecondweek aftertreatment,thebloodglucoselevelswasfoundtobereduced in PMb treated diabetic rats (P<0.05) against DC group. It is noteworthythat,fromthethirdweektosixweekonwards,both PMaandPMbhadconsiderablydecreasedthebloodglucoselevel (P<0.05)comparedtotheuntreateddiabeticrats(DCgroup).

EffectofPMonbiochemicalparameters

TheeffectofPMonseruminsulinlevelsindiabeticandnormal ratsareillustratedinFig.3A.Withouttreatment,theseruminsulin levelwasfoundtobeelevatedinDCgroupcomparedtoNCgroup

atweek2and6(P<0.05).However,during2ndand6thweeks,the PMaandPMbtreateddiabeticratshaveshownsigniﬁcantrisein seruminsulin levelscomparedtoDCgroup.Ontheotherhand, Fig.3BhasdisplayedtheeffectofPMonC-peptidelevelsinthe serum of normal and treated diabeticrats.Like insulin, theC- peptidelevelwasreducedinDCgroupduring2ndand6thweeks when compared toNCgroup(P<0.05). Animprovement inC- peptidelevelwasobservedin2ndweekinPMbtreateddiabetic rats(P<0.05)andfromweek6,thePMaandPMbtreateddiabetic rats showed signiﬁcant rise in C-peptide levels against theDC group indicating that PM improves glycemic control dose- dependently(P<0.05).

EffectofPMonantioxidantstatus

Fig.3illustratestheantioxidantpropertiesofPMbydetermin- ingtheGSHandLPOlevelsinnormalanddiabeticrats,treatedwith PMaandPMbatweek2and6.TherewasasigniﬁcantfallofGSH level in DC group when compared with NC group (P<0.05).

However,theGSHlevelwasimprovedinthePMaandPMbtreated diabeticratsin 2ndand 6thweeks (P<0.05).Theeffect ofthe extractwasfoundtobehigherinweek6comparedtoprevious weeks (Fig. 3C). In contraryto GSH, (Fig. 3D) demonstrates a marked rise in the LPO level in DC group against NC group (P<0.05).TreatmentwithPMbresultedindeclineofLPOlevelat week2comparedtoDCgroup.Inaddition,atweek6,bothPMaand PMbsigniﬁcantlyinhibitedtheLPOexpressionlevelagainsttheDC group(P<0.05).

EffectofPMonTGF-

b

¹^level

Fig.3Ereﬂectsacertainincreaseintheoxidativestressmarkers (LPO) and TGF-

b

¹ concentrations after diabetes induction in serumofDCratscomparedtoNCat2ndand6thweek.Treatment withPMaandPMbdidnotshowanysigniﬁcantchangesinserum TGF-

b

¹ concentrations for two weeks compared to DC rats Fig.2.EffectsofPMextractonbodyweight(A)andglucoselevel(B)innormalanddiabeticrats.AllthevaluesareexpressedasthemeanSD.‘a’indicatesthemean differenceissigniﬁcantwhencomparedtotheNCgroup(P<0.05).‘b’indicatesthemeandifferenceissigniﬁcantwhencomparedtotheDCgroup(P<0.05).

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(P<0.05).On theotherhand,whenboth thedosesofPMwere administeredforanextendedperiodi.e.6weeks,theconcentra- tionofTGF-

b

¹^was^found^to^be^decreased^against^DC^rats.

EffectofPMonpro-inﬂammatorycytokines

Fig. 4 illustrates the effect of two doses of PM on pro- inﬂammatorycytokinesi.eTNF-

a

^,^IL-

b

¹ândÎL-6ⁱⁿ^normalând

diabeticrats.Higherpro-inflammatorycytokinesconcentrations weredetectedinDCgroupcomparedtoNCgroupduring2ndand 6thweeks(P<0.05).Nevertheless,theadministrationofPMbhad significantlyreducedthepro-inflammatory cytokinesatweek2 comparedtoPMaagainsttheDCgroup(P<0.05).Inaddition,the levelofpro-inflammatorycytokineswascompletelyreducednear tonormallevelinPMaandPMbtreateddiabeticratsatweek6as opposedtoDCgroup(P<0.05).

