Rev. bras. farmacogn. vol.25 número2

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w w w . s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

Stevia

rebaudiana

loaded

titanium

oxide

nanomaterials

as

an

antidiabetic

agent

in

rats

Ariadna

Langle

a

_,

_Marco

_Antonio

_{González-Coronel}

b

_,

_Genaro

_{Carmona-Gutiérrez}

a

_,

José

Albino

Moreno-Rodríguez

a

_,

_Berenice

_Venegas

c

_,

_Guadalupe

_Mu˜noz

b

_,

_Samuel

_Trevi˜no

d

_,

Alfonso

Díaz

b,∗

a_Departamento_de_Química_General,_Facultad_de_Ciencias_Químicas,_Benemérita_Universidad_Autónoma_de_Puebla

b_Departamento_de_Farmacia,_Facultad_de_Ciencias_Químicas,_Benemérita_Universidad_Autónoma_de_Puebla

c_Departamento_de_Análisis_Clínicos,_Facultad_de_Ciencias_Químicas,_Benemérita_Universidad_Autónoma_de_Puebla

d_Departamento_de_Biología_y_Toxicología_de_la_{reproducción,}_Instituto_de_Ciencias,_Benemérita_Universidad_Autónoma_de_Puebla

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received25July2014 Accepted4March2015 Availableonline30March2015

Keywords:

Diabetes

Steviarebaudiana

Nanomaterials Hypoglycemic Antihyperlipidemic

a

b

s

t

r

a

c

t

Steviarebaudiana(Bertoni)Bertoni,Asteraceae,isaplantwithhypoglycemicandantihyperlipidemic properties.S.rebaudiana(SrB)hasbecomealeadcandidateforthetreatmentofthediabetes melli-tus.However,chronicadministrationsofS.rebaudianaarerequiredtocausethenormoglycemiceffect. Importantly,nanomaterialsingeneralandtitaniumdioxide(TiO2)inparticularhavebecomeeffective

toolsfordrugdelivery.Inthiswork,weobtainedTiO2nanomaterialswithSrBatdifferent

concentra-tions(10,20and30␮M)bysol–gelmethod.AfterthisnanomaterialswerecharacterizedbyFourier transforminfraredspectroscopyandtransmissionelectronmicroscopy.Whereitwasdemonstrated,the presenceoftheS.rebaudianainTiO2nanomaterials,whichwereobservedashemisphericalagglomerated

particlesofdifferentsizes.Thenanomaterialswereevaluatedinmaleratswhosediabetes mellitus-phenotypewasinducedbyalloxan(200mg/kg,i.p.).Theco-administrationofTiO2-SrB(20and30␮M)

inducedasignificantandpermanentdecreaseintheglucoseconcentrationsince4h,until30days post-administration.Likewise,theconcentrationsofinsulin,glycosylatedhemoglobin,cholesterol,and triacylglyceridesshowedasignificantrecoverytobasallevels.Themajorfindingofthestudywasthat theTiO2-SrB(20and30␮M)hasapotentandprolongedactivityantidiabetic.TiO2canbeconsideredlike

Introduction

Traditionalmedicineprincipallyderivedfromplantsand cur-rentlyrepresentsatherapeuticpotentialforDiabetesmellitus(DM) (Laitiffetal.,2010;Malviyaetal.,2010).

Steviarebaudiana(Bertoni)Bertoni(SrB),alsoknownas“sweet herb,”isa shrubbyperennialplantbelongingtotheAsteraceae family(Soejartoetal.,1982).SrBsheetscontainingapproximately 4–15%stevioside,whichhavebeenisolatedandcharacterizedby chemicalandspectralstudies(Starrattetal.,2002).

SrBhasvariouspharmacologicalpropertiesincluding antioxi-dantactivity,antihypertensive,antihyperlipidemicand cardiovas-cularprotector (Chan et al., 1998).Recent studies have shown thattheSrBstimulatesinsulinsecretionfrompancreatic␤-cells

∗ Correspondingauthor.

