ACUTE AND SUBCHRONIC EFFECTS OF COCHLOSPERMUM VITIFOLIUM IN BLOOD GLUCOSE LEVELS IN NORMOGLYCEMIC...

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ACUTE AND SUB-CHRONIC EFFECTS OF COCHLOSPERMUM VITIFOLIUM IN BLOOD GLUCOSE LEVELS IN NORMOGLYCEMIC AND STZ-NICOTINAMIDE-INDUCED DIABETIC RATS

ROLFFY ORTIZ-ANDRADEa,� , MARIANA TORRES-PIEDRAa, JUAN CARLOS SÁNCHEZ-SALGADOa, SARA GARCÍA-JI-MÉNEZa, RAFAEL VILLALOBOS-MOLINAb, MAXIMILIANO IBARRA-BARAJASb, ITZELL GALLARDO-ORTIZb AND SAMUEL ESTRADA-SOTOa,*

(Received May 2009; Accepted August 2009)This paper is dedicated to Professor Doctor Rachel Mata for her 60th birthday

ABSTRACT

The aim of the present study was to determine the acute and sub-chronic hypo-glycemic and antidiabetic effects of methanol extract from Cochlospermum vitifo-lium (MECv), a medicinal plant used in Mexican folk medicine for the treatment of hypertension, diabetes and liver damage-related diseases. Intragastric admin-istration of MECv (100 mg/Kg) induced a significant decrease on plasma glucose in normoglycemic and STZ-nicotinamide-induced diabetic rats in acute and sub-chronic models compared to control (p< 0.05). The increase in plasma glucose after sugars (glucose and sucrose) administration was partially suppressed by MECv (p< 0.05). Moreover, extract induced in vitro α-glucosidase activity inhibition in a concentration-dependent manner (IC50= 1.9 mg/mL). Furthermore, biochemical profiles as Glucose, total cholesterol, HDL and triglycerides (TG) were modified in serum blood after sub-chronic MECv treatment on normoglycemic and diabetic rats. Finally, it was established the pre-clinical security and tolerability parameters of MECv through toxicological evaluation in mice and rats using OECD protocols (LD50> 5000 mg/Kg). Findings suggest that MECv might exert its hypoglycemic and anti-diabetic effect by extra-pancreatic action through both partial suppression of carbohydrate absorption on intestine lumen and acting as α-glucosidase inhibitor to reduce post-prandial rise of blood glucose.

Keywords: acute toxicity; α-glucosidase inhibitor; antidiabetic; Cochlospermum vitifolium; Mexican medicinal plants.

RESUMEN

El objetivo del presente proyecto fue determinar el efecto hipoglucemiante y antidia-bético in vivo del extracto metanólico íntegro de Cochlospermum vitifolium (EMCv),

aFacultad de Farmacia, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001 Col. Chamilpa, C.P. 62209 Cuernavaca, Morelos, México.bUnidad de Biomedicina, Facultad de Estudios Superiores-Iztacala, Universidad Nacional Autónoma de México, Av. De los Barrios No. 1, Los Reyes Iztacala, C.P. 54090, Tlalnepantla, Estado de México.*Corresponding author: [email protected], Tel/fax: + (52 777) 3 29 70 89 (S. Estrada-Soto)� Taken in part from the PhD thesis of R. Ortiz-Andrade and sabbatical stay of Dr. S. Estrada-Soto

