Rev. bras. farmacogn. vol.26 número5

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

Original

article

Hypoglycemic

effect

of

formulation

containing

hydroethanolic

extract

of

Calophyllum

brasiliense

in

diabetic

rats

induced

by

streptozotocin

Helison

de

Oliveira

Carvalho

_,

_Belmira

_Silva

_Farias

_e

_Souza

_,

_Igor

_Victor

_Ferreira

_dos

_Santos

_,

Rafael

Lima

Resque

_,

_Hady

_Keita

_,

_Caio

_Pinho

_Fernandes

_,

_José

_Carlos

_Tavares

_Carvalho

a_{LaboratóriodePesquisaemFármacos,DepartamentodeCiênciasBiológicasedaSaúde,ColegiadodeFarmácia,UniversidadeFederaldoAmapá,Macapá,AP,Brazil}

b_{ProgramadePós-graduac¸ãoemCiênciasdaSaúde,DepartamentodeCiênciasBiológicasedaSaúde,ColegiadodeFarmácia,UniversidadeFederaldoAmapá,Macapá,AP,Brazil} c_{ProgramadePós-graduac¸ãoemInovac¸ãoFarmacêutica,DepartamentodeCiênciasBiológicasedaSaúde,ColegiadodeFarmácia,UniversidadeFederaldoAmapá,Macapá,AM,}

Brazil

d_{LaboratóriodeNanobiotecnologiaFitofarmacêutica,DepartamentodeCiênciasBiológicasedaSaúde,ColegiadodeFarmácia,UniversidadeFederaldoAmapá,Macapá,AP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received9February2016 Accepted24April2016

Keywords: Calophyllum Hypoglycemic Granulated Diabetesmellitus Polyphenols

a

b

s

t

r

a

c

t

Diabetesmellitusisachronicandseveremetabolicdysfunction,it’sslowandprogressiveevolution

inter-feresdirectlyinthemetabolismofcarbohydrates,fatsandproteins,causinghyperglycemia,glycosuria,

polydipsia,hyperlipidaemia,amongothers.Theaimofthisstudywastoevaluatetheantidiabeticeffectof

hydroethanolicextractandgranulatedofCalophyllumbrasilienseCambess.,Clusiaceae,speciesindiabetic

ratsaswellasit’sbiochemicalparameters.Theresultsdemonstratedthatboththepharmaceuticalforms,

hydroethanolicextractandgranulated,wereabletoreducesignificantly(p<0.001)hyperglycemiaand

glycosuria,inadditiontoimprovepolydipsia,polyuria,andweightloss.Treatmentsusing

hydroethano-licextractandgranulatedwerealsoabletoreducesignificantlylevelsoftriacylglycerides,cholesterol

andlow-densitylipoprotein,aswellasthetransaminases,ureaandcreatininelevels.Therefore,itis

concludedthatthesepharmaceuticalformshaveanti-diabeticeffectandactimprovingthe

biochemi-calparameters,thiseffectisprobablyduetothehighcontentofpolyphenoliccompoundsfoundinthe

formulations.

©2016SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Thisisanopen

accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

CalophyllumbrasilienseCambess.(Cb)speciesbelongs to Clu-siaceaefamily;it canbefoundspontaneously throughoutLatin America, with predominance of thespecies in regions such as AmazonandAtlanticForest.Pharmacologically,isusedforseveral diseasessuchasdiabetes,bronchitis,liverdisorders, gastrointesti-naldiseases, pain,inflammation,hypertension and rheumatism (Silvaetal.,2001).

Diabetesmellitus(DM)isachronicandseveremetabolic dys-function,it’sslowandprogressiveevolutioninterferesdirectlyin themetabolismofcarbohydrates,fatsandproteinsand is char-acterizedbyabsentordecreasedproductionofinsulinand/orits failuretoproperlyexercisetheireffectsoncells,leadingmainlyto hyperglycemia,glycosuria,polyuria,polydipsia,amongothers.DM isnotasinglediseasebutagroupofseveralmetabolicdisorders

∗ Correspondingauthor.

E-mail:[email protected](J.C.Carvalho).

thathaveincommonhyperglycemiaanddyslipidemia,andleadto seriouscomplicationscausingdamagetomanyorgans,especially theeyes,kidneys,nerves,heartandbloodducts,makingDMthe seventhcauseofdeathindevelopedcountries(Ada,2011;Sacks etal.,2002).

Thereisagrowingdemandbytheworldpopulationfor medici-nalplants,whereabout65–80%ofthepeopleuseplantsbecauseof poverty,precarioushealthsystemortheeasyaccesstothese nat-uralproductsthataresoldinstreetmarketsandpopularmarkets (Calixto,2000).Therefore,studiesofnewanti-DMdrugshavebeen conductedwithspecialfocusonmedicinalplants;agood exam-pleis theplantGalegaofficinalisthat ledtothedevelopmentof Metformin,anoralhypoglycemicdrug(Noeletal.,1997).

Phytotherapicsaredevelopedbytechnologicalprocessesfrom vegetable raw materials, and among the main pharmaceutical formsproduced, lyophilizeddryextractsand granulated,which canbeintermediateorfinalformulationsforobtainingtabletsand capsules,arefound(Carvalhoetal.,2013).

