Isolation, Purification and Characterization of Vinblastine and Vincristine from Endophytic Fungus Fusarium oxysporum Isolated from Catharanthus roseus
Endophytic fungi reside in a symbiotic fashion inside their host plants, mimic their chemistry and interestingly, produce the same natural products as their hosts and are thus being screened for the production of valuable compounds like taxol, camptothecin, podophyllotoxin, etc. Vinblastine and vincristine are excellent anti-cancer drugs but their current production using plants is non-abundant and expensive. In order to make these drugs readily available to the patients at affordable prices, we isolated the endophytic fungi from Catharanthus roseus plant and found a fungus AA-CRL-6 which produces vinblastine and vincristine in appreciable amounts. These drugs were purified by TLC and HPLC and characterized using UV-Vis spectroscopy, ESI-MS, MS/MS and 1H NMR. One liter of culture filtrate yielded 76 μg and 67 μg of vinblastine and vincristine respectively. This endophytic fungal strain was identified as Fusarium oxysporum based upon its cultural and morphological characteristics and internal transcribed spacer (ITS) sequence analysis.
References
[1]
Yabuta T, Sumiki Y, Uno S (1939) The biochemistry of the “bakanae fungus”. IV. Cultural conditions of Gibberella fujikuroi for the production of gibberellin and fusaric acid. J Agr Chem Soc 15: 1209–1220.
[2]
Stierle A, Strobel GA, Stierle D (1993) Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of pacific yew. Science 260: 214–216 Available: http://www.ncbi.nlm.nih.gov/pubmed/80970?61.
[3]
Deng BW, Liu KH, Chen WQ, Ding XW, Xie XC (2009) Fusarium solani, tax-3, a new endophytic taxol-producing fungus from Taxus chinensis. World J Microbiol Biotechnol 25: 139–143 DOI http://link.springer.com/content/pdf/10.?1007/s11274-008-9876-2.pdf">10.1007s1127400898762 Available httplink.springer.comcontentpdf10.1007s1?127400898762.pdf/s11274-008-9876-2Available: http://link.springer.com/content/pdf/10.?1007/s11274-008-9876-2.pdf.
[4]
Strobel GA, Yang XS, Sears J, Robert K, Sidhu RS, et al. (1996) Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallachiana. Microbiology 142: 435–440 Available: http://www.ncbi.nlm.nih.gov/pubmed/89327?15. doi:10.1099/13500872-142-2-435.
[5]
Rangarajulu SK, Hyung JK, Byung-Ki H (2010) Taxol promising fungal endophytes, Pestalotiopsis species isolated from Taxus cuspidate. J Biosci Bioeng 110: 541–546 Epub 2010 Jul 15. Available: http://www.ncbi.nlm.nih.gov/pubmed/20634?132. doi:10.1016/j.jbiosc.2010.06.007.
[6]
Sreekanth D, Syed A, Sarkar S, Sarkar D, Santhakumari B, et al. (2009) Production, purification and characterization of taxol and 10DAB III from a new endophytic fungus Gliocladium sp. isolated from the Indian yew tree, Taxus baccata. J Microbiol Biotechnol 19: 1342–1347 Available: http://www.ncbi.nlm.nih.gov/pubmed/19996?685. doi:10.4014/jmb.0904.04041.
[7]
Wall ME, Wani MC, Cook CE, Palmer KH, McPhail AT, et al. (1966) The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata A. J Am Chem Soc 88: 3888–3890 Available: http://www.citeulike.org/group/7862/arti?cle/3740780.
[8]
Kusari S, Zühlke S, Spiteller M (2009) An endophytic fungus from Camptotheca accuminata that produces camptothecin and analogues. J Nat Prod 72: 2–7 Available: http://www.ncbi.nlm.nih.gov/pubmed/19119?919. doi:10.1021/np800455b.
[9]
Eyberger AL, Dondapati R, Porter JR (2006) Endophyte fungal isolates from Podophyllum peltatum produce podophyllotoxin Amy. J Nat Prod 69: 1121–1124 Available: http://www.ncbi.nlm.nih.gov/pubmed/16933?860. doi:10.1021/np060174f.
[10]
Paulo RH, Heijden R, Verpoorte R (1995) Cell and tissue cultures of Catharanthusroseus: A literature survey. Plant Cell Tiss Organ Cult 42: 1–25 Available: http://link.springer.com/article/10.1007?%2FBF00037677.
[11]
Aniruddha D, Srivastava PS (1997) Variation in vinblastine production by Catharanthus roseus, during in vivo and in vitro differentiation. Phytochem 46: 135–137 Available: http://dx.doi.org/10.1016/S0031-9422(97)?00165-9. Available: http://www.sciencedirect.com/science/art?icle/pii/S0031942297001659.
[12]
MiuraY, Hirata K, Kurano N, Miyamoto K, Uchida K (1988) Formation of vinblastine in multiple shoot culture of Catharanthus roseus. Planta Med 54: 18–20 Available: http://www.ncbi.nlm.nih.gov/pubmed/33753?31. doi:10.1055/s-2006-962321.
[13]
Azra AA, Babak DH, Hassan E, Ahmad M (2008) High in vitro production of anti-canceric indole alkaloids from periwinkle (Catharanthus roseus) tissue culture. Afr J Biotechnol 7: 2834–2839 Available: http://www.academicjournals.org/AJB. Available: www.ajol.info/index.php/ajb/article/down?load/59172/47477.
