摘要
粘细菌是一种革兰氏阴性、单细胞、杆状细菌,有独特的形态分化过程和社会性行为,因其能够产生广谱次级代谢产物而被人们所熟知,已经成为继放线菌、芽孢杆菌之后的第三大药源菌。
本文对粘细菌菌株Corallococcus coralloides 085B04的生物学特性及其次级代谢产物进行了研究。通过显微观察和发酵培养等方法,掌握了粘细菌的生长习性及生活史,获得了粘细菌发酵液的乙酸乙酯萃取物。
使用高效液相色谱法对乙酸乙酯萃取的粗产物和培养基的乙酸乙酯萃取物进行对比,通过分析发现,粗产物的色谱峰比VY-2培养基多12个。
将发酵液粗提物通过硅胶色谱(石油醚/丙酮)梯度洗脱分离后,多次使用硅胶色谱、凝胶色谱和HPLC等技术对各组分进行分离纯化,最终从中分离得到粘细菌次级代谢产物19个。通过1H-NMR,13C-NMR、二维核磁数据、质谱、红外和紫外数据分析,对分离到的化合物进行了结构确证,其中化合物2、15和19是新化合物,它们分别是1-(1H-pyrrolo[3,2-c]quinolin-4-yl)ethanone ,5-(hydroxyimino)-1,3- dimethyl-1H-pyrrol-2(5H)-one和8-(色原烷-4’-甲酰)-环(S-脯氨酸-R-亮氨酸)。
通过MTT法对得到的部分化合物作了抗肿瘤活性分析,得到了1个对前列腺癌细胞(PC3)和Hela YFP细胞抗性较好的化合物16,其IC50分别为7.8μg/mL和10.3μg/mL;1个对乳腺癌细胞(MCF-7)细胞抗性较好的化合物18,其IC50为13.7μg/mL。其中某些化合物,可能对其它肿瘤细胞具有很好的抗性,但需要进一步的研究。
The myxobacteria are Gram-negative, unicellular bacteria with rod-shaped vegetative cells, which have the unique morphological differentiation and amazing social behaviors. Besides the readily established microbial secondary metabolite producers actinomycetes and bacillus, myxobacteria have become the third largest one, which are known for their ability to produce a broad spectrum of secondary metabolites and form fruiting bodies upon starvation.
In this thesis, the biological characteristics and secondary metabolites of Corallococcus coralloides 085B04 have been studied. By the methods of microscopic observation and fermentation, the growth characters and life cycle of myxococcus have been discovered, and the acetic ether extracts of the myxobacterial fermentation broth has been obtained.
It is found that there are 12 more peaks in residue than that in acetic ether extracts of the VY-2 medium, by camparing with their HPLC procedure.
Separation of the crude residue by column chromatography over Silica gel by consecutively employing petroleum ether and acetone as eluents gave some fractions. Then, fractions were isolated and purificated by Silica gel chromatography, Sephadex LH-20 gel chromatography and HPLC techniques. Finaly, 19 metabolites of the myxobacteria were obtained. The chemical structures were confirmed by 1H-NMR, 13C-NMR, 2D-NMR, MS, IR and UV. Compounds 2, 15 and 19 are novel, which are named 1-(1H-pyrrolo[3,2-c]quinolin-4-yl)ethanone, 5-(hydroxyimino)-1,3-dimethyl -1H–pyrrol-2(5H)-one and 8-(chroman-4’-yl)-cyclo(S) -Pro-(R)-Leu, respectively.
The antitumor bioactivities of the prepared samples were evaluated with the MCF-7, Pc3 and YFP cell lines using a MTT assay. The results show that, compound 18 exhibited moderate activity with IC50 13.7μg/mL against MCF-7, and compound 16 exhibited high cytotoxicity against human tumor PC3 and YFP cell lines, with their IC50 7.8μg/mL and 10.3μg/mL, respectively. Moreover, some of these compounds may exhibite more significantly against other tumor cells, and this project needs more further study.