E-mail:[email protected](A.Díaz).

and cause normolipidemia and normoglycemia in diabetic rats (Raskovicetal.,2004;Chenetal.,2005).Inthissense,SrBprovides evidenceofhistraditionaluseinthecontrolofDM.However,ithas beendemonstratedthatarerequiredcontinuousadministrations ofSrBtoachievenormoglycemiceffect(CurryandRoberts,2008). Onewaytoaddressthisproblemis,todevelopacontrolledrelease systemofSrB,inordertoreducethedoseandmakeavailableSrB inthetargetsitesandkeepitsprolongedactivity.Avarietyof sys-temshavebeendevelopedandused,includingliposomes,micelles, dendrimers,andcopolymers.

Currently, nanomaterials of titanium dioxide (TiO2) have attractedattentionasdeliverypotentialsystems(Barbetal.,2004). TheTiO2nanomaterialsarechemicallyinert,possesshydrophilic features and its synthesis isn’t complicated, in addition, these presenthighmechanicalstrengthandlowtoxicity(Sonetal.,2007). Recentlythenanomaterialshavebeenimplantedintheamygdalaof ratswithepilepsyandobtainedoptimalresults,andfoundthatthe porousTiO2matrixreleasetheircontentsinacontrolledmanner

http://dx.doi.org/10.1016/j.bjp.2015.03.004

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A.Langleetal./RevistaBrasileiradeFarmacognosia25(2015)145–151

foraperiodapproximatelyof500h(h)(Lópezetal.,2010a,b).Here, weperformedthesynthesisofTiO₂nanomaterialsfromsolutions oftitaniumn-butoxideandtheaqueousextractofSrBconcentrate obtainedfromthedriedleavesoftheplant.Thiswiththepurposeof usingitasacontrolledreleasesystemSrBextract.Fortheabove,this studyaimedtoevaluatetheantidiabeticactivityofintraperitoneal injectionofTiO2-SrBnanomaterialsinadiabeticratmodel.

Materialsandmethods

PreparationofSrB-extract

Steviarebaudiana(Bertoni)Bertoni,Asteraceae,wasgrownon campus grounds of the Benemérita Universidad Autónoma de Puebla(BUAP),México.Dr.AlbinoMoreno-Rodríguezconfirmed the identification,additionally; a sample was deposited in the botanicalgardenof the BUAP(file21-09/14). Leaves SrB (10g) washedanddriedinanElisa-550ovenfor24h.Thenitpulverized andmaceratedwithasolutionofethanolandwater(witharatioof 80:20).Thesolutionobtainedfromthemashingprocessisfiltered andtheSrBaqueousextractisplacedonarotaryevaporatorofthe ESEVE-402-2brandtoeliminateto80%ofsolvent(waterand alco-hol)underreducedpressure.ThusweobtaintheSrBconcentrated extractto80wt.

PreparationofTiO2-SrBnanomaterialsbysol–gelmethod

The homogeneous solutions were prepared separately with differentconcentrationsoftheSrB concentratedextract (10,20 and 30␮M). Each homogeneous solution containing 150ml of anhydrous1-butanol(99.8%,Sigma–Aldrich),10ml ofdeionized waterand0.5gofpolyvinylpyrrolidone(withanaveragemolecular weightof40,000,Sigma–Aldrich)andtherequiredconcentration oftheSrBconcentratedextract(10,20and30␮M).

Foreachhomogeneoussolutionwasadded21.5mloftitanium

n-butoxide(97%,Sigma–Aldrich)inarefluxsystemat70◦_C_with constantagitation.Thefinalsolutionwithpropertiesofgelwas immersedinacontainerwithicefor15minat3◦_C._The_solvent_was removedonarotaryevaporatorat50◦_C_under_vacuum_conditions tofinallyobtaintheTiO2-SrBnanomaterials(Lópezetal.,2010a,b).

