Preliminary Study On Anti-AD's Activity Of Endophytic Fungus From Huperzia Serrata

Alzheimer's disease(AD)is neuronal degeneration with irreversible process.It is a common disease of middle-aged and aged.At present,When the world's population gradually aging,AD is growing.Research indicated that the pathogenesis of AD is a series of cascades in brain inducing neuronal damage and neuronal apoptosis.The AChE in Ach-system,the excess ROS releasing from the ETC of mitchondria and the accumulation of Aβsoluble oligomers possiblely play a critical role in cascades.Huperzia serrata is belonged to Huperzia of fern,which is a kind of valuable medicinal plants.The Huperzine A,which is isolated from it and successfully delays and treats AD,is developed by China's own drug.It treats AD not only by efficiently and reversibly inhibiting AChE,but also by multi-channel and multi-target treatment. For its weakly reproductive capacity and over-harvested,the wildlife resources of Huperzia serrata are less and less,or even been exhausted.Therefore,we must search a new effective way to resolve this contradiction.The endophytic fungus of plants can produce a large number of new natural products and some kinds of endophytic fungus even can produce the same or similar metabolites with the host plant.They are become potentially important resources of natural products.This study used modified inhibiting AChE activity's test in vitro and H₂O₂-induced SH-SY5Y nerve cell injury for the screening models.By multi-activity screening with endophytic fungal metabolites of Huperzia serrata,we gradually focused on the strains which have active role.Then we made preliminary study with the kinetic relations,physical and chemical stability factor and acute toxicity of the metabolites.Finnally,we identified active ingredients in the metabolites of the strain by GC-MS analysis.This research is for the purpose of exploring for endophytes from Huperzia serrata to produce active components of delaying and treating AD, developing the new active screening method,providing the idea for studies the new natural products for AD's treatment.Main contents of this thesis as follows:1,DTNB coloration method was used to screen endophytic fungi from Huperzia serrata which have AChE inhibitory activity in vitro.The results show that the inhibition ratios to AChE of g5,xg,g8 were higher than the others and the IC₅₀were 434.17μg/mL,293.53μg/mL and 285.62μg/mL,respectively.And it was indicated that g5,g8,xg were reversible inhibitory type.HPLC method on g5,xg,g8 broths showed that the HupA wasn't exist in their broths.2,The preliminary study is for g5,xg,g8 endophytic fungal metabolites antagonizing againest H₂O₂-induced nerve cell damage.The results of MTF method and cell observation showed that prioring to given 0.5～5000μg/mL dosage of g5 metabolites' alcohol extract samples could significantly antagonize againest H₂O₂-induced nerve cell damage.But the same dosage of xg,g8 metabolites' alcohol extract samples had no protective effect.3,The result of preliminary identification of the g5 morphology showed that g5 belongs to Penicillium.rugulosum.4,According to initially analysis of enzyme kinetics,it was showed that g5 was admixed competitive reversible inhibitory type,and the inhibit-constant K_I against free E and K_ISagainst comoles compound of ES was 0.0789mL and 1.1352mL, respectively.5,We also have studied physical and chemical stability of g5 metabolites.The result showed that active material of inhibiting AChE could resist heat and radiation of UV.The acute toxicity test shows that g5 strain broth is the low toxicity.6,Under the guiding of the tracking activiy principles,active systematic separation portions of g5 strain were traced in order to determined active intensity with DTNB coloration method and SH-SY5Y cells MTT method.The result shows that the parts of ethyl ether and ethyl acetate had significantly the activity of inhibiting AChE.And the results of MTT method and cell observation shows that the parts of ethyl ether and Ethyl acetate could significantly protective effect for H₂O₂-induced nerve cell damage,but they no treat effect for it.7,We have done the preliminary separation and analysis of g5 metabolites.By extracting with ethyl acetate from g5 metabolites,preparing the active Y-e component with PTLC and analysis with GC-MS method,we have identified the mevalonic acid and nicotinic acid in the Y-e component and the part of ethyl acetate.8,The results of MTT method showed that 0.5～5000μg/mL dosage of Y-e component could significantly antagonize againest H₂O₂-induced nerve cell damage. The mechanisms by which Y-e component protected neuron cells from oxidative stress included the increasing the level of SOD and GSH activity;downregulation of LDH leakage of extracellule.