Doctorate Work

PhD Project

Title: Interaction of Natural Plant Constituents and Commercial Oral Hypoglycemic Drugs

Advisor: Prof. Mukesh Doble, Prof. Department of Biotechnology, IIT Madras, Chennai, India

Diabetes mellitus (DM) is a metabolic disorder characterized by increased blood glucose level and the only cause is insufficient or inefficient insulin secretary response. Plants have always been an exemplary source of drugs and many of the currently available drugs have been derived directly or indirectly from them. The ethnobotanical information report says that there are about 800 plants that have anti-diabetic properties. Humankind has a long history in the use of herbal medicines. India unquestionably occupies the topmost position in the use of herbal drugs since ancient times. A number of genes and their product have been involved in diabetes and its complications. If we regulate those genes with natural product which are effective, with fewer side effects and comparatively less costly, it will be a safe and easy way to manage diabetes. My work is to find out the hypoglycemic mechanism of action of some of the selected natural plant constituents and see the interaction of these natural compounds with oral hypoglycemic drugs. .

Nine phytochemicals (Arecoline, berberine, caffeic acid, chlorogenic acid, cinnamic acid, coumaric acid, eugenol, ferulic acid and vanillic acid) have been selected for the current study based on the antidiabetic activity reported by their corresponding plant sources. In the case of diabetes, due to hyperglycemia generation of free radicals and secondary complications are important concerns. The phytochemicals are tested for their antioxidant activity with four commonly used methods. Few of them show good antioxidant activity and are better than the commercial OHDs and standards (such as BHT and ascorbic acid). Phytochemicals with hydroxyl group have higher antioxidant activity that the phytochemicals having methoxy group. Presence of conjugation increases their activity.

All the nine phytochemicals do not exhibit toxicity towards L6 myotubes and 3T3-L1 adipocytes, at a concentration of 50 µM. 75-96% of the cells are viable after 24 hours and 67-81% are viable after 48 hours of treatment with phytochemicals or OHDs when compared to control. These compounds reduce the accumulation of lipid contents.

In both the cell lines, phytochemicals and the OHDs increase 2-deoxy glucose (2DG) uptake in dose and time dependent manner. The uptake is higher in 3T3-L1 adipocytes than in L6 myotubes. In the later cell lines, THZ and metformin, increase 2DG uptake by 3.05- and 2.75-fold (when compared to control) at a concentration of 10 μM and 15 μM respectively. 25 μM of arecoline, berberine, caffeic acid, chlorogenic acid, coumaric acid, eugenol and ferulic acid show a maximum 2DG uptake of 2.95-, 2.91-, 3.07-, 3.03-, 2.99, 2.99- and 2.97- fold respectively in the same cell line whereas cinnamic acid and vanillic acid show a maximum 2DG uptake of 3.02- and 2.15-fold at 50 and 20 μM respectively when compared to control. THZ and metformin at a concentration of 15 μM and 20 μM increase 2DG uptake in 3T3-L1 adipocytes by 3.3- and 2.95- fold respectively. Maximum 2DG uptake in 3T3-L1 adipocytes is observed with coumaric and ferulic acids (~3.3 fold) and minimum is observed with vanillic acid (2.15-fold). 2DG uptake with THZ, chlorogenic acid and eugenol reach the maximum 2DG uptake at 3 hours of incubation with L6 myotubes. With the other compounds maximum 2DG uptake is observed at 4^th hours. The interaction of phytochemicals with OHDs in 2DG uptake in both the cell lines is studied with two different methods, namely isobologram and combination index (CI). Interactions between arecoline, caffeic acid, chlorogenic acid, coumaric acid, eugenol and ferulic acid with both the OHDs in both the cell lines are synergistic. Cinnamic acid exhibits additive behavior while vanillic acid with metformin shows antagonistic behaviour.

In the case of diabetes the main problem is utilization of the blood glucose, which may be caused because of impaired glucose transport or reduced GLUT4 translocation. PPAR gamma agonist and PI3K play a very important role in glucose transportation inside the cells. Based on reverse transcriptase polymerase chain reaction (RTPCR) it is concluded that THZ increases the expressions of PPARγ and GLUT4 and, metformin increases that of AMPK and GLUT4. Activation of PI3K, AMPK or PPARγ by its agonist increases glucose uptake in both the cell lines. All these three molecular targets, namely PI3K, AMPK and PPARγ, are downstream signaling molecules in the insulin cascade which enhance the glucose uptake by the cell via the activation of GLUT4 and its translocation to the plasma membrane. Four cinnamic acid derivatives, namely ferulic acid, eugenol, coumaric acid and caffeic acid significantly increase the expressions of GLUT4 and PI3K. Whereas arecoline, chlorogenic acid and cinnamic acid have a significant effect on the expressions of PPARγ and GLUT4 genes. Berberine and vanillic acid significantly increase the expressions of AMPK and GLUT4 genes. The phytochemicals significantly reduce the expressions of FAS and HMG CoA reductase when compared to control and this reduction is more than those observed with OHDs.

Theoretical calculations indicate that these phytochemicals possess good ADME (Absorption, digestion, metabolism and excretion) and drug likeness properties and none of them (except chlorogenic acid) violates the Lipinski’s rule of five and Jorgensen’s rule of three (chlorogenic acid violates one rule in both the cases) indicating that they will possess good oral bioavailability.

The interaction of ferulic acid with both the OHDs was studied in streptozotocin induced rats. Ferulic acid is seen to act in synergy with both the OHDs and reduce the blood glucose level, lipid profile and improve the kidney as well as liver function. Histopathology study indicates that ferulic acid has the capacity to increase the islet cells mass in the diabetic rats when compared to non-diabetic control rats. Increase in the β-cell mass will increase the secretion of insulin, which will lead to the enhanced peripheral utilization of glucose.

The current research has shown that these phytochemicals increase 2DG uptake via translocation of GLUT4 which may be activated either by PI3K, PPARγ or AMPK. ADME calculation also suggests that these phytochemicals can be used as drugs. A reduction in the quantity of the use of OHD could lead to a reduction in the side effects and toxicity caused due to their excess usage. Proper precaution and care should be taken to avoid the severe hypoglycemia that may occur due to combination of these phytochemicals and OHD.