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Viabilityassessmentofpancreaticand hepaticcellsuponstimulationwithplants extractsusedasdiabetictreatment adjuvants

Lidiannie OteroDeLen AMPSummerInternship Mentor:Claribel LucianoMontalvo,PhD

Diabetes
Diabetesmellitus
isagroupofmetabolicdiseasescharacterizedbyhigh bloodsugar(glucose)levelsorhyperglycemia

Diabetesisclassifiedintwotypes:
Type1
calledinsulindependentdiabetesmellitus

Type2
noninsulindependentdiabetesmellitus

Diabetescomplications
Hyperglycemia:
Impairimmunesystem Patientssufferofincreasedlevelsofbacterialinfections (Stoeckle etal2008). Damagesthecardiovascularsystem diabeticsshowedincreaselevelsofhypercholesterolemia andhypertension(Khamaisi etal2003).

Theoverwhelmingtreatmentofdiabetesisoneofthe reasonsmanygroupsinvestigatealternativemedicines totreatorcurediabetes.

PreviousStudies
Gymnema Studies Gymnema sylvestre isstudiedforitsknownanti hyperglycemicproperties
Theextractincreasesthefunctionofbetacells(Persaud et al1999)

Liuetal2008,showedaconcentrationof0.5mg/mL ofGymnema sylvestre showednocellviability impairment.


Theytestedinsulinsecretionofbetacellexposedto extractconcentrationsrangingfrom0.062mg/mL

Previousstudies
Ethnobotanical surveyconductedinCayey region,literature,andbooksinPRidentified fourplantsusedinPuertoRicoasdiabetes therapyadjuvant.
TheseareTapeinochilus ananassae,Costus spp, Rhoeo spathacea andSyzygium jambos Bothmethanolic andaqueousextractswere tested Gymnema sylvestre extractwasusedaspositive control

Relevance
Thisisthefirstassessmentofthesafetyatthe cellularlevelofthesefourextracts
Weusedtwocellmodelsofbetacells(BetaTC6) andtheHepaticcellline(Hep G2)

CellModel:BetaCells

CellModel:Hep Cells

MTTAssay
Invitro assayforthemeasurementofcell proliferation Itdeterminesareductionincellviabilityaftera particulartreatment Cellsareculturedinflatbottomed,96welltissue cultureplates Thetetrazolium compoundMTT(3[4,5 dimethylthiazol2yl]2,5diphenyltetrazolium bromide)isthecompoundusedtomeasure viability

MTTAssay
MTTisreducedbythemitochondrial dehydrogenase,presentinactivecells Reductionallowstheformationofinsoluble purpleformazan dyecrystals Adetergentisaddedtothewellstosolubilize the crystals Absorbanceisreadusingaspectrophotometer RateofMTTreductionisproportionaltotherate ofcellproliferation

ResearchDesign

~ 3 months UPR-MSC Dr. Cadillas lab

ResearchDesign

24h Incubation
Extracts (0.5 mg/mL)

ResearchDesign
24h

ResearchDesign
Absorbance 570 nm

Results

Results

Results

Results

Conclusions
Noneoftheextractspreparationstestedin Betacellsshoweddecreasecellviability Alloftheaqueousandmethanolic extracts testedwithHep cellsshowednodecreased cellviability. Theextractsrepresentpromisingcandidates forfurthertherapeutical analysesastheydo notinducecelldeath.

FutureExperiments
Testadditionalconcentrationsoftheextracts Examinetheeffectoflongtermexposure Futureinvivo assaywillbenecessaryto establishthetherapeutical benefitsofthese extractsinbetacellfunctionandin improvementofdiabetes

References
Persaud SJ,AlMajed H,RamanA,JonesPM.Gymnema sylvestre stimulatesinsulin releaseinvitrobyincreasedmembranepermeability.JEndocrinol.1999 Nov;163(2):20712. LiuB,AsareAnane H,AlRomaiyan A,HuangG,Amiel SA,JonesPM,Persaud SJ. Characterisation oftheinsulinotropic activityofanaqueousextractofGymnema sylvestre inmousebetacellsandhumanisletsofLangerhans.CellPhysiol Biochem.2009;23(13):12532. Stoeckle M,Kaech C,Trampuz A,Zimmerli W.Theroleofdiabetesmellitusin patientswithbloodstreaminfections.SwissMedWkly.2008Sep6;138(35 36):5129. Khamaisi M,WexlerID,Skrha J,Strojek K,Raz I,Milicevic Z.Cardiovasculardisease intype2diabetics:epidemiology,riskfactorsandtherapeuticmodalities.Isr Med AssocJ.2003Nov;5(11):8016.

Thanks
Collaborators BiologyDepartmentandtechnicians Nivea Franco AngieCastell RISEprogram Yadira Ortiz MSC BiochemistryDepartment Dr.CarmenCadilla Otherresources NIHRIMIProgram#1P2MD0011120 AMPprogram InstitutodeInvestigacionesInterdisciplinarias

Thanks

Questions???

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