蛇足石杉内生真菌抗AD症活性的初步研究
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摘要
阿尔茨海默病(AD)是一种进行性神经退行性疾病,是中老年常见病。目前,世界人口正逐渐迈入老龄化,AD病也日益增多。研究表明,其发病机理是由于一系列级联反应引起的神经元损伤和神经细胞凋亡。其中,乙酰胆碱能系统中的乙酰胆碱酯酶(AChE)、线粒体电子传递链(ETC)中释放出来的活性氧(ROS)以及Aβ可溶性寡聚体的聚集可能在级联反应中处于较为重要的地位。
蛇足石杉(Huperzia serrata(Thunb.)Trev.)属于石杉属(Huperzia),是一种珍贵的药用蕨类植物。从中分离得到的石杉碱甲(Huperzine A,Hup A)是国内自主开发的成功治疗和延缓AD病的药物,它以高效可逆抑制AChE为主要作用,多靶点多途径治疗AD病症。但由于蛇足石杉繁殖能力弱,过度采摘造成其野生资源减少,甚至枯竭,但HupA的市场需求量大,因此解决其原料矛盾就是需要另辟新路。
植物内生真菌能产生大量新的天然产物,有些内生真菌还可以产生与宿主植物相同或相近的代谢产物,已成为天然产物潜在的重要资源。据此,本研究采用改良体外AChE抑制活性试验以及H_2O_2诱导的SH-SY5Y神经细胞损伤为筛选模型,对野生蛇足石杉的内生真菌代谢产物进行多活性初筛,逐步聚焦活性菌株。对活性菌株代谢产物进行初步的动力学关系、理化因素稳定性以及急性毒性的研究。最后运用活性检测指导下的靶向跟踪,确定该菌株代谢产物中的活性部位,并做初步的分析。本文为探索性研究,旨在从蛇足石杉内生真菌代谢产物中发现缓解和治疗AD病的活性成分,开拓新的活性筛选方法,为研究新的AD病天然药物提供思路。
主要研究内容如下:
1、采用改良后的DTNB法对蛇足石杉内生真菌代谢产物进行了体外AChE抑制活性筛选。结果表明:从根部分离得到内生真菌g5、xg、g8具有显著抑制AChE的活性,其IC_(50)分别达到:434.17μg/mL、293.53μg/mL、285.62μg/mL,且初步判断其抑制类型为可逆抑制。HPLC法对g5、xg、g8发酵液进行检测,发现其中不存在HupA。
2、采用MTT法检测了蛇足石杉内生真菌g5、xg、g8代谢产物拮抗由H_2O_2诱导的神经细胞氧化应激损伤。检测结果及细胞形态学观察表明,预先给予0.5～5000μg/mL剂量的g5菌株代谢产物醇提液,均能有效的对抗H_2O_2诱导的神经细胞氧化应激损伤,同样剂量下的xg、g8发酵醇提液样品却无保护作用。
3、对g5菌株做了初步的形态学鉴定,结果表明g5菌株为半知菌目丛梗孢科青霉属褶皱青霉组(Penicillium.rugulosum)。
4、对g5菌株代谢产物做了初步的AChE抑制动力学分析,确定其对AChE的抑制作用为混合竞争型可逆抑制。对游离酶抑制常数K_I与对酶底物络合物的抑制常数K_(IS)分别为0.0789mL和1.1352mL。
5、对g5菌株代谢产物的理化性质进行了初步的研究,结果表明,代谢产物中抑制AChE的活性物质耐热及耐紫外辐射;急性毒性试验表明,g5菌株发酵液为低毒毒性。
6、以活性检测指导下的靶向跟踪为手段,对g5菌株代谢产物进行了系统分离。结果显示,乙醚及乙酸乙酯部位具有显著抑制AChE的活性,且MTT及形态观察结果表明乙醚及乙酸乙酯部位对H_2O_2诱导的神经细胞氧化应激损伤均具有显著的保护作用,但对该损伤无治疗作用。
7、对g5菌株代谢产物进行了初步分离及分析。乙酸乙酯萃取g5菌株代谢产物中的活性成分,PTLC分离制备活性组分Y-e,GC-MS初步分析Y-e中含有甲羟戊酸类及烟酸等活性物质,有报道称其具有抗AChE的作用。
8、对活性组分Y-e拮抗H_2O_2诱导的神经细胞氧化损伤进行了初步的研究。MTT结果表明503μg·mL~(-1)～50.3ng·mL~(-1)的组分Y-e能有效拮抗由H_2O_2诱导的神经细胞氧化损伤,在一定剂量下的组分Y-e可上调胞内SOD的活性水平及GSH的产生效率,显著降低LDH的胞外漏出量。
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 H_2O_2-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 IC_(50)were 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 H_2O_2-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 H_2O_2-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_(IS)against 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 H_2O_2-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 H_2O_2-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.