EffectofPMonpancreastissue

Fig. 5 representstheimpactof PMonpancreasstructure in normalanddiabeticrats.Thepancreassectionswerestainedwith

hematoxylin and eosin and were determined after 45 days of treatment.Panel (A)pancreaticsectionshowednormal isletsof Langerhans of pancreatic structure.Panel (B) STZ-Nicotinamide treatmentelicitedsevereinjuryinpancreatic

b

^-cells,^such^as,^cell

damageandnecroticchanges.Panel(C)STZ-Nicotinamideinduced ratstreatedwithPMa,reﬂectedgradualrestorationofpancreatic endocrine cells, degeneration of the pancreatic acini was still observedandmoderateexpansionofpancreaticislets.Panel(D), treatedwithPMbhadshownreductioninvacuolationandnormal architectureofisletcells,suchlesionswereconsiderablyreduced.

After 6 weeks of glibenclamide treatment period, panel (E), demonstrated the most interesting aspect of the examined pancreaticsectionsbyshowingnormalLangerhansislets,absence ofdilationandprominenthyperplasticofislets.

EffectofPMoninsulinexpressionlevel

Fig. 6 illustrates the changes in insulin expression level in normalanddiabeticratstreatedwithPM.TheDCgroupshowed reducedexpressionofinsulincomparedtoNCgroup(Fig.6A–D).

However,theinsulinexpressionlevelwasupregulatedinPMaand Fig.3. EffectsofPMextractonInsulin(A),C-peptide(B),GSH(C),LPO(D)andTGF-b¹^(E)^levelsⁱⁿ^normalând^diabetic^ratsât^2ndând^6th^weeks.Âll^the^valuesâreêxpressed asthemeanSD.‘a’indicatesthemeandifferenceissignificantwhencomparedtotheNCgroup(P<0.05).‘b’Indicatesthemeandifferenceissignificantwhencomparedto theDCgroup(P<0.05).

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PMbtreateddiabeticratsagainsttheDCgroup.Theresultswere conﬁrmedbyhistomorphometric study(Fig.6E).TheDC group showedsigniﬁcantreductionintheinsulinexpressionlevelagainst NCgroup(P<0.05).Nevertheless,bothPMaandPMbdisplayed higherinsulin expressionconcentrationswhen comparedtoDC group(P<0.05).

Enzymelinkedimmunosorbent(ELISA)assay

Fig. 7 demonstrates the expression level of three glucose transporter proteins (GLUT-1, GLUT-2 and GLUT-4) inside the pancreastissueinnormal,PMaandPMbtreateddiabeticrats.The resultsrevealedthat,DCgroupshowedsigniﬁcantdecreaseofall theseproteins against NCgroup(P<0.05).Conversely, GLUT-1, GLUT-2andGLUT-4weresigniﬁcantlyincreasedinthePMaand PMbtreatedgroupscomparedtoDCgroup(P<0.05).

Discussion

Inspite ofthetremendous useoforal hypoglycemicagents, plant products play an important role in the management of diabetes although few have been scientifically claimed. The existenceof bioactive chemical componentsor phytochemicals withinterestingtherapeuticeffectsinthePseuduvariaspeciesof theAnnonaceaefamilyhasledresearcherstoinvestigatedifferent aspects of the pharmacological properties that this species possesses. The current study assessed the antihyperglycemic effectof thebark methanolicextractofPseuduvariamacrophylla (PM) (200 and 400mg/kg) using STZ-nicotinamide induced diabeticratsinviewofthesupportingdatafromtheantioxidant propertyofthisextract.Ourfindingshaveshownthatdestruction ofthepancreascausedbySTZ-nicotinamidewasmarkedlyreduced indiabeticratsafterreceivingPMtreatment.Thisresultcouldbe duetotheantioxidant,anti-inflammatory,andantihyperglycemic potential ofthe extract.Themajoractiveconstituents,suchas,