Acuteandsub-chroniceffectsofCochlospermum vitifolium Rev. Latinoamer. Quím. 37/2(2009)123

plantautilizadaenlamedicinatradicionalmexicanaparaeltratamientodelahiper-tensión,ladiabetesyenfermedadesrelacionadasconelhígado.Laadministraciónintragástrica de EMCv (100 mg/Kg) provocó una disminución significativa de la glucosaplasmáticaenratasnormoglucémicasydiabetizadasconestreptozotocinaynicotinamida,enlosmodelosagudosysub-crónicoscomparadosconelcontrol(p<0.05).Elincrementodelosnivelesplasmáticosdeglucosadespuésdelaad-ministracióndeglucosaysucrosafueparcialmenteinhibidoporEMCv(p<0.05).Adicionalmente,elextractoinhibiólaactividadenzimáticain vitrodelasα-glucosi-dasasdemaneradependientedelaconcentración(CI50=1.9mg/mL).Másaún,losniveles plasmáticos de glucosa, colesterol, HDL y triglicéridos (TG) fueron modifi-cadosenlasratasnormoglucémicasydiabéticastratadasdemanerasub-crónicaconEMCv.Finalmente,seestablecieronlosparámetrospreclínicosdeseguridadytolerabilidaddelEMCvatravésdelosprotocolostoxicológicosestablecidosporlaOECD en ratas y ratones (DL50>5000mg/Kg).LosresultadosobtenidossugierenqueelEMCvproducesuefectohipoglucemianteyantidiabéticovíamecanismosdeacciónextrapancreáticosatravésdeunainhibiciónparcialdelaabsorcióndeglucosa en intestino e inhibiendo la actividad de las α-glucosidasas, y consecuen-temente,disminuyendolosnivelesplasmáticosdeglucosapostpandrial.

INTRODUCTION

Diabetes mellitus (DM) is a chronic disease caused by inherited and/or acquired defi-ciencyofinsulinproductionbypancreasaswell as modification of insulin sensitivity on tissuessuchasmuscle,liverandadiposetissue.Thesemetabolicalterationstriggertoxicglucoseaccumulationinserumbloodcausing hypertriglyceridemia, impairedHDL/LDL ratio, blindness, impaired blood coagulationandweight loss. Inaddition,long-term hyperglycemic process is animportantriskfactorfordevelopmentandprogressionofmicro-andmacrovascularcomplications for cardiovascular diseaseoutcome (Altan, 2003; Adisakwattana et al.,2005).Theunderlyinggoalofpharma-cological therapy against DM is to maintain anadequateplasmaticglucoseconcentra-tionduringextendedtimeperiods.Inthiscontext, thereexist fourmajorclassesoforalantidiabeticagentsforthetreatmentof these illnesses: insulin secretagogues,(sulfonylureas, meglitinide class agents,glucagon-like peptide 1 analogs, etc.)antihyperglycemic (biguanides), insulinsensitizers (thiazolidinediones) and α-glu-

cosidase inhibitors (Davis, 2005). These typeofdrugsworkondifferenttargetsitesincluding insulin secretion stimulationin beta pancreatic cells, suppression ofgluconeogenesisprocessinliver,increaseofinsulinsensitivityinperipheraltissuesas muscle, liver and adipose tissue andreducing intestinal absorption of starch,dextrin, and disaccharides by inhibitingthe α-glucosidase in the intestinal brush border, respectively. Thus, ethnomedicalstudies in Mexico have reveal that someplantremedieshavebeenusedtoimprovediabetes and its complications (Monroy-Ortiz and Castillo, 2007; Andrade-Cettoand Heinrich, 2005). However, only few medicinal plants have been scientifically evaluated to confirm its therapeutic pro-pertiesandtoxicologicalsecurityinanimalmodelsinordertoestablishpharmacologi-cal/toxicologicalbasistoextrapolateforfu-turehumanclinicaltrials(Andrade-Cettoand Heinrich, 2005; Ortiz-Andrade et al.,2005;2007).