Theaimofthisstudywastoevaluatetheantidiabeticeffect of hydroethanolic extract and granulated of Cb species and

http://dx.doi.org/10.1016/j.bjp.2016.04.004

biochemicalparameters ofnormal anddiabeticratsinducedby streptozotocin.

Materialsandmethods

Plantmaterial

StembarksofCalophyllumbrasilienseCambess.,Clusiaceae,was collectedinthemunicipalityofFerreiraGomes,AmapáState,Brazil (0.859831Nand−51.158938W).Thefertilematerialwasidentified intheHerbariumoftheInstitutodeEstudosePesquisasdoEstado doAmapá,with0598APasnumberofvoucherspecimen.

ObtainingtheC.brasiliensehydroethanolicextract

ToobtaintheC.brasiliensehydroethanolicextract(ECb),2kg ofcrushedandmilledhullsweresubjectedtomacerationin70% hydroethanolsolutionat45◦_{Cfor4daysinaratioof1:8(w/v).The}

extractivesolutionwasfilteredthroughfilterpaperand concen-tratedonrotaevaporatorModelQ.218.2(QuimusLtda,SãoPaulo, Brazil)atatemperatureof40◦_{Cuntilcompleteevaporationofthe}

solvent,yielding32.80%.Lateritwaslyophilizedtocomplete elim-inationofwater,withfinalyieldof7%.

ObtaininggranulatedofC.brasilienseextract(GCb)

Thegranulewasobtainedbymanualmixing forgranulating usingthefollowingcombinationofexcipientsandextract:21.80% Avicel®_{cellulose(Sigma–AldrichCo.,St.Louis,USA),3.87%}

magne-siumstearateRiedeldeHaën®

(Sigma–AldrichCo.,St.Louis,USA), 33.35%lactosemonohydrateD-Vetec®_{(VetecFineChemicalsLtd.,}

RiodeJaneiro,Brazil)6.9%cornstarchDuryea® _{(UnileverBrazil}

IndustrialLtda,Pernambuco,Brazil),28.5%waterand26.64%dry ECb(Carvalhoetal.,2013).

QuantitativeanalysisofpolyphenolsandtotaltanninsinECband GCb

In analysis of polyphenols and total tannins 0.750g of lyophilizedECb and 2.81g of GCb equivalentto0.750gof ECB wereused.Subsequentlyweusedthereductionof phosphomolyb-dotungstic acidtechnique in an alkaline medium (20%sodium carbonate). Later the absorbance was measured at 760nm in UV-VISmodelUVmini-1240spectrophotometer(Shimadzu Corpo-ration,Kyoto,Japan)(Sáetal.,2015).

Levels ofpolyphenols and tannins werecalculated fromthe absorbance submittedto the equationof thestraight obtained bythestandardcurveofthepyrogallicacidinconcentrationsof 0.02–0.10mgml−1 _{inreactionwiththephosphomolybdotungstic} acidin an alkaline medium,lyophilized bovine serum albumin wasusedtocomplexationwithtannin.Thepercentageof polyphe-nolsandtanninswasobtainedapplyingtheformuladescribedby Carvalhoetal.(2013).

%Pf= x(mg/ml)·FD·100

m(mg)

where %Pf=polyphenols percentage; x=sample concentration obtainedinstraightequation;DF=dilutionfactorofthesolution; m=massofthesample.

Animalsusedinthestudy

Wistarmaleratswereused,weighingaround210±30g, dur-ing the animals activities were placed in individual metabolic cagesstainlesssteel,measuring60cm×50cm×22cm,keptin air-conditionedenvironmentwithtemperaturearound25±3◦_Cand

humidityof50±10%,photoperiodof12hlightanddark,fedwith standard feedforrodentsand water adlibitum. Thisstudywas approved bythe Ethics Committeeof theFederalUniversity of AmapáundertheProtocol002A/2012August6,2012.

Oraltestforglucosetolerance(OTGT)

Todetermineglucosetolerance,hyperglycemiawasinducedin normoglycemicanddiabeticratswith16hoffastingby admin-isteringbymouthofaglucosesolutionof4g/kgofbodyweight 30minaftertreatment.Bloodglucoselevelswereassessedat0,30, 60,90,120and180min.Theanimalsweredividedintofivegroups (n=5),adiabeticgrouptreatedwithdistilledwater0.5ml/animal (DTA), thegrouptreatedwithglibenclamidebymouth 3mg/kg (GBC),diabeticgrouptreatedwithGCbbymouth500mg/kg, dia-beticgrouptreated withECbbymouth500mg/kg,non-diabetic groupofanimalstreatedwithdistilledwater0.5ml/animal(NDC).

