Isolation, purification and characterization of Trichothecinol-A produced by endophytic fungus Trichotheciumsp. and its antifungal, anticancer and antimetastatic activities

Abstract

Abstract Background 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. Results 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. Conclusion 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.