Isolation, purification and characterization of Trichothecinol-A produced by endophytic fungus Trichotheciumsp. and its antifungal, anticancer and antimetastatic activities
Tóm tắt
A total of 30 endophytic fungi (AAP-PS 1–30) were isolated from the medicinal herb Phyllanthus amarus and screened for the production of Trichothecinol-A. Out of all the endophytic strains screened for Trichothecinol-A production, the culture filtrate of AAP-PS-1 extracted with ethyl acetate yielded Trichothecinol-A extracellularly in appreciable amounts. Trichothecinol-A was purified, quantified and completely characterized by different standard chromatographic and spectroscopic techniques including reverse phase HPLC, 1D and 2D NMR spectroscopy, etc. The compound was tested for antifungal activity against filamentous fungi and yeast, apoptotic activity against B16F10 cells, anticancer activity against MDA-MB-231, HeLa and B16F10 cells as well as antimetastatic activity against MDA-MB-231 cell line. The endophyte producing Trichothecinol-A was identified as Trichothecium sp. by morphological, cultural and molecular methods. RP-HPLC analyses performed on a Waters model using a C18 symmetry pack column with a flow rate of 0.5 ml/min and the eluting compounds were detected by a dual mode wavelength detector set at 220 nm and 240 nm. The 1D (1H, 13C) and 2D NMR (COSY, NOESY, TOCSY, DEPT, 13C–1H HMBC, 13C–1H HSQC), ESI-MS, HRMS, IR and UV–vis show conclusively that the isolated compound was Trichothecinol–A. One liter of Trichothecium sp. yielded 4.37 mg of Trichothecinol-A. Trichothecinol-A exhibited antifungal activity against Cryptococcus albidus (NCIM 3372) up to 20 μg/ml. Cytotoxicity studies indicate that Trichothecinol-A causes 50% cell death at 500nM concentration in HeLa and B16F10 cells and induces apoptosis in later. Inhibition of wound migration assay performed on MDA-MB-231 cells reveals that 500nM of Trichothecinol-A was able to inhibit wound migration by 50% indicating its remarkable antimetastatic property. The compound Trichothecinol-A has previously been isolated from Trichothecium roseum and characterized by various standard techniques. Anti-cancer studies conducted on Trichothecinol-A showed that it significantly inhibits cancer cell migration and can thus be developed as a new class of anti-metastatic drug. Here, we for the first time report the anti-metastatic as well as anti-fungal activity exhibited by Trichothecinol-A isolated by us from the endophytic fungus Trichothecium sp. of medicinal plant Phyllanthus amarus. Trichothecinol-A also exhibited apoptotic activity.
Tài liệu tham khảo
Tan RX, Zou WX: Endophytes: a rich source of functional metabolites. Nat Prod Rep. 2001, 18: 448-459. 10.1039/b100918o.
Gunatilaka AAL: Natural products from plant-associated microorganisms: distribution, structural diversity, bioactivity, and implications of their occurrence. J Nat Prod. 2006, 69: 509-526. 10.1021/np058128n.
Zhou L, Zhao J, Xu L, Huang Y, Ma Z, Wang JJW: Antimicrobial compounds produced by plant endophytic fungi. In Fungicides: Chemistry. 2009, New York: Publishers, 91-119.
Bretz M, Beyer M, Cramer B, Knecht A, Humpf HU: Thermal degradation of the Fusarium mycotoxin deoxynivalenol. J Agric Food Chem. 2006, 54: 6445-6451. 10.1021/jf061008g.
Hazel CM, Patel S: Influence of processing on trichothecene levels. Toxicol Lett. 2004, 153: 51-59. 10.1016/j.toxlet.2004.04.040.
Middlebrook JL, Leatherman DL: Specific association of T-2 toxin with mammalian cells. Biochem Pharmacol. 1989, 38: 3093-3102. 10.1016/0006-2952(89)90020-8.
Wannemacher RW, Winer SL: Trichothecene Mycotoxins. Medical Aspects of Chemical and Biological Warfare. Edited by: Sidell RR, Takafuji ET, Franz DRE. 1977, Washington DC: Office of the Surgeon General at TMM Publications, 655-676.
Bamburg J: Biological and biochemical actions of trichothecene mycotoxins. Prog Mol Subcell Biol. 1983, 8: 41-110. 10.1007/978-3-642-69228-4_3.