InductionofDiabetesmellitus

Theinductionofdiabeteswasperformedinanimalsafter16h fastingperiod,byintraperitonealinjectionofstreptozotocin(STZ) (SIGMA-AldrichInc.,St.Louis,MO,USA)dissolvedin0.01Msodium citratebuffer(pH 4.5),witha doseof55mg/kgin a volumeof 1ml/kgbodyweight.FourdaysafterSTZinjection,animalswere considered diabeticwith blood sugar >300mg/dlurine glucose >100mg/dl, polydipsia and polyuria. The animals weredivided into five groups (n=5), a diabeticgroup treated with distilled water0.5ml/animal(DTA),thegrouptreatedwithglibenclamide by mouth 3mg/kg (GBC), diabetic group treated with GCb by mouth 500mg/kg, diabetic group treated with ECb by mouth 500mg/kg,animalsandnon-diabeticgrouptreatedwithdistilled water0.5ml/animal(NDC).

Developmentandexperimentalevaluation

Diabeticanimalswerekeptinmetaboliccagesduringthe30 daysoftreatment,wheretheywereevaluateddaily,bodyweight, water intake, foodintake, and urine volume. Glyemiaand gly-cosuriawereevaluatedeveryfivedays,thebloodcollectionwas performedbyretrorbitalplexusandglucoselevelsevaluatedby photocolorimetric methodglucose-oxidase(Glucox 500, Doles®

Reagentsand EquipmentLab. Ltda.,Goiânia-GO, Brazil). On the 30thdayoftreatmentwasheldbloodcollectionforbiochemical analysisoftotalprotein,totaltriacylglycerides,totalcholesterol, highdensitylipoprotein(HDL),low-densitylipoprotein(LDL),urea, creatinine,transaminaseglutamicoxaloaceticandglutamic pyru-victransaminase.AlltestswereperformedusingreagentsDoles®

ReagentsandEquipmentLabindustry.Ltda.(Goiânia,GO,Brazil) and the samplesanalyzed in UV-VIS model UVmini-1240 (Shi-madzuCorporation,Kyoto,Japan).

Statisticalanalysis

We used analysisof variance (ANOVA) followed by Tukey’s test,resultswithsignificancelevelofp<0.05wereconsidered sta-tisticallysignificant.GraphPadInstat® _andPrism®_{(version5.03)}

softwareswereusedforanalyzes.

Results

Quantificationofpolyphenolsandtotaltanins

0.00 0.04 0.08 0.12 0.0

0.4 0.8 1.2 1.6

Concentration (mg ml–1₎

Ab

s

.(

U

.A

)

Fig.1.Standardcurvepyrogallicacidbyspectrophotometry(=760nm)at con-centrationsfrom0.02to0.10mgml−1_{,straightlineequationy=10.64x−0020,the}

correlationcoefficientr2_=0.9964.

0 30 60 90 120 150 180

80 160 240 320 400 480 560

ECb GCb DTA GBC

NDC

*** ***

***

*** _*** *** ***

**

*** ***

*** **

*** ***

*** *** ***

***

Time (min)

Bood glucose levels (mg dl

–1

)

Fig.2. Effectoftreatmentsonthebloodglucoselevelsofdiabeticratsand non-diabeticintheTOTG.Significance:**p<0.01and***p<0.001,ComparedwithDTA group.Valuesexpressthemean±SD(n=5/group).

theECbwere0.025±0.0019mgml−1 _{representing8.36%oftotal} polyphenolsand0.016±0.0014mgml−1_{inoftanninswith5.33%.} InGCbpolyphenolsandtotaltanninscontentswere0.021±0.0023 and 0.014±0.0017mgml−1 _{with percentages of 7% and 4.66%} respectively.

Oraltestforglucosetolerance(OTGT)

IntheOTGTperformedinnormalanddiabeticmice(Fig.2),all groupspresentedareductionofbloodglucoselevelsinallanalyzed timeswithexceptionoftheDTAgroup.Thegroupsofdiabeticrats treatedwithGCbandECbreducedbloodsugarlevelssignificantly (p<0.001) when compared to the DTA group, reaching 28.98% (144±6.9mgdl−1_{)and41.86%(208}

±13.58mgdl−1_)ofreduction intheendoftheanalysisperiodrespectively.Thegrouptreated withthestandarddrug(GBC)alsopresentedstatisticallysignificant (p<0.001),withglucosereductionvaluesof288±13.46mgdl−1_, equivalentto57.95%.TheNDCgrouppresentedthecharacteristic profileofnon-diabeticanimals,withpeakglucoselevelsat60min andthendecreaseinsubsequenttimes.

Treatmenteffectonclinicalparameters

TheDTAgrouppresentedawidevariationintheevaluated clin-icalparameters(Table1),comparedtotheNDCgroupisobserved

0 5 10 15 20 25 30

0 100 200 300 400 500 600

DTA

GCb NDC GBC

ECb

*** *** *** *** *** *** ***

*** **

*** *** *** *** _***

*** ** _**

***

Treatment days

Urinary glucose (mg dl

–1

)

Fig.3.Effectoftreatmentsonglucoselevelsinurineofdiabeticandnon-diabetic mice.Significance:**p<0.01and***p<0.001ComparedwithDTAgroup.Values expressthemean±SD(n=5/group).