Cochlospermum vitifolium(Willd.)Spren-gel isamedicinal treewidelydistributedalong rain-dry forest in Sierra de Huautla, Morelos. The bark is commonly used to

124 R. ORtiz-AndRAde, M. tORRes-PiedRA, et. Al.,

improvehighbloodpressure,diabetesandhepatobiliarydiseasesasjaundiceandcir-rhosis(Monroy-OrtizandCastillo-España,2007; Castillo-España et al., 2009; Sán-chez-Salgadoet al.,2007).Previousstudiesperformedbyourresearchgroupshowedthat methanolic (MECv) and hexanic (HECv) extractsfromC. vitifoliumbarkwereabletodecrease noradrenaline-induced contrac-tiononrataorticringsabout80and90%,respectively. Furthermore, both extractsdecreasebloodglucoselevelsinnormogly-cemicratmodelabout15%comparedwithcontrol group. HECv was more potent than MECv since decreased glucose levels first hour after oral administration while theothershowedahypoglycemiceffectuntil5 hours after administration (Sánchez-Salgadoet al.,2007).Theseexperimentalevidences suggest that C. vitifolium barkconstitutedanaturalsourceforbioactivecompounds that might improve diabetesand hypertension. Additionally, previouschromatographicanalysisofMECvlettheisolation of a pure crystalline compoundidentified as naringenin by X-ray diffraction and NMR techniques (Sánchez-Salgado et al., 2007), a flavonoide with pharmaco-logicaleffectindiabetesaswedescribedinpreviousexperiments(Ortiz-Andradeet al.,2008). Recent HPLC analysis allowed us to establishthatMECvcontain5%ofnarin-genin(Sánchez-Salgadoet al.,2009).

In this context, current study aimedtoevaluatehypoglycemicandantidiabeticactivities of methanolic extract from C. vitifolium (MECv) in normoglycemic andstreptozotocin-nicotinamide-induced dia-beticratstoestablishitspossiblemodeofactionaswellastodetermineitstoxicologi-cal profile.

MATERIALS AND METHODS

MaterialsGlibenclamide, nicotinamide, streptozoto-cin(STZ),glucoseandsucrosewerepur-

chasedfromSigma-Aldrich(St.Louis,MO,USA). Pentobarbital (Anestesal) was ob-tained from Glaxo-Smithkline (Mexico City, Mexico).Testkitsforenzymaticend-pointdeterminations for glucose, cholesterol,triglycerides (TG) and high density lipopro-tein (HDL) were obtained from Wiener Labs (MexicoCity,Mexico).

Plant material and preparation of MECvCochlospermum vitifolium(Willd.)Sprengelbarkwascollectedin October 2005. PlantinOctober2005.Plantmaterial was collected from its naturalhabitatindeciduousdryforestintheState of Morelos, Mexico and identified by Dr. P. Castillo-España. A voucher specimen(14628) has been deposited into CEAMISH-Herbarium (HUMO), Universidad Autóno-madelEstadodeMorelos,inCuernavaca,Morelosasreferencematerial.Theair-driedplant material was ground into powder.Crudeextractwaspreparedbymacerationof 2 Kg at room temperature in 4 liters of methanol for 72 h. After filtration, the ex-tractwasconcentratedin vacuo at 40°C and w/wpercentageyieldswasdetermined.

AnimalsMaleWistarrats(200-250g)wereobtainedfromFacultaddeEstudiosSuperiores(FES)Iztacalaanimalfacility,UniversidadNacio-nalAutónomadeMéxico(UNAM).Allratsweremaintainedunderstandardlaboratoryconditions (12-h light/dark cycle, 25 ± 2 °C and humidity 45-65 %). Prior to experimen-tation,ratswerefedwithstandardrodentdietandtapwaterad libitumduringoneweekinordertoadaptthemtolaboratoryconditions. All animal procedures wereconductedinaccordancewithourFederal Regulations for Animal Experimentation and Care (SAGARPA, NOM-062-ZOO-1999, México),andapprovedbytheInstitutionalAnimalCareandUseCommittee.