alkaloids,polyphenols,andcaffeicacid,whichwereidentiﬁedin PMbyLCMS-QTOFandNMRaftertheacutetoxicitystudies,might have contributedtothedownregulation ofoxidativestress and inﬂammatory response in the pancreas during the course of diabetes,and,eventually,recoveredthepancreaticdegeneration byupregulatingtheinsulinandGLUTproteinexpressionsinthe pancreas.

Whenthe biochemical parametersof the diabeticratswere analyzedinthis study,weobserveda drasticriseinthefasting blood glucose levels accompanied by reduced insulin and C- peptidelevels.However,thesealterationswerereversedsigniﬁ- cantlybytheoraladministrationofPMdose-dependentlybecause the PMb (400mg/kg) showed a higher reduction in the blood glucose levelsand improvedsecretionof insulin and C-peptide levelscomparedtoPMa(200mg/kg).Thereasonfortherecovery ofnormalbloodglucoselevelsinthePMtreateddiabeticratscould bedirectlyattributabletotheupregulatedinsulinandC-peptide levelsinourstudy.Itiswellunderstoodthatinsulinistheonly pancreatic

b

^-cell^hormone^known^to^reduce^blood^glucose^levels

(Brezaretal.,2011),andtheC-peptidereleasedfromthe

b

^cell^is

usuallyusedasamarkerforthe

b

^cell^functionândânîndex^for

insulin secretion (Palmer et al., 2004). The elevated insulin is responsibleforbringingtheserumandtissueproteinstoanormal level by reducing protein catabolism and thus enhancing its synthesis(Aryaetal.,2012b).Inadditiontotheantihyperglycemic activity,PMwasalsoabletoimprovetherecoveryofnormalbody weight in diabetic ratsdue toits similareffect to insulin. The breakdownofstructuralproteinsthatusuallycontributetobody weight due to insulin deﬁciency leads to a reduction in body weight(RameshandPugalendi,2006).TheobservedPMﬁndings ofimproved insulinandC-peptidelevelsresemblethereported resultsofpolyphenolic-richnutraceuticals,quercetin,andquinic acidfromPseuduvariamonticola(Tahaetal.,2014).

TheelevatedseruminsulinandC-peptidelevelsrepresentthe regeneration of the

b

^-cell ^function ^that ^is ^associated ^with^the

Fig.4.EffectsofPMextractonpro-inflammatorycytokinesTNF-a^(A),ÎL-b¹^(B)ândÎL-6^(C)concentrationsinnormalanddiabeticratsat2ndand6thweek.Allthevaluesare expressedasthemeanSD.‘a’indicatesthemeandifferenceissignificantwhencomparedtotheNCgroup(P<0.05).‘b’Indicatesthemeandifferenceissignificantwhen comparedtotheDCgroup(P<0.05).

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antioxidativeandfreeradicalscavengingpotentialoftheextract, as demonstrated in our study by a reduction in the lipid peroxidationlevel(LPO),oxidativestressmarkers(TGF-

b

^1),^and

anincreaseintheantioxidantenzyme(GSH)level.Thepresenceof activeconstituents, suchas caffeic acid, a polyphenolicin PM, might havecontributed tothe reduction of oxidative stressby downregulatingtheLPOlevelandupregulatingtheGSHlevel.The phenolic compounds, such as resveratrol, and caffeic acid are knowntohaveapotentantioxidantandantihyperglycemiceffect byameliorating oxidative stress in type-2 diabetic mice (Chao etal.,2009;Jungetal.,2006;Ramaretal.,2012).Similarly,the excessiveoxidativestressmarker(TGF-

b

¹⁾ⁱⁿ^the^serum^of^diabetic

ratswaspotentiallyreducedbyPMconﬁrmingitsantioxidantand protectivenature.Thereleaseof transforminggrowthfactor-

b

¹

(TGF-

b

¹⁾^bymacrophagesandimpairedtissuebetweensurfaces (interstitium) induced by oxidative stress leads to diabetic glomerularinjury (Klahrand Morrissey,2000), which produces swellingof theneighboringtissue, or acommoninﬂammation, withelevated pro-inﬂammatorycytokines(Trojanowska,2012).

