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.
引文
[1]李瑞.药理学[M].北京:人民卫生出版社,2006:310
[2]林煜.对AD治疗研究的反思[J].医学与哲学,2000,3(1):14-16
[3]Brookmeyer,R.,Gray,S.,Kawas,C.Projections of Alzheimer's disease in the United States and the public health impact of delaying disease onset[J].Public Health 88,1337-1342
[4]Moon Ho Park,Sangmee Ahn Jo.Awareness of putative risk factors for Alzheimer's disease among elderly Koreans[J].Acta Neuro psychiatrica,2008,20(1):20-24
[5]盛树力.老年痴呆发病机理研究进展和药物治疗未来战略[J].中国医学科学院学报,2004,26(2):101-103
[6]赵芳,马文雯.上海南汇区养老院老年痴呆症流行病学调查[J].现代预防医学,2007,34(15):2864-2866
[7]刘泉开.老年性痴呆病研究进展[J].现代诊断与治疗,2001,12(2):65-67
[8]Selkoe,D.J.,Abraham,C.R.,Podlisny,M.B.,& Duffy,L.K.Isolation of low-molecular-weight proteins from amyloid plaque fibers in Alzheimer' s disease[J].Journal of Neurochemistry,1986,46,1820-1834.
[9]Kang,J.,Lemaire,H.G.,Unterbeck,A.,Salbaum,J.M.,Masters,C.L.,Grzeschik,K.H.,et al.The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor[J].Nature,1987,325,733-736
[10]Lue,L.F.,Kuo,Y.M.,Roher,A.E.,Brachova,L.,Shen,Y.,Sue,L.,et al.Soluble amyloid beta peptide concentration as a predictor of synaptic change in Alzheimer's disease[J].American Journal of Pathology,1999,155,853-862
[11]McLean,C.A.,Cherny,R.A.,Fraser,F.W.,Fuller,S.J.,Smith,M.J.,Beyreuther,K.,et al.Soluble pool of Aβ amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease[J].Annals of Neurology,1999,46,860-866.
[12]Dahlgren,K.N.,Manelli,A.M.,Stine,W.B.J.,Baker,L.K.,Krafft,G.A.,& LaDu,M.J.Oligomeric and fibrillar species of amyloid-β peptides differentially affect neuronal viability[J].Journal of Biological Chemistry,2002,277,32046-32053.
[13]1Haass,C.,& Selkoe,D.J.Soluble protein oligomers in neurodegeneration:lessons from the Alzheimer' s amyloid betapeptide.Nature Reviews in Molecular Cell Biology,2007,8,101-112.
[14]Lambert,M.P.,Barlow,A.K.,Chromy,B.A.,Edwards,C.,Freed,R.,Liosatos,M.,et al.Diffusible,nonfibrillar ligands derived from Abetal-42 are potent central nervous system neurotoxins[J].Proceedings of the National Academy of Science USA,1998,95,6448-6453.
[15]Lacor,P.N.,Buniel,M.C.,Furlow,P.W.,Clemente,A.S.,Velasco,P.T.,Wood,M.,et al.Abeta oligomer-induced aberrations in synapse composition,shape,and density provide a molecular basis for loss of connectivity in Alzheimer' s disease[J].Journal of Neuroscience,2007,27,796-807.
[16]盛一珉.乙酰胆碱酯酶抑制剂的研究进展[J].海峡药学,2005,17(4):10-13
[17]韩大雄.老年痴呆症的分子病理机制[J].化学通报,2003,2:95-102
[18]Paul T Francis,Alan M Palmer,Michael Snape,et.al.The cholinergic hypothesis of Alzheimer's disease:a review of progress[J].Neurol Neurosurg Psychiatry,1999,66:137-147
[19]Soreq H,Seidman S.Acetylcholinesterase-new roles for an old actor.Nat Rev Neurosci,2001,2:294-302
[20]Calderon F H,von Bernhardi R,De Ferrari G,et al.Toxic effects of acetylcholinesterase on neruronal andglial-like cells in vitro.MolPsychiatry,1998,3:247-255
[21]Rees T,Hammond P I,Soreq H,et al.Acetylcholinesterase promotes beta-amyloid plaques in cerebral cortex.Neurobiol Aging,2003,24:777-787
[22]徐恩斌,张忠兵.乙酰胆碱酯酶的研究进展[J].国外医学生理、病理科学与临床分册,2003,23(1):73-75
[23]Melo J B,Agostinho P,Oliveira C R.Involvement of oxidative stress in the enhancemen to face tylcholinesterase activity induced by amyloid beta-peptide.Neurosci Res,2003,45:117-127
[24]Hirai,K.,Aliev,G.,Nunomura,A.,Fujioka,H.,Russell,R.L.,Atwood,C.S.,et al.Mitochondrial abnormalities in Alzheimer' s disease.Journal of Neuroscience,2001,21,3017-3023.
