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

cellPreproinsulin

Endoplasmic Reticulum

Secretary granules

cytoplasm

Proinsulin

Insulin

C-peptide

Golgi

Insulin Secretion

IRS 1/2

P110

P85

Raf

MEK

MAPK

PIP3

PKC

PDK 1

Akt/PKB

RasGDP GTP

Gene expression

PI3 kinase

GSGS

Glycogen Synthesis

GSK 3

PP1

GLUT

GLUTPIPPIP2

Present Targets and their ValidationInsulin Trafficking

GLUT 4 RT-PCR, Western

GLUT 1 RT-PCR

PI3 Kinase RT-PCR

PKC RT-PCR

Insulin Sensitizer

PPAR RT-PCR

Glucose Storage

Glycogen Synthase RT-PCR

Potential future targetGSK-3 (Glycogen Synthase

Kinase- 3)RT-PCR

Plant MaterialHexane

DCM

Ethyl Acetate

Methanol

Active Extract

Bio Assay

Column Chromatography

Thin layer chromatograph

y

Pooling

Active Fraction

Bio Assay

Repeated till a single lead molecule is obtained

Lead Molecule

(Further Purification)HPLC

(Structure Elucidation)

NMR & Mass Spectroscopy

VALIDATIONRT-PCR, Western blotting

Incr

ea

sin

g P

ola

rity

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 phasePrinciple:

Column Chromatography- POLARITY ADSORPTION

Thin Layer Chromatography CAPILLARY 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

+ -

3. 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, 37oC, 5% CO2 )

Incubation ( 20 min, 37oC, 5% CO2 )

Termination of the Glucose Uptake

Spin [ 8000 rpm, 7 min ]

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] GlucoseDMEM medium

KRPH buffer

Ice cold KRPH buffer

buffer

STV buffer

KRPH

SDS

Supernatant

Supernatant