Gymnopilus junonius and Rhodofomitopsis lilacinogilva: In vitro evaluation of antimycobacterial activity, safety assessment and isolation of active compounds
- Authors: Didloff, Jenske
- Date: 2024-12
- Subjects: Antibacterial agents , Plants, Useful -- South Africa , Medicinal plants -- South Africa
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10948/68840 , 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. , Thesis (PhD) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2024
- Full Text:
- Date Issued: 2024-12
- Authors: Didloff, Jenske
- Date: 2024-12
- Subjects: Antibacterial agents , Plants, Useful -- South Africa , Medicinal plants -- South Africa
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10948/68840 , 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. , Thesis (PhD) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2024
- Full Text:
- Date Issued: 2024-12
Biological activity of macrofungi in South Africa against respiratory and lung disease
- Authors: Didloff, Jenske
- Date: 2018
- Subjects: Macrofungi , Microbiology Medical microbiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/30138 , vital:30835
- Description: Macrofungi represent an untapped source of natural bioactive compounds for various diseases, which have been targeted as potential therapeutic agents. The medicinal uses of macrofungi discovered to date include anticancer, antidiabetic, antioxidant, antimicrobial, and immunomodulatory properties. The knowledge regarding the medicinal uses of macrofungi in Africa is limited; however, it is believed that Africa may contain a large number of unidentified species of macrofungi. The objectives of this study were to: (i) screen the macrofungal extracts for antimicrobial activity against microorganisms responsible for respiratory diseases (e.g. Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Mycobacterium tuberculosis, HIV-1), (ii) determine the effect of macrofungal extracts on bacterial morphology, (iii) investigate the cytotoxicity of macrofungal extracts against human lung carcinoma cells, and to elucidate the mechanism/s of action of cytotoxicity/anticancer activity. In vitro bioassays for antimicrobial activity included: ρ-iodonitrotetrazolium chloride assays and microplate alamar blue assay (MABA), while the HIV-1 reverse transcriptase colorimetric ELISA and HIV-1 protease fluorometric assay kits were used for anti-HIV activity. Cytotoxicity of the macrofungal species against A549 lung cancer cell line was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and the IC50 values determined for the most cytotoxic macrofungal spp. The mechanism of cytotoxicity was investigated by cell cycle analysis and fluorescent staining to observe morphological and biochemical changes (i.e. acridine orange, caspase activation). Ethanol extracts of Amanita foetidissima, Gymnopilus junonius, Pisolithus tinctorius, Fomitopsis lilacinogilva, Stereum hirsutum and Pycnoporus sanguineus showed higher antimicrobial activity against the Gram-positive bacteria than aqueous extracts, with S. pneumoniae being the most susceptible. The ethanol extracts of Agaricus campestris, Chlorophyllum molybdites, Gymnopilus penetrans, Pseudophaeolus baudonii and Laetiporus sulphureus exhibited anti-TB (tuberculosis) activity with minimum inhibitory concentrations (MICs) ranging between 500-1,000 μg/mL. C. molybdites ethanol extract inhibited HIV-1 protease activity (IC50: 49.7 μg/mL). The macrofungal extracts did not inhibit HIV-1 reverse transcriptase activity. Ethanol extracts of F. lilacinogilva, G. junonius, P. sanguineus and the aqueous extract iv of P. baudonii were cytotoxic against A549 lung cancer cells at IC50 values of 69.2±3.6, 57±5, 7.4±1.1 and 53.6±1.1 μg/mL, respectively. Cell cycle arrest was observed in the G2 phase for both P. sanguineus and P. baudonii, and G2/M and early M phases for G. junonius and F. lilacinogilva, respectively. Apoptosis induced by macrofungal extracts was confirmed by fluorescent staining. Morphological and biochemical changes included chromatin condensation, membrane blebbing, loss of cytoskeletal structure, caspase activation and phosphatidylserine translocation. This study demonstrates the biological activities of selected macrofungal extracts and their potential mechanisms of action. Isolation and identification of active compounds require further analysis.
- Full Text:
- Date Issued: 2018
- Authors: Didloff, Jenske
- Date: 2018
- Subjects: Macrofungi , Microbiology Medical microbiology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/30138 , vital:30835
- Description: Macrofungi represent an untapped source of natural bioactive compounds for various diseases, which have been targeted as potential therapeutic agents. The medicinal uses of macrofungi discovered to date include anticancer, antidiabetic, antioxidant, antimicrobial, and immunomodulatory properties. The knowledge regarding the medicinal uses of macrofungi in Africa is limited; however, it is believed that Africa may contain a large number of unidentified species of macrofungi. The objectives of this study were to: (i) screen the macrofungal extracts for antimicrobial activity against microorganisms responsible for respiratory diseases (e.g. Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Mycobacterium tuberculosis, HIV-1), (ii) determine the effect of macrofungal extracts on bacterial morphology, (iii) investigate the cytotoxicity of macrofungal extracts against human lung carcinoma cells, and to elucidate the mechanism/s of action of cytotoxicity/anticancer activity. In vitro bioassays for antimicrobial activity included: ρ-iodonitrotetrazolium chloride assays and microplate alamar blue assay (MABA), while the HIV-1 reverse transcriptase colorimetric ELISA and HIV-1 protease fluorometric assay kits were used for anti-HIV activity. Cytotoxicity of the macrofungal species against A549 lung cancer cell line was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and the IC50 values determined for the most cytotoxic macrofungal spp. The mechanism of cytotoxicity was investigated by cell cycle analysis and fluorescent staining to observe morphological and biochemical changes (i.e. acridine orange, caspase activation). Ethanol extracts of Amanita foetidissima, Gymnopilus junonius, Pisolithus tinctorius, Fomitopsis lilacinogilva, Stereum hirsutum and Pycnoporus sanguineus showed higher antimicrobial activity against the Gram-positive bacteria than aqueous extracts, with S. pneumoniae being the most susceptible. The ethanol extracts of Agaricus campestris, Chlorophyllum molybdites, Gymnopilus penetrans, Pseudophaeolus baudonii and Laetiporus sulphureus exhibited anti-TB (tuberculosis) activity with minimum inhibitory concentrations (MICs) ranging between 500-1,000 μg/mL. C. molybdites ethanol extract inhibited HIV-1 protease activity (IC50: 49.7 μg/mL). The macrofungal extracts did not inhibit HIV-1 reverse transcriptase activity. Ethanol extracts of F. lilacinogilva, G. junonius, P. sanguineus and the aqueous extract iv of P. baudonii were cytotoxic against A549 lung cancer cells at IC50 values of 69.2±3.6, 57±5, 7.4±1.1 and 53.6±1.1 μg/mL, respectively. Cell cycle arrest was observed in the G2 phase for both P. sanguineus and P. baudonii, and G2/M and early M phases for G. junonius and F. lilacinogilva, respectively. Apoptosis induced by macrofungal extracts was confirmed by fluorescent staining. Morphological and biochemical changes included chromatin condensation, membrane blebbing, loss of cytoskeletal structure, caspase activation and phosphatidylserine translocation. This study demonstrates the biological activities of selected macrofungal extracts and their potential mechanisms of action. Isolation and identification of active compounds require further analysis.
- Full Text:
- Date Issued: 2018
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