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DIABETES

DIABETES. Gestational Diabetes. Insulin Dependent Diabetes Mellitus. Non-Insulin Dependent Diabetes Mellitus. Glucose Intolerance Trunal Obesity Hypertension Dyslipidemia Increased risk of coronary artery disease, etc. Gestational Diabetes.

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DIABETES

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  1. DIABETES Gestational Diabetes Insulin Dependent Diabetes Mellitus Non-Insulin Dependent Diabetes Mellitus • Glucose Intolerance • Trunal Obesity • Hypertension • Dyslipidemia • Increased risk of coronary artery disease, etc.

  2. Gestational Diabetes This develops during pregnancy and may disappear later. The patients develop a high risk of Type II Diabetes Insulin Dependent Diabetes Mellitus The  cells of the Pancreas fail to produce insulin So insulin is administered sub cutaneous to the patient Non-Insulin Dependent Diabetes Mellitus This is due to Insulin Resistance i.e., low secretion of Insulin or insulin insensitivity of the cells.

  3. Type II Diabetes mellitus Current Therapeutic Agents •  Glucosidase Inhibitors • Sulfonyl Ureas • Metformin • Thiazolidinediones • Insulin Injections Side Effects • Weight gain • Hypoglycemia • Gastrointestinal disturbances • Lactic acidosis • Edema • Anemia

  4. Insulin Secretion Secretary granules Preproinsulin  cell Endoplasmic Reticulum Proinsulin Insulin Golgi cytoplasm C-peptide

  5. Ras P110 GDP P85 IRS1/2 Raf PDK 1 MEK MAPK GS Gene expression PIP2 PIP PIP3 GLUT GTP PI3 kinase PKC Akt/PKB GSK 3 GS GLUT Glycogen Synthesis PP1

  6. Present Targets and their Validation Insulin Trafficking Insulin Sensitizer Glucose Storage Potential future target

  7. Plant Material Increasing Polarity Hexane Bio Assay DCM Active Extract Ethyl Acetate Methanol Column Chromatography Thin layer chromatography RT-PCR, Western blotting Repeated till a single lead molecule is obtained VALIDATION Pooling NMR & Mass Spectroscopy (Structure Elucidation) Bio Assay Active Fraction HPLC (Further Purification) Lead Molecule

  8. Techniques Involved Purification (a) Extraction Hexane 0.0 Dichloromethane 3.1 Ethyl Acetate 4.4 Methanol 5.1 3 days each (b) Chromatography Solid – liquid Technique 2 phases Stationary phase Mobile phase Principle: Column Chromatography- POLARITY ADSORPTION Thin Layer Chromatography CAPILLARY ACTION ADSORPTION

  9. POLYMERASE CHAIN REACTION CYCLING REACTIONS Denaturation 94 o C n cycles Standardized temperature Annealing Extension 72 o C

  10. Agarose Gel Electrophoresis + - Agarose Very small amount of Ethidium Bromide melt 1. Pour into Gel plate 2. Allow to solidify TEB 3. Place the plate in the gel tank Load samples mixed with Orange G Set Voltage & Current RUN THE GEL

  11. Glucose uptake Assay L 6 myotubes Dilutions of the plant extract Incubation ( 24 hr, 37oC, 5% CO2 ) DMEM medium 2 Deoxy D[3H] Glucose KRPH buffer Incubation ( 20 min, 37oC, 5% CO2 ) Ice cold KRPH buffer buffer Termination of the Glucose Uptake STV buffer Spin [ 8000 rpm, 7 min ] KRPH Supernatant Spin [ 8000 rpm, 7 min ] Supernatant SDS Incubate the lysate [Room Temp., overnight ] Scintillation Count Plot the Graph

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