Comparing The Hypoglycemic Activity Of Constituents From Mulberry Leaves And Its Mechanism Study

Objective:Mulberry leave is a traditional hypoglycemic Chinese herbal medicine, a large number of studies have shown that the alkaloids, flavonoids and polysaccharides from mulberry leaves can reduce blood glucose、blood pressure、 blood lipid etc. This research utilize the animal model of type 2 diabetes and the cell model of insulin resistance to screen the hypoglycemic active parts in mulberry leaves, also the hypoglycemic mechanism is studied. The result will provide the experimental basis for clinical therapy of diabetes mellitus using mulberry leaves and lay foundation for the exploration of novel diabetes drugs.Methods:1. Using KKAy mice with type 2 diabetes to get the optimal concerntration of the constituents in the pre-experiment. And then, dividing animals into normal control group, KKAy model group, rosiglitazone group, Shenqi Jiangtang Capsule group, the alkaloid of mulberry leaves group, the flavone of mulberry leaves group, the polysaccharide of mulberry leaves group and water extract of mulberry leaves group. During the experiment, observe the different signs of mice, and record the relevant pharmacodynamic index.2. Induce HepG2 cells with high glucose and high insulin medium to form the model of insulin resistance. After the growth curve of HepG2 cells was measured and the model was made successfully, constituents from mulberry leaves were compared with Rosiglitazone and Shenqi Jiangtang Capsule by evaluating the effect on glucose consumption of insulin resistance HepG2 cells. Then using the kit to detect the HK’s activity and PK’s activity in IR-HepG2 cell to explore the hypoglycemin mechanism.3. To observe the relationship between the constituents from mulberry leaves and JNK pathway, the real-time fluorescence quantitative PCR (RT-qPCR) method was utilized to detect these relative genes expression of JNK pathway in the IR-HepG2 cells,4. Using the Western Blot technique to detect the expression of c-Jun N-terminal kinase (JNK)、insulin receptor substrate 1 serine (IRS-lser) and protein kinase B (Akt), and to study how the mulberry leaves effect the key protein’s expression in JNK pathway.Results:1.The optimal concentration for each experimental group are as follows: rosiglitazone group(2mg · kg-1), Shenqi Jiangtang Capsule group(1300mg · kg-1), the alkaloid of mulberry leaves group(30mg · kg-1), the flavone of mulberry leaves group (1000mg · kg-1), the polysaccharide of mulberry leaves group(1500mg · kg-1) and water extract of mulberry leaves group(1300mg · kg-1). After drug intervention for 5 weeks, the experimental mice’s body weight and fasting blood glucose level were decreased, oral glucose tolerance test showed that the alkaloid of mulberry leaves group were restored with a best state.2. The rosiglitazone group(2.5～10 μg·L-1), Shenqi Jiangtang Capsule group (125～1000 mg · L-1), the alkaloid of mulberry leaves group (2～12 mg · L-1), the flavone of mulberry leaves group(10～100 mg · L-1), the polysaccharide of mulberry leaves group (50～250 mg · L-1) and water extract of mulberry leaves group(250～1000mg ·L-1) have no effect on HepG2 cells and IR HepG2 cells(P>0.05). And the hypoglycemic parts of mulberry leaves can increase the glucose consumption and the activity of HK and PK, especially the alkaloid of mulberry leaves group has the strongest effect.3. In QPCR results, all of the groups have the influence on the expression of JNK related genes. It can decrease the expression of JNK mRNA and increase the expression of IRS-1 mRNA、AKT mRNA.4. In Western Blot results, the alkaloid of mulberry leaves group, the flavone of mulberry leaves group, and the polysaccharide of mulberry leaves group also have the influence on the expression of JNK related protein, the result is the same with the QPCR.Conclusion:1. In the animal experiments, the hypoglycemic effective parts of mulberry leaves can reduce the weight and the the oral glucose tolerance, and improve the fasting blood glucose and the insulin sensitive index of diabetic mice. The alkaloid of mulberry leaves has the best effect, and then is the flavone of mulberry leaves and the polysaccharide of mulberry leaves.2. In the cell experiments, the hypoglycemic effective parts of mulberry leaves can improve the glucose consumption and significantly increase the activity of HK and PK in the IR-HepG2 cells. This is the same with the animal’s results.3. The QPCR’s result shows that the hypoglycemic effective parts of mulberry leaves can influence the gene’s expression of JNK signaling pathway, this may be related to insulin resistance.4. The Western Blot’s result shows that the hypoglycemic effective parts of mulberry leaves also can influence the protein’s expression of JNK signaling pathway.