CYTOTOXIC METABOLITES OF ALTERNARIA ALTERNATA, AN ENDOPHYTE OF THE MEDICINAL PLANT BIDENS BIPINNATA
Keywords:Alternariol, Sterigmatocystin, Tentoxin, Endophytes, Alternaria alternata, Bidens bipinnata
Objective: Endophytes are widely spread in the plant kingdom and represent a very promising source of biologically active natural products. The medicinal plant Bidens bipinnata Lin. (Asteraceae) which is known for its anti-inflammatory, antifungal and antitumor effects has been chosen for the investigation of its endophyte to search for bioactive metabolites.
Methods: An endophytic Alternaria alternata species was isolated from the leaves of the plant B. bipinnata Lin. To investigate the metabolic profile of this endophytic fungus it was cultivated in several culture media as static and shaken culture. The antimicrobial and cytotoxic activities of the ethyl acetate extracts of the fungus were examined. Extracts exhibiting highest antimicrobial activities in agar diffusion assay and cytotoxicity against HeLa cancer cell line were subjected to activity-guided chromatographic fractionation for the identification of bioactive metabolites. A cytotoxic assay was performed on the isolated compounds against HeLa cancer cell lines as well as cytostatic activity tests against HUVEC and K-562 cell lines.
Results: Chromatographic fractionation resulted in the isolation and identification of alternariol and tentoxin from the extract of the fungus cultivated in medium M5 while sterigmatocystin was isolated in addition to alternariol and tentoxin from the extract of the fungus grown in medium M25. Both alternariol and sterigmatocystin proved to be of moderate cytotoxicity and weak cytostatic activity with alternariol showing higher cytotoxic activity than sterigmatocystin. Highest cytotoxicity against HeLa cell lines was observed for tentoxin with a CC50 of 22.5 µg/ml.
Conclusion: This study presents the isolation and identification of the bioactive metabolites alternariol, sterigmatocystin and tentoxin from the endophyte A. alternata in addition to the antifungal activity of the strain extract as well as the cytotoxic and cytostatic activities of the isolated metabolites against HeLa, HUVEC and K-562 cell lines, respectively.
Guo ZF, TT Guo, M Guo. Preparation of molecularly imprinted adsorptive resin for trapping of ligustrazine from the traditional Chinese herb ligusticum chuanxiong Hort. Anal Chim Acta 2008;612:136-43.
Bose P, U Gowrie. Assessment of bioactive metabolites from the root endophyte isolated from casuarina junghuhniana miq. Asian J Pharm Clin Res 2017;10:137-43.
Devi BRK, E Elizabeth. Pharmacophoric screening of various endophytic fungal metabolites. Asian J Pharm Clin Res 2017;10:140-6.
Strobel G, B Daisy, U Castillo, J Harper. Natural products from endophytic microorganisms. J Nat Prod 2004;67:257-68.
Yan Boa. Total flavonoids of Bidens bipinnata L. a traditional Chinese medicine inhibits the production of inflammatory cytokines of vessel endothelial cells stimulated by sera from henoch–schonlein purpura patients. J Pharm Pharmacol 2012;64:882-7.
Wang X, CG, Pan C, Deng Z, Ge J, Li N, et al. Polyacetylenes from bidens bipinnata L. and their biological activities. Phytochem Lett 2014;7:198-201.
Yang QH. Study on in vitro anti-tumor activity of bidens bipinnata l. extract. Afr J Tradit Complement Altern Med 2013;10:543-9.
Abdou R, Shaker K. Bioactive metabolites of the endophyte Khuskia oryzae isolated from the medicinal plant Bidens bipinnata. Asian J Pharm Life Sci 2013;3:137-45.
Thomma BPHJ. Alternaria spp: from general saprophyte to specific parasite. Mol Plant Pathol 2003;4:225–36.
RNS. Phytotoxins produced by microbial plant pathogens. Nat Prod Rep 2007;24:127-44.
Strange RN. Phytotoxins produced by plant pathogens. Nat Prod Rep 2007;24:127–44.
Mobius NHC. Fungal phytotoxins as mediators of virulence. Curr Opin Plant Biol 2009;12:390–8.
Brase SEA, Keck J, Nising CF. Chemistry and biology of mycotoxins and related fungal metabolites. Chem Rev 2009;109:3903-90.
Tsuge T. Host-selective toxins produced by the plant pathogenic fungus Alternaria alternata. FEMS Microbiol Rev 2013;37:44–66.
Horiuchi M. Porritoxins, metabolites of Alternaria porri, as anti-tumor-promoting active compounds. Nat Prod Res 2006;20:161–6.