引文
[1] Rolf Jansen, Hans Reichenbach, Gerhard H?fle, et al. Apicularen A and B, Cytotoxic 10-Membered Lactones with a Novel Mechanism of Action from Chondromyces Species (Myxobacteria): Isolation,Structure Elucidation, and Biosynthesis [J]. Eu. J Org Chem, 2000: 913-919
[2] S Frykman, H Tsuruta, J Lau, et al. Modulation of epothilone analog production through media design [J]. Journal of Industrial Microbiology & Biotechnology, 2002, 28: 17-20
[3] Kim Jiyoung, Jung Nam Choi, Choong Hwan Lee, et al. LC-MS/MS Profiling -Based Secondary Metabolite Screening of Myxococcus xanthus [J]. J Microbiol Biotechnol, 2009, 19(1): 51–54
[4] Li YZ, Hu W, Zhang YQ, et al. A simple method to isolate salt-tolerant myxobacteria from marine samples [J]. J Microbiol Meth, 2002, 50: 205-209
[5] Iizuka T, Jojima Y, Fudou R, et al. Isolation of myxobacteria from the marine environment [J]. FEMS Microbiol Lett, 1998, 169: 317-322
[6] De-Ming Jiang, Zhi-Hong Wu, Yue-Zhong Li, et al. Fruiting and non-fruiting myxobacteria: A phylogenetic perspective of cultured and uncultured members of this group [J]. Molecular phylogenetics and evolution, 2007, 44(2): 545–552
[7] Kaiser, D. Coupling cell movement to multicellular development in myxobacteria [J]. Nat. Rev. Microbiol, 2003, 1: 45-54
[8] Reichenbach, H. Myxobacteria: a most peculiar group of social prokaryotes, In Myxobacteria, edited by E. Rosenberg. 1984 Springer-Verlag, Berlin
[9] Schneiker S, O Perlova, O Kaiser, et al. Complete genome sequence of the myxobacterium Sorangium cellulosum [J]. Nat. Biotechnol, 2007, 25: 1281-1289
[10] Shimkets LJ, M Dworkin, D Kaiser. Myxobacteria II. American Society for Microbiology. The myxobacterial Genome, 1993: 85-108.
[11] Goldman BS, WC Nierman, D Kaiser, et al. Evolution of sensory complexity recorded in a myxobacterial genome [J]. Proc. Natl Acad Sci, 2006, 103: 15200-15205
[12] Kaiser D. Signaling in myxobacteria [J]. Annu Rev Microbiol, 2004, 58: 75-98
[13] Bode HB, Müller R. Analysis of myxobacterial secondary metabolism goes molecular [J]. J. Ind. Microbiol. Biotechnol, 2006, 33: 577-588
[14] Gerth K, Pradella S, Perlova O, et al. Myxobacteria: Proficient producers of novel natural products with various biological activities–past and future biotechnological aspects with the focus on the genus Sorangium [J]. J.Biotechnol, 2003, 106: 233-253
[15] Reichenbach H. Myxobacteria, producers of novel bioactive substances [J]. J. Ind.Microbiol. Biotechnol, 2001, 27: 149-156
[16] Gerth K, Irschik H, Reichenbach H, et al. Myxothiazol, an antibiotic from Myxococcus fulvus (myxobacterales). I. Cultivation, isolation, physico-chemical and biological properties [J]. J Antibiot (Tokyo), 1980, 33: 1474-1479
[17] Thierbach G, Reichenbach H. Myxothiazol, a new antibiotic interfering with respiration [J]. Antimicrob Agents Chemother, 1981, 19: 504-707
[18] Thierbach G, Reichenbach H. Myxothiazol, a new inhibitor of the cytochrome b-c1 segment of the respiratory chain [J]. Biochim Biophys Acta, 1981, 638: 282-289
[19] Gerth K, Jansen R, Thierbach G, et al. The myxalamids, new antibiotics from Myxococcus xanthus (myxobacterales)Ⅰ.Production, physico-chemical and biological properties,and mechanism of action [J]. J Antibiot (Tokyo), 1983, 36: 1150-1156