thattheanimalsinDTAgrouplostweight,increasedfeedintake (polyphagia), water intake(polydipsia)and urination(polyuria) significantly(p<0.05 andp<0.001),thesearetypicalsymptoms of diabetes in untreated individuals. From the treatment with standard drug (GBC) and oral formulations, extract (ECb) and granulated(GCb),itwasnotedthatthesetreatmentswereable to improve the clinical parameters of diabetic animals signifi-cantly(p<0.001)whencomparedwiththeDTAgroup.Regarding theweightdevelopment,an increaseof body weightof 11.76% (GBC),18.09%(GCb)and14.02%(ECb)wasobserved,therewereno significantfindingsaboutpolyphagiabecausethediabeticanimals hadfoodconsumptionvaluesveryclose.Regardingpolydipsiaand polyuria,significantreductions(p<0.001)wereobserved,where theGBCgrouppresentedreductionsof41.81%and63.10%,theGCb grouppresented44.27%and55.33%andtheECbgroup47.55%and 53.39%respectively.

Effectoftreatmentonglycosuriaandglycemia

Fromthedetermination ofglucoselevelsin urine(Fig.3), it was observed that the non-diabeticgroup (NDC) presented an averageof7.12±3.51mgdl−1_{,buttheDTAgroupshowedhigh} val-uesofaverage, presenting442.33±44.6mgdl−1_.WhenECband GCbtreatedgroupsarecomparedtoDTA,asignificantreduction (p<0.001) in glucose excretion could be observed after treat-ment, with reductions of 42.80% and 34.33% respectively. The grouptreatedwithGBCalsopresentedasignificantreductionwith p<0.001,representingadecreaseof64.83%.Theevaluationofblood glucose(Fig.4)showedthattheNDCgrouppresentedaglycemia meanof160.77±08.6mgdl−1_{whereasDTAgrouppresentedvery} highbloodglucoselevelswitha meanof455.08±18.4mgdl−1_. Whencompared totheGBCgroups,GCband ECbwiththeDTA groupaftertreatment,itwasobservedthattherewasasignificant reductioninbloodglucoselevelswithp<0.001,correspondingto 32.19%reductions,18.88%and20.89%,respectively.

Effectoftreatmentsonbiochemicalparameters

Table1

Effectoftreatmentsfor30daysonMDclinicalparametersinanimalsinducedwithstreptozotocin(weightdevelopment,polyphagia,polydipsiaandpolyuria).

Clinicalparameters Groups

NDC DTA GBC GCb ECb

Bodyweight(g/day) 275±6.4a ₂₂₁

±5.8 247±5.1a ₂₆₁

±7.3a ₂₅₂

±6.4a

ConsumptionRation(g/day) 30±5.2b ₄₀

±4.1 35±3.3 36±3.7 37±5.5

Waterintake(ml/day) 46±5.3a _{122±15.2 71±13.4}a _68±8.1a _64±9.7a

Urinevolume(ml/day) 12±5.7a _{103±8.9 38±6.2}a _46±5.8a _48±6.5a

Thedatarepresentmean±SD(n=5/group).

a_{p<0.001representsastatisticallysignificantresultscomparedwiththeDTAgroup.} b_{p<0.05representsastatisticallysignificantresultscomparedwiththeDTAgroup.}

0 5 10 15 20 25 30

100 170 240 310 380 450 520

GBC

GCb DTA

NDC ECb

** *

***

*** *** *** **

* *

**

***

*** ***

***

*** _*** ***

*** *** ***

Treatment days

Bood glucose levels (mg dl

–1

)

Fig.4. Effectoftreatmentsonthebloodglucoselevelsofdiabeticratsand non-diabetic.Significance:*p<0.05and**p<0.01,***p<0.001.ComparedwithDTA group.Valuesexpressthemean±SD(n=5/group).

levels,creatinineandurea,actedincreasingserumlevelsoftotal protein.InthegrouptreatedwithGBCwasalsopossibletoobserve significantimprovement(p<0.001)inthebiochemicalparameters, withtheexceptionoftransaminaseAST.

Discussion

Severalstudieshavebeendevelopingandstandardizing formu-lationsbasedonplantextractsinordertomakesuchextractsa moretechnologicalpharmaceuticalproductandoptimizeits phar-macologicaleffects(Lindenetal.,2000;Sartorietal.,2003;Carvalho etal.,2013).

In thisstudy,the ECbandGCb formulationswere standard-izedasthedeterminationofpolyphenolsandtotaltannins,where showedhighcontentofthesesubstancescollaboratingwiththe resultsobtainedby Carvalhoet al.(2013),but thegranule pro-ductiontechnique(GCb)fromECbdemonstratedaslightdecrease

of 1.36% in this content,this decrease waspossiblydue to the greatoxidativeeaseofthesephenolicsubstances(Robardsetal., 1999).

Thegreatantioxidantpotentialofmedicinalplantsespecially those with largecontent of polyphenoliccompounds has been proven,theseantioxidantplantshasgainedanimportantroleas asourceoftreatmentfordiseasesthatpresenthighproductionof freeradicals(FR),especiallymetabolicandgeneticdisordersrelated diseasesasdiabetes,dyslipidemiaandcancer.Amongthe antiox-idant phenolic compounds we can highlight, phenolic acids, flavonoids, tannins, coumarins and carotenoids (Marles and Farnsworth,1995;Perezetal.,1998;Ojewole,2002;Aslanetal., 2010).