[25]Yah,S.D.,Fu,D.,Soto,C.,Chert,X.,Zhu,H.,Al-Mohanna,F.,et al.An intracellular protein that binds amyloid-β peptide and mediates neurotoxicity in Alzheimer' s disease.Nature,1997,389,689-695.
[26]Falkevall,A.,Alikhani,N.,Bhushan,S.,Pavlov,P.F.,Busch,K.,Johnson,K.A.,et al..Degradation of the amyloid beta-protein by the novel mitochondrial peptidasome,PreP.Journal of Biological Chemistry,2006,281,29096-29104.
[27]Aleardi,A.M.,Benard,G.,Augereau,O.,Malgat,M.,Talbot,J.C.,Mazat,J.P.,et al.Gradual alteration of mitochondrial structure and function by beta-amyloids:importance of membrane viscosity changes,energy deprivation,reactive oxygen species production,and cytochrome c release.Journal of Bioenergetics and Biomembranes,2005,37,207-225.
[28]Fukuyama H,Ogawa M,Yamauchi H,et al.Altered cerebral energy metabolism in Alzheimer's disease:a PET study[J].J Nucl Med,1994,35:1-6.
[29]代其林,张跃飞.线粒体与细胞的衰老[J].绵阳师范高等专科学校学报,2002,21(5):57-60
[30]Arlt,S.,Beisiegel,U.,& Kontush,A.Lipid peroxidation in neurodegeneration:newinsights into Alzheimer' s disease.Current Opinion in Lipidology,2002,13,289-294.
[31]盛树力.老年性痴呆的药物治疗和药物作用靶位[J].中国医药导刊,2003,28(6):402-404
[32]叶未设.中医学对老年性痴呆症病因机学认知及治疗[J].中国临床康复,2002,6(15):2311-2312
[33]陈莲珍,王育琴.老年痴呆症的药物治疗与进展[J].中国临床康复,2002,6(21):3269
[34]余红英.草药蛇足石杉的研究进展[J].中草药,2001,32(3):279-282
[35]Tang XC,Han YF.Pharmacological profile of huperzine A,a novel acetylcholine esterase inhibitor from Chinese herb[J].CNS Drugs Rev,1999,5:281-300
[36]李沛琳,郭水良.石杉科植物研究综述[J].兴安师范学院学报,2005,11(1):56-62
[37]Zhi-fei Wang,Xi-can Tang.Effects of huperzine A on memory deficits and neurotrophic factors production after transient cerebral ischemia and reperfusion in mice[J].Pharmacology,Biochemistry and Behavior,2006,83:603-611
[38]Li-Li Tang,Xi-Can Tang.Huperzine A protects SH-SY5Y neuroblastoma cells against oxidative stress damage via nerve growth factor production[J].European Journal of Pharmacology,2005,519:9-15
[39]Xiao Qiu Xiao,Xi Can Tang.Protective effects of huperzine A on β-amyloid25-35 induced oxidative injury in rat pheochromocytoma cells[J].Neuroscience Letters,2000,286:155-158
[40]Xiao Qiu Xiao,Xi CanTang.Huperzine A protects rat pheochromocytoma cells against hydrogen peroxide-induced injury[J].Neuroscience Letters,1999,275:73-76
[41]张磊等.石杉碱甲的研究进展[J].中草药,2006,36(9):1422-1426
[42]吴荭等.中国蛇足石杉资源调查与评估[J].自然资源学报,2005,20(1):59-67.
[43]黎万奎,胡之璧.内生菌与天然药物[J].中国天然药物,2005,3(4):193-200
[44]Stierle A,Strobel G,Stierle D·Taxol and taxane production by Taxomyces andreanae,an endophytic fungus of Paific yew[J].Science,1993,260(5105):214-216
[45]Petrini O.Fungal Endophytes of Tree Leaves In:Andrews JH,Hirano SS eds.Microba Ecology of Leaves[M].New York:Springer-Yerlag,1991,179-197
[46]Stone J K,Bacon C W,White J F Jr.An overview of endophytic microbes:endophytism defined[]].Microbial Endophytes.New York:Marcel Dekker,2000,3-29
[47]郭良栋.内生真菌研究进展[J].菌物系统,2001,20:148-152
[48]Bruchman,H.Vber die Prothallien und die Keimpflanzen mehrerer europiischer Lycopodien[m].Gotha,1898:119.Treub,M.Etudes sur les lycopodiacees.I.Le prothalle du Lycopodium cernuum L[J].Ann.Jard.Bot.Buitenz.1884,4:107-138.
