鬼臼毒素高产菌株的诱变选育及发酵条件的研究
|
摘要
鬼臼毒素类物质因具有显著的抗病毒、抗肿瘤活性,在医药领域具有重要的应用价值。目前该类物质主要从植物中提取获得,但天然资源有限,无法满足市场需求。由于植物内生真菌具有产生与宿主相同或相似代谢物的特性,且易培养、易控制,并可通过诱变育种等手段来提高其有效成分产量,真菌发酵为获得鬼臼毒素类药物资源提供了新途径。目前这一领域的研究多集中于对有价值菌株的筛选,在菌种改造、发酵条件等方面有待进一步深入研究。
目的对可产生鬼臼类物质的内生真菌采取复合诱变处理,进一步对选育获得的高产株进行发酵条件的研究,以期为解决鬼臼类抗癌药物短缺提供新途径;通过研究获得微生物发酵生产鬼臼类物质的工艺数据,为实现工业化生产奠定基础。方法以药用真菌Ty为研究对象,采用紫外线及He-Ne激光复合诱变处理,获得优势变异菌株;利用薄层层析、薄层色谱扫描、高效液相色谱分析确定高产菌株的发酵培养物中含有目的产物;设计适于该菌株生长的半合成发酵培养基,并优化其组分。结果紫外诱变选育获得的高产菌株Ty4-16,较同批发酵培养的出发菌株生物量提高31%,产物量提高约25%。在此基础上进行激光诱变,最终获得高产株Ty4-16-26,较Ty4-16菌株的生物量提高17%,产物量提高约20%。确定该诱变菌株可产生鬼臼毒素。经优化的半合成发酵培养基配方为:2%淀粉、2%蔗糖、0.1%蛋白胨、0.1%NH_4(SO_4)_2、0.1%酵母膏、0.03%K_2HPO_4、0.03%Nacl、0.03%MgSO_4,水100mL,pH6.5。采用该发酵培养基,28℃培养7天,菌株生物量提高约20.3%,鬼臼毒素产量提高约9.7%。菌株5L发酵罐放大试验表明菌体生长较好,产物合成稳定。结论得到鬼臼毒素高产菌株的诱变选育方法以及用微生物发酵法获得鬼臼毒素类物质的技术路线,该研究具有实现工业化生产的潜力。
Podophyllotoxin is of great important in the field of medicine because of its antivirus and anti-cancer activities. It was derived mainly from the Podophyllotoxin plants. But the natural sources were limited. Endophytic fungi have the activity that can produce same or similar compounds to the plant. They are easy to be raised and controlled and their effective component output may be raised through mutation breeding. It is a new method by fermentation of endophytic fungui. So far,many research focused on separating valuable endophytic fungi which can produce Podophyllotoxin. Through compound mutation to producing-srains,then sudy on the optimizational fermentation conditions of high production strains,in the hope of providing some theoretic reference on producing botanic activity compouds by microbiology fermentation.
Objectives:Compound mutation to producing-srains,then sudy on the optimizational fermentation conditions of high production strains in order to provide a new method obtaining anti-cancer material——podophyllotoxin and the technical data obtained from research on producing botantic activity compounds by microbiology fermentation. This will be lay the foundation for industrialization. Methods:High producing strain is attained by UV and He-Ne laser .The components were identified by thin layer chromatographic(TLC),Thin layer chromatographic scanning,high performance liquid chromatographic(HPLC) that consist in fermentation of high producing strain;The thesis designed semi-synthesized medium compatibhe to the strain, and optimized composition respectively. Results:Ty4-16-26 was attained after UV mutation with its biomass enhancing 31% and production-biomass enhancing 25%. Laser mutation based on this carries obtained high production Ty4-16-26, with biomass enhancing 17% and production-biomass enhancing 20%. This theis confirm that the mutated strain can produce podophyllotoxin. The optimizational ingredient of the semi-synthesized medium were as follows: 2% soluble srarch, 2 %sucrose, 0.1% peptone, 0.1% yeast extract, 0.1% NH_4(SO4)_2, 0.03% K_2HPO_4, 0.03% Nacl , 0.03% MgSO_4, pH6.5. The biomass increase 20.3% and the production-biomass ratio increase 9.7% by using the optimizational medium at temperature of 28℃for 7d. The scale-up trial in 5L fermentor showed that mycelium grew well and products produced steadily. Conclusions:The method that obtain high-producing with mutation breeding;The technics that obtain podophyllotoxin with fermentation and the tenique for extraction were gained. This research has the potential of industrialization.
