Study Of The Anti-AD Chemical Ingredients In The EtOAc Fraction Of Bupleurum Smithii Wolff

Objective:To study the chemical constituents in the Et OAc extraction from Bupleurum smithii Wolff and inverstigate the effect of extraction and separated compounds on AD mouse model and BV-2 microglia cells.Method:1.The root of Bupleurum smithii Wolff were extracted by Et OH under refluxition.The crude extraction was concentrated from the Et OH distillate in a vacuum to obtain,which was further extracted with PE,Et OAc and n-Bu OH successively.The pharmacodynamic effect of the Et OAc extraction of Bupleurum smithii（EOB）was investigated in the ICR AD mice model.To the model group,positive control group,the low,medium and high-dose EOB groups mice,LPS were injected into the lateral ventricles to model.While the control group mice were injected with the same amount of saline after environmental adaptation.Intragastric administration was abided by the 400,200,100 mg/kg for high,medium and low dose group of extraction,and positive control group mice received donepezil（5mg/kg/d）by intragastric administration,while the control group and model group mice were give N.S.according to the mice weigh（0.2m L/10g）after injection.NOR experiment was performed 8 days after injection.Y maze experiment was performed 16 days after injection.The Morris water maze experiment was performed 20 days after injection.ELISA test of the hippocampus in mice was taken after the behavioral experiments.2.The Et OAc extraction was separated by different methods（TLC,HPLC,etc）and further purified by recrystallization and prep-HPLC to yield pure compounds.The structures of isolated compounds were identified by spectroscopic methods(¹H and ¹³C NMR)and comparison with the literature data.3.The effect of three isolated compounds on microglia inflammatory cytokines release in mice BV-2 microglia were detected by the ELISA test.Results:1.Fourteen compounds were isolated from the Et OAc extraction of Bupleurum smithii Wolff,including:（1）saikogenin A,（2）saikogenin C,（3）saikogenin D,（4）saikogenin F,（5）saikogenin Q,（6）kaemferol,（7）quercetin,（8）isorhamnetin,（9）3,5,7-trihydroxy-3’,4’-dimethoxyflavone,（10）caffeic acid,（11）ferulic acid,（12）gallic acid,（13）4-methoxycinnamic acid,（14）3,4-dihydroxybenzoic acid.2.Compared with the control group,model group showed remarkable damage in learning and memory ability.And there were more inflammation factors release in brain hippocampus.Compared with model group,learning and memory ability of the positive control and high,medium,low dose EOB group mice were obviously improved after the treatment,and inflammatory factors in to the mouse in the release of has obvious inhibitory effect.The three compounds from Et OAc extraction show remarkable inhibitory effect on the release of inflammatory factors on mice BV-2 microglia cells.Conclusion:1.Fourteen compounds were isolated from the Et OAc extraction of Bupleurum smithii Wolff.This is the first time to report the isolation of compounds 2,3,4,5,9,10,13 and 14 from Bupleurum smithii Wolff.2.It is showed that Et OAc extraction from Bupleurum smithii Wolff has significant protection effect to learning and memory ability damage and significant inhibitory effect in the release of inflammatory factor on AD mice.The saikogenins may be the active anti-AD compounds in Bupleurum smithii Wolff.