CharacterizedofTiO2-SrBnanomaterials

Infraredspectroscopy(FTIR)

The TiO₂-SrB nanomaterials were mixed with KBr (5wt %) and pressed in transparent wafers. Fourier transform infrared spectroscopywasrecordedusingaPerkin-Elmer1600 spectropho-tometer(Perkin-Elmer,Shelton,CT)inthe4000–400cm−1_range, and32scanswererunforeachmeasurement.

Scanningelectronmicroscopy(SEM)

Theparticlesize wasmeasuredusingconventional Scanning electron microscopy (SEM, Zeiss; Carl Zeiss, Oberkochen, Ger-many),operatedat100kV,withentrygoniometeratthesideand 0.4nmpoint-to-pointresolution,andattachedtoaCCDcamera (MegaVision,SantaBarbara,CA).

Animals

AdultmaleLongEvansrats(230–250g)wereobtainedfrom Bio-terio“ClaudeBernard”BUAP.Animalswereindividuallyhoused in anenvironment withcontrolledtemperature, humidity, and lightconditions(12hlight:12hdarkcycle), withfreeaccessto foodandwater.Allproceduresdescribedinthisstudywere per-formedin accordancetotheMexicanLaw ofAnimalTreatment andProtectionGuidelines(NOM-062-ZOO-1999)andtheResearch

Committeeusesoflaboratoryanimalsof theBUAP (VIEP-3450-2013).

Inductionofdiabetesinrats

The animals were injected with alloxan dissolved in 0.1M citratebuffer,pH4.5atadose200mg/kgbodyweight(i.p.).The alloxandosewasselectedbasedonvariousreports(Raskovicetal., 2004;Szkudelski,2001).Threedayafteroftheadministrationof alloxan,bloodsamplesweretakenandbloodglucoselevelswere determined.Animals that had higherconcentrations of glucose (150mg/dl)wereconsidereddiabetic.

SrB-TiO2administrationprotocols

Intheexperimentatotaloffortyratswereused,whichwere dividedinfivegroups(n=8pergroup).

Group1:normal ratstreatedwithTiO2 nanomaterials (vehicle group)

Group2:diabeticgrouptreatedwithTiO₂nanomaterials Group3:diabeticratswithTiO2-SrBnanomaterialsto10␮M Group4:diabeticratswithTiO2-SrBnanomaterialsto20␮M Group5:diabeticratswithTiO2-SrBnanomaterialsto30␮M

Thenanomaterialswereinjectedbyi.p.Therouteinallanimals dependingoftreatment.

Subsequently, both theempty TiO2 nanomaterials (1g/kgin sterilewaterat37◦_C)_and_SrB_(10,₂₀_and₃₀_␮_M)₍₁_g/kg_in_sterile waterat37◦_C)_were_{administered,}_{respectively.}

Methodfordeterminationofplasmabloodglucoselevel

Toevaluatetheplasmaticconcentrationofglucosewithrespect atcourseoftimeintheanimalsinjectedwithSrBnanomaterials,the animalsweredeprivedofthefoodfor5hbeforethedetermination. Thebloodglucoselevelsweredeterminedat0,4,8and24aswell asat5,10,15and30daysafteradministrationofeachtreatments. Intheexperimentthebloodglucoseleveloftheanimalswere esti-matedbyGlucoseOxidase–PeroxidaseEnzymaticMethodusinga digitalglucometer(ACCU-CHEKbrandactive).Bloodfromthetail veinwascollected.

EvaluationoftheeffectofTiO2-SrBnanomaterialson hyperglycemiaandhyperlipidemiainducedbyalloxan

At31dayspost-injectionofTiO₂andTiO₂-SrB,theanimalsof each experimentalgroup weresacrificed by dislocationand by cardiacpuncture.Thebloodsampleswerecollectedtomeasure glucose,insulin,glycatedhemoglobin,cholesteroland triacylglyc-erides.Collectedsampleswerecentrifugedat2×gfor5min.Serum determinationsofglucose,cholesterolandtriacylglycerideswere developedbyenzymaticcolorimetricassay,followingtheprotocols respectivelylabeledaccordingtheBioSystemkitsandanalyzedina semi-automatedspectrophotometer(BayerRA-50).Plasmainsulin concentrationwasdeterminedbyanELISAimmunoassay (Diagnós-ticaInternacional),andantibody-antigencomplexwasdetermined at415nminaStatfax2600platereader(WinerLabgroup).The glycatedhemoglobinmeasuringwascarriedoutby immunofluo-rescencemethod,followingtheprotocolaccordingthei-Chroma kitanditwasanalyzedinadetectorofthesamebrand.

