海洋来源的放线菌Z3001次级代谢产物抗肿瘤活性成分研究
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摘要
本文采用小鼠乳腺癌细胞tsFT210，以细胞周期抑制、细胞凋亡诱导及细胞坏死为活性指标，在考察了海洋来源的放线菌Z3001生产活性物质的发酵条件的基础上进行了大量生产发酵，对此菌株发酵产物的抗肿瘤活性成分进行了生物活性筛选。结果表明，菌株Z3001的乙酸乙酯提取部分具有细胞坏死性细胞毒和周期抑制作用。我们采用活性追踪分离的方法对上述菌株的抗肿瘤活性成分进行了初步研究。
对上述活性部分采用减压硅胶柱层析、凝胶柱层析(Sephadex LH-20)、反相硅胶柱层析和反相制备高效液相(HPLC)等分离手段，从菌株Z3001分离得到11个单体化合物。利用理化性质和光谱学方法(UV、IR、MS、1D-NMR和2D-NMR)，10个单体化合物分别鉴定为：正十六烷酸(1)，3′-软脂酸甘油单酯(2)，3，6-二烯-2，8-(8-羟基-9-甲基)癸基-1，5-二氧辛烷(4)，3，4-二羟基-2-甲基-吡啶(5)，环-(缬-脯)(6)，环-(脯-亮)(7)，4-羟基苯乙醇(8)，1H-3-羧酸-吲哚(9a)，1H-3-甲酰基-吲哚(9b)，次黄嘌呤核苷(10)。
生物活性测试结果表明，化合物(1)(2)(4)对tsFT210小鼠乳腺癌细胞具有显著的坏死性细胞毒活性，化合物(4)还具有显著得诱导细胞调亡活性。化合物(1)(10)具有G_2／M周期抑制活性。
本文首次发现化合物(4)，在体外对小鼠乳腺癌温敏型tsFT210细胞有诱导细胞凋亡活性；化合物(10)在体外对小鼠乳腺癌温敏型tsFT210细胞有G_2／M期抑制活性。化合物(3)结构有待进一步确定。
本文通过活性追踪分离的方法，成功地分离得到代表菌株Z3001次级代谢产物的抗肿瘤活性成分，证明这一模型及筛选方法可以快速准确找到海洋微生物中的活性成分，是寻找潜在药用价值活性化合物的有效手段。
We have undertaken the screening for novel cell cycle inhibitors and apoptosis inducers from the marine-derived actinomycetes, Z3001 by use of a mouse cdc2 mutant cell line, tsFT210. In anticipation of achieving the above objectives, marine-derived actinomycetes, Z3001 have been screened by using this model, the EtOAC extracts of strain Z3001 showed bioactivities of inhibiting the cell cycle progression and cytoclasis of tsF210 cells. Thus, we selected them for further investigation.
Eleven compounds were isolated from the bioactive part, EtOAC extract of strain Z3001, by repeated column chromatography on Sephadex LH-20, silica gel and RP18, followed by reverse-phase high performance liquid chromatragraphy. The structures of compounds were elucidated mainly by use of spectroscopic method (UV, IR, MS, 1D-NMR, 2D-NMR) and according to their physicochemical properties: palmitic acid(l), 2,3-dihydroxypropyl palmitate (2), 3,6-diene-2,8(8-hydroxy-9-methyl) decyl-l,5-dioxocane(4), 3, 4-Dihydroxy-2-methylpyridine (5), cyclo-(Val-Pro)(6), cycle- (Pro-Leu) (7), 4- (2-hydroxyethyl)phenol(8), 1H-indole-3-carboxylic acid(9a), lH-indole-3-carboxamide (9b), 2-(6-hydroxy-9H-purin-9-yl) -5- (hydroxymethyl) -tetrahydrofuran- 3,4-diol( 1 0). The structure of compound ( 3 ) is still in affirming.