TheOTGTisatestthatevaluatestheabilityofantidiabeticdrugs toreducesharplythepostprandial glycemia,examplesofdrugs thatreducebloodglucoseinthistestarethosewhoworkdirectly inthepancreas␤-cellssecretinginsulinsuchassulfonylureasor drugsthatinhibitglucoseabsorptionbythegastrointestinaltract andincreasethesensitivitytoinsulininperipheraltissuessuchas thebiguanides(Souzaetal.,2009).

AccordingtoSouzaetal.(2009)innormoglycemicrats,the ele-vation ofpostprandial glycemia afterglucose overload,and the consequentnormalizationtobasallevelsafterabout120min, fea-turinganormalfunctioninglucosemetabolism.

InOTGTitwaspossibletoobservethatboththeECbandtheGCb presentedeffectonglucosemetabolism,withasignificant reduc-tionofbloodglucosewhencomparedwithDTA,standardGBCdrug alsosignificantlyreduced,butthisreductiondidnotleadtobasal levelsasobservedin normoglycemicgroup(NDC),this reduced effectispossiblyduetodestructionofpancreatic␤cellsbythe actionofSTZ,sothereaislittleamountinsulintobesecretedby GBCeffect.

Thereductionofglycemiaindiabeticratsbymedicinalplants containingpolyphenolsanddetectedbyOTGThasbeendescribed inseveralstudies(PandaandKar,2007;Jiaetal.,2009;Lietal., 2015)aswellastheresultsobtainedinthisstudyusing formula-tionsofC.brasiliensespecies.

Table2

Effectoftreatmentsfor30daysonbiochemicalparametersofdiabetesinSTZ-inducedanimals.(Weightdevelopment,polyphagia,polyuriaandpolydipsia.).

Biochemicalparameters Groups

NDC DTA GBC GCb ECb

Totalproteins(g/dl) 6.80±0.12a _{4.73±0.27 6.89±0.11}a _7.03±0.07a _6.93±0.11a

Triacylglycerides(mg/dl) 82.4±8.8a _241.2

±24.7 163.4±15.4a _131.7

±10.8a _159.4 ±12.3a

Urea(mg/dl) 45.7±4.9a _93.2

±13.2 62.3±9.4a _64.8

±7.5a _59.5

±9.5a

Creatinine(mg/dl) 0.56±0.03a _0.94

±0.08 0.64±0.05a _0.69

±0.03a _0.59

±0.05a

TotalCholesterol(mg/dl) 59.7±5.5a _165.1

±16.4 115.5±11.1a _98.3

±9.3a _96.4

±11.6a

HDL(mg/dl) 37.1±4.6 34.1±8.9 30.3±6.8 39.8±6.1 35.6±7.2

LDL(mg/dl) 20.1±6.1a _{72.7±7.5 52.5±6.6}a _32.2±5.9a _28.9±6.3a

AST(U/dl) 54.5±4.4a _{79.2±6.7 75.7±3.6 59.3±4.8}a _65.9±5.1b

SGPT(U/dl) 65.4±6.7a _94.3

±5.4 76.6±6.1b _79.1

±7.1b _74.3

±6.3a

Thedatarepresentthemean±standarddeviation(n=5/group).

Authors report several mechanisms of action proposed for plantscontainingpolyphenoliccompoundandthesecaninvolve, protection of the pancreatic ␤-cells from oxidative damage, increasedinsulinsecretion,increasedsensitivityofperipheral tis-suesinresponsetoinsulinandreduced gastrointestinalglucose absorption(Seziketal.,2005;PandaandKar,2007).

Indiabeticindividuals,lackofinsulinleadstovariousclinical signsandsymptoms, includingchronichyperglycemia,elevated bloodglucoselevelinurine,constantthirst(polydipsia),increased urination(polyuria),weightloss,andseverestarvation (polypha-gia)(Mahendranetal.,2014).

Insulindeficiencycauseshyperglycemiaand whentheblood glucoselevelis higherthan therenalfiltrationthreshold, there isthepresenceofglucoseinurine,aswellasincreasedexcreted urinevolumeduetoosmoticimbalance,hyperosmolarity,because ofhighlevelsof circulating glucose,causeswater topassfrom intracellular toextracellular medium in order to maintain this osmoticequilibrium, intracellular dehydration is recognized by brain osmoreceptors generating a response triggering intense thirst,characteristic of diabetes (Lerco et al.,2003; Mahendran etal.,2014).

Insulinisahormonethatfacilitatestheglucosetransportinto muscle cells and adipocytes, increases the synthesis and stor-ageofcellularproteins,muscleglycogenandtriacylglyceridesin adipocytes,anddecreaseproteincatabolism(MayandBuse,1989). Lackof insulin causes intense catabolism process of structural proteinsand␤-oxidationoffattyacidstoformsubproductsto glu-coneogenesis,thelossorbreakdownofthesestructuralproteins directlyreflectinreduced bodyweight(RameshandPugalendi, 2006),thisprocesscanbeobservedindiabeticanimalstreatedwith distilledwater(DTA).