Induction of DiabetesSTZ was dissolved in citrate buffer (pH 4.5), andnicotinamidewasdissolvedinnormal


physiological saline solution. Diabetes was inducedinovernightfastedratsbyasingleintraperitoneal(i.p.)injectionof65mg/kgSTZ,15minafterthei.p.administrationof120mg/kgofnicotinamide(Masielloet al., 1998). Hyperglycemia was confirmed by theelevatedglucose levels inplasma,determined after 10 days. Animals withblood glucose concentration higher than150mg/dlwereusedforthestudy.

Experimental protocolThirty rats were divided into six groups,each consisting of five animals. Test sam-ples were suspended in 0.05% of Tween80 in saline solution (vehicle) and wereadministered orally by intragastric (i.g.)route using an intragastric tube. Rats were dividedintothefollowinggroups:• Group 1: Normoglycemic control.

Received only vehicle (0.5% Tween 80) (5mL/Kg).

• Group 2: Normoglycemic reference. Glibenclamide was given at dose of 5 mg/Kg

• Group 3: Normoglycemic treated. MECv wasgivenatdoseof100mg/Kg.

• Group 4: Diabetic control. Received only vehicle(0.5%Tween80)(5mL/Kg).

• Group 5: Diabetic reference. Glibenclamide was given at dose of 5 mg/Kg.

• Group 6: Diabetic treated. MECv was givenatdoseof100mg/Kg.

Acute experimental modelAprotocolpreviouslydescribedwasusedtoevaluatehypoglycemiceffect(Ortiz-An-dradeet al., 2008).Sixteenhoursbeforethe experiments, rats were fasted withfreeaccesstowater.Thebodyweightandfastingbloodglucoselevelsofallratsweredeterminedbeforethestartoftheexperi-ment.Bloodsampleswerecollectedfromthecaudalveinat0,1,3,5,and7haftervehicle,MECvandglibenclamideadminis-tration.Bloodglucoseconcentrationwasdetermined by using a glucose oxidase-

basedcommercialglucometer (AccutrendGCT, Roche Diagnostic®). The percentage variationofglycemia foreachgroupwascalculatedinrelationtoinitial(0h)level,accordingtotheequation:

WhereG0wereinitialglycemiavaluesandGx wereglycemiavaluesat+1,+3,+5and+7h,respectively(Verspohl,2002;Ortiz-Andradeet al.,2007).

Sub-chronic experimental modelThe samples (vehicle, test samples andglibenclamide) were administered 5 daysconsecutively. Blood glucose levels weredetermined 1 h after test samples wereadministeredandbodyweightwasdeter-minedbefore theadministration.On thefifth day, all animals were sacrificed and thetotalbloodcollectedformeasurementofbiochemicalparameters.Thepercentagevariationofglycemia foreachgroupwascalculated in relation to initial (first day) day of experimentaccording to the equation:

%Variationofglycemia=Dayx - Day1X100

Day1

Where Day1wastheinitialglycaemiavalueand Dayx were the glycaemia values at+secondday,+thirdday,+fourthdayand+fifth day respectively (Verspohl, 2002; Ortiz-Andradeet al.,2007).

Oral Glucose Tolerance TestsThirtyminutesafteradministrationoftestsamples, a dose of 2 g/kg of substrate(glucoseandsucrose)solutionwasadmi-nistered toeachrat.MECv (100mg/kg),vehicleandacarbosa(3mg/kg)weread-ministeredtoratsinthesamevolumeofsolution.Bloodsampleswerecollectedfromthe tail tip at 0 (before oral administra-

%Variationofglycemia=Gx − G0 X 100

G0


tion), 1, 1.5, 2, 2.5, 3 and 4 h after vehicle, positivecontrolandextractadministration.Bloodglucoseconcentrationwasestimatedasdescribed.

Biochemical ProfileBlood samples were centrifuged at 3000g for 10 min at 4 °C. Then, serum was removed for the respective analytical de-terminations. The serum glucose, totalcholesterol, HDL and TG concentrations were determined using commercial kits(WienerLabs,Mexico)byenzymaticpho-tocolorimetricmethods.