目的对可产生鬼臼类物质的内生真菌采取复合诱变处理,进一步对选育获得的高产株进行发酵条件的研究,以期为解决鬼臼类抗癌药物短缺提供新途径;通过研究获得微生物发酵生产鬼臼类物质的工艺数据,为实现工业化生产奠定基础。方法以药用真菌Ty为研究对象,采用紫外线及He-Ne激光复合诱变处理,获得优势变异菌株;利用薄层层析、薄层色谱扫描、高效液相色谱分析确定高产菌株的发酵培养物中含有目的产物;设计适于该菌株生长的半合成发酵培养基,并优化其组分。结果紫外诱变选育获得的高产菌株Ty4-16,较同批发酵培养的出发菌株生物量提高31%,产物量提高约25%。在此基础上进行激光诱变,最终获得高产株Ty4-16-26,较Ty4-16菌株的生物量提高17%,产物量提高约20%。确定该诱变菌株可产生鬼臼毒素。经优化的半合成发酵培养基配方为:2%淀粉、2%蔗糖、0.1%蛋白胨、0.1%NH_4(SO_4)_2、0.1%酵母膏、0.03%K_2HPO_4、0.03%Nacl、0.03%MgSO_4,水100mL,pH6.5。采用该发酵培养基,28℃培养7天,菌株生物量提高约20.3%,鬼臼毒素产量提高约9.7%。菌株5L发酵罐放大试验表明菌体生长较好,产物合成稳定。结论得到鬼臼毒素高产菌株的诱变选育方法以及用微生物发酵法获得鬼臼毒素类物质的技术路线,该研究具有实现工业化生产的潜力。
Podophyllotoxin is of great important in the field of medicine because of its antivirus and anti-cancer activities. It was derived mainly from the Podophyllotoxin plants. But the natural sources were limited. Endophytic fungi have the activity that can produce same or similar compounds to the plant. They are easy to be raised and controlled and their effective component output may be raised through mutation breeding. It is a new method by fermentation of endophytic fungui. So far,many research focused on separating valuable endophytic fungi which can produce Podophyllotoxin. Through compound mutation to producing-srains,then sudy on the optimizational fermentation conditions of high production strains,in the hope of providing some theoretic reference on producing botanic activity compouds by microbiology fermentation.
Objectives:Compound mutation to producing-srains,then sudy on the optimizational fermentation conditions of high production strains in order to provide a new method obtaining anti-cancer material——podophyllotoxin and the technical data obtained from research on producing botantic activity compounds by microbiology fermentation. This will be lay the foundation for industrialization. Methods:High producing strain is attained by UV and He-Ne laser .The components were identified by thin layer chromatographic(TLC),Thin layer chromatographic scanning,high performance liquid chromatographic(HPLC) that consist in fermentation of high producing strain;The thesis designed semi-synthesized medium compatibhe to the strain, and optimized composition respectively. Results:Ty4-16-26 was attained after UV mutation with its biomass enhancing 31% and production-biomass enhancing 25%. Laser mutation based on this carries obtained high production Ty4-16-26, with biomass enhancing 17% and production-biomass enhancing 20%. This theis confirm that the mutated strain can produce podophyllotoxin. The optimizational ingredient of the semi-synthesized medium were as follows: 2% soluble srarch, 2 %sucrose, 0.1% peptone, 0.1% yeast extract, 0.1% NH_4(SO4)_2, 0.03% K_2HPO_4, 0.03% Nacl , 0.03% MgSO_4, pH6.5. The biomass increase 20.3% and the production-biomass ratio increase 9.7% by using the optimizational medium at temperature of 28℃for 7d. The scale-up trial in 5L fermentor showed that mycelium grew well and products produced steadily. Conclusions:The method that obtain high-producing with mutation breeding;The technics that obtain podophyllotoxin with fermentation and the tenique for extraction were gained. This research has the potential of industrialization.