Treatment for 30 days withECb and GCb formulations sig-nificantlyreducedhyperglycemia(20.89%and18.88%)andurine glucose(42.80%and34.33%)ofthediabeticratsrespectively,and actedalsoreducingpolydipsia,polyuriaandlossofbodymass.

Theimprovementofsymptomsandclinicalsignsduring30days oftreatmentwithECbandGCb,reinforcetheantidiabetic activ-ityofCbspecies,aspossiblythisimprovementisduetothehigh contentofpolyphenoliccompounds,wherethissubstanceswould actrestoringthe␤-pancreaticcellsfromoxidativedamagecaused bySTZandconsequentlyincreasingtheinsulinproduction(Sezik etal.,2005),theseresultsconfirmtheresultsobtainedbyseveral authors,whoconcludethatnaturalantioxidantsubstancessuch aspolyphenoliccompoundshavegreatpotentialforanti-diabetic activity(Sabuetal.,2002;Houetal.,2003;PandaandKar,2007; Jiaetal.,2009;Aladeetal.,2012).

Liver and kidneys are the main organs responsible for metabolismandexcretion ofendogenous substancesand xeno-biotics, the dysfunction of these organs leads to changes in biochemicalparameters,beingthemarkerstransaminasesASTand ALTwhodeterminehepatocytesdamageandelevationof creati-nineandureaindicatingrenaldysfunction(AlmdalandVilstrup, 1988;Ohaeri,2001).

InDTAgroup,alargeincreaseinlevelsofAST,ALT,creatinineand ureawereobservedwhencomparedtotheNDCgroup,theseresults indicatethatthe animalsintheDTA groupshowpossibleliver andkidneydysfunction.Thesechangesarejustifiedbecausethe inductionofdiabetesbystreptozotocinandtheresultingchronic hyperglycemia,arefactorsthatleadtoformationofreactiveoxygen species(ROS),whichinturncauseslipidperoxidationanddamage tocellmembranes,theseROSareresponsiblebysecondary com-plicationsofdiabetesmellitussuchaskidney,liver,retina,blood vesselsandnervedamage(Huntetal.,1988).

TreatmentoftheanimalsusingECbandGCbwereableto sig-nificantlyreduce(p<0.001andp<0.05)creatinine,urea,ASTand ALTlevels,theseresultsdemonstratethattherewasasignificant

reductioninliverdamageandareinlinewithresultsobtainedby (Ohaeri,2001;Rameshetal.,2010).Theimprovementofrenal dys-functioncanbejustifiedbytheincreaseintotalproteinlevels,as indiabeticindividualsnephropathyisthemainfactorforprotein excretioninurine,theseresultsareconsistentwiththoseobtained byBakris(1993)andTuvemoetal.(1997).

Theresultsofrenalandhepaticbiochemicalparametersalso reinforcethelackoftoxicityofthedosesoftheformulationsused inthisstudy,andcorroboratewiththeresultsobtainedbyOliveira etal.(2014),whichdemonstratethatthetreatmentfor30days with500mg/kgofECB,noshowedsignsoftoxicityinrats.

Diabetes is a disease that has a great influence on lipid metabolismcausingincreasesinserumtriacylglycerides, choles-terol and lipoproteins, this fact can beobserved in DTA group whereispossibletoseeaverysignificantincrease(p<0.001)in triacylglyceride,cholesterolandLDLserumvalues.Treatmentwith ECbandGCbwereabletoreducetheincreaseofthe triacylglyc-erideslevels,cholesterolandLDL, buttherewasnosignificance intheresultsofHDL,similarresultswereobtainedwith experi-mentaldiabeticmiceaftertreatmentwithplantextractscontaining polyphenols,whereasignificantreductioninlipidlevelscouldbe observed(Ramadanetal.,2009;Islam,2011).

Theincreaseinlipidlevelsondiabetesaremainlyresponsiblefor mediatingtheformationofRLbyperoxidationofunsaturatedfatty acids,cholesterol and lipoproteins, increasedlipid peroxidation leadstomembranedamageandconsequentlyorgansdysfunction beingthisanimportantriskfactorforatherosclerosisandcoronary arterydisease(Maghranietal.,2004;Alfyetal.,2005).

Decreaseonlipidlevelsandconsequentlythereductionoflipid peroxidationis improveddue tothehighantioxidantpotential ofpolyphenoliccompoundsthatactbymechanismsofreaction inhibition in the peroxidationchain and can reduce complica-tionsresultingfromdiabetes(KamalakkannanandPrince,2006; Mahendranetal.,2014).

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thattheproceduresfollowedwereinaccordancewiththe regula-tionsoftherelevantclinicalresearchethicscommitteeandwith thoseoftheCodeofEthicsoftheWorldMedicalAssociation (Dec-larationofHelsinki).

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat nopatientdataappearinthisarticle.