α-glucosidase activity assayEach animal (n= 4) was sacrificed by cer-vicaldislocation.Thesmallintestinewasobtained and flushed several times with ice-cold 0.9% NaCl, sodium phosphatebuffer 10 mM (pH 7.0) and 1 mg/mL of ampicillin. The intestine was cleaned ofadiposetissueandcutlongitudinally.Then,themucosawasscrapedwithaglassslideontoanice-coldglasssurface.Thematerialcontainingα-glucosidasewashomogenizedwith 10 strokes of a Teflon pestle, aliquoted in 1.8 mL cryotubes and stored at -25 °C untilrequired(Ortiz-Andradeet al.,2007).TotalproteinwasdeterminedbytheLowrymethod(Lowryet al., 1951). The α-glucosi-daseactivitywasassayedwithcornstarchas substrate (12.5 mg/mL, S-4126; Sigma-Aldrich,St.Louis,MO,USA)in0.1Mso-dium phosphate buffer (pH 7.0) in a volume of250µl.SubstrateswerepremixedwithMECvatvariousconcentrations(0.01–20mg/mL),thereactionstartedbyadditionof10mLofcrudeenzymeandthemixtureincubated at 37 °C for 10 min. The reac-tionwasstoppedbyadditionofacarbose(12.5 mg, enough for 99% inhibition) inan ice-water bath. Released glucose was quantified with a commercial glucose oxi-dase–based assay (Glucose GOD-PAP, GL 2614; Randox Laboratories, Antrim, UK) followingthemanufacturer’sinstructionsand was read at 492 nm and 630 nm for

turbiditycorrectioninamicroplatereader(StatFax 2100; Awareness Technology,LosAngeles,CA,USA).Thereactionwaslinearuntil15min.Inhibitoryactivitywasreportedaspercentageofinhibition.Assayswereperformedinquadruplicate(Ortiz-An-dradeet al.,2007).

LD50 estimationThe determination of LD50wascarriedoutusingthe‘AcuteToxicClass’method[Or-ganizationforEconomicCooperationandDevelopment (OECD) 423] as described in a previouswork(Ortiz-Andradeet al.,2008).Twelvemalemicewereused (30 -35g).Thestudywasconductedwithastartingdoseof5mg/kg.Eachdosewastestedonthreemice(fourgroups),andthenextstepwas selected depending on the mortalityobserved during the first 24 h of exposure. In the second step, 12 male Wistar rats(250–300g)wereused.Thistoxicologicalstudywasstartedatadoseof50mg/kg.Threeanimalswereusedfortestedgroups.Finally, the range of LD50 (category) wasestimated (OECD, 1996).

Statistical analysisAlldataareexpressedasmeanofexperi-ments±standarderrorofmean (S.E.M).Concentration–responsecurveswereplot-tedandexperimentaldatawereadjustedby the non-linear, curve fitting program Mi-crocal®Origin6.0(MicrocalSoftwareInc.,USA). Statistical analysis was performedbyonewayanalysisofvariance(ANOVA)and Dunett’s test (sample versus control). p-valueslessthan0.05wereconsideredtobe statically significant.

RESULTS AND DISCUSSION

We have published that hexanic extractfrom C. vitifolium (120 mg/kg) elicits asignificantly decrease in blood glucose in normoglycemic rats (about 15%) at first hourafteroraladministration.Therefore,