引文
[1]陈毓亨.我国鬼臼类植物资源的研究[J].药学学报,1979,14(2):101-107
[2]孙文基,谢世昌.天然药物成分定量分析[M].北京:中国医药科技出版社,2003年1月,365
[3]杨明,纳绍荣.鬼臼毒素质量标准研究[J].云南师范大学学报,1999,19(2):49-50
[4]李国峰,许重远,孟庆礼等.鬼臼毒素具有荧光性[J].第一军医大学学报,1996,16(3):268-269
[5]汪世龙,姚思德,李义久等.鬼臼毒素及其衍生物的光敏性研究[J].化学物理学报,1997,10(5):432-435
[6]高蓉,田暄,张兴等.鬼臼毒素类生物活性物质的研究概况[J].农药学学报,2000,2:1-6
[7]朱承根,杨晶,李弟灶等.天然抗肿瘤药鬼臼毒素及其衍生物研究进展[J].药品评价,2004,1(4):306-309
[8]Kaplan I W.Condylomata acuminate.New Orleans Med Surg J,1942,94:388-390
[9]Cannel C.,Moraes R.M.,Dayan F.E.,Ferreira D.,Phytochemistry,2000,54:115-120
[10]TF lmbert.Discovery of podophyllotoxin.Biochimie,1998,80:207-222
[11]冯国宜.抗癌植物资源与天然产物研究.湖北民族学院学报(自然科学版).2001,19(3):26-32
[12]贾红光,曹长年,陈建军等.鬼臼毒素类抗肿瘤药物的研究[J].青海医学院学报,2002,23(2):52-55
[13]杨新波.鬼臼毒素抗肿瘤作用研究进展[J].国外医药·植物药分册,1996,11(4):158-159
[14]马维勇,张椿年.鬼臼类抗肿瘤药物的研究进展.中国医药工业杂志,1997,28(2):65-69
[15]潘建林,王彦广,陈耀祖.表鬼臼毒素羧酸酯的合成及体外抗肿瘤活性.药学学报,1997,32(12):898-901
[16]贾红光,贾长年,陈建军等.鬼臼类抗肿瘤药物的研究.青海医学院学报,2002,23(2):52-55
[17]王彦广,陶兰,潘建林等.抗癌活性鬼臼毒素衍生物的合成与构效关系的研究.高等学校化学学报,1997,18(7):1061-1066
[18]WANG Jun-Zhi,TSUMURA Hideki,SHIMURA Keishiro etal.Effect of spin labeled derivatives of podophyllotoxin on cell cycle and macromolecular synthesis in human lymphoid leukemia Molt 4B cells.中国药理学报,1998,19(16):501-505
[19]潘晓辉,王琳,王浩东.鬼臼类木脂素抗肿瘤药物研究进展[J].安康师专学报,1999,11(3):48-53
[20]何峰,刘宗潮,罗一帆等.抗肿瘤药鬼臼毒素衍生物构效关系的研究[J].癌症,2001,20(4):368-372
[21]Wilson L,Friedkin M.the biochemical events of mitosis ll.the in vivo and vitro binding of colchicine in grasshopperembryos and its possible relation to inhibition of mitosis[J].Biochemistry,1967,6(10):3126
[22]Grace LC,Liu Y,Thomas CR,et al.Nonintercalative Anti-tumor Drugs Interfere with the Breakage Reunion Reaction of Mammalian DNA Topoisomerase 11[J].The Journal of Biological Chemistry,1984,259(21):13560
[23]John DL,Susan BH.Effect of VP-16-213 on the Intracelular Degradation of DNA in HeLa Cells[J].Biochemistry,1976,15(25):5443
[24]汪世龙,姚思德,王玫等.鬼臼毒素及其衍生物的化学活性研究[J].辐射研究与辐射工艺学报,1997,15(2):86-90
[25]Kalyanaraman B,Nemec J,Sinha BK.Characterization of free radical produced during oxidation of etoposide(VP-16)and its catehol and quinine[J].Biochemistry,1989,28(11):4839-4846
[26]Yoshihiko lnamori,Mayuri Kubo.Himishi Tsujibo,et al,The biological activities of podophyllotoxin compounds[J].Chen Pharm Bukk,1986,34(9):3928-3932
[27]Kozawa M.,Baba K.,Matsuyma Y.et al.Chen.Pharm.Bull.,1982,30(8):2885-2888
[28]张兴,冯俊涛,陈安良等.砂地柏杀虫作用研究概况[J].西北农林科技大学学报(自然科学版),2002,30(4):130-134
[29]刘艳青,张守刚,程洁等.鬼臼毒素类物质生物活性的研究[J].医学研究生学报,2006,19(3):205-209
[30]田喧,高蓉,张兴.鬼臼毒素类似物结构与杀虫活性关系初探[J].西北农业大学学报,2000,28(6).19-24
[31]Inamori Y..Chem.Pharm.Bull.,1984,32(5):2015-2019
