- Title
- Gymnopilus junonius and Rhodofomitopsis lilacinogilva: In vitro evaluation of antimycobacterial activity, safety assessment and isolation of active compounds
- Creator
- Didloff, Jenske
- Subject
- Antibacterial agents
- Subject
- Plants, Useful -- South Africa
- Subject
- Medicinal plants -- South Africa
- Date Issued
- 2024-12
- Date
- 2024-12
- Type
- Doctoral theses
- Type
- text
- Identifier
- http://hdl.handle.net/10948/68840
- Identifier
- vital:77122
- Description
- Tuberculosis remains a significant public health challenge due to the complexity and compliance issues of its treatment, the prevalence of latent infections, the high rate of HIV co-infection, and the spread of drug-resistant strains. This highlights the need to search for new antimycobacterial drugs. Natural products have historically been used as sources for the discovery of pharmaceuticals. Although medicinal macrofungi have been exploited in traditional folk medicines for the treatment of various diseases, research into their antimycobacterial activity remains limited.The aim of this study was to isolate, identify, and characterise the antimycobacterial compound(s) present in ethanol fruiting body extracts of Gymnopilus junonius (Fr.) P.D. Orton (Hymenogastraceae) and Rhodofomitopsis lilacinogilva (Berk.) B.K. Cui, M.L. Han, & Y.C. Dai (Fomitopsidaceae), as well as to investigate their mechanism of action and potential toxicity using in vitro and in vivo screening methods.Bioassay-guided fractionation was conducted using column chromatography and preparative thin-layer chromatography to isolate the active antimycobacterial fraction/compound from ethanol extracts of G. junonius and R. lilacinogilva. Antimycobacterial activity against M. tuberculosis H37Rv was screened using a resazurin microplate assay (REMA). The chemical structure was elucidated employing 1D-(1H NMR) and 2D NMR spectroscopy (HSQC, HMBC) techniques, along with High- Performance Liquid Chromatography High-Resolution Electron Spray Ionisation Mass Spectrometry (HPLC-HRESI-MS). Transmission electron microscopy (TEM) was utilised to observe the ultrastructural changes in M. tuberculosis induced by the active fraction/compound. Toxicity assessment was performed on African green monkeykidney (Vero) and human hepatoma (C3A) cell lines using various staining techniques to observe the potential risk for hepatotoxicity (CellRox orange, TMRE, and MTG staining) and genotoxicity (NucRed staining). In vivo toxicity of extracts was evaluated using a zebrafish embryo/larvae model to observe developmental and teratogenic effects.The bioassay-guided fractionation of G. junonius and R. lilacinogilva extracts yielded an active compound and a fraction displaying inhibitory activity against M. tuberculosis, with MIC values of 31.25 μg/mL and 250 μg/mL, respectively. Gymnopilene was identified as the main active compound isolated from G. junonius, while fraction 8 from R. lilacinogilva contained a predominant constituent resembling a pachymic acid derivative. Transmission electron microscopy revealed ultrastructural damage to M. tuberculosis cells upon treatment with gymnopilene and fraction 8, which was observed as the disruption and disintegration of the cell wall. In vitro toxicity assessment indicated a reduction in the cell density in both Vero and C3A cell lines following exposure to the fractions/gymnopilene. Although there were no significant alterations in the production of reactive oxygen species, changes in mitochondrial dynamics were observed. Genotoxic assessment showed no significant changes. In vivo toxicity assessment showed no significant effects for G. junonius against the zebrafish embryos/larvae, whereas R. lilacinogilva remained toxic and induced developmental abnormalities over a 72-hour time period.The findings of this study highlight the potential of macrofungi as a valuable source of bioactive compounds, confirming the significance of natural products in drug discovery. Notably, this research represents the first to demonstrate that the predominant compound, gymnopilene, isolated from G. junonius exhibits inhibitory activity against M. tuberculosis, contributing to the novelty of the study. Furthermore, the study expands our understanding of the antimycobacterial properties of the selected macrofungal species, G. junonius and R. lilacinogilva, by demonstrating the ultrastructural damage to M. tuberculosis cell wall induced by treatment. However, it is important to recognise the potential toxicity identified through in vitro and/or in vivo screening assays. The observed toxicity may hamper their suitability formdrug development. Future investigations could explore the inclusion of additional in vitro based models, perhaps more complex and physiologically relevant models alongside the in vivo screening model to assess the therapeutic potential of isolated fractions/compounds.
- Description
- Thesis (PhD) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2024
- Format
- computer
- Format
- online resource
- Format
- application/pdf
- Format
- 1 online resource (213 pages)
- Format
- Publisher
- Nelson Mandela University
- Publisher
- Faculty of Science
- Language
- English
- Rights
- Nelson Mandela University
- Rights
- All Rights Reserved
- Rights
- Open Access
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Collections
NMU School of BioMolecular and Chemical Sciences
| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | SOURCE1 | DIDLOFF, J.pdf | 2 MB | Adobe Acrobat PDF | View Details Download |