In vitro evaluation of antimycobacterial activity, safety assessment and isolation of an active compound from Gymnopilus junonius

Background: Tuberculosis remains a significant public health challenge due to complex treatment regimens, compliance issues, latent infections, high HIV co-infection rates, and drug-resistant strains. This highlights the need for new antimycobacterial drugs. Natural products have historically been valuable in pharmaceutical discovery. Although medicinal macrofungi have long been used in traditional medicine, research into their antimycobacterial activity is still limited. The aim of this study was to isolate, identify, and characterise the antimycobacterial compound present in an ethanol fruiting body extract of Gymnopilus junonius (Fr.) P.D. Orton (Hymenogastraceae), as well as to investigate their mechanism of action and potential toxicity using in vitro and in vivo screening methods. Methods: Bioassay-guided fractionation was conducted using column chromatography and preparative thin-layer chromatography to isolate the active antimycobacterial compound from an ethanol extract of G. junonius. 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. Toxicity assessment was performed on African green monkey kidney (Vero) and human hepatoma (C3A) cell lines using various staining techniques to observe the potential risk for hepatotoxicity and genotoxicity. In vivo toxicity of the crude extract was also evaluated using a zebrafish embryo/larvae model to observe developmental and teratogenic effects. Results: Bioassay-guided fractionation of G. junonius resulted in the isolation of an active compound exhibiting inhibitory activity against M. tuberculosis (MIC: 31.25 µg/mL). Gymnopilene was identified as the main active compound. Transmission electron microscopy revealed ultrastructural damage to M. tuberculosis cells upon treatment with gymnopilene 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 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 the crude extract of G. junonius against the zebrafish embryos/larvae over a 72-hour time period. Discussion: The research represents the first to demonstrate that the predominant compound, gymnopilene, from G. junonius inhibits M. tuberculosis and causes cell wall damage. However, identified toxicity may limit drug development suitability. Future research should include more complex in vitro models alongside the in vivo screening model to assess the therapeutic potential of gymnopilene. Conclusion: The study highlights the potential of macrofungi as a source of bioactive compounds, confirming the significance of natural products in drug discovery.