Bioassay results indicated that (1)(2)(4) show cell cycle inhibition and cytoclasis of tsFT210 cells. Compound (4) showed a strong apoptosis and cytoclasis of tsFT210 cells. Compound (1)(4)(10) show cell cycle inhibition and cytoclasis of tsFT210 cells, the were the main antitumor active compounds of EtOAC extract of strain Z3001.
Bioassay results indicated that we isolated the main antitumor active compounds of strainZ3001 successfully which showed that this antitumor model and the method were exact and available in achieving bioactive components from marine microbes.
对上述活性部分采用减压硅胶柱层析、凝胶柱层析(Sephadex LH-20)、反相硅胶柱层析和反相制备高效液相(HPLC)等分离手段，从菌株Z3001分离得到11个单体化合物。利用理化性质和光谱学方法(UV、IR、MS、1D-NMR和2D-NMR)，10个单体化合物分别鉴定为：正十六烷酸(1)，3′-软脂酸甘油单酯(2)，3，6-二烯-2，8-(8-羟基-9-甲基)癸基-1，5-二氧辛烷(4)，3，4-二羟基-2-甲基-吡啶(5)，环-(缬-脯)(6)，环-(脯-亮)(7)，4-羟基苯乙醇(8)，1H-3-羧酸-吲哚(9a)，1H-3-甲酰基-吲哚(9b)，次黄嘌呤核苷(10)。
生物活性测试结果表明，化合物(1)(2)(4)对tsFT210小鼠乳腺癌细胞具有显著的坏死性细胞毒活性，化合物(4)还具有显著得诱导细胞调亡活性。化合物(1)(10)具有G_2／M周期抑制活性。
本文首次发现化合物(4)，在体外对小鼠乳腺癌温敏型tsFT210细胞有诱导细胞凋亡活性；化合物(10)在体外对小鼠乳腺癌温敏型tsFT210细胞有G_2／M期抑制活性。化合物(3)结构有待进一步确定。
本文通过活性追踪分离的方法，成功地分离得到代表菌株Z3001次级代谢产物的抗肿瘤活性成分，证明这一模型及筛选方法可以快速准确找到海洋微生物中的活性成分，是寻找潜在药用价值活性化合物的有效手段。
We have undertaken the screening for novel cell cycle inhibitors and apoptosis inducers from the marine-derived actinomycetes, Z3001 by use of a mouse cdc2 mutant cell line, tsFT210. In anticipation of achieving the above objectives, marine-derived actinomycetes, Z3001 have been screened by using this model, the EtOAC extracts of strain Z3001 showed bioactivities of inhibiting the cell cycle progression and cytoclasis of tsF210 cells. Thus, we selected them for further investigation.
Eleven compounds were isolated from the bioactive part, EtOAC extract of strain Z3001, by repeated column chromatography on Sephadex LH-20, silica gel and RP18, followed by reverse-phase high performance liquid chromatragraphy. The structures of compounds were elucidated mainly by use of spectroscopic method (UV, IR, MS, 1D-NMR, 2D-NMR) and according to their physicochemical properties: palmitic acid(l), 2,3-dihydroxypropyl palmitate (2), 3,6-diene-2,8(8-hydroxy-9-methyl) decyl-l,5-dioxocane(4), 3, 4-Dihydroxy-2-methylpyridine (5), cyclo-(Val-Pro)(6), cycle- (Pro-Leu) (7), 4- (2-hydroxyethyl)phenol(8), 1H-indole-3-carboxylic acid(9a), lH-indole-3-carboxamide (9b), 2-(6-hydroxy-9H-purin-9-yl) -5- (hydroxymethyl) -tetrahydrofuran- 3,4-diol( 1 0). The structure of compound ( 3 ) is still in affirming.
Bioassay results indicated that (1)(2)(4) show cell cycle inhibition and cytoclasis of tsFT210 cells. Compound (4) showed a strong apoptosis and cytoclasis of tsFT210 cells. Compound (1)(4)(10) show cell cycle inhibition and cytoclasis of tsFT210 cells, the were the main antitumor active compounds of EtOAC extract of strain Z3001.
