Study On Anti-Cancer Effects And Mechanism Of Polyphenol Extract From Elaeagnus Angustifolia

Objective: To screen antioxidant activities of different separation fractions of Elaeagnus Angustifolia,to investigate proliferation and apoptosis activities of human hepatoma Bel-7404 cell lines,human hepatoma HepG-2 cell lines,human esophageal cancer ECA-109 cell lines,human lung cancer A549 cell lines,human gastric cancer SGC–7901 cell lines and cervical cancer Siha cell lines.In further to study antitumor mechanisms on molecular levels of polyphenol Elaeagnus Angustifolia.Method: 1)Use Gallic acid as reference substance to determine total polyphenol contents of each separated fraction of E.angustifolia separated fractions by the Folin-Ciocalteu method;2)Use ascorbic acid as a positive control,to observe antioxidant activities of each separated fractions using the iron ion reducing power,hydroxyl free radical and DPPH free radical scavenging methods;3)Observe the inhibition effects of different fractions on six tumor cell lines by MTT assay;4)Screened MR-20 and prepared low(50 ?g/mL),medium(100 ?g/mL)and high(150 ?g/mL)concentrations,detect the apoptosis induction of 6 kinds of tumor cells,with AinexinV-FITC/PI double staining and flow cytometry instrument method.Nucleus morphology was observed by Hoechst33258 staining;5)Use cisplatin as a positive control to observe the mitochondrial membrane potential affected by MR-20 with Rhodamine-123 staining assay.Observe the cell cycle affected by MR-20 by PI staining.Observe expression level of cell cycle regulation genes CyclinB1,CyclinE and cell autophagy related gene LC3 B,Beclin1.Observe expression level of cell cycle regulatory proteins by Western blot method.Result: 1)Different separation fractions of E.Angustifolia have different antioxidant activities,their activities and total polyphenols contents showed Dose-effect relationship,the strongest active fraction is MR-20 which contains highest content of polyphenol.2)Each separated fraction showed varying degrees of proliferation inhibition effects on six kinds of tumor cells,among them Siha cell proliferation was stronger,the fraction with strongest activity was MR-20.After 72 h intervention,IC50 value of Siha cell proliferation inhibition was 56.2 ?g/mL.Three dose groups of MR-20 have induction apoptosis effect to six kinds of cells,the early apoptosis rate is higher than blank group.The more polyphenols concentration of MR-20 was,the higher apoptosis rate was.3)After processing Siha cells with MR-20,mitochondrial membrane potential is decreased.Cell cycles G2 phase is retardant,expression levels of cycle regulation genes CyclinB1,CyclinE were lowered(P<0.05),expression levels of cell autophagy related genes LC3 B,Beclin1 were lowered(P<0.05),expression levels of cell cycle regulatory proteins CyclinB1,CyclinE were decrease while P21 was increase(P<0.05).Expression level of cell autophagy related protein Beclin1 was lowered(P<0.05).Conclusion: Total polyphenol of E.Angustifolia has antioxidant activity.It shows different degree of inhibition activity to tumor cell proliferation to induce its apoptosis.Total polyphenols can induce cell cycle block,its mechanism may be related to up-regulating P21 expression and down-regulating Cyclin B1,Cyclin E expression.