MECvalsoshowedanimportantdecreaseinplasmaticglucoseconcentration3hoursafteroraladministration(Sánchez-Salgadoet al.,2007).Basedonlastresults,wede-cidetodeterminetheeffectofMECv(100mg/kg)inacutenormoglycemicandSTZ-nicotinamideinduceddiabeticrats.Tables1-2showthati.g.administrationofMECvinduced a significant decrease in plasma glucose concentration in normoglycemicrats, during both acute and subchronictime periods compared with control rats(p<0.05).Inacutemodel,MECvreduced7%bloodglucose(Table1),whichdidnotreturntonormalat7hafteradministration(-13%).Inaddition,sub-chronictreatmentwith MECv (5 days) also induced a signifi-canthypoglycemiceffectatday2(approxi-mately-8%)andwassustainedduringthenextthreedaysoftreatment(-13%)(Table2). In DM rats, i.g. administration of MECv (100 mg/kg) also induced a significant reduction in plasma glucose at first hour,

and effect was preserved during all timeperiodoftheacuteexperimentcomparedtocontrol(p<0.05).Moreover,antidiabeticeffectwas comparable thanproducedbyglibenclamide(positivecontrol).Similarly,sub-chronictreatmentwithMECv(5days)produced a significant antidiabetic effect after first day of treatment (-9%). Effect was maintained, and also was significant until the endof the treatment comparedwith the control group (p< 0.05) (Table 4). Aswecansee,MECvwasmorepotentinDM than in normoglycemic rats. These fin-dingssuggestmetabolicregulationinliverprocessessimilartosomeapprovedanti-hyperglycemicdrugsasmetformin,whosedoesnothasplasmaticglucosereductioninnormoglycemicpatientsbuthigherglu-cosedecreaseindiabeticpeople.Becauseofmetformin suppress liver glucosepro-duction in gluconeogenesispathwayandincreaseinsulinactioninmuscleandfat,thisdrugonlycanactinpathologicalpro-

Table 1. Hypoglycemic effect of acute intragastric administration on MECv, glibenclamide and vehicle in normoglycemicrats

Percentage of variation of glycemia (mean ±S.E.M.) (mg/dL)Test samples Dose (mg/Kg) Zero hour First hour Third hour Fifth hour Sevent hourControl - 0±0.0 10.9±3.3 1.4±5.6 1.2±3.6 -3.6±2.7Glibenclamide 5 0±0.0 -8.3±1.9* -21.7±3.9* -24.8±1.0* -23.8±0.9*MECv 100 0±0.0 1.25±5.15* 1.67±3.55 -7.01±3.42* -13.43±2.5*

Table 2. Hipoglycemic effect of sub-chronic intragastric administration of MECv, glibenclamide and vehicle innormoglycemicrats

Percentage of variation of glycemia (mean ± S.E.M.) (mg/dL)Test samples Dose (mg/Kg) Day zero First day Second day Third day Fourth day Fifth dayControl - 0±0.0 27.2±5.3 21.9±2.2 20.0±0.9 23.0±1.7 28.9±1.4Glibenclamide 5 0±0.0 -23.51±2.1* -28.7±2.1* -33.5±3.5* -29.8±2.9* -30.1±2.4*MECv 100 0±0.0 5.36±3.08* -8.22±2* -8.32±2.4* -11.86±2.93* -12.66±2.52*


cesses (Davis, 2007). In this context, MECv antidiabeticmodeofactioncouldberelatedwithanantihyperglicemicactiontroughanenhancinginsulinaction(ligandsforper-oxisome-proliferator activated receptors,retinoid X receptor modulators and protein tyrosinephosphatase1Binhibitors)and/orinhibitorofhepaticglucoseproduction(in-hibitorsofpyruvatedehydrogenasekinase,liver-selective glucocorticoid receptor an-tagonists,11b-HSD1 inhibitors, adenosine A2Breceptorantagonists,glucagonreceptorantagonists, glycogen phosphorylase in-hibitors,glucose-6-phosphataseinhibitors,fructose-1,6-bisphosphatase inhibitorsandglycogensynthasekinase-3inhibitors)(SarabuandTilley,2005).Anotherpossiblerelatedmechanismcouldbetheantihyper-glycemic action by regulation of glucoseuptakefromintestinallumen,becauseofinhibition of carbohydrate digestion andabsorption,orretardationofpostprandialglucoseincreasebyinhibitionofα-gluco-sidases(Ortiz-Andradeet al.,2008).Thus,in order to establish if MECv possessedan in vivo antihyperglycemic effect, weevaluatedtheeffectoftheextractonoralglucose tolerance test (OGTT) in normo-

