Biflavonoids From Selaginella Uncinata (Desv.) Spring And Their Hypoglycemic Activities

Objective:The aim of this thesis was to isolate and identify the biflavonoids from the 80%ethanol extract of Selaginella uncinata（Desv.）Spring,and to detect their hypoglycemic effects in enzyme,cell and protein levels.Also,the hypoglycemic mechanism of the new compound was investigated in order to provide reference data for the further development and utilization of S.uncinata.Methods:1.The 80%ethanol extract of the S.uncinata was extracted by the petroleum ether,ethyl acetate and n-butanol,respectively.The compounds were isolated from the ethyl acetate extract by means of chromatography on silica gel,ODS column,Sephadex LH-20column,MCI column and semi-preparative HPLC.The structures were elucidated by using1D and 2D NMR,MS,UV and IR analyses.2.All the biflavonoids were evaluated in vitro for inhibitory activities against PTP1B.Additionally,a molecular docking software AutoDock Tools-1.5.6 was used to study the binding site.3.The insulin resistance model of HepG2 cells was successful created by high insulin induction.The effect of biflavonoids on cell proliferation was detected by MTT assay,and the glucose uptake was detected by glucose kit.4.Western blot was used to examine the effect of compounds 1 on the protein expression levels of key signaling molecules in IRS-1/PI3K/Akt signaling pathways in insulin resistance HepG2 cells.Results:1.Fourteen biflavonoids were isolated from 80%ethanol extract of Selaginella uncinata（Desv.）Spring,and were identified as（2S,2′′S）uncinatabiflavone C-7-methyl ether（1）,（2R）2,3-dihydroamentoflavone（2）,（2S）2,3-dihydro-5,5",7,7",4’-pentahydroxy-6,6"-dimethyl-[3’-O-4’’’]-biflavone（3）,（2′′S）2′′,3′′-dihydroamentoflavone（4）,2,3-dihydrorobustaflavone（5）,7,7’’-di-O-methylrobustaflavone（6）,sotetsuflavone（7）,chrysocauloflavone I（8）,delicaflavone（9）,robustaflavone-7-methyl ether（10）,2′′,3′′-dihydrorobustaflavone-7,4′-dimethylether（11）,bilobetin（12）,robustaflavone-4′-methyl ether（13）and amentoflavone（14）.2.Eight compounds were found to exhibit inhibitory activities on PTP1B with IC₅₀values range of 4.7-15.8μM.Additionally,molecular docking results showed that the biflavonoids bind to the allosteric site and the second binding site of the PTP1B enzyme.3.The model of insulin resistance was successfully established by insulin induction in HepG2 cells for 48 hours.The compounds were not cytotoxic for HepG2 cells detected by MTT assay.The results of hypoglycemic activity test in vitro showed that compounds1-14 increased the glucose consumption of insulin resistance HepG2 cells in various degrees.4.Western blot results showed that the compound 1 dose-dependently increased the expression of p-IRS-1,p-PI3K and p-Akt in insulin-resistant HepG2 cells,while the protein expressions of total IRS-1,PI3K and Akt were unchanged.Conclusion:Compounds 1 and 2 were new compounds,and compounds 3,4,6,7,8 and 9 were firstly found in this plant.The biflavonoids inhibited PTP1B by binding to the allosteric site and the second binding site of the enzyme.The hypoglycemic experiments showed that biflavonoids could improve insulin resistance in HepG2 cells and influence the protein expression of key signaling molecules in the IRS-1/PI3K/Akt signaling pathway in model cells.