Monneret C. Histone deacetylase inhibitors. Eur J Med Chem 2005;40:1–13.
Lakshmi AI. Histone deacetylase inhibitors in cancer therapy: an update. Int J Pharm Sci Rev Res 2011;10:38–44.
Stierle A, G Strobel, D Stierle. Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of pacific yew. Science 1993;260:214-6.
Liebermann BER, Pinet E. Isotentoxin, a conversion product of the phytotoxin tentoxin. Phytochem Lett 1996;42:1537–40.
Sanodiya BS. Isolation and characterization of tenuazonic acid produced by alternaria alternata, a potential bioherbicidal agent for control lantana camara. J Plant Prot Res 2010;50:133–9.
Bills GF. Analyses of microfungal diversity from a user's perspective. Can J Bot 1995;73:S33-S41.
Knight V. Diversifying microbial natural products for drug discovery. Appl Microbiol Biotechnol 2003;62:446-58.
Zhang HW, YC Song, RX Tan. Biology and chemistry of endophytes. Nat Prod Rep 2006;23:753-71.
Afonin S. 4-fluorophenylglycine as a label for 19F NMR structure analysis of membrane-associated peptides. ChemBioChem 2003;4:1151-63.
Wayne PA. Reference method for broth dilution susceptibility testing of filamentous fungi. Approved Standard M38-A, USA, NCCls; 2002. p. 22.
Raistrick H, CE Stickings, R Thomas. Studies in the biochemistry of microorganisms. 90. Alternariol and alternariol monomethyl ether, metabolic products of Alternaria tenuis. Biochem J 1953;55:421-33.
Griffin GF, FS Chu. Toxicity of the Alternaria metabolites alternariol, alternariol methyl ether, altenuene, and tenuazonic acid in the chicken embryo assay. Appl Environ Microbiol 1983;46:1420-2.
Doupnik BJr, EK Sobers: mycotoxicosis: toxicity to chicks of Alternaria longipes isolated from tobacco. Appl Microbiol Biotechnol 1968;16:1596-7.
Ostry V. Alternaria mycotoxins: an overview of chemical characterization, producers, toxicity, analysis and occurrence in foodstuffs. World Mycotoxin J 2008;1:175–88.
Bensassi F. Cell death induced by the alternaria mycotoxin alternariol. Toxicol In Vitro 2012;26:915–23.
J Lou, L Fu, Y Peng, L Zhou. Metabolites of alternaria fungi and their bioactivities. Molecules 2013;18:5891-935.
Kollarova J, Cenk E, Schmutz CMD. The mycotoxin alternariol suppresses lipopolysaccharide-induced inflammation in THP-1 derived macrophages targeting the NF-κB signalling pathway. Molec Toxicol 2018;92:3347-58.
Lillehoj EB, A Ciegler. Biological activity of sterigmatocystin. Mycopathol Mycol Appl 1968;35:373-6.
Purchase IF, JJ Van Der Watt. Acute toxicity of sterigmatocystin to rats. Food Cosmet Toxicol 1969;7:135-9.
Van Der Watt JJ, IF Purchase. Subacute toxicity of sterigmatocystin to rats. S Afr Med J 1970;44:159-60.
Dickens F, HE Jones, HB Waynforth. Oral, subcutaneous and intratracheal administration of carcinogenic lactones and related substances: the intratracheal administration of cigarette tar in the rat. Br J Cancer 1966;20:134-44.
Scott PM, W Van Walbeek, B Kennedy, D Anyeti: mycotoxins (ochratoxin A, citrinin, and sterigmatocystin) and toxigenic fungi in grains and other agricultural products. J Agric Food Chem 1972;20:1103-9.
Shigenori Kumazawa, Mok Ryeon Ahn, Takunori Fujimoto, M Kato. Radical-scavenging activity and phenolic constituents of propolis from different regions of argentina. Nat Prod Res 2010;24:804-12.
KI-Bong Oh, Yasuo Tajima, Mikako Saito, IL-Moo Chang, H Matsuoka. Cytotoxicity of extracts from alternaria alternata against cultured tobacco by-2cells. Biocontrol Sci 2002;7:127-30.
Steyn PS. Mycotoxins, general view, chemistry, and structure. Toxicol Lett 1995;82-83:843-51.
Schulz B, C Boyle. The endophytic continuum. Mycol Res 2005;109:661-86.
Kogel KH, P Franken, R Huckelhoven. Endophyte or parasite-what decides? Curr Opin Plant Biol 2006;9:358-63.