[49]Lang,W.H.The prothallus of Lycopodium clavatum[J].Ann.Bot,1899,13:279-318.
[50]皱文欣 谭仁祥 植物内生菌研究新进展[J].植物学报,2001,43:881-892
[51]Freeman S,Rodriguez R J.Geneticconversion of a fungal plant pathogen to a nonpathogenic,endophytic mutualist.Science,1993,260:75-78.
[52]Sieber Canavesi F,Petrini O,Sieber TN.Endophytic Lep tostroma specieson Picea abies,Abies alba and Abies balsamea:a cultural,biochemical,biochemical,and numerical sthdy[J].Mycologia,1991,83:89-96
[53]Vujanvic V,St-Arnaud M.Leptomelanconium abietis sp.nov.on needles of Abies balsamea[J].Mycologia,2001,93:212-215
[54]张集慧,王春兰.兰科药用植物的5种内生真菌产生的植物激素[J].中国医学科学院学报.1999,12:460-465
[55]Lu H,Zou W X,Meng J C.New bioactive metabolites produced by Colletotrichum sp.,an endophytic fungus in Artemisia annua[J].Plant Sci,2000,151:67-73.
[56]赵杨景,郭顺星,高薇薇,等.三种内生真菌与大花蕙兰共生对矿质营养吸收的影响[J].园艺学报,1999,26(2):110-115
[57]兰琪,姬志勤,顾爱国,师宝君,吴文君.苦皮藤内生真菌中杀虫杀菌活性物质的初步研究[J].西北农林科技大学学报(自然科学版),2004,32(10):79-84.
[58]Ball O J P,Barker G M,Prestidge R A.endophytes of grasses:their potential as biocontrol agent[J].Journal of Chemical Ecology,1997,23:1420-1449.
[59]Spiers D E,Zhang Q,Eichen P A.Endophyte the evolution of tem,and clarification of its use and definitionEJ].J Anim Sci,1995,73:1954-1961.
[60]Ball O J P,Prestidge R A,Sprosen J M.Fungal endophytes of grasses[J]..Applied and Environmental Microbiology,1995,61:1527-1533.
[61]梁宇,高玉葆.内生真菌对植物生长发育及抗逆性的影响[J].植物学通报,2000,17(1):52-59
[62]杨海莲,孙晓璐,宋未.植物内生细菌的研究[J].微生物学通报.1998,25(4):224-227
[63]ZouW-X,TanR-X.Biological and chemical diversity of endophytes and their potential applictions.LiC-S[D].Advances in Plant Sciences.Vol 2.Beijing:China Higher Education Press,1999.183-190.
[64]任安芝,高玉蕉.植物内生真菌——一类应用前景广阔的资源微生物[J].微生物学通报.2001,28(6):90-93
[65]范黎,郭顺星,徐锦堂.我国部分兰科植物菌根的内生真菌种类研究[J].山西大学学报(自然科学版),1998,21(2):169-177.
[66]Anorld A E,Maynard Z,Gilbert G S,Coley P D,Kursar T A.Are tropical fungal endophytes hyperdiverse?[J].Ecol Lett,2000,3:267-274
[67]Strobel G A.Endophytes as sources of bioactive products[J]Microbes Infect.2003,5(6):535-544
[68]李桂玲,王建锋,黄耀坚.几种药用植物内生真菌抗真菌活性的初步研究[J].微生物学通报,2001,28(6):64-68
[69]Zou W X,Meng J C,Lu H,etal.Metabolites of Colletotrichum gloeosporioides,an endophytic fungus in Artemisia mongolica[J].J Nat Prod,2000,63:1529-1530
[70]Rodrigues K F.The foliar fungal endophytes of the Amazonian palm Euterpe oleracea[J].Mycologia,1994,86:376-385.