Statisticalanalysis

Thedatawereexpressedasmean±standarderror(SE)forall

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4000 3500 3000 2500

Wave number (cm–1₎

Wave number (cm–1)

2000 1500 1000 500 TiO2

SrB10-TiO2

SrB20-TiO2

SrB30-TiO2

Intensity (a.u.)

4000 3500 3000 2500 2000 1500 1000 500

B

A

Fig.1.(A)FTIRspectrumofSrBextract80wt%.(B)FTIRspectraofTiO2,SrB/TiO2-10, SrB/TiO2-20andSrB/TiO2-30nanomaterials.

andaconfidencelevelof95%and,whennecessary,themeanswere comparedusingtheBonferronitest.

Results

CharacterizationofTiO2-SrBnanomaterials

InfraredSpectroscopy(FTIR)

The FTIR spectrum of SrB extract is presented in Fig. 1A. The absorptionbandlocated at 3383cm−1_, _it _{corresponding}_to thestretchingvibrationmode␯OH,whichidentifiesthehydroxyl groups(OH−_),_of_water_(H _OH),_and_the_alcohol_(ethanol,_R _OH). Thestretchingvibrationmodes␯CHCH,ofchemicalspeciesC Hare locatedto2988cm−1_._Mode_␯_CH_of_asymmetric_stretching vibra-tionitlocatesat2900cm−1_._The_vibration_band_{corresponding}_to thebendingtype interactionsmode␯OH oftheOH-groups,it is assigned ofthesurfacehydroxyl groupsof thematerial,to sol-ventandthecoordinateddeformation␦HOHofWaterbelocated at1630cm−1_._The_wavenumber_to₁₄₂₅_cm−1_and₁₄₁₀_cm−1_,_they corresponding to the symmetrical stretching vibration ␯COO of theCOO groups and deformation ␦CH3 vibration of modes.The

vibrationbandscorrespondingtotheasymmetricstretching vibra-tiontype␯COOofcarboxylateionsarelocatedat1593cm−1.

TheIRspectrumofTiO2 nanomaterialsshowsintheFig.1B. Theabsorptionbandat3,339.1cm−1_,_correspond_to_the_stretching vibrationmode␯OH,whichidentifiesthehydroxylgroups(OH−)of water(H OH),thesolvent(butylalcohol,R OH)andhydroxylation ofgel(Ti OH).TheasymmetricstretchingvibrationmodesofCHis locatedat2939.2cm−1_,_correspond_to_the_chemical_species_of_the methyl( CH3)(Ti O CH3)andethoxygroups( Ti OCH2CH3).

Thebendingmode(␯OH)localizedto1635.9cm−1,correspond toOHgroupsofwater,whicharepresentonthesurfaceofTiO2 nanomaterial.Theyare mainlyassociated withthehumidityof thenanomaterial,the solventand thecoordinateddeformation modeofwater.Intherange(from ∼1400cm−1 _to_∼₁₃₀₀_cm−1₎ ofelectromagneticradiation,arelocalizedthebendingvibrationof symmetricaltypeoftheCOO− _groups,_the_vibration_asymmetric deformationoftheCHgroupsandthevibrationmodeofthe defor-mationscissorofthe␦CH3oftheCH3groups.Inthenearinfrared region,thevibrationmodes␯Ti–Otypebendingofthemetal-oxygen interaction, theylocalized at 745.3cm−1_, _to _640.2_cm−1 _and _to 498.8cm−1_._The_TiO₂_{nanomaterials}_doped_with₂₀_␮_M_and₃₀_␮_M oftheextractSrB,presentsimilarstretchingvibrationmodes:(␯OH) and (␯CH)and bending vibrationmodes: (␯OH), (␦ HOH), (␯COO), (␯CH),(␯CH3),(␦CC),(␯CO)and(␯Ti–O),asthanthoseobservedinthe TiO2nanomaterialundoped.