蛇足石杉(Huperzia serrata(Thunb.)Trev.)属于石杉属(Huperzia),是一种珍贵的药用蕨类植物。从中分离得到的石杉碱甲(Huperzine A,Hup A)是国内自主开发的成功治疗和延缓AD病的药物,它以高效可逆抑制AChE为主要作用,多靶点多途径治疗AD病症。但由于蛇足石杉繁殖能力弱,过度采摘造成其野生资源减少,甚至枯竭,但HupA的市场需求量大,因此解决其原料矛盾就是需要另辟新路。
植物内生真菌能产生大量新的天然产物,有些内生真菌还可以产生与宿主植物相同或相近的代谢产物,已成为天然产物潜在的重要资源。据此,本研究采用改良体外AChE抑制活性试验以及H_2O_2诱导的SH-SY5Y神经细胞损伤为筛选模型,对野生蛇足石杉的内生真菌代谢产物进行多活性初筛,逐步聚焦活性菌株。对活性菌株代谢产物进行初步的动力学关系、理化因素稳定性以及急性毒性的研究。最后运用活性检测指导下的靶向跟踪,确定该菌株代谢产物中的活性部位,并做初步的分析。本文为探索性研究,旨在从蛇足石杉内生真菌代谢产物中发现缓解和治疗AD病的活性成分,开拓新的活性筛选方法,为研究新的AD病天然药物提供思路。
主要研究内容如下:
1、采用改良后的DTNB法对蛇足石杉内生真菌代谢产物进行了体外AChE抑制活性筛选。结果表明:从根部分离得到内生真菌g5、xg、g8具有显著抑制AChE的活性,其IC_(50)分别达到:434.17μg/mL、293.53μg/mL、285.62μg/mL,且初步判断其抑制类型为可逆抑制。HPLC法对g5、xg、g8发酵液进行检测,发现其中不存在HupA。
2、采用MTT法检测了蛇足石杉内生真菌g5、xg、g8代谢产物拮抗由H_2O_2诱导的神经细胞氧化应激损伤。检测结果及细胞形态学观察表明,预先给予0.5～5000μg/mL剂量的g5菌株代谢产物醇提液,均能有效的对抗H_2O_2诱导的神经细胞氧化应激损伤,同样剂量下的xg、g8发酵醇提液样品却无保护作用。
3、对g5菌株做了初步的形态学鉴定,结果表明g5菌株为半知菌目丛梗孢科青霉属褶皱青霉组(Penicillium.rugulosum)。
4、对g5菌株代谢产物做了初步的AChE抑制动力学分析,确定其对AChE的抑制作用为混合竞争型可逆抑制。对游离酶抑制常数K_I与对酶底物络合物的抑制常数K_(IS)分别为0.0789mL和1.1352mL。
5、对g5菌株代谢产物的理化性质进行了初步的研究,结果表明,代谢产物中抑制AChE的活性物质耐热及耐紫外辐射;急性毒性试验表明,g5菌株发酵液为低毒毒性。
6、以活性检测指导下的靶向跟踪为手段,对g5菌株代谢产物进行了系统分离。结果显示,乙醚及乙酸乙酯部位具有显著抑制AChE的活性,且MTT及形态观察结果表明乙醚及乙酸乙酯部位对H_2O_2诱导的神经细胞氧化应激损伤均具有显著的保护作用,但对该损伤无治疗作用。
7、对g5菌株代谢产物进行了初步分离及分析。乙酸乙酯萃取g5菌株代谢产物中的活性成分,PTLC分离制备活性组分Y-e,GC-MS初步分析Y-e中含有甲羟戊酸类及烟酸等活性物质,有报道称其具有抗AChE的作用。
8、对活性组分Y-e拮抗H_2O_2诱导的神经细胞氧化损伤进行了初步的研究。MTT结果表明503μg·mL~(-1)～50.3ng·mL~(-1)的组分Y-e能有效拮抗由H_2O_2诱导的神经细胞氧化损伤,在一定剂量下的组分Y-e可上调胞内SOD的活性水平及GSH的产生效率,显著降低LDH的胞外漏出量。
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 H_2O_2-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 IC_(50)were 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 H_2O_2-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 H_2O_2-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_(IS)against 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 H_2O_2-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 H_2O_2-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.
引文
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