[32]Inamori Y.,Kubo M.,Tsujibo H..Mechanisms of insecticidal action of deoxypodophyllotoxin.Chen.Pharm.bull.,1986,34(5):2247-2250
[33]Mac Rae W D,Hudson J B,Towers G H.The anvirus action of lignans[J].Planta Med,55,531-535
[34]杨璐璐,秦兴卫,杨倩.桃耳七的研究现状及开发利用[J].解放军药学学报,2000,16(1):51-52
[35]李建军.不同浓度鬼臼毒素涂膜剂治疗尖锐湿疣疗效观察[J].中国麻风皮肤病杂志,2003,19(1):77-78
[36]M.Gordaliliza,P.A.Garcia,J.M.Miguel del Corral et al.podophyllotoxin:distribution,sources,applications and new cytotoxin derivatives[J].Toxicon,2004,44(4):441-459
[37]Xiao-kang Zhu,Jian Guan,Zhiyan Xiao,et al.Anti-AIDS agents.Part 61:Anti-HIV activity of new podophyllotoxin derivatives[J].Bicorfanic & Medicinal Chemistry,2004,12(15):4267-4273
[38]畅行若,胡之壁.草药窝耳七化学成分研究.药学学报,1980,15(3):158-161
[39]郭增军主编.陕西七药[M].陕西科学技术出版社,2003:355
[40]贾正平,张培琰,梁重栋.44(2”2”6”6”-四甲基哌啶氮氧自由基)氨基4-去甲表鬼臼毒素对小鼠免疫功能的影响[J]中国药理学报,1993,14(3):221-224
[41]徐成东.川八角莲内生及根际真菌和产鬼臼毒素类似物菌株的研究[D].2000,云南大学硕士论文:3-5
[42]马绍宾,胡志浩,小檗科鬼臼亚科的地理分布与系统发育[J].云南植物研究,1997,19(1):48-56
[43]刘长军,侯嵩生.抗癌活性物质鬼臼木脂素的研究进展[J].天然产物研究与开发,1997,(3):81-87
[44]李忠超,王小兰,葛学军.濒危药用植物桃儿七的生物学特性及其保育措施[J].广西植物,2005,25(2):179-185
[45]中国医学科学院药物研究所等.中药志(第一册)[M].北京:人民卫生出版社,1979:246-249
[46]徐国钧,何宏贤,徐络珊等.中国药材学(上册)[M].北京:中国医药科技出版社,1996,536
[47]马辰,罗淑荣.鬼臼类植物中木脂素类化合物的研究进展[J].中草药,1992,23(5):271
[48]董晓萍,曾莉.川产八角莲中鬼臼毒素的分离鉴定及含量测定.天然产物研究与开发,1994,6(2):17
[49]贾忠建,杜枚,王梦林.薄层扫描法测定桃儿七鬼臼毒素含量.中草药,1988,19(7):13
[50]余自成,王丽平,郁人海等.HPLC法测广西八角莲中鬼臼毒素的含量.药物分析杂志,1997,17(1):43-44
[51]白亚民,徐建中,桃儿七中鬼臼脂素的高效液相色谱法分离与含量测定[J].中药通报,1988,13(5):39
[52]陈新谦.我国抗肿瘤药物研究的新进展[J].药学通报,1987,22(4):193-197
[53]Shang Mingying,Xu Guojun,Luoshan,et al.Quantitative analysis of the podophyllotoxin,4-demethyldeoxypodophyllotoxin and deoxypodophtllotoxin in fruit,root and rhizome of podophyllum plants by HPLC[J].Zhongguo yaoke Daxue xuebao.1996,27(4):219-222
[54]MaSB,XuZY,HuZH.A contribution to the reproductive biology of Sinopodophyllum hexandrum(Royle)Ying.A cta B ot B oreali-Occid ent Sin[J],1997,17(1):49-55
[55]Prasad P.Impact of cultivation on active constituents of the medicinal plants Podophyllum hexandrum and A conitum heterophyllum in Sikkim[J].Plant Genetic Res New sletter,2000,124:33-35
[56]N adeem M,Palni L M S,Puroh it A N,et al.Propagation and conservation of Podophyllum hexandrum Royle:an important medicinal herb[J].Biol Conserv,2000,92:121-129
[57]李广民.介绍一种药用植物--桃儿七.植物学杂志[J],1975,2(2):28
[58]Ward R S.鬼臼毒及其有关化合物的合成.国外医药·植物药分册[M],1993,8(5):206-210
[59]许重远,贾江滨,陈振德.4'-去甲表鬼臼毒素衍生物4β-取代结构修饰研究进展.[J]中草药,2000,31(5):389-392
[60]Canel C,Moraes R M,Dayan F.et al.Molecules of Interest Podophyllotoxin[J].Phytochemistry,2000,54:115-120
[61]Berkowitz,David B.,Maeng Jun-ho,Dantzig Anne H,et al.Synthesis of 3',4',5'-Tridemethoxypodophyllotoxin[J].Chem.Soc.1996,118(39):9424-9427