Thedecreaseinintensityoftheabsorptionbands,mainlyofOH− vibrationmodescorrespondingtothehumidity(at1635.9cm−1₎ ofSRB-TiO2 with10,20 and30␮Mnanomaterials,maybedue totheinteractiontoSrBaqueousextractwiththesurfaceofTiO2 nanoreservoir,asshowninschemeofFig.2.Itisalsoimportant tonotethat under theseexperimentalconditions isimpossible totalkabouttheformationofpolymorphsofTiO2 relativetothe concentrationoftheaqueousextractofSrB.

Scanningelectronmicroscopy(SEM)

TheFig.3A,showtheSEMstudiesofTiO2nanomaterialwitha tracedareato100,000magnifications.ThetextureoftheTiO2has showntheformofagglomeratedparticleswithanaverageparticle diameterof100nm.ThemicrographofTiO2nanomaterialwith SrB-10␮M;presentinasweptareato100,000×.Themicrographofthe

TiO2-SrB-10␮Mnanomaterialpresentinasweptareaof100,000 magnifications,formlessparticleswithanaverageareaof70nm

Fig.3B,showthemicrographofthenanomaterialofTiO2with 20␮lof SrB (SrB-TiO₂-20␮l), which presented a swept areato 100,000magnifications,itshowedhemisphericaltypeparticlewith anaverageareaof60nm.Similarly,theFig.3Cshowthemicrograph ofSrB/TiO₂-30 nanomaterialwithanaveragesphericalparticles sizeof4nmthatarelocatedwithinthematrixofTiO2.InFig.3D, theincreasingintheconcentrationoftheaqueousextractofSrB inTiO₂nanomaterials,wenotethatdecreasestheaverageparticle sizeofnanomaterialsof100nm(forTiO2)to4nm(forSrB-Ti2-30␮l andweconcludedthatthemoleculesoftheaqueousextractofSrB werepresentinthemeshofTiO2.

EffectofTiO2-SrBnanomaterialstreatmentabouthyperglycemia inducedbyalloxan

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Tiδ+ _Tiδ+ _Tiδ+ _Oδ– _Tiδ+ _Oδ– _Tiδ+ _Tiδ+ _Tiδ+

Stevioside Steviol

CH3

H3C 3H_C

CH3

CH

3

CH

3

HO O

OH

HO

HO HO

HO

HO O

O

O O

O OH

OH

OH OH Stevioside

Oδ– _Oδ–

OH– OH–

–_HO

TiO₂

OH– CH₂ H

δ+ δ– δ+ δ– δ+ δ– δ+

CH 2 CH₂

H H

H O H

O

Oδ– _Oδ–

Fig.2.SchemeofinteractiontoSrBwiththesurfaceofTiO2nanomaterials.

In treatedrats withalloxantheblood glucose levelsat 4, 8 or24h,increased(160–180mg/dl),respecttothevehiclegroup (80.43mg/dl)(Fig.4B).TreatmentwithTiO2-SrBnanomaterialsin alloxan-treatedanimalsshowedaprogressivedecreaseinglucose levelsat4,8 and24hafteradministration,which, whenitwas comparedwiththealloxan+TiO2 treatedgroup,wasobserveda significantdifferenceintheanimalstreatedwithTiO2-SrB20and 30␮M(Fig.4B).(OnewayANOVA,Bonferronipost-test,p<0.05).