[71]Findlay J A,Li G,Penner P E.Novel diterpenoid insect toxin from a conifer endophyte[J].J Nat Prod,1995,58:197-200
[72]Ju Y et al.Bioactive Favonolds from endophyte infected blue grass(Poaampla)[J].J Agric Food Chem.1998,46:3785-3788
[73]ZouW-X,TanR-X.Biological and chemical diversity of endophytes and their potential applictions.LiC-S[D].Advances in Plant Sciences.Vol 2.Beijing:China Higher
[74]张玲琪,郭波,李海燕,等.长春花内生真菌的分离及其发酵产生药用成分的初步研究[J].中草药,2000,31(11):805-807
[75]郭波,李海燕,张玲琪.一种产长春碱真菌的分离[J].云南大学学报(自然科学版),1998,20(3):214-215
[76]李海燕,王志军,张玲琪,等.一种桃儿七内生真菌的分离初报[J].云南大学学报(自然科学版),1999,21(3):243。
[77]邹文欣,谭仁祥.植物内生菌研究新进展[J].植物学报,2001,43(9):881-892
[78]张玲琪,谷苏,邵华,等.发酵产鸢尾酮真菌的分离鉴定及生香特性的初步研究[J].菌物系统,1999,18(1):49-54
[79]黎万奎,周吉燕,林子为,胡之璧.蛇足石杉内生真菌2F09P03B产石杉碱甲发酵条件的研究[J].中国医药生物技术,2007,2(4):254-259
[80]Nisbet L J.,Fox F.M.The importance of microbiodiversity to biotechnology.Hawksworth D L(ed)The Biodiversity of Microorganismsand Invertebrates:Its Role in Sustainable Agriculture,1991,229-244
[81]Roth J,et al.The evolutionary origions of intercellular communication and the Maginot Lines of the mind[J].Ann New York Academy Sci,1986,436:1-11
[82]杜晓华.观赏植物马尾杉内生菌及其次生代谢产物对AChE的抑制活性[D].西北农林科技大学,2003
[83]Naoki Nagatani,Akihiro Takeuchi.Rapid and sensitive visual detection of residual pesticides in food using acetylcholinesterase-based disposable menbrancs chips[J].Food control,2007(18):914-920
[84]Ellman GL,Courtney KD,Andre V,et al.A new and rapid colorimetric determination of acetylcholinesterase activity[J].Blochem Pharmocol,1961,7:88-95
[85]B.Yinutha,D.Prashanth,et al.Screening of selected Indian medicinal plants for acetylcholinesterase inhibitory activity[J].Journal of Ethmopharmacology,2007,109:359-363
[86]杨宏云,徐国兵,夏铁安.人血清假性胆碱酯酶连续监测法测定方法学的建立及其在肝功能评价中的应用[J].中国实验诊断学,2003,7(2):83-87
[87]董杰.乙酰胆碱酯酶抑制法快速检测蔬菜农药残留技术的研究[D].山东农业大学,2003,125-127
[88]郭勇,郑穗平.酶学[M].广州:华南理工大学出版社,2000,95-97
[89]王峻,潘胜利.HPLC法测定6种石杉科植物中石杉碱甲的含量[J].中草药,2003,(34)7:607-608
[90]Rosenfeld C,Kousba A,SuLtatos LG.Interactions of Rat Brain Acetylcholinesterase with the Detergent Triton X-100 and the Organophosphate Paraoxon[J].Toxicol Sci.,2001,(63):208-213
[91]Kazuhiko Otoguro,Satoshi Omura.Arisugacins,selective acetylcholinesterase inhibitors of microbial origin[J].Pharmacology & Therapeutics,1997,76:45-54
[92]魏景超.真菌鉴定手册[M].第1版.上海:上海科学技术出版社,1979.501-512
[93]龚仁敏.酶可逆抑制作用中线性混合型抑制的动力学[J].安徽师大学报,1998,21(1):46-49
[94]郭晶,高菊芳,唐振华.乙酰胆碱酯酶的动力学机制及其应用[J].农药,2007,46(1):18-22
[95]周立国.药物毒理学[M].湖北:中国医药科技出版社,2003(第1版),146-161
[96]李寿祺.毒理学原理与方法[M].成都:四川大学出版社,2003(第2版),77-83
[97]louis CD,Hayes A W.Acute toxicity and eye irritancy.In:Hays A W edited,Principles and methods of toxicology.Fourth edition,2001:853-916
[98]袁勤生.现代酶学[M].第1版:华东理工大学出版社,2001.31-36
[99]林敏,何群,陈清西.没食子酸衍生物对蘑菇酪氨酸酶的抑制机理[J].厦门大学学报.2006,45(4):567-569
[100]沈建忠.动物毒理学[M].中国农业出版社,2002(第1版),84-95
[101]姚新生.中药天然药物活性成分的研究方法[J].药学服务与研究,2003,3(4):205-209.
