Isolation, purification and characterization of vinblastine and vincristine from endophytic fungus Fusarium oxysporum isolated from Catharanthus roseus

Abstract

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 (1)H 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.

Figure 1. Morphological features of endophytic fungus Fusarium oxysporum.

Colony shape: (a) Mycelium, (b) Chlamydospores (c) Macroconidia, microconidia and chlamydospores formed on PDA.

Figure 2. TLC analysis.

(a) TLC of crude fungal vinblastine from culture filtrates along with standard Vinblastine 1: Vinblastine standard, 2: Crude sample, 3: Vincristine standard, Detection: Ceric ammonium sulphate reagent. (b) TLC of partially purified fungal vinblastine from culture filtrates along with standard vinblastine on silica gel using chloroform∶methanol (8∶2) solvent system. A: Standard vinblastine B: Partially purified vinblastine C: Partially purified vincristine, Detection: Ceric ammonium sulphate reagent. (c) TLC of fungal vinblastine purified from culture filtrates along with standard vinblastine on silica gel using chloroform∶methanol (8∶2) solvent system. 1: Purified fungal vinblastine 2: Standard vinblastine, Detection: Ceric ammonium sulphate reagent. (d) TLC of fungal vincristine purified from culture filtrates along with standard vincristine on silica gel using chloroform∶methanol (8∶2) solvent system. 1: Standard vincristine, 2: Purified fungal vincristine, Detection: Ceric ammonium sulphate reagent.

Figure 3. HPLC and UV spectrum.

(a) HPLC profile of pure fungal vinblastine with retention time of 36.6 min. (b) HPLC profile of pure fungal vincristine with retention time of 34.9 min. (c) UV absorption spectrum of standard vinblastine and fungal vinblastine. (d) UV absorption spectrum of standard vincristine and fungal vincristine.

Figure 4. ESI-MS spectrum.

(a) Molecular mass determination of the fungal vinblastine by ESI-MS. (b) Molecular mass determination of the fungal vincristine by ESI-MS.

Figure 5. MS-MS spectrum.

(a) MS-MS spectrum of the purified fungal vinblastine. (b) MS-MS spectrum of the purified fungal vincristine.

Figure 6. ¹H NMR spectrum.

(a) 400 MHz ¹H NMR spectra of fungal vinblastine (A) and standard vinblastine sulphate (B). The signals marked with ‘s’ is coming from the residual solvent (CHCl₃). The signals marked with * and x are due to water (from the solvent) and contamination form n- hexane, respectively. The broad signal marked ú′ at ∼6 ppm in A is an unidentified impurity. (b) 500 MHz ¹H NMR spectra of fungal vincristine (A) and standard vincristine sulphate (B). The signals marked with ‘s’ are coming from the residual solvent (Methanol-d₄). The signals marked with * and x are due to contamination from trace amounts of n-hexane and ethanol, respectively.