Authors’contributions

HOC,BSFS,IVFS,andHKcontributedtothepreparationof for-mulationsandexecutionofexperimentaltests.HOC,RLR,CPFand JCTC,contributedexecutionofexperimentaltestsandto develop-mentandcriticalreadingofthemanuscript.Allauthorsreadand approvedthefinalmanuscriptsubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

EFP-00007609,theCAPESforthegrantbestowed,andGuyamazon Project(FAPEAP–AIRD)andCNPqProcessnumber407768/2013-0.

References

ADA-AmericanDiabetesAssociation,2011.Standardofmedicalcarein Diabetes-2011.DiabetesCare34,11–61.

Alade, G.O., Adebajo,A.C.,Omobuwajo, O.R., Proksch, P., Verspohl, E.J., 2012. Quercetin,aminorconstituentoftheantihyperglycemicfractionofBauhinia

monandraleaf.J.Diabetes4,439–441.

Alfy,A.,Ahmed,A.,Fatani,A.,2005.Protectiveeffectofredgrapeseed sproantho-cyanidinsagainstinductionofdiabetesbyalloxaninrats.Pharmacol.Res.52, 264–270.

Almdal,T.P.,Vilstrup,H.,1988.Strictinsulintreatmentnormalizestheorganic nitro-gencontentsandthecapacityofurea-Nsynthesisinexperimentaldiabetesin rats.Diabetologica31,114–118.

Aslan,M.,Orhan,N.,Orhan,D.D.,Ergun,F.,2010.Hypoglycemicactivityand antioxi-dantpotentialofsomemedicinalplantstraditionallyusedinTurkeyfordiabetes. J.Ethnopharmacol.128,384–389.

Bakris,G.L.,1993.Diabeticnephropathy.Whatyouneedtoknowtopreservekidney function.Postgrad.Med.93,89–90.

Calixto,J.B.,2000.Efficacy,safety,qualitycontrol,marketingandregulatory guide-linesforherbalmedicines.Braz.J.Med.Biol.Res.33,179–189.

Carvalho,H.O.,Medeiros,B.J.L.,Sá,B.M.,Araújo,J.T.C.,Kawakami,M.Y.M.,Favacho, H.A.S.,etal.,2013.Studyofdissolutionprofilesanddesintegrationofcapsules containingthedriedhydroethanolicextractofCalophyllumbrasiliense.Rev.Bras. Farmacogn.23,194–199.

Hou,C.C.,Lin,S.L.,Cheng,J.T.,Hsu,F.L.,2003.Antidiabeticdimericguianolidesand alignanglycosidefromLactucaindica.J.Nat.Prod.66,625–629.

Hunt,J.V.,Dean,R.T.,Wolff,S.P.,1988.Hydroxylradicalproductionand autoxida-tiveglycosylation.Glucoseautoxidationasthecauseofproteindamageinthe experimentalglycationmodelofdiabetesandaging.Biochem.J.256,205–212. Islam,S.,2011.Effectsoftheaqueousextractofwhitetea(Camelliasinensis)ina

streptozotocin-induceddiabetesmodelrats.Phytomedicine19,25–31. Jia,Q.,Liu,X.,Wu,X.,Wang,R.,Hu,X.,Li,Y.,etal.,2009.Hypoglycemicactivityofa

polifenolicoligomer-richextractofCinnamomumparthenoxylonbarkinnormal andstreptozotocin-induceddiabeticrats.Phytomedicine16,744–750. Kamalakkannan,N.,Prince,P.,2006.Antihyperglycaemicandantioxidanteffectof

rutin,apolyphenolicflavonoid,instreptozotocin-induceddiabeticWistarrats. BasicClin.Pharmacol.Toxicol.98,97–103.

Lerco,M.M.,Spadella,C.T.,Machado,J.L.,Schellini,M.S.A.,Padovani,C.R.,2003. Caracterizac¸ãodeummodeloexperimentaldediabetesmellitus,induzidopor aloxanaemratos:estudoclínicoelaboratorial.ActaCir.Bras.18,132–142. Li,L.,Xu,J.,Mu,Y.,Han,L.,Liu,R.,Cai,Y.,etal.,2015.Chemicalcharacterizationand

antihyperglycaemiceffectsofpolyphenolenrichedlongan(Dimocarpuslongan

Lour.)pericarpextracts.J.Funct.Foods13,314–322.

Linden,R.,GonzálezOrtega,G.,Petrovick,P.R.,Bassani,V.L.,2000.Responsesurface analysisappliedtothepreparationoftabletscontainingahighconcentrationof vegetablespray-driedextract.DrugDev.Ind.Pharm.26,441–446.

Maghrani,M.,Lemhadri,A.,Zeggwagh,N.,Amraoui,M.,Haloui,M.,Jouad,H.,etal., 2004.EffectsofanaqueousextractofTriticumrepensonlipidmetabolismin normalandrecent-onsetdiabeticrats.J.Ethnopharmacol.90,331–337. Mahendran,G.,Manoj,M.,Murugesh,E.,SathishKumar,R.,Shanmughavel,P.,

Rajen-draPrasad,K.J.,etal.,2014.Invivoanti-diabetic,antioxidantandmolecular dockingstudiesof1,2,8-trihydroxy-6-methoxyxanthoneand 1,2-dihydroxy-6-methoxyxanthone-8-O-d-xylopyranosyl isolated from Swertia corymbosa. Phytomedicine21,1237–1248.

