Abstract
The current study focuses on the isolation and in vitro characterization of bioactive metabolites produced by endophytic fungi isolated from the Himalayan yew (Taxus wallichiana Zucc.). The endophytic fungi were isolated on artificial media from inner tissues of bark and needles. Antimicrobial and antioxidant activity, along with total phenolic- and flavonoid-content assays were used in the evaluation of bioactivity of the fermented crude extracts. The ability of the endophytes to produce the anticancer compound Taxol was also analyzed using thin-layer chromatography (TLC) and reverse-phase high-performance liquid chromatography (RP-HPLC). A total of 16 fungal morphotypes were obtained from asymptomatic inner tissues of the bark and needles of T. wallichiana. Among the 16 isolates, the ethyl acetate (EA) fraction of isolate MUS1, showed antibacterial and antifungal activity against all test-pathogens used (Streptococcus faecalis ATCC 19433, Staphylococcus aureus ATCC 12600, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Salmonella enterica ATCC 13076, Pseudomonas aeruginosa ATCC 27853, and Candida albicans). MUS1 showed significant inhibition against Pseudomonas aeruginosa ATCC 27853 (minimum inhibitory concentration (MIC): 250 µg/ml) and the pathogenic yeast, Candida albicans (MIC: 125 µg/ml). Antioxidant activity, total phenolic, and total flavonoid content as well as in vitro Taxol production were evaluated for EA fraction of isolate MUS1. Antioxidant activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. At a concentration of 100 µg/ml, the % DPPH radical scavenging activity was 83.15 ± 0.40, 81.62 ± 0.11, and 62.36 ± 0.29, for ascorbic acid, butylated hydroxytoluene (BHT), and the EA fraction of MUS1, respectively. The DPPH-Half maximal inhibitory concentration (DPPH-IC50) value for the EA fraction was 81.52 ± 0.23 µg/ml, compared to BHT (62.87 ± 0.08 µg/ml) and ascorbic acid (56.15 ± 0.19 µg/ml). The total phenolic and flavonoid content in the EA fraction were 16.90 ± 0.075 µg gallic acid equivalent (GAE) and 11.59 ± 0.148 µg rutin equivalent (RE), per mg of dry crude extract, respectively. TLC and RP-HPLC analysis showed that the isolate MUS1 also produces Taxol (282.05 µg/l of fermentation broth). Isolate MUS1 was identified as Annulohypoxylon sp. by internal transcribed spacer (ITS) sequencing. Having the ability to produce antimicrobial and antioxidant metabolites, as well as the anticancer compound Taxol, makes Annulohypoxylon sp. strain MUS1, a promising candidate for further study of naturally occurring bioactive metabolites.
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Abbreviations
- ATCC:
-
American Type Culture Collection
- BLAST:
-
Basic Local Alignment Search Tool
- BLASTn:
-
Nucleotide BLAST
- HIV:
-
Human immunodeficiency virus
- MEGAX:
-
Molecular Evolutionary Genetics Analysis Version X
- NCBI:
-
National Center for Biotechnology Information
- UV–Vis:
-
Ultraviolet–Visible
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Acknowledgments
We would like to acknowledge the Department of Biotechnology, Kathmandu University (KU), Nepal for the facilitation of the research works. We would also like to acknowledge Mr. Basant Awasthi, Teaching Assistant, Department of Geomatics Engineering, KU for his contribution to making the location map of Mustang. Further, we want to thank Prof. Dr. Janardan Lamichhane, Department of Biotechnology, KU, and Associate Professor Dr. Rajani Shakya, Department of Pharmacy, KU for the HPLC analysis.
Funding
We are grateful to the Swedish Research Council for their grant (Ref/348-2012-6138/Agreement/C0613801) for this project.
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DPG designed the study, carried out the majority of research activities, wrote the Manuscript. HV and AA equally contributed to conducting research activities and manuscript editing. JO, KL, ME are involved in molecular analysis of isolated and purified endophytes and editing the manuscript. MRGG participated in designing the research, guiding DPG to carry out the research, and provided financial support. All authors read and approved the final manuscript.
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The sequence obtained in this study was deposited in the NCBI GenBank database (Accession number: MN699475).
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Gauchan, D.P., Vélëz, H., Acharya, A. et al. Annulohypoxylon sp. strain MUS1, an endophytic fungus isolated from Taxus wallichiana Zucc., produces taxol and other bioactive metabolites. 3 Biotech 11, 152 (2021). https://doi.org/10.1007/s13205-021-02693-z
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DOI: https://doi.org/10.1007/s13205-021-02693-z