[20] Irschik H, Reichenbach H. The mechanism of action of myxovalargin A, a peptide antibiotic from Myxococcus fulvus [J]. J Antibiot (Tokyo), 1985, 38(9): 1237-1245
[21] Irschik H, Gerth K, Kemmer T, et al. The myxovalargins, new peptide antibiotics from Myxococcus fulvus (Myxobacterales). I. Cultivation, isolation, and some chemical and biological properties [J]. J Antibiot (Tokyo), 1983, 36: 6-12
[22] Sasse F, Steinmetz H, Hofle G, et al. Rhizopodin, a new compound from Myxococcus stipitatus (myxobacteria) causes formation of rhizopodia-like structures in animal cell cultures. Production, isolation, physico-chemical and biological properties [J]. J Antibiot (Tokyo), 1993, 46: 741-748
[23] Gregor Hagelueken, Simone C. Albrecht, Wolf-Dieter Schubert, et al. The absolute configuration of rhizopodin and its inhibition of actin polymerization by dimerization [J]. Angew Chem Int Ed, 2008, 48(3): 595-598
[24] Rolf Jansen, Heinrich Steinmetz, Florenz Sasse, et al. Isolation and structure revision of the actin-binding macrolide rhizopodin from Myxococcus stipitatus (Myxobacteria) [J]. Tetrahedron Letters, 2008, 49: 5796-5799
[25] H Irschik, K Gerth, W Kohl, et al. The myxopyronins, new inhibitors of bacterialRNA synthesis from Myxococcus fulvus [J]. J Antibiot, 1983, 36: 1651-1658
[26] Herbert Irschik, Klaus Gerth, Hans Reichenbach, et al. Saframycin Mx1, a new natural saframycin isolated from a myxobacterium(Myxococcus xanthus) [J]. J Antibiot, 1988, 41: 993-998
[27] Andreas Pospiech, Jürg Bietenhader, Thomas Schupp. Two multifunctional peptide synthetases and an O-methyltransferase are involved in the biosynthesis of the DNA-binding antibiotic and antitumour agent saframycin Mx1 from Myxococcus xanthus [J]. Microbiology, 1996, 142: 741-746
[28] Rosenberg E, Vaks B, Zuckerberg A. Bactericidal action of an antibiotic produced by Myxococcus xanthus [J]. Antimicrob Agents Chemother, 1973, 4: 507-513
[29] Rosenberg E, Dworkin M. Autocides and a paracide, antibiotic TA, produced by Myxococcus xanthus [J]. Journal of Industrial Microbiology, 1996, 17: 424-431
[30] D Zafrifi, E Rosenberg, D Mirelman.Mode of action of Myxococcus xanthus antibiotic TA [J]. Antimicrob Agents Chemother, 1981, 19: 349-351
[31] K Gerth, H Irschik, W Trowitzsch. The myxovirescins, a family of antibiotics from Myxococcus virescens (Myxobacterales) [J]. J Antibiot, 1982, 35: 1454-1459
[32] Norimasa Onishi, Kazuo Izaki, Hajime Takahashi. A macrocyclic antibiotic M-230B produced by Myxococcus xanthus isolation and characterization [J]. J Antibiot, 1983, 37: 13-19
[33] Peter Meiser, Helge B.Bode, Rolf Müller. The unique DKxanthene secondary metabolite family from the myxobacterium Myxococcus xanthus is required for developmental sporulation [J]. PNAS, 2006, 103: 19128-19133
[34] Birgit Ohlendorf, Stefan Kehraus, Gabriele M K?nig. Myxochromide B3, a new member of the myxochromide family of secondary metabolites [J]. J Nat Prod, 2008, 71: 1708-1713