[62]陈士云,侯嵩生。植物细胞培养生产抗癌药物研究进展.天然产物研究与开发,1993,5 (1):61-65
[63]Takemoto Masumi,Yamamoto Yuichi,Achiwa Kazuo,et al.The synthesis of podophyllotoxin in derivatives etc.with plant cell cultures.Tennen Yuki Kagobutsr Toronkai Koen Yoshishu,1996,38:643-648
[64]兰小中,罗勇,叶东平.发根农杆菌转化与西藏药用植物的开发研究[J]西藏科技,2002,10期(总第114期)33-35
[65]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
[66]文才艺,吴元华,田秀玲.植物内生菌研究进展及其存在的问题[J].生态学杂志 2004,23(2):86-91
[67]梁宇,高玉葆.内生真菌对植物生长发育及抗逆性的影响.植物学通报,2000,17(1):52-59
[68]邱德有.一种云南红豆杉内生真菌的分离.真菌学报,1994,13(4)
[69]王伟,贺雄雪等.南方红豆杉内生真菌及紫杉烷类产物的初步鉴定.中山大学学报(自然科学版),1999,38(3)
[70]郭仕平,徐成东,张玲琪等.川八角莲内生真菌的分离及其发酵产生药用成分的的初步研究[J].发酵工程学科的进展,2002:124-127
[71]刘仕平,曾松荣,郭仕平等.中华山荷叶内生真菌的分离及其发酵产生药用成分的初步研究[J].中国药物与临床,2002,3(3):227-228
[72]李海燕,曾松荣,张玲琪.云南桃儿七植株地下茎内生真菌多样性及有价值菌株的筛选[J].西南农业学报,1999,12(4):123-125
[73]张晓瑞.一株桃儿七内生菌产鬼臼类化合物的研究[D].2005,西北大学硕士学位论文
[74]张琨.一株产鬼臼毒素内生真菌发酵条件的研究[D].西北大学硕士学位论文
[75]曹莉.内生真菌产鬼臼毒素类物质发酵条件的研究[D].西北大学硕士学位论文
[76]施巧琴,吴松刚.工业微生物育种[M].科学出版社,2003,156
[77]雷肇祖,钱志良,章健.工业菌种改良评议.工业微生物[J],2004,34(1):39-51
[78]Thom,C.and Steinberg,R.A:Proc.Nat.Acad.Sci.Wash.,25,329,1939
[79]Richard,P.and Marlin,A.Applications of microbial genetics to industrial fermentation process[M].Academic Press,1976
[80]Zvenigorodskii Ⅵ,Tyaglov BV,Voeikova TA.Isolation of components of the peptide antibiotic virginiamycin and breeding of their producer,Streptomyces virginiae[J].Applied biochemistry and microbiology,2001,37(3):260-266
[81]Lee SH,Rho YT.Improvement of tylosin fermentation by mutation and medium optimization[J].Letters in applied microbiology,1999,28(2):142-144
[82]Aharonowitz Y,Cohen G The microbial Production of pharmaceuticals[J].Scientific American.1981,245(3):141-152
[83]Bhaskara,et al.Use of microwave energy for the eradiation of seedborne diaporthe phaseolorum in soybean and its effect on seed quality[J].The Journal of Microwave Power and Elecromagnetic Energy,1995,30:199-204
[84]Prakash A,et al.Assessment of microwave sterilization of food using intrinsic chemical markers[J].The Journal of Microwave Power and Electromagnetic Energy,1997,32:50-51
[85]宫春波,刘鹭,谢丽源等.离子注入微生物诱变育种研究进展[J].生物技术,2003,13(2):47-49
[86]陈义光,李铭刚,徐丽华等.新型物理诱变方法及其在微生物诱变育种中的应用进展[J].长江大学学报(自科版),农学卷25(2):46-48
[87]石义萍,曾洪梅.武夷霉素高产菌株的选育[A].中国抗生素杂志社.第九次全国抗生素(微生物药物)学术会议论文汇编[C].上海出版社.2001:111
[88]曹福祥编著.次生代谢及其产物生产技术[M].长沙:国防科技大学出版社,2003年6月,141-167
[89]李越中主编.药物微生物技术[M].北京:化学工业出版社,现代生物技术与医药科技出版中心,2004年4月,64-101
[90]叶勤.现代生物技术原理及其应用.北京:中国轻业出版社,2003:212-214
[91]刘锡,白金凯译.真菌学基础[M].北京:科学出版社,1989,3-5
[92]沈萍,范秀容,李广武主编.微生物学实验[M].高等教育出版社,1999年6月第3版,214-215
[93]宋国安,马铃薯的营养价值及开发利用前景[J].河北工业科技,2004,21(4):55-58
[94]俞梅敏,马树义编.生物化学实验指导[M].北京:北京大学生物系生物化学教研室,1979:22-24