glycemic rats using glucose and sucroseassubstrates.Inbothexperiments,MECvinduced a significant suppression in the increaseofplasmaglucoseaftersubstrateadministration(Figures1and2)comparedto control (p< 0.05), indicating that themechanismofactionofMECvisthroughananti-hyperglycemiceffect,mediatedbytheregulationofglucoseuptakefromtheintestinallumen,throughtheinhibitionofcarbohydratedigestionorabsorption(Or-tiz-Andradeet al.,2007).Thiscouldbepos-siblebyretardingthepostprandialglucoseby inhibition of glucose co-transporters(SGLT) of by the inhibition of intestinal α-glucosidasecomplex(Ortiz-Andradeet al.,2008).Inordertotestthelasthypothesis,wedeterminedMECv’sabilitytoinhibittheα-glucosidases. The extract inhibited α-glucosidaseactivityin vitro inaconcentra-tion-dependentmanner(IC50of1.9mg/mL)(Figure3).Consequently,within vivo andin vitro experiments, we established thatthe inhibitionofα-glucosidases isoneofthemodeofactionofMECvasantidiabeticagent.Finally,theantihyperglycemiceffectinducedbyMECvseemstobemuchmoreeffectivein vivo thanin vitro.Thisobserva-

Table 3.Antidiabetic effectofacute intragastricadministrationonMECv,glibenclamideandvehicle instreptozotocin-nicotinamidediabeticrats

Percentage of variation of glycemia (mean±S.E.M.) (mg/dL)Test samples Dose (mg/Kg) Zero hour First hour Third hour Fifth hour Sevent hourControl - 0±0.0 9.4±5.4 0.1±5.9 4.7±1.6 -13.9±5.4Glibenclamide 5 0±0.0 8.4±7.8 -21.1±4.5* -28.2±6.7* -20.9±6.7*MECv 100 0±0.0 -9.1±6.39* -19.23±1.49* -7.18±7.44* -18.73±3.67*

Table 4.AntidiabeticeffectofsubchronicintragastricadministrationofMECv,glibenclamideandvehicleinstreptozotocin-nicotinamidediabeticrats

Percentage of variation of glycemia (mean±S.E.M.) (mg/dL)Test samples Dose Day zero First day Second day Third day Fourth day Fifth day (mg/Kg) Control 0±0.0 27.2±5.3 21.9±2.2 20.0±0.9 23.0±1.7 28.9±1.4Glibenclamide 5 0±0.0 -23.51±2.1* -28.7±2.1* -33.5±3.5* -29.8±2.9* -30.1±2.4*MECv 100 0±0.0 -9.1±4.84* -15.26±8.93* -26.98±8.81* 26.9±8.5* -31.09±7.89*


tioncouldberelatedwithapossiblebloc-kade of glucose co-transporters as Glut-2 and Glut-4, main carriers of glucose from intestinetocirculation(Liet al.,2006).Thislastasseverationisbasedonthepresenceof naringenin (NG) on the methanolic ex-tract of C. vitifolium (Sánchez-Salgado et al., 2007). NG exhibited a strong inhibitory actiononglucoseuptakeinnormoglycemicratsandindiabeticratsrevertedintestinal

sleevesthroughtheinhibitionofintestinalandrenalNa+-GLU co-transporters (SGLT) (Liet al.,2006).Therefore,wesuggestthatthemainantidiabeticprinciplefromMECvis naringenin, since its pharmacologicalpropertieshavebeenrelatedwithantidia-betic activity. So, it was reported that NG induced hypoglycemic and antidiabeticactivities in STZ-nicotinamide-induceddiabetic rat models, and also, inhibited

Figure 1.EffectofMECvonglycemiaafterasingleoraladministrationof2g/kgofglucoseinmaleWistarrats.Eachplotrepresentsthemeans±S.E.M.(n=5);*p<0.05;SSvs.control.