Thus, our results are in parallel with the study conducted by Rajaveletal.(2012),whodemonstratedthattheinhibitionofTGF-

b

¹ⁱⁿ^palm^oiladministereddiabeticratswasduetothehighlevel ofphenoliccompoundspresentintheoil.Moreover,inlinewith

ourantioxidantstudy,thephenoliccompoundsidentiﬁedinthe extract have been shown to suppress the secretion of pro- inﬂammatorycytokines detected in the serum of diabetic rats treatedwithPM.Theseassociateandcorrelatetheactivationof manytranscriptionfactorsincludingNF-

k

^B^and^pro-inﬂammatory cytokines(TNF-

a

^,^IL-1

b

^,ÎL-6)êxpression^generated^by^T^cells ôr

macrophages(Reuteretal.,2010).Basedontheseﬁndings,itcanbe suggestedthattheabilityofPMtoinhibitoxidativestressmight lead to a reduction in pro-inﬂammatory cytokines. Similarly, besides being antihyperglycemic, the caffeic acid played a protectiveroleinattenuatingdiabetes-relatedkidneydiseaseby reducing TNF-

a

^, ^IL-1

b

^and ^IL-6 dose-dependently, thereby supportingourstudyandindicatingthattheseactivecomponents existing in our extract are most likely responsible for the documentedeffectofthisplantextract(Chaoetal.,2009).

The impairedstateof

b

^-celldegenerationin diabeticratsis causedbythechronicoxidativestressandinﬂammatoryresponse withinthemitochondriaduetoglucosetoxicityandlipotoxicity (Montane et al., 2014). Similarly, our histopathology analysis displayed degranulation of

b

^-cells ^followed ^by ^the ^damaged

structuralintegrity ofthecells, reductionin isletnumbers,and necroticchangesinthediabeticrats.Interestingly,the

b

^-cell^mass

waseffectivelypreserved,and anormalarchitectureoftheislet Fig.5.H&Estainingofdiabeticratpancreas(400x).(A):NC(B)DC(C)PMa(D)PMb(E)PC.

xxx–xxx

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Fig.6.EffectofPMoninsulinexpressionlevelinnormalanddiabeticpancreasrats(400x)A:NC;B:DC:C:PMa;D:PMbandE:PC.(E)Insulinexpressionlevelwerequantified byImageJanalysissoftwareasmeanSDandanalyzedbyone-wayANOVA(Tucky),‘a’indicatessignificantdifferenceP<0.05fromNC(P<0.05).‘b’indicatessignificant differenceP<0.05fromDC(P<0.05).

Fig.7.Pancreastissuepreparedandsubjectedtoenzymelinkedimmunosorbent(ELISA)assayusinganantibodyspecificforGLUT-1,GLUT-2andGLUT-4.Thepercentage proteinlevelwasmeasureandpresentedasmeanS.D(B).‘a’indicatessignificantdifferencescomparedwithsampleofNC(P<0.05).‘b’indicatessignificantdifferences comparedwithsampleofDC(P<0.05).

xxx–xxx

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cellswasobservedwithafairlyhighersurfaceareaandreduced lesions in the PM treated groups. The present ﬁndings are in accordancewiththeresultsobtainedfrompreviousstudiesonthe caffeicacid and alkaloidsco-existing in ourextract,where the pancreas histology was restored by reducing the glucose and increasingtheinsulinlevel(Gandhietal.,2011;Karthikesanetal., 2010). The immunohistochemical observation of islets in the presentdataconﬁrmedtheapparent

b

^-cellregenerationinthePM treateddiabeticratsbyshowingincreasedinsulin-immunoreac- tive expression. The pancreatic-cell disorder associated with insulin resistance is one of the symptoms of type 2 diabetes, which can be recovered by escalating the secretion of insulin (Robertsonetal.,1992).Therefore,PMhadrestoredthepancreas histologybyalleviatingoxidativestressandinﬂammationresult- ing in improved insulin secretion from theregenerated

b

^-cell,

whichisintunewiththeantidiabeticstudyconductedbyChung andShin(2007).