Moreover,comparingtheglucoseconcentrationat5,10,15and 30days inthehyperglycemic ratsafter administrationofTiO2 -SrB,wasobserved,a significantdecreaseinthetreatedanimals withTiO2-SrBnanomaterialsat20and30␮Mwithrespecttothe alloxan+TiO2group(Fig.4C).Inthisregard,thedecreaseof glu-coselevelswasobservedduring30daysafteradministrationof

TiO₂-SrBataconcentrationof20–30␮M(one-wayANOVA, Bon-ferronipost-test,p<0.05).

Laboratorystudies,developed31daysafteradministrationof treatments,showasignificantincreaseintheconcentrationof glu-cose (86%) of thehyperglycemic group+TiO2, compared tothe normalratstreatedwithTiO2 (Fig.5AandB).Consequently,the concentration of glycosylated hemoglobin in the alloxan+TiO2 group,washigher(38%)comparedtothenormalrats+TiO2(Fig.5C) (one-wayANOVA,post-Bonferronitest,p<0.05).

Moreover,theanimalsofgroupsofalloxan+TiO2-SrB20and 30␮M,hadalowerconcentrationofglucose(20%and56%)and glycatedhemoglobin(25%and41%)comparedtothealloxan+TiO2 group (Fig. 5A and C). Also, groups with alloxan+TiO2-SrB 20 and30␮Mshoweda higherinsulin concentrationcomparedto

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300

A

275

Vehicle + Ti2O

### ###

*** Alloxan + Ti2O

Week

Alloxan + TiO2-SrB (10μM)

Alloxan + TiO2-SrB (20μM)

Alloxan + TiO₂-SrB (30μM)

Vehicle + Vehicle

Hours Alloxan + TiO₂

Alloxan + TiO2-SrB (10 μM)

Vehicle + Vehicle Alloxan + TiO2

Alloxan + TiO₂-SrB (10 μM) Alloxan + TiO2-SrB (20 μM)

250

225

W

eight (g)

200

1 2 3 4

0 4 8 24

0 4 10 20 25 30

Days 15 175 150 200

B

150 100 50 [Glucose] mg/gl 0 200

C

150 100 50 [Glucose] mg/gl 0 ## ### ### ### *** ***

Fig.4. EffectofadministrationofTiO2-SrBnanomaterialsontheweightandthe timecourseoftheglycemiclevelsintreatedratswithalloxan.Theanimalswere weighedeveryweekfor4weeks(A).Thebloodglucoselevelsweredeterminedat 0,4,8and24h(B),aswellas5,10,15and30days(C)afteradministrationofthe nano-TiO2SrB(10,20and30␮M)indiabeticrats(alloxan,200mg/kg).Thevehicle groupwereadministeredwiththevehicle,andwithemptynanomaterials(Vehicle andalloxan+TiO2)underthesameexperimentalconditions(n=8pergroup).The datashowtheaverageweight(g)andglucoseconcentration(mg/dl)±SEofeach grouprespectively.(One-wayANOVA,Bonferronipost-test,***p<0.001:Vehiclevs alloxan+TiO2;# #p<0.01and###_p_<_0.001:_alloxan₊_TiO

2-SrBvsalloxan+TiO2).

the group with alloxan+TiO2 (80% and 100%) (Fig. 5B). (One wayANOVA,Bonferronipost-test,p<0.05).Thisshowedforthe firsttime,thatnanomaterialsTiO2-SrB(20and30␮M)reversed hyperglycemic effect inducedby alloxan, due tothe biochemi-calparametersmeasured(glucose,glycosylatedhemoglobinand insulin)arestatisticallysimilartothevehiclegroup+TiO₂.