Figure 2.EffectofMECvonglycemiaafterasingleoraladministrationof2g/kgofsucroseinmaleWistarrats.Eachplotrepresentsthemeans±S.E.M.(n=5);*p<0.05;MECvvs.control.


Figure 3.Concentration-responsecurveoftheinhibitoryactivityofMECvoninvitroactivityofintestinalα-glucosidases. Each point represents the means ± S.E.M. (n = 4.)

Figure 4. Blood biochemical profiles in normoglycaemic and streptozotocin (STZ)–nicotinamide (Nc) diabetic ratstreatedwithMECv,glybenclamideandvehicleduring5days.*p<0.05;MECvvs.control.


11β-hidroxysteroid dehydrogenase in vi-tro activity type 1 (Ortiz-Andrade et al.,2008). Moreover, Harmon and Patel (2003) described that NG inhibited insulin-stimu-latedglucoseuptakein3T3-L1adipocytes,inadose-dependentmannerbyinhibitingthephosphoinositide3-kinase,akeyregu-latorofinsulin-inducedglucosetransporter(GLUT-4) translocation. Taking these data, weproposethatMECvinduceditsantidia-betic effectbyextrapancreatic eventsviainhibition of α-glucosidase Activity, and alsobyapossibleblockadeofglucoseco-transporters.

Ontheotherhand,STZ-nicotinamide-induceddiabeticratstreatedsub-chroni-cally with MECv were anesthetized, andbloodwaswithdrawnfromtheheart.Then,thefollowingbiochemicalmetabolitesweredetermined: glucose, total cholesterol, HDL cholesterol and triglycerides (TG) (Figure 4). InSTZ-nicotinamide-induceddiabeticratsglucose blood concentration was signifi-cantlyreducedtocontrolvalues(p<0.05);while TG were unexpectedly increased (p< 0.05).Ontheotherhand,innormoglyce-mic rats HDL values were augmented than normoglycemiccontrolgroup(p<0.05).

Finally,apreliminaryoraltoxicologicalstudy of MECv was conducted. The LD50estimationusingthe ‘AcuteToxicClass’methodrequired todetermine theeffectofMECvatdosesof2000,300,50and5

mg/kg. Following 24 h of observation at eachofthesedoses,allanimalssurvived.LD50wasdeterminedandwasassignedtocategory5, that is>5000mg/kg.MECvpossesses a high LD50atdosesgivenandcanbeconsideredasanon-toxiccandi-dateforfuturedrugdetectionandisola-tion.

In conclusion, findings allowed us to establish C. vitifolium as an importantanti-diabetic agent. Furthermore, inhibi-tion of intestinal α-glucosidase enzymesbyMECv,suggestthatthisplantspeciesis an alternative for the control of DM, and alsoitseemstobenontoxicforitsusesintraditionalmedicine.Finally,basedonits capability to augment HDL-cholesterol in normal rats, and by the presence of NG that is able to diminish TG levels and to inhibit activity of 11βHSD type 1, MECv couldbeusedforpreventionandcontrolofthemetabolicsyndrome.

ACKNOWLEDGMENTS

This study was financed by a grant from CONACyT (FORMACIÓN DE DOCTORES) and FONDO DE CONSOLIDACIÓN-UAEM (FOLIO 4). R.R. Ortiz-Andrade is grateful to CONACyTandECOESSantanderUniversiaforthescholarshipgrants(No.175295).

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ACUTE AND SUBCHRONIC EFFECTS OF COCHLOSPERMUM VITIFOLIUM IN BLOOD GLUCOSE LEVELS IN NORMOGLYCEMIC...

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