[2]孙文基,谢世昌.天然药物成分定量分析[M].北京:中国医药科技出版社,2003年1月,365
[3]杨明,纳绍荣.鬼臼毒素质量标准研究[J].云南师范大学学报,1999,19(2):49-50
[4]李国峰,许重远,孟庆礼等.鬼臼毒素具有荧光性[J].第一军医大学学报,1996,16(3):268-269
[5]汪世龙,姚思德,李义久等.鬼臼毒素及其衍生物的光敏性研究[J].化学物理学报,1997,10(5):432-435
[6]高蓉,田暄,张兴等.鬼臼毒素类生物活性物质的研究概况[J].农药学学报,2000,2:1-6
[7]朱承根,杨晶,李弟灶等.天然抗肿瘤药鬼臼毒素及其衍生物研究进展[J].药品评价,2004,1(4):306-309
[8]Kaplan I W.Condylomata acuminate.New Orleans Med Surg J,1942,94:388-390
[9]Cannel C.,Moraes R.M.,Dayan F.E.,Ferreira D.,Phytochemistry,2000,54:115-120
[10]TF lmbert.Discovery of podophyllotoxin.Biochimie,1998,80:207-222
[11]冯国宜.抗癌植物资源与天然产物研究.湖北民族学院学报(自然科学版).2001,19(3):26-32
[12]贾红光,曹长年,陈建军等.鬼臼毒素类抗肿瘤药物的研究[J].青海医学院学报,2002,23(2):52-55
[13]杨新波.鬼臼毒素抗肿瘤作用研究进展[J].国外医药·植物药分册,1996,11(4):158-159
[14]马维勇,张椿年.鬼臼类抗肿瘤药物的研究进展.中国医药工业杂志,1997,28(2):65-69
[15]潘建林,王彦广,陈耀祖.表鬼臼毒素羧酸酯的合成及体外抗肿瘤活性.药学学报,1997,32(12):898-901
[16]贾红光,贾长年,陈建军等.鬼臼类抗肿瘤药物的研究.青海医学院学报,2002,23(2):52-55
[17]王彦广,陶兰,潘建林等.抗癌活性鬼臼毒素衍生物的合成与构效关系的研究.高等学校化学学报,1997,18(7):1061-1066
[18]WANG Jun-Zhi,TSUMURA Hideki,SHIMURA Keishiro etal.Effect of spin labeled derivatives of podophyllotoxin on cell cycle and macromolecular synthesis in human lymphoid leukemia Molt 4B cells.中国药理学报,1998,19(16):501-505
[19]潘晓辉,王琳,王浩东.鬼臼类木脂素抗肿瘤药物研究进展[J].安康师专学报,1999,11(3):48-53
[20]何峰,刘宗潮,罗一帆等.抗肿瘤药鬼臼毒素衍生物构效关系的研究[J].癌症,2001,20(4):368-372
[21]Wilson L,Friedkin M.the biochemical events of mitosis ll.the in vivo and vitro binding of colchicine in grasshopperembryos and its possible relation to inhibition of mitosis[J].Biochemistry,1967,6(10):3126
[22]Grace LC,Liu Y,Thomas CR,et al.Nonintercalative Anti-tumor Drugs Interfere with the Breakage Reunion Reaction of Mammalian DNA Topoisomerase 11[J].The Journal of Biological Chemistry,1984,259(21):13560
[23]John DL,Susan BH.Effect of VP-16-213 on the Intracelular Degradation of DNA in HeLa Cells[J].Biochemistry,1976,15(25):5443
[24]汪世龙,姚思德,王玫等.鬼臼毒素及其衍生物的化学活性研究[J].辐射研究与辐射工艺学报,1997,15(2):86-90
[25]Kalyanaraman B,Nemec J,Sinha BK.Characterization of free radical produced during oxidation of etoposide(VP-16)and its catehol and quinine[J].Biochemistry,1989,28(11):4839-4846
[26]Yoshihiko lnamori,Mayuri Kubo.Himishi Tsujibo,et al,The biological activities of podophyllotoxin compounds[J].Chen Pharm Bukk,1986,34(9):3928-3932
[27]Kozawa M.,Baba K.,Matsuyma Y.et al.Chen.Pharm.Bull.,1982,30(8):2885-2888
[28]张兴,冯俊涛,陈安良等.砂地柏杀虫作用研究概况[J].西北农林科技大学学报(自然科学版),2002,30(4):130-134
[29]刘艳青,张守刚,程洁等.鬼臼毒素类物质生物活性的研究[J].医学研究生学报,2006,19(3):205-209
[30]田喧,高蓉,张兴.鬼臼毒素类似物结构与杀虫活性关系初探[J].西北农业大学学报,2000,28(6).19-24
[31]Inamori Y..Chem.Pharm.Bull.,1984,32(5):2015-2019