Marles,R.J.,Farnsworth,N.R.,1995.Antidiabeticplantsandtheiractiveconstituents. Phytomedicine2,137–189.

May,M.E.,Buse,M.G.,1989.Effectsofbranched-chainaminoacidsonprotein turnover.DiabetesMetab.Rev.5,227–245.

Noel,P.H.,Pugh,J.A.,Larme,A.C.,Marsh,G.,1997.Theuseoftraditionalplant medicinesfornon-insulin-dependentDiabetesmellitusinsouthTexas. Phy-tother.Res.11,512–517.

Ohaeri,O.C.,2001.Effectofgarlicoilonthelevelsofvariousenzymesintheserum andtissueofstreptozotocindiabeticrats.Biosci.Rep.21,19–24.

Ojewole, J.A.O., 2002. Hypoglycemic effect of Clausena anisata (Willd) Hook methanolicrootextractinrats.J.Ethnopharmacol.81,231–237.

Oliveira,M.A.,Lemos,L.M.S.,Oliveira,R.G.,Dall’Oglio,E.L.,SousaJunior,P.T.,Martins, D.T.O.,2014.EvaluationoftoxicityofCalophyllumbrasiliensestembarkextract

byinvivoandinvitroassays.J.Ethnopharmacol.155,30–38.

Panda, S., Kar, A., 2007. Apigenin (4′_{,5,7-trihydroxyflavone) regulates} hyper-glycemia,thyroiddysfunctionandlipidperoxidationinalloxaninduceddiabetic mice.J.Pharm.Pharmacol.59,1543–1548.

Perez,R.M.,Zavala,G.M.A.,Perez,S.G.,Perez,C.G.,1998.Antidiabeticeffectof com-poundsisolatedfromplants.Phytomedicine5,55–75.

Ramadan,G.,El-Beih,N.M.,AbdEl-Ghffar,E.A.,2009.Modulatoryeffectsofblackv. greenteaaqueousextractonhyperglycaemia,hyperlipidaemiaandliver dys-functionindiabeticandobeseratmodels.Br.J.Nutr.102,1611–1619. Ramesh,B.,Pugalendi,K.V.,2006.Antihyperglycemiceffectofumbelliferonein

streptozotocin-diabeticrats.J.Med.Food9,562–566.

Ramesh,B.K.,Maddirala,D.R.,Vinay,K.K.,Shaik,S.F.,Tiruvenkata,K.E.G.,Swapna, S., et al., 2010. Antihyperglycemic and antihyperlipidemic activities of methanol:water(4:1)fractionisolatedfromaqueousextractofSyzygium

alterni-foliumseedsinstreptozotocininduceddiabeticrats.FoodChem.Toxicol.48,

1078–1084.

Robards,K.,Prenzler,D.K.,Tucker,G.,Swatsitang,P.,Glover,W.,1999.Phenolic com-poundsandtheirroleinoxidativeprocessesinfruits.FoodChem.66,401–436. Sá,B.M., Lima,C.S., Silca, U.D.A.,Carvalho, H.O.,Fernandes, C.P.,Resque,R.L., etal.,2015.Subchronictoxicityevaluationofthehydroethanolicextractfrom

Endopleurauchi(Huber)CuatrecinWistarrats.Afr.J.Pharm.Pharmacol.9,

223–229.

Sabu,M.C.,Smitha,K.,RamadasanKuttan,2002.Anti-diabeticactivityofgreentea polyphenolsandtheirroleinreducingoxidativestressinexperimentaldiabetes. J.Ethnopharmacol.83,109–116.

Sacks,D.B.,Bruns,D.E.,Goldstein,D.E.,Maclaren,N.K.,Mcdonald,J.M.,Parrott,M., 2002.Guidelinesandrecommendationsforlaboratoryanalysisinthediagnosis andmanagementofDiabetesmellitus.Clin.Chem.48,436–472.

Sartori,L.R.,Ferreira,M.S.,Perazzo,F.F.,MandalhoLima,L.,Carvalho,J.C.T.,2003. AtividadeantiinflamatóriadogranuladodeCalendulaofficinalisL.eMatricaria

recutitaL.Rev.Bras.Farmacogn.13,17–19.

Sezik,E.,Aslan,M.,Yesilada,E.,Ito,S.,2005.HypoglycemicactivityofGentianaolivieri

andisolationoftheactiveconstituentthroughbioassay-directedfractionation techniques.LifeSci.76,1223–1238.

Silva,K.L.,Santos,A.R.S.,Matos,P.E.O.,Yunes,R.A.,Delle-Monache,F.,Cechinel-Filho, V.,2001.ChemicalcompositionandanalgesicactivityofCalophyllumbrasiliense. Therapie56,431–434.

Souza,V.H.,Barbosa,A.P.O.,Cardoso,G.C.,Marreto,R.N.,Barreto-Filho,J.A.S., Anto-niolli,A.R.,etal.,2009.Avaliac¸ãodoPotencialAntidiabéticodeCincoPlantas MedicinaisemRatos.Lat.Am.J.Pharm.28,609–612.