Isolation and characterization of endophytic fungi as a source of anticancer compounds from Catharanthus roseus / Farah Wahida Ayob

Farah Wahida , Ayob (2017) Isolation and characterization of endophytic fungi as a source of anticancer compounds from Catharanthus roseus / Farah Wahida Ayob. PhD thesis, University of Malaya.

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Abstract

The potential anti-carcinogenic endophytic fungi that could produce the same alkaloids vinblastine and vincristine were isolated from wild Catharanthus roseus (purple and white) plants. Four endophytic fungi have been identified as Colletotrichum gloeosporioides, Macrophomina phaseolina, Nigrospora sphaerica and Fusarium solani based on both molecular and morphological techniques. In order to confirm these fungi were endophytes, they were tested for antimicrobial properties and hydrolytic enzyme test. The antibacterial properties of these endophytic fungi were tested against Staphylococcus aureus and Escherichia coli where N. sphaerica showed a positive result against S. aureus of plug sample. While CSE of F. solani showed a positive result against S. aureus. Meanwhile, the antifungal test showed that C. gloeosporioides, M. phaseolina, N. sphaerica and F. solani were positive against Aspergillus niger and Candida albicans. These endophytic fungi also positively present of qualitative enzyme test of cellulase, amylase and protease except M. phaseolina which only positive on cellulase. Through liquid chromatography-mass spectrometry (LCMS), crude mycelia extract (CME) of N. sphaerica was determined for vinblastine (0.868 μg/mL) and CME of C. gloeosporioides for vincristine (<0.001 μg/mL). The CME of these two potential endophytic fungi has been purified using high performance liquid chromatography (HPLC) before being tested for cytotoxicity assay. These alkaloids, produced by the CME of N. sphaerica and C. gloeosporioides showed a positive result against breast cell line cancer (MDA-MB 231) where N. sphaerica had a half maximal inhibitory concentration (IC50) with value of > 32 μg/mL compared to the vinblastine from a CLE of C. roseus > 350 μg/mL. While the highest percentage of inhibition (POI) of C. gloeosporioides was 50.45 % of the concentration 50 μg/mL against breast cell line cancer. The apoptosis assay showed this cell line cancer treated with alkaloids purified from N. sphaerica, C. gloeosporioides, C. roseus (white and purple) have a percentage of early apoptosis 3.1 %, 1.61 %, 1.20 % and 1.34 %, and a percentage of late apoptosis 4.57 %, 7.82 %, 4.15 % and 3.16 % respectively. These endophytic fungi have been tested for antioxidant properties using different assays; DPPH radical-scavenging activity, ferric reducing antioxidant power (FRAP) and ferrous ion chelating activity (FCA). There was no result on the half maximal concentration (IC50) for both DPPH and FCA assays of all fungal extracts. However, through FRAP assays the results ranged from 0.336±0.01 to 0.477±0.11 mmol Fe2+/g extract where N. sphaerica extract had the highest FRAP value. This fungus also showed the highest TPC and TFC levels, which were 0.030±0.000 (mg GAE/g) and 0.038±0.001 (mg QE/g) respectively. Even though the crude mycelia extract of N. sphaerica and C. gloeosporioides were shown positive for vinblastine and vincristine production, they did not perform on polyphenolic test and it revealed that the polyphenolic content did not affect the anticancer activity of the crude fungal extract.

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