Therestorationof

b

^-cell^massândîmprovedînsulinsensitivity inthestudywasfurtherassessedbymeansofanenzyme-linked immunosorbent(ELISA) assay,asillustrated bytheupregulated expression of GLUT-1, GLUT-2, and GLUT-4 proteins, which is attributedto the insulin-mediatedglucose uptake and thereby reduces the blood glucose levels. The enhanced expression of pancreatic insulin from the existing beta cells of islets is responsible for the upregulation of these GLUT proteins, as discussedin thestudyofHajiaghaalipour etal. (2015).The PM enrichedwithalkaloids,flavonoid,andpolyphenoliccompounds couldhavecausedthiselevationofglucosetransporterproteinsby enhancing the insulin signaling cascade. Our results are in agreementwiththestudyof Changet al.(2016)and Ongetal.

(2011) who stated that alkaloids and phenolic compounds demonstratedanantihyperglycemiceffectinSTZ-induceddiabetic ratsbyincreasingtheGLUT-4expressionandtherebyenhancing theglucoseuptakeandinsulin-stimulatedglucoseconsumption.

Also, caffeic acid has been shown to enhance the GLUT-2 expressionin TNF-

a

^induced ^insulin ^resistance ⁱⁿ ^mouse ^liver

(FLB83B)cells(Huangetal.,2009).Inpancreatic

b

^-cells,^glucose^is

theprimaryphysiologicalstimulusforinsulinsecretionanditis ingestedintothecellthroughGLUT-2andGLUT-4intheplasma membrane of the cells (Schuit et al., 2001). Interestingly, the ﬁndingsofLeeetal.in2009displayedthatgenisteinderivatives elevatedtheexpressionsofGLUT-1andGLUT-4inL6myotubes, indicatingtheantidiabeticpotentialoftheiso-ﬂavones.Thus,the modulationoftheseglucosetransporterproteinscouldbeoneof themechanismsinvolvedwiththeanti-diabeticactivityofthePM extract.

Itisevidentfromthefindingsofourpresentinvestigationthat PMpossessesantioxidantandhypoglycemicpotentialresultingin downregulationoftheelevatedoxidativestressand pro-inflammatory cytokines. The anti-inflammatory response of PM was furtherevidencedbyareductioninthetransforminggrowthfactor beta-1(TGF-

b

^1).Furthermore,theimprovedregulationofinsulin andGLUT-1,GLUT-2,andGLUT-4accountfortheprotectiveand regenerativerole of PMon the pancreatic

b

^-cell ^mass ^of ^STZ-

nicotinamide induced diabetic rats. Nevertheless, the detailed mechanistic approach of PM on insulin signaling pathway is warranted.

Conclusion

The present study has shown potential role of PM in the managementoftype-2diabetesbydownregulatinghyperglycemia andelevatedlevelofpro-inﬂammatorycytokines.Moreover,PM has ameliorated diabetes-induced pancreatic degeneration by improving antioxidant defense system and pancreatic protein expressions.

Conﬂictofinterests

Theauthorshavenoconﬂictofintereststodeclare.

Acknowledgements

The accomplishment of this researchwas made possible by HighImpactResearchGrant(UM-MOHEUM.C/625/1/HIR/MOHE/

09) and the Fundamental Research Grant (FRGS):FP021-2014A fromtheMinistryofHigherEducation,Malaysia.

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