TiO2-SrBnanomaterialstreatmenteffectonhyperlipidemiceffects inducedbyalloxan

The resultsshow that the diabetic group treated withTiO2 increasedthecholesterol and triacylglycerides levelsrespectto

Vehicle *** ## ### [glucose] mg/cl 200

A

150 100 50 0 Alloxan + TiO2

Alloxan + TiO2

SrB (10μM)

Alloxan + TiO2

SrB (20μM) Alloxan

+ TiO2

SrB (30μM)

Vehicle Alloxan + TiO2

Alloxan + TiO2

SrB (10μM) Alloxan

+ TiO2

SrB (20μM)

Alloxan + TiO2

SrB (30μM)

Alloxan + TiO2

SrB (10μM)

Alloxan + TiO2

SrB (20μM) Alloxan

+ TiO2

SrB (30μM) [Insuline] mg/cl 40

B

30 20 10 0

% Glycated Hb (HbA1c)

10

C

8 6 2 4 0 *** ## ## *** # ##

Fig.5. EffectofadministrationofTiO2-SrBnanomaterialsonglucose,insulinand glycosylatedhemoglobininadministeredratswithalloxan.Quantificationwas per-formedinplasmaat31daysafteradministrationofTiO2-SRB(10,20and30␮M)or TiO2intreatedratswithalloxan(200mg/kg)orvehicle.Thegraph(A)glucose con-centration(mg/ml)inplasmaandshowningraphs(B)and(C)insulinconcentration (mU/ml),andthepercentageofglycosylatedhemoglobin,respectivelyareshown. Thedatashowthemean±SEofeachgrouprespectively.(One-wayANOVA, Bon-ferronipost-test,***p<0.001:Vehiclevsalloxan+TiO2and##p<0.01,###_p_<_0.001: alloxan+TiO2-SrBvsalloxan+TiO2).

normalratstreatedwithTiO2(227%and185%,respectively)(Fig.6). Moreover the treatmentwith TiO₂-SrB nanomaterials to 20 or 30␮M in diabetic rats decreased the cholesterol (62 and 64%, respectively)andtriacylglycerideslevels(59and63%,respectively) respecttodiabeticratswithTiO2(Fig.6AandB).(OnewayANOVA, Bonferronipost-test,p<0.05).TreatedanimalswithTiO2-SrB nano-materialsto10␮Mdidnotmodifythebiochemical parameters evaluated.

Discussion

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Vehicle ***

##

[T

riglycer

ides] mg/cl

[Cholesterol] mg/cl

300

A

B

200

100

0

300

200

150 250

100

50

0

Alloxan + TiO2

SrB (10μM)

Alloxan + TiO2

SrB (20μM) Alloxan

+ TiO2

SrB (30μM)

Alloxan + TiO2

SrB (10μM)

Alloxan + TiO2

SrB (20μM) Alloxan

+ TiO2

SrB (30μM) ***

### ###

Fig.6.EffectofadministrationofTiO2-SrB nanomaterialsonconcentrationof cholesterolandtriacylglyceridesinadministeredratswithalloxan.Quantification wasperformedinplasmaat31daysafteradministrationofTiO2-SrB(10,20and 30␮M)orTiO2intreatedratswithalloxan(200mg/kg)orvehicle.Thegraph (A)isshowncholesterolconcentration(mg/dl)andinthegraph(B) triacylglyc-erides(mg/dl)areshownrespectively.Datashowthemeanconcentration±SEof eachgrouprespectively.(One-wayANOVA,Bonferronipost-test,***p<0.Vehiclevs alloxan+TiO2and##p<0.01,###_p_<_0.001:_alloxan₊_TiO

2-SrBvsalloxan+TiO2).

TiO2-SrBnanomaterialsindiabeticratmodelinducedwithalloxan, which is a toxic hepato-pancreaticagent,togenerate a similar metabolicchangestotheDM.Thealloxanisemployedasan exper-imentaldiabetogenictoevaluatetheeffectivenessofnewdrugs withhypoglycemicproperties(Szkudelski,2001).