Bioassay results indicated that we isolated the main antitumor active compounds of strainZ3001 successfully which showed that this antitumor model and the method were exact and available in achieving bioactive components from marine microbes.
引文
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4. Fenical W. Chemical studies of marine bacteria : Developing a new resource J. Chem Rev,1993,1673
5．阎莉萍 等．海洋微生物合成的抗肿瘤活性物质 J．国外医药抗生素分册，2003，(24)：213-217
6．林丽于 高霞灵 吴萍茹 方金瑞 海洋药物资源的微生物分离技术 J 中国海洋药物 1999，(3)：15-18
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8．邵世和，孙丽媛，母润红，平世荣，曹翠红．SRB法检测4HPR对Hela细胞生长的影响．吉林药学院学报，1998，18(4)：15—16
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16．袁瑾，胡旺云．3’—长直链饱和羧酸甘油酯的~(13)C，~1H NMR的研究．波谱学杂志，1993，
10(2)：185—188
16a．于德全．分析化学手册(第七分册)．北京：化学工业出版社，1999，246、700
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19．丁中涛，保志娟，杨雪琼，江明．金铁锁根中的3个环二肽．中国中药杂志，2003，28，(4)：337—339
20．王有初，周俊，谭宁华，丁中涛，江芯．五味子的环二肽及其合成．药学学报，1999，34(1)：19—22
21. Steven D. Bull, Stephen G. Davies, Richard M. Parkin and Francisco Sánchez-Sancho . The biosynthetic origin of diketopiperazines derived from D-proline. J. Chem. Soc. , Perkin Trans. 1, 1998 2313—2320
22. Olga P. Serdyuk, Lidiya D. Smolygina, Eugine N. Muzafarov, Vladimir M. Adanin,Michael U. Arinbasarov. 4-Hydroxyphenethyl alcohol-a new cytokinin-like substance from the phototrophic purple bacterium Rhodospirillum rubrum 1R. FEBS Letters, 1995(365): 10-12
23. Pei-Lin Wu,Fu-Wen Lin et. , Cytotoxic and Anti-HIV Principles from the Phizomes of Begonia nantoenisi. Chem Pharm Bull. 2004, 52(3):345-349
24. Toyokazu Yoshida, Kohei Fujita, Toru Nagasawa. Novel Reversible Indole-3-carboxylate Decarboxylase Catalyzing Nonoxidative Decarboxylation . J Biosci. Biotechnol. Biochem. ,2002,66(11):2388-2394
25. Yoshio Saito, Thomas A. Zevaco and Luigi A. Agrofoglio. Chemical synthesis of ~(13)C labeled anti-HIV nucleosides as mass-internal standards. J Tetrahedron. 2002,(58):9593-9603
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27. Murray H. G. Munro, John W. Blunt, Eric J. Dumdei, Sarah J. H. Hickford, Rachel E. Lill et al. The discovery and development of marine compounds with pharmaceutical potentialJ. J. Biotech 1999,70:15-25
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2. Flam F. Chemical prospectors scour the seas for promising drugs J. Science , 1994, (266):1324
3. Berna V S, Greenstein M, Maiese W M. A new depsipeptide with antitumor activity produced by a marine micromonospora J. Adv Appl Microbiol, 1997,(43): 57
