The Flavonoids From Ziziphus Jujuba Mill.Var.Spinosa(Bunge)Hu Ex H.F.Chow Process Optimization And Anti Bacterial Biofilm And Inhibitory Effect On The Human Brain Glioma U251 Cells Research

Objective:This study aims to find the optimal technology of total flavonoids extracted from the Ziziphus jujuba Mill.var.spinosa(Bunge)Hu ex H.F.Chow(FZM)by different methods,and establish stability checking method of Thin-layer Chromatography(TLC)and High Performance Liquid Chromatography(HPLC)about FZM,further observation FZM antibacterial antitumor activity and discuss about FZM brain glioma U251 cell apoptosis,cell cycle and the impact of autophagy.Methods:(1)Solid-liquid ratio,ethanol concentration and extraction time were selected as the index,analyse and optimize the extraction technology.(2)The cell counting method was applied to determine the influence of wall-broken time on the wall-broken rate,then analyse and optimize the extraction technology.(3)TLC:With Ethyl Acetate-Acetone-Methanol-Formic acid-Wate as developing agent,HPLC:With acetonitrile-water(volume fraction:20%-80%)as mobile phase,detectioned at 258 nm.(4)Cultivation the bacterial biofilms of staphylococcus aureus and Streptococcus mutans in vitro,after the intervention of FZM,respectively using crystal violet assays method,optical microscope examination,confocal laser scanning microscope examination,scanning electron microscope observation to detect the S.aureus BBF.(5)FZM intervention after U251 cells,used MTT method to detect cell vitality,used flow cytometry to detect the various concentrations of FZM induced the expression of human brain U251 glioma cell apoptosis,used q-PCR to detect the FZM on the lever of MAP1LC3B/BECN1 mRNA in U251 cells.Results:(1)The average content of total flavonoids was 1.540 mg.g-1,.(2)The average content of total flavonoids was 1.435 mg.g-1,contrast the conventional reflux extracting method the FZM content was increased by 2.24%.(3)The Rf of Rutin,Spinosin is 0.56,0.45.HPLC:the average content of rutin was 0.078%,the average content of spinosin was 0.048%.(4)FZM can not inhibit the formation of S.mutans biofilms in vitro,FZM can effectively inhibit the formation of S.aureus biofilms in vitro.(5)The inhibitory rate of FZM increased with increasing concentration.And the impact of FZM on U251 cell apoptosis also was dose-dependently increasedc.In addition,the autophagy related factor MAP1LC3B mRNA was up-regulated,and the difference was significant;BECNl mRNA was up-regulated,and the difference was not significant.Conclusion:The validation test shows that the method is simple and feasible.Wall-broken extraction operation suitable for FZM efficient extraction.Established a stable detection method of TLC and HPLC about FZM,the methods can be used for quality control of FZM,accurately and reliably.FZM reduced the adherence of S.aureus BBFs by removing the exopolysaccharides on the surface of the films and damaging the channels for material exchange,depriving bacteria of nutrients.The inhibitory effect of FZM on the human glioblastoma cell line U251 may be related to the non-classical autophagy pathway.