[32]Inamori Y.,Kubo M.,Tsujibo H..Mechanisms of insecticidal action of deoxypodophyllotoxin.Chen.Pharm.bull.,1986,34(5):2247-2250
[33]Mac Rae W D,Hudson J B,Towers G H.The anvirus action of lignans[J].Planta Med,55,531-535
[34]杨璐璐,秦兴卫,杨倩.桃耳七的研究现状及开发利用[J].解放军药学学报,2000,16(1):51-52
[35]李建军.不同浓度鬼臼毒素涂膜剂治疗尖锐湿疣疗效观察[J].中国麻风皮肤病杂志,2003,19(1):77-78
[36]M.Gordaliliza,P.A.Garcia,J.M.Miguel del Corral et al.podophyllotoxin:distribution,sources,applications and new cytotoxin derivatives[J].Toxicon,2004,44(4):441-459
[37]Xiao-kang Zhu,Jian Guan,Zhiyan Xiao,et al.Anti-AIDS agents.Part 61:Anti-HIV activity of new podophyllotoxin derivatives[J].Bicorfanic & Medicinal Chemistry,2004,12(15):4267-4273
[38]畅行若,胡之壁.草药窝耳七化学成分研究.药学学报,1980,15(3):158-161
[39]郭增军主编.陕西七药[M].陕西科学技术出版社,2003:355
[40]贾正平,张培琰,梁重栋.44(2”2”6”6”-四甲基哌啶氮氧自由基)氨基4-去甲表鬼臼毒素对小鼠免疫功能的影响[J]中国药理学报,1993,14(3):221-224
[41]徐成东.川八角莲内生及根际真菌和产鬼臼毒素类似物菌株的研究[D].2000,云南大学硕士论文:3-5
[42]马绍宾,胡志浩,小檗科鬼臼亚科的地理分布与系统发育[J].云南植物研究,1997,19(1):48-56
[43]刘长军,侯嵩生.抗癌活性物质鬼臼木脂素的研究进展[J].天然产物研究与开发,1997,(3):81-87
[44]李忠超,王小兰,葛学军.濒危药用植物桃儿七的生物学特性及其保育措施[J].广西植物,2005,25(2):179-185
[45]中国医学科学院药物研究所等.中药志(第一册)[M].北京:人民卫生出版社,1979:246-249
[46]徐国钧,何宏贤,徐络珊等.中国药材学(上册)[M].北京:中国医药科技出版社,1996,536
[47]马辰,罗淑荣.鬼臼类植物中木脂素类化合物的研究进展[J].中草药,1992,23(5):271
[48]董晓萍,曾莉.川产八角莲中鬼臼毒素的分离鉴定及含量测定.天然产物研究与开发,1994,6(2):17
[49]贾忠建,杜枚,王梦林.薄层扫描法测定桃儿七鬼臼毒素含量.中草药,1988,19(7):13
[50]余自成,王丽平,郁人海等.HPLC法测广西八角莲中鬼臼毒素的含量.药物分析杂志,1997,17(1):43-44
[51]白亚民,徐建中,桃儿七中鬼臼脂素的高效液相色谱法分离与含量测定[J].中药通报,1988,13(5):39
[52]陈新谦.我国抗肿瘤药物研究的新进展[J].药学通报,1987,22(4):193-197
[53]Shang Mingying,Xu Guojun,Luoshan,et al.Quantitative analysis of the podophyllotoxin,4-demethyldeoxypodophyllotoxin and deoxypodophtllotoxin in fruit,root and rhizome of podophyllum plants by HPLC[J].Zhongguo yaoke Daxue xuebao.1996,27(4):219-222
[54]MaSB,XuZY,HuZH.A contribution to the reproductive biology of Sinopodophyllum hexandrum(Royle)Ying.A cta B ot B oreali-Occid ent Sin[J],1997,17(1):49-55
[55]Prasad P.Impact of cultivation on active constituents of the medicinal plants Podophyllum hexandrum and A conitum heterophyllum in Sikkim[J].Plant Genetic Res New sletter,2000,124:33-35
[56]N adeem M,Palni L M S,Puroh it A N,et al.Propagation and conservation of Podophyllum hexandrum Royle:an important medicinal herb[J].Biol Conserv,2000,92:121-129
[57]李广民.介绍一种药用植物--桃儿七.植物学杂志[J],1975,2(2):28
[58]Ward R S.鬼臼毒及其有关化合物的合成.国外医药·植物药分册[M],1993,8(5):206-210
[59]许重远,贾江滨,陈振德.4'-去甲表鬼臼毒素衍生物4β-取代结构修饰研究进展.[J]中草药,2000,31(5):389-392
[60]Canel C,Moraes R M,Dayan F.et al.Molecules of Interest Podophyllotoxin[J].Phytochemistry,2000,54:115-120
[61]Berkowitz,David B.,Maeng Jun-ho,Dantzig Anne H,et al.Synthesis of 3',4',5'-Tridemethoxypodophyllotoxin[J].Chem.Soc.1996,118(39):9424-9427