4. Fenical W. Chemical studies of marine bacteria : Developing a new resource J. Chem Rev,1993,1673
5．阎莉萍 等．海洋微生物合成的抗肿瘤活性物质 J．国外医药抗生素分册，2003，(24)：213-217
6．林丽于 高霞灵 吴萍茹 方金瑞 海洋药物资源的微生物分离技术 J 中国海洋药物 1999，(3)：15-18
7．金静君，魏一生，何萍，曹祖蕊．白血病细胞株化疗药物敏感性试验SRB法的研究．福建医药杂志，1998，20(5)：108—109
8．邵世和，孙丽媛，母润红，平世荣，曹翠红．SRB法检测4HPR对Hela细胞生长的影响．吉林药学院学报，1998，18(4)：15—16
9．王青青，余海．SRB显色法用于抗癌药物敏感性试验的研究．科技通报，2000，16(2)：104—107
10．邵世和，李明成，董丽萍，宋文纲，张晓刚，曹翠红．SRB法检测4HPR对KB、SCB—5e细胞的生长影响．吉林药学院学报，1998，18(3)：5—6
11．金静君，魏一生，何萍，曹祖蕊．SRB试验最适工作条件探讨。福建医药学杂志，1998，20(2)：92—94
12．刘彬，谷静丽，王玉兰．SRB染色法定量测定EGF、IGE—1及TNF—α对细胞增殖的影响．河南肿瘤学杂志，1995，8(4)：254—255
13．金静君，魏一生，何萍，龙瑞斌．SRB法急性白血病体外药敏试验适用性研究．福建医药杂志，1999，2l(4)：84—85
14．董爱森，林剑锋，翁建南，金静君．雷公藤内酯醇对K_(562)细胞的抑制作用研究．福建医药杂志，1999，21(5)：87—88
15．凌云，张永林，万峰，等．中药大藻化学成分的研究．中国中药杂志，1999，24(5)：289-291
16．袁瑾，胡旺云．3’—长直链饱和羧酸甘油酯的~(13)C，~1H NMR的研究．波谱学杂志，1993，
10(2)：185—188
16a．于德全．分析化学手册(第七分册)．北京：化学工业出版社，1999，246、700
17. Kontoghiorghes, G. J. Inorg. Chim. ACTA, 1987, 136: 11
18. Faouzi Fdhila, Victorizno Vázquez. DD-Diketopipetazines: Antibiotics Active against Vibrio anguillarum Isolated from Cultures of Pecten maximus. J. J. Nat. Pro. 2003,66,1299:1301
19．丁中涛，保志娟，杨雪琼，江明．金铁锁根中的3个环二肽．中国中药杂志，2003，28，(4)：337—339
20．王有初，周俊，谭宁华，丁中涛，江芯．五味子的环二肽及其合成．药学学报，1999，34(1)：19—22
21. Steven D. Bull, Stephen G. Davies, Richard M. Parkin and Francisco Sánchez-Sancho . The biosynthetic origin of diketopiperazines derived from D-proline. J. Chem. Soc. , Perkin Trans. 1, 1998 2313—2320
22. Olga P. Serdyuk, Lidiya D. Smolygina, Eugine N. Muzafarov, Vladimir M. Adanin,Michael U. Arinbasarov. 4-Hydroxyphenethyl alcohol-a new cytokinin-like substance from the phototrophic purple bacterium Rhodospirillum rubrum 1R. FEBS Letters, 1995(365): 10-12
23. Pei-Lin Wu,Fu-Wen Lin et. , Cytotoxic and Anti-HIV Principles from the Phizomes of Begonia nantoenisi. Chem Pharm Bull. 2004, 52(3):345-349
24. Toyokazu Yoshida, Kohei Fujita, Toru Nagasawa. Novel Reversible Indole-3-carboxylate Decarboxylase Catalyzing Nonoxidative Decarboxylation . J Biosci. Biotechnol. Biochem. ,2002,66(11):2388-2394
25. Yoshio Saito, Thomas A. Zevaco and Luigi A. Agrofoglio. Chemical synthesis of ~(13)C labeled anti-HIV nucleosides as mass-internal standards. J Tetrahedron. 2002,(58):9593-9603
26. Christine D. Jardetzky and Oleg Jardetzky. Investigation of the Structure of Purines,Pyrimidines,Ribose Nucleosides and Nucleotides by Proton Magnetic Resonance. 1960,(22)238-245.
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