Cytotoxicity study using MTT assay (Includes 5 different concentrations with triplicate test along with respective images of treated cells)

Post MTT anti-proliferative studies [LDH, Real-Time PCR (Caspase, P53, Bcl2 gene expression)]

Caspase-3 calorimetric Assay (Exclusive of Cell culture and treatment)

Dual staining (AO/EB) (Includes only control and cells treated with IC50 concentrations)

DAPI staining (Includes only control and cells treated with IC50 concentrations)

DCFDA staining for detection of ROS (Includes only control and cells treated with  IC50 concentrations)

DNA fragmentation (Includes only control and cells treated with IC50 concentrations)

Scratch wound or wound-healing assay  [2 durations (24 and 48 hrs)]

In-vitro anti neural damage study using SHSY5Y cell line model (Note: needs to be finalized after detailed discussion)

Invitro Hepatoprotective activity assay using HepG2 cell line model (Note: needs to be finalized after detailed discussion)

The antidiabetic study will include the following array of studies (if the sample shows a positive effect, it will be carried on to the next individual step).

Step 1. Cytotoxicity assay on 3T3 fibroblast  (5 concentrations as triplicate)

Step 2. Differentiation of 3T3 fibroblast to adipocyte and subsequent treatment with test compound and oil red staining (1 concentration as triplicate)

Step 3. Glucose uptake study(2 concentrations as triplicate with 2 duration)

Step 4. Gene expression study (PPAR?,PI3K and Glut4 gene expression)

An anti-inflammatory study using LPS induced RAW cell line:

Step 1. Cytotoxicity assay on RAW macrophage cell line (5 concentrations as triplicate)

Step 2. LPS induction, MTT assay, and NO quantification (Five different conc as triplicate)

Step 3. Gene expression study (TNF?, IL6 and IL1 gene expression)