Separation And Screening Of PPARγ Agonists In Mulberry Leaves

Diabetes is a metabolic disease, and the main characteristics is hyperglycemia.Diabetic patients are in a state of high blood sugar for a long time which will lead avarious injury and dysfunction to tissues and organs, especially kidneys, heart, eyes,blood vessels, nerve chronic injury. Mulberry offers a good therapeutic effect ofdiabetes.China is a big country in the production of mulberry. The extraction materialof mulberry was widely research in hypoglycemic effect, such as total polysccharides,flavonoids and alkaloids. In addition, mulberry often cooperates with other traditionalChinese medicine to treat diabetes melltius.PPARγ, a member of nuclear receptor superfamily, can promote adipocytedifferentiation. Actived PPARγ can promote the secretion of adiponectin, andincreased the insulin sensitivity in human. Therefore, improving insulin resistancethrough PPARγ signaling pathway is one of effective ways to study anti diabetesdrugs. It is important to establish a fast and efficient screening method and the modelof high-throughput screening for looking for effective and low toxic PPARγ activator.PPAR and RXR will form the heterogeneous dimers when PPAR combines withligand and is activated. PPRE that is PPAR reaction components in the upper reachesof target gene promoter regulate and control luciferase reporter gene expression level.This study will establish a simple, sensitive and efficient Cos-7 cells model of PPARγactivator for high-throughput screening the ligand, and it applys to screening ofactive substances in mulberry leaves.Two vectors of p EGFP-N2-PPARγ and p GL4-PPRE4-luc were constructed andwere co-transfected into the cells using lipofectamine 2000. Finally, the criteria of theoptimized cell model were revealed, namely 70% of cells convergence degree, 2:1 ofplasmid concentration between p EGFP-N2-PPARγ and p GL4-PPRE4-luc, 20 h forthe transfection incubation, and 24 h for drug incubation. Using PPARγ positiveligand rosiglitazone to test the stability and specificity of the model, the resultsshowed that the stability of cell model is good, and only have an activation effect onrosiglitazone which presented dose-response relationship within a certain range.The effective components were roughly extracted useing N-hexane in mulberryleaves. Forty components, namely PE-1 to PE-40, were separated from the extractionuseing column chromatography on silica gel and thin layer chromatography. Of these,6 components were screened as the activators of PPARγ through the Cos-7 cellsmodel. We made the six samples perform alkaline derivation, and GC-MS used toanalyze the composition of fatty acid. 17 kinds of fatty acids were obtained. UsingCos-7 cells model to analyze fatty acids, we found that myristic acid, pentadecanoicacid, linolenic acid all can active PPARγ, but other fatty acids had no effect. Theactivation would reach to the maximums of 1.51, 2.12, 2.48 and 2.12 using myristicacid of 100μM, pentadecanoic acid of 50μM, linolenic acid of 150μM and linoleicacid of 100μM, respectively. We concluded that there are 1.91% linolenic acid,0.94% linoleic acid and 0.11% myristate in dry mulberry leaves using the standardcalibration curve.Our study could help us for fastly and conveniently searching a new naturalinsulin sensitizer and synthesis of novel partial agonits, and also provides a referencefor natural medicine research in China.