Novel anti diabetic phytochemicals: isolation, protein target and action mechanism

We have evaluated the anti-diabetic activity of several medicinal plants in vitro. Recently, we have tested the role of glucose transporter-4 (GLUT4) in the anti-diabetic activity of Ocimum basilicum (OB) and Tucrium polium (TP) methanol-, hexane- and dichloromethane- extracts. Phytochemical analysis of the extracts by GC/MS revealed cohort new compounds. GLUT4 translocation to the plasma membrane was elevated by 2 and 3.5 folds in the absence of insulin as well as 3 and 7 folds in the presence of insulin post treatment with TP and OB extracts respectively. The rationale of the present study is to isolate anti-diabetic phytochemicals from OB and TP and test their mechanism of action in vitro. Our target cells will be those with known relevance in diabetes with an emphasis on muscle cells. Protocols well-recognized in our laboratory for functional cell assays will be utilized, such as glucose transport, ROS production, cytotoxicity, protein/gene expression in metabolism pathways, and oxidative stress. The anti-diabetic crude extracts of OB and TP will be subjected to fractionation using flash chromatography and preparative HPLC in order to purify the active compound(s)/fraction. The resulted fractions will be subjected to MTT and LDH test in order to detect the nontoxic concentrations on L6 muscle cells. The anti-diabetic activity (enhancing GLUT4 translocation to the plasma membrane) of the fractions will be tested in L6 cells as described in the methods section. The chemical composition of the active fraction will be evaluated by GC/MS. A next round of purification will be applied for the mostly active fractions. We believe that the combined approach which utilizes biological, biochemical and chemical tools in one project is unique and could serve as a basis for fast isolation and identification of the bioactive compounds, unraveling their mechanism of action and their targeted proteins related to the metabolic disorders especially diabetes.