Konishi K, Iida A, Kaneko M, Tomioka K, Tokuda H, Nishino H, Kumeda Y: Cancer preventive potential of trichothecenes from Trichothecium roseum. Bioorg Med Chem. 2003, 11: 2511-2518. 10.1016/S0968-0896(03)00215-3.
Chopra RN, Nayar SL, Chopra IC: Glossary of Indian Medicinal Plants. 1956, National Institute of Science Communication and Information Resources, Council of Scientific & Industrial Research: New Delhi, India
Kirtikar KR, Basu BD: Indian Medicinal Plants. 2001, Uttranchal, India: Oriental Enterprises, 3068-3069. 2
Reddy KR: Folk medicine from Chittoor district, Andhra Pradesh, India, used in the treatment of jaundice. Int J Crude Drug Res. 1988, 26: 137-140.
Satyavati GV, Gupta AK, Tandon N: Medicinal plants of India. 1987, New Delhi: Indian Council of Medical Research, 405-411.
Barnett H: Illustrated genra of imperfect fungi. 1969, Minneapolis: Burgess publishing company, 66-67. Second
Hedley BD, Winquist E, Chambers AF: Therapeutic targets for antimetastatic therapy. Expert Opin Ther Targets. 2004, 8: 527-536. 10.1517/14728222.8.6.527.
Wyckoff JB, Jones JG, Condeelis JS, Segall JE: A critical step in metastasis: in vivo analysis of intravasation at the primary tumor. Cancer Research. 2000, 60: 2504-2511.
Ahmad A: Investigations on the grassy-shoot disease of lemongrass (Cymbopogon flexuosus) and characterization of toxic metabolites produced by the causal agent Balansia sclerotica. 1991, Lucknow: University of Lucknow
Bills GF, Redlin SC, Carris LM: Endophytic fungi in grasses and woody plants: systematics, ecology, and evolution. Isolation and analysis of endophytic fungal communities from woody plants. Edited by: Redlin SC, Carris LM. 1996, St Paul: American Phytopathological Society Press, 31-65.
Janardhanan KK, Ahmad A, Gupta ML, Hussain A: Grassy- shoot, a new disease of lemongrass caused by Balansia sclerotica (Pat) Hohnel. J Phytopathology. 1991, 133: 163-168. 10.1111/j.1439-0434.1991.tb00149.x.
Strobel GA, Hess WM, Ford E, Sidhu RS, Yang X: Taxol from fungal endophytes and the issue of biodiversity. J Ind Microbiol. 1996, 17: 417-423. 10.1007/BF01574772.
Stierle A, Strobel G, Stierle D: Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew. Science. 1993, 260: 214-216. 10.1126/science.8097061.
Sorenson WG, Sneller MR, Larsh HW: Qualitative and quantitative assay of trichothecin: a mycotoxin produced by Trichothecium roseum. Appl Microbiol. 1975, 29: 653-657.
Neumann B, Pospiech A, Schairer HU: Rapid isolation of genomic DNA from gram-negative bacteria. Trends Genet. 1992, 8: 332-333.
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning, Volume 68. 1989, New York: Cold Spring Harbor Laboratory Press, 1232-1239.
Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA. 1977, 74: 5463-5467. 10.1073/pnas.74.12.5463.
Cavalieri SJ: Manual of Antimicrobial Susceptibility Testing. 2005, Washington DC: American Society for Microbiology, 53-62.
Amsterdam D: Susceptibility testing of antimicrobials in liquid media. Antibiotics in Laboratory Medicine, Volume 4. Edited by: Williams LV. 2005, Baltimore: Wilkins, 61-143.
Valli C, Paroni G, Di Francesco AM, Riccardi R, Tavecchio M, Erba E, Boldetti A, Gianni’ M, Fratelli M, Pisano C, Merlini L, Antoccia A, Cenciarelli C, Terao M, Garattini E: Atypical retinoids ST1926 and CD437 are S-phase-specific agents causing DNA double-strand breaks: significance for the cytotoxic and antiproliferative activity. Mol Cancer Ther. 2008, 7: 2941-2954. 10.1158/1535-7163.MCT-08-0419.
Chakraborty G, Jain S, Kale S, Raja R, Kumar S, Mishra R, Kundu G: Curcumin suppresses breast tumor angiogenesis by abrogating osteopontin-induced VEGF expression. Mol Med Rep. 2008, 1: 641-646.