[62]陈士云,侯嵩生。植物细胞培养生产抗癌药物研究进展.天然产物研究与开发,1993,5 (1):61-65
[63]Takemoto Masumi,Yamamoto Yuichi,Achiwa Kazuo,et al.The synthesis of podophyllotoxin in derivatives etc.with plant cell cultures.Tennen Yuki Kagobutsr Toronkai Koen Yoshishu,1996,38:643-648
[64]兰小中,罗勇,叶东平.发根农杆菌转化与西藏药用植物的开发研究[J]西藏科技,2002,10期(总第114期)33-35
[65]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
[66]文才艺,吴元华,田秀玲.植物内生菌研究进展及其存在的问题[J].生态学杂志 2004,23(2):86-91
[67]梁宇,高玉葆.内生真菌对植物生长发育及抗逆性的影响.植物学通报,2000,17(1):52-59
[68]邱德有.一种云南红豆杉内生真菌的分离.真菌学报,1994,13(4)
[69]王伟,贺雄雪等.南方红豆杉内生真菌及紫杉烷类产物的初步鉴定.中山大学学报(自然科学版),1999,38(3)
[70]郭仕平,徐成东,张玲琪等.川八角莲内生真菌的分离及其发酵产生药用成分的的初步研究[J].发酵工程学科的进展,2002:124-127
[71]刘仕平,曾松荣,郭仕平等.中华山荷叶内生真菌的分离及其发酵产生药用成分的初步研究[J].中国药物与临床,2002,3(3):227-228
[72]李海燕,曾松荣,张玲琪.云南桃儿七植株地下茎内生真菌多样性及有价值菌株的筛选[J].西南农业学报,1999,12(4):123-125
[73]张晓瑞.一株桃儿七内生菌产鬼臼类化合物的研究[D].2005,西北大学硕士学位论文
[74]张琨.一株产鬼臼毒素内生真菌发酵条件的研究[D].西北大学硕士学位论文
[75]曹莉.内生真菌产鬼臼毒素类物质发酵条件的研究[D].西北大学硕士学位论文
[76]施巧琴,吴松刚.工业微生物育种[M].科学出版社,2003,156
[77]雷肇祖,钱志良,章健.工业菌种改良评议.工业微生物[J],2004,34(1):39-51
[78]Thom,C.and Steinberg,R.A:Proc.Nat.Acad.Sci.Wash.,25,329,1939
[79]Richard,P.and Marlin,A.Applications of microbial genetics to industrial fermentation process[M].Academic Press,1976
[80]Zvenigorodskii Ⅵ,Tyaglov BV,Voeikova TA.Isolation of components of the peptide antibiotic virginiamycin and breeding of their producer,Streptomyces virginiae[J].Applied biochemistry and microbiology,2001,37(3):260-266
[81]Lee SH,Rho YT.Improvement of tylosin fermentation by mutation and medium optimization[J].Letters in applied microbiology,1999,28(2):142-144
[82]Aharonowitz Y,Cohen G The microbial Production of pharmaceuticals[J].Scientific American.1981,245(3):141-152
[83]Bhaskara,et al.Use of microwave energy for the eradiation of seedborne diaporthe phaseolorum in soybean and its effect on seed quality[J].The Journal of Microwave Power and Elecromagnetic Energy,1995,30:199-204
[84]Prakash A,et al.Assessment of microwave sterilization of food using intrinsic chemical markers[J].The Journal of Microwave Power and Electromagnetic Energy,1997,32:50-51
[85]宫春波,刘鹭,谢丽源等.离子注入微生物诱变育种研究进展[J].生物技术,2003,13(2):47-49
[86]陈义光,李铭刚,徐丽华等.新型物理诱变方法及其在微生物诱变育种中的应用进展[J].长江大学学报(自科版),农学卷25(2):46-48
[87]石义萍,曾洪梅.武夷霉素高产菌株的选育[A].中国抗生素杂志社.第九次全国抗生素(微生物药物)学术会议论文汇编[C].上海出版社.2001:111
[88]曹福祥编著.次生代谢及其产物生产技术[M].长沙:国防科技大学出版社,2003年6月,141-167
[89]李越中主编.药物微生物技术[M].北京:化学工业出版社,现代生物技术与医药科技出版中心,2004年4月,64-101
[90]叶勤.现代生物技术原理及其应用.北京:中国轻业出版社,2003:212-214
[91]刘锡,白金凯译.真菌学基础[M].北京:科学出版社,1989,3-5
[92]沈萍,范秀容,李广武主编.微生物学实验[M].高等教育出版社,1999年6月第3版,214-215
[93]宋国安,马铃薯的营养价值及开发利用前景[J].河北工业科技,2004,21(4):55-58
[94]俞梅敏,马树义编.生物化学实验指导[M].北京:北京大学生物系生物化学教研室,1979:22-24