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

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

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

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

IRS 1/2 P110 P85 RafMEK MAPK PIP 3 PKC  PDK 1 Akt/PKB Ras GDP GTP Gene expression PI3 kinase GS Glycogen Synthesis GSK 3 PP1 GLUT PIPPIP 2

Present Targets and their Validation Insulin Trafficking GLUT 4 RT-PCR, Western GLUT 1RT-PCR PI3 KinaseRT-PCR PKC  RT-PCR Insulin Sensitizer PPAR  RT-PCR Glucose Storage Glycogen SynthaseRT-PCR Potential future target GSK-3 (Glycogen Synthase Kinase- 3) RT-PCR

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

Techniques Involved Purification (a) Extraction 3 days each Hexane 0.0 Dichloromethane3.1 Ethyl Acetate4.4 Methanol5.1 (b) Chromatography Solid – liquid Technique 2 phases Stationary phase Mobile phase Principle: Column Chromatography- POLARITY ADSORPTION Thin Layer ChromatographyCAPILLARY ACTION ADSORPTION

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

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

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