[102]肖崇厚主编.中药化学[M].上海科学技术出版,2002
[103]罗健东,管锦霞.甲羟戊酸通路对细胞生长调控作用的研究进展[J].生命科学,1999,11(5):212-216
[104]刘克杉,黄晓星等.辅酶Q的生物合成途径以及相关的酶和基因[J].中国医药工业杂志,2006,37(2):140-144
[105]Bonakdar RA,Guarneri E.Coenzyme Q_(10)[J].Am Fam Physician,2005,72(6):1065
[106]张继中,迟莉丽等.辅酶Q_(10)的生产及在医学领域中的应用[J].上海应用技术学院学报,2004,4(4):31
[107]王怀颖,石少慧.辅酶Q_(10)对脑衰老影响的实验研究[J].中国公共卫生,2004,20(11):1346-1347
[108]Storch A.Coenzyme Q(10)in Parkinson's disease[J].NERVENARZT,2007,78(12):1378-1382
[109]文立,蒋春笋等.线粒体呼吸链电子传递载体——辅酶Q与运动能力[J].中国运动医学杂志,2001,20(1):68-71
[110]Kenjiro Ono,Kazuhiro Hasegawa,et al.Preformed β-amyloid fibrils are destabilized by coenzyme Q_(10)in vitro[J].Biochemical and Biophysical Research Communications,2005,330:111-116
[111]罗健东,管锦霞.甲羟戊酸通路对细胞生长调控作用的研究进展[J].生命科学,1999,11(5):212-216
[112]汤德良.植物抗虫德次生代谢物质[J].世界农业,1999,239(3):32-33
[113]钟寿民.食用植物中甲羟戊酸衍生物的抗肿瘤作用[J].医学研究通讯,1999,28(6):22-23
[114]Goldstein JL.甲羟戊酸途径的调节[J].国外医学分子生物学分册,1990,12(6):271-276
[115]RoulletJB,Xue H,RoulletCM,etal.Mevalonate availability affects human and rat resistance vessel function.J Clin Invest,1995,96 1:239-244
[116]李艳云,尹振晏等.烟酸的研究进展[J].北京石油化工学院学报,2006,14(1):59-64
[117]Morris M.C.,Evans D.A.et al.Dietary niacin and the risk of incident Alzheimer' s disease and of cognitive decline[J].Clinical Neurology,2005,1(1):16-17
[118]Cheesman KH,Slater TF.An introduction to free radical biochemistry.New York:Chrchill Livingstone;1993,481-93.
[119]Al-OmarMA,BeedhamC,Alsarra IA.Pathological roles of reactive oxygen species and their defence mechanisms.Saudi Pharm J 2004;12:1-18.
[120]Spickett CM,Jerlich A,Panasenko OM,et al.The reactions of hypochlorous acid,the reactive oxygen species produced by myeloperoxidase,with lipids.Acta Biochim Pol 2000;47:889-99.
[121]Forman HJ,Torres M.Reactive oxygen species and cell signaling:respiratory burst in macrophage signaling.Am J Respir Crit Care Med 2002;166:4-8.
[122]Rosen GM,Pou S,Ramos CL,Cohen MS,Britigan BE.Free radicals and phagocytic cells.FASEB J 1995;9:200-9.
[123]G.Cohen,Enzymatic/nonenzymatic sources of oxyradicals and regulation of antioxidant defenses,Ann.N.Y.Acad.Sci.738(1994),pp.8-14.
[124]Yoshikawa et al.,2006 A.Yoshikawa,Y.Saito and K.Maruyama,Lignan compounds and 4,4′ -dihydroxybiphenyl protect C2C12 cells against damage from oxidative stress,Biochem.Biophys.Res Commun.344(2006),394-399.
[125]Hensley K,Maidt ML,Yu Z,Sang H,Markesbery WR,Floyd RA.Electrochemical analysis of protein nitrotyrosine and dityrosine in the Alzheimer brain indicates region-specific accumulation.J Neurosci.1998 Oct 15;18(20):8126-32.
[126]Butterfield DA,Castegna A,Lauderback CM,Drake J.Evidence that amyloid beta-peptide-induced lipid peroxidation and its sequelae in Alzheimer's disease brain contribute to neuronal death.Neurobiol Aging.2002,23(5):655-664.
[127]冯波,王蓉,盛树力.神经退行性疾病研究中拟神经细胞模型:人神经母细胞瘤株SH-SY5Y的来源特性及应用[J].中国临床康复.2006,10(6):121-123
[128]郑志弘,林铃.神经细胞培养理论与实践[M].科学出版社,2001:18
[129]张卓然.培养细胞学与细胞培养技术[M].上海科技出版社,2004