[14]
Satoshi Y, Toshihiro U, Satoshi K, Ayato S, Takeshi K, et al. (2002) Stereocontrolled Total Synthesis of (+)-Vinblastine. J Am Chem Soc 124: 2137–2139 Available: http://pubs.acs.org/doi/abs/10.1021/ja01?77049. doi:10.1021/ja0177049.
[15]
Kharwar RN, Verma VC, Strobel G, Ezra D (2008) The endophytic fungal complex of Catharanthus roseus (L.). G Don Curr Sci 95: 228–233 Available: http://www.currentscience.ac.in/Download?s/article_id_095_02_0228_0233_0.pdf.
[16]
Zhang LB, Gou LH, Zeng SV (2000) Preliminary study on the isolation of endophytic fungus of Catharanthus roseus and its fermentation to produce product of therapeutic value. Chinese Trad Herb Drugs 11: 805–807.
[17]
Tung CY, Yang DB, Gou M (2002) A preliminary study on the condition of the culture and isolate of endophytic fungus producing Vincristine. J Chuxiong Normal Univ 6: 39–41.
Nelson PE, Dignani MC, Anaissie EJ (1994) Taxonomy, biology, and clinical aspects of Fusarium species. Clin Microbiol 7: 479–504 Available: http://www.ncbi.nlm.nih.gov/pmc/articles?/PMC358338/.
[20]
Guttman A, Khandurina J, Budworth P, Xu W, Hou Y, et al. (2004) Analysis of combinatorial natural products by HPLC and CEZLGGC. Europe 17: 39–41 Available: http://www.chromatographyonline.com/lcgc?/data/articlestandard//lcgceurope/072004?/84873/article.pdf.
[21]
Volkov SK (1996) Methods of analysis of some antitumor- active alkaloids from Catharanthus roseus. Pharm Chem J 30: 391–399 Available: http://link.springer.com/article/10.1007?%2FBF02219327.
[22]
Tikhomiroff C, Jolicoeur M (2002) Quantification of indole alkaloids and irridoid precursor in Catharanthus roseus hairy roots by HPLC. J Chromatogr A 955: 87–93.
[23]
Olaf VT, Jos HB, William JN (1991) Analytical methods for its determination of vinca alkaloids in biological specimens: a survey of the literature;. J Pharm Biomed Anal 9: 1077–1082 Available: http://dx.doi.org/10.1016/0731-7085(91)8?0047-D. Available: http://www.ncbi.nlm.nih.gov/pubmed/18221?76.
[24]
Chu I, Bodnar JA, White EL, Bowman RN (1996) Quantification of vinblastine and vincristine in Catharanthus roseus plant by capillary zone electrophoresis. J Chromatogr A 755: 281–288 b Goldsmith Seeds, Inc., P.O. Box 1349, Gilroy, CA 95021, USA. Available: http://dx.doi.org/10.1016/S0021-9673(96)?00614-0. Available: http://www.sciencedirect.com/science/art?icle/pii/S0021967396006140.
[25]
Favretto D, Piovan A, Filippini R, Caniato R (2001) Monitoring the production yield of vincristine and vinblastine in Catharanthus roseus from somatic embryogenesis. Semi quantitative determination by flow injection spray ionization mass spectrometry. Rapid Commun Mass Spectrom 15: 364–369 Available: http://www.ncbi.nlm.nih.gov/pubmed/11241?768.
[26]
Zhou H, Tai Y, Sun C, Pan Y (2005) Rapid identification of vinca alkaloids by direct- injection electrospray ionization tandem mass spectrometry and confirmation by HPLC-MA. Phytochem Anal 16: 328–333 Available: http://www.ncbi.nlm.nih.gov/pubmed/16223?089. doi:10.1002/pca.852.
[27]
Elena GG, Valensin NJ, Stolovich H, Williams J, Scott AI (1992) Conformation of vinblastine in aqueous solution determined by 2-D proton and C-13 –NMR spectroscopy. J Nat Prod 55: 285–293 eurekamag.com//conformation-vinblastine-?aqueous-solution-determine Available: www.researchgate.net//239706794_Conforma?tional_analysis_of_trans-Re.
[28]
Ishikawa HH, David AC, Shigeki S, Porino V, Annie T, et al. (2009) Total Synthesis of vinblastine, vincristine, Related Natural Products, and Key Structural Analogues. J Am Chem Soc 131: 4904–4916 Available: http://www.ncbi.nlm.nih.gov/pubmed/19292?450. doi: 10.1021/ja809842b.
[29]
Dennison JB, Kulanthaivel P, Barbuch RJ, Renbarger JL, Ehlhardt WJ, et al. (2006) Selective metabolism of vincristine in vitro by CYP3A5. Drug Metab Dispos 34: 1317–1327 Available: http://www.ncbi.nlm.nih.gov/pubmed/16679?390. doi:10.1124/dmd.106.009902.
[30]
Dubrovay Z, Háda V, Béni Z, Szántay C Jr (2012) NMR and mass spectrometric characterization of vinblastine, vincristine and some new related impurities - Part I. J Pharm Biomed Anal Available: http://dx.doi.org/10.1016/j.jpba.2012.08?.019. Available: http://www.sciencedirect.com/science/art?icle/pii/S0731708512004785.
[31]
Barthe L, Ribet JP, Pélissou M, Degude MJ, Fahy J, et al. (2002) Optimization of the separation of Vinca alkaloids by nonaqueous capillary electrophoresis. J Chromatogr A 968: 241–50 Available: http://www.sciencedirect.com/science/art?icle/pii/S0021967302009548.