[35] A El Akoum, M Vijayalakshmi, P Cardon, et al. Myxococcus xanthus produces an extracellular glycopeptide that displays blood anticoagulant properties [J]. Enzyme and Microbial Technology, 1987, 9(7): 426-429
[36] Akoum A, Guidoin R, King MW, et al. A new bioactive molecule for improving vascular graft patency: exploratory trials in dogs.Clin Invest Med, 1992, 15(4): 318-330
[37] Trowitzsch-Kienast, W Forche, G H?fle, et al. Antibiotics from gliding bacteria, 45-Phenylamides, new HIV-1 inhibitors from Myxococcus stipitatus Mx s40 [J].LIEBIGS ANN. CHEM, 1992, 7: 659-664
[38] M Poza, C Sieiro, L Carreira, et al. Production and characterization of the milk-clotting protease of Myxococcus xanthus strain 422 [J]. Ind Microbiol Biotechnol, 2003, 30: 691-698
[39] BS Goldman, WC Nierman, D Kaiser, et al. Evolution of sensory complexity recorded in a myxobacterial genome [J]. PNAS, 2006, 103: 15200-15205
[40] Starks CM, Zhou Y, Liu F, et al. Isolation and characterization of new epothilone analogues from recombinant Myxococcus xanthus fermentations [J]. J Nat Prod, 2003, 66 (10): 1313-1317
[41] Smith WB. The carbon-13 specta of steroids on the way to ecdysone [J].Organic Magnetic Resonance,1997, 9(11): 644-648
[42]高锦明,董泽军,刘吉开.蓝黄红菇的化学成分[J].云南植物研究, 2000, 22(1): 85-89
[43] Bettina B?hlendorf, Edgar Forche, Gerhard H?fle, et al. Antibiotics from Gliding Bacteria, LXXIII Indole and Quinoline Derivatives as Metabolites of Tryptophan in Myxobacteria [J]. Liebigs Ann, 1996, 49-53
[44] QIN Yue, CJ Miller, MD Richardson,et al. Isolation and characterization of fungal inhibitors from Epichlo? festucae [J]. J Agr Food Chem, 2000, 48(10): 4687-4692
[45] Lee Joo-Sang, Ma Chao-Mei, Park Dong-Ki, et al. Transformation of ergosterol peroxide to cytotoxic substances by rat intestinal bacteria [J]. Biological & Pharmaceutical Bulletin, 2008, 31(5): 949-954
[46] Pichan Sawangwong, Rawiwan Wattanadilok, Werner Herz, et al. Secondary metabolites from a marine sponge Cliona patera [J]. Biochemical Systematics and Ecology, 2008, 36: 493-496
[47] Faouzi Fdhila, Victoriano Vázquez, Ricardo Riguera, et al. DD-Diketopiperazines: Antibiotics Active against Vibrio anguillarum Isolated from Marine Bacteria Associated with Cultures of Pecten maximus [J]. Journal of Natural Products, 2003, 66(10): 1299-1301
[48] Adamczeski M, Reed AR, Crews P. New and Known Diketopiperazines from the Caribbean Sponge, Calyx cf. podatypa [J]. J. Nat. Prod., 1995, 58 (2): 201-208
[49] Xie HH, Dan Y, Wei Xiao-Yi. Diketopiperazines and nucleosides from mycelial cultures of Dichomitus squalens [J]. Zhongguo Tianran Yaowu, 2008, 6(5): 395-39
[50] Markus Gautschi, Joachim P Schmid, Terry L Peppard, et al. ChemicalCharacterization of Diketopiperazines in Beer [J]. J Agric Food Chem. 1997, 45: 3183-3189
[51] Zhang CHSH, Albermann Christoph, Thorson Jon S, et al. RebG- and RebM-catalyzed indolocarbazole diversification [J]. Chem Bio Chem, 2006, 7(5): 795-804
[52] Perez-Prieto Julia, Galian Raquel E, Miranda Miguel A, et al. Steady-state and time-resolved studies on the formation of skatolyl radicals photosensitized by 2-benzoylthiophene [J]. Photochemical & Photobiological Sciences, 2003, 2(11): 1200-1204