Glycemia and lipemiaevaluations,determines thedegree of metabolicdamagethatexistsinexperimentalanimals.Thisstudy showsthatthetreatmentwithTiO2alonehavenoeffectonglucose andlipidsvalues,whileTiO₂-SrBat20or30␮Mcauseda hypo-glycemicand antihyperlipidemiceffect indiabetic ratsinduced withalloxan,which isa toxic glucoseanalog,whichselectively destroys insulin-producing beta cells in the pancreas when is administeredtorodents(Jeppesenetal.,2000),andcausesthat secretedinsulinareinsufficienttoregulatebloodglucose, there-foreinthisstudytheratsexhibitedcontinuoushyperglycemia.This modelproducesadecreaseintheinsulinlevelsintheanimals, simi-lartothereportedinseveralstudies.Whentheratsaretreatedwith TiO2-SrBto20or30␮Mproduceahypoglycemiceffectfrom4hto 31daysafterthetreatment.Theseresultsrevealtheimportance ofTiO2nanomaterialsascontinuousandsustainedreleasedrugs deliverysystemintothebloodstream.

According to Jeppesen et al. (2000) the SrB exerts a hypo-glycemicaction whichis analogoustosulfonylureas, becauseit blocksK+_channels_in_pancreatic_␤_-cells_remaining,_depolarized_the membraneandpromoteschannelopeningofvoltage-dependent Ca+2_. _Wherein _the _increase _of _{intracellular} _Ca+2 _is _crucial _to induceinsulin secretionfrompancreatic␤-cellsand thuscause thedecreaseofbloodsugar,reachingnormalvaluesbiologically (Raskovicetal.,2004).

Accordingourresultsit appearsthatthetreatmentwithSrB intoTiO2 nanomaterials ondiabeticratsstimulateofremaining

␤-pancreaticcellsandinducetheinsulinsecretionconstantly.This insulinpromotesthedecreaseofglucoseandglycatedhemoglobin levels in theplasma of the diabeticrats (Park and Cha, 2010). It is noteworthy that interaction,SrB, withnanomaterials TiO2 decreasesprogressively,oncetheyarein theaqueousmedium, whichprovidesthebloodstream,therefore,theSrBisreleasedand distributedperipherallytoreachingthesitesofactionandexercise therespectivepharmacologicaleffects.Accordingassuggestedby someworkwithnanomaterialsofTiO2(Lópezetal.,2010a,b,2015). However,itisnecessarytodemonstratethisprocessinthefuture. In DM, highlipid levelsare present and play an important toxicrole.Thepresentstudyshowhighlipidlevelsindiabeticrats inducedwithalloxanbutthetreatmentwithnanomaterialsofTiO2 -SrBnormalizetheprofilelipidsdemonstratingthehypolipidemic effectof SrB. However,themolecularmechanism to regulating glucoseorlipidsoftheSrBareunknown.Atthemoment, stud-ies indicate that SrB has antioxidant and antiapoptotic effects (Shivannaetal.,2013),whichprobablyhelpstoenhancetheSrB therapeuticeffect.AccordingtoMisraetal.(2011)suggestthat Ste-viaextractproducedgoodantidiabeticeffectstogetherwithlesser lossinbody weightandproposethatcanbeusedinthe prepa-rationof coughsyrupsand coldbeveragesfor diabetespatients (Himanshuetal.,2011).

Foraboveexpose,thisstudydescribesforthefirsttimesthatis necessarythedeliverysystemlikeTiO2nanomaterialstorelease SrB and generate regulation on hyperglycemia and hyperlipi-demiaindiabeticratsinduced withalloxan.Ourresultsopena new opportunity for the treatment of theDM; however more studiesareneededtobetterunderstandthepotentialuseofTiO2 -nanomaterialsandSrBinmetabolicdiseases.

Authorcontributions

AL,MGC,ST,AD,BV,GI,GCandAMRdesignthestudyandwrote theprotocol.AL,ADandSTperformedtheexperiments.AL,AD, AMR,ST,BVandGCmanagedtheliteraturesearchesand analy-sis;MGCandADundertookthestatisticalanalysis.Allcontributing authorshaveapprovedthefinalmanuscript.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TheauthorsthankDr.CarlosEscamillaforhishelpwiththe ani-malcareandthankstoThomasEdwards,PhDforeditingtheEnglish languagetext.

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