Biological activities of tulbaghia violacea against cryptococcus species

Cryptococcus neoformans and Cryptococcus gattii, which are environmental yeasts, are the etiological agents of cryptococcosis in both immunocompromised and immunocompetent individuals, and account for high mortality and morbidity rates in sub-Saharan Africa. The current antifungal agents used for treatment of cryptococcal infections either target the fungal cell wall (β-(1,3) glucan and chitin) or cell membrane directly or ergosterol biosynthetic pathways or fungal DNA and RNA. Gaps in antifungal therapy include the unavailability and exhorbitant costs of these drugs especially to patients in the developing world. Drug resistance to conventional drugs is also an ever-increasing problem. It is therefore essential to find alternative natural compounds from medicinal plants that are safer, cheaper and more widely available. Tulbaghia violacea, also known as wild garlic, has been used as a traditional remedy in South Africa for the treatment of several ailments. Aqueous and organic extracts of the plant have been demonstrated to have antimicrobial and antifungal activity against several pathogens. However, there is a huge gap in our current knowledge in that the mechanism/s of action/s of these extracts have not been fully investigated. The focus of the current study therefore was to determine whether T. violacea extracts from the roots, rhizome, leaves and tubers exhibited antifungal activity against C. neoformans and C. gattii and to evaluate the ability of the rhizome extract to induce changes in key fungal virulence factors. Three mechanisms (ergosterol, β-(1,3) glucan and chitin production) regulating the antifungal activity of the rhizome extract were also examined. In the current study, phytochemical analysis of aqueous extracts of the roots, leaves, rhizomes and tubers showed that the rhizomes had the highest phenolic, saponin and tannin content when compared to the other plant organs. Fingerprinting by GC-MS revealed identical compounds in the different plant parts with the detection of 4 H Pyran-4-one DDMP (known antifungal), previously unreported in studies on T. violacea. The bulk of the extract comprised of 40% sulphur-containing and 20% furan-containing compounds. The remaining minor compounds comprised of 2x alcohols (13.3%), 1x pyran (6.7%), 1x ketone (6.7%), 1x halogen (6.7%) and 1x acid (6.7%) compounds. The rhizomes also had highest content of 2 methyl methioacetic acid, benzophenone and chloromethyl methylsulfide compared to the other plant parts. The rhizomes were found to be more potent against both pathogenic fungi tested here with an MIC and MFC of 1.25 mg/ml. Nystatin was included as a positive control when determining the MIC’s and MFC’s of the different plant extracts. The antifungal nature of the T. violacea extracts in the current study may be due to the synergistic effects of the sulphide, furan, pyran and ketone compounds present in the extracts, but this still remains to be verified in future studies. An investigation of the effects of an aqueous rhizome extract of T. violacea on Cryptococcus virulence factors showed that phospholipase activity of C. neoformans and C. gattii remained unaffected with increasing sub-lethal doses of the plant extract. There was significant reduction in urease production in both fungi in a dose dependent manner relative to the untreated cultures after 24 hr exposure to the extract. However, urease production reverted to normal after 48 hr post exposure implying that the cultures were able to recover due to temporary inhibition of urease activity. A significant decrease in melanin production was observed in both C. neoformans and C. gattii with increasing sub-inhibitory concentrations of the rhizome extract. Investigation of the effect of the plant extract on the ultrastucture of the fungi via Transmission Electron Microscopy showed the induction of cytomorphological changes in C. neoformans and C. gattii. Changes included thickening of the cell wall, an increase in the number of vacuoles, mitochondrial swelling and occasional detachment of the membrane from the cell wall. These changes suggest the activation of possible defence mechanisms to compensate for the loss of cellular materials or an effort to sequester toxic T. violacea components or toxic intermediates generated from inhibited cellular pathways. The capsule size and architecture remained unaltered in the presence of sub-lethal doses of the rhizome extract. To study the mechanism of action of the rhizome extract on ergosterol biosynthesis, total sterols were extracted and ergosterol, squalene, 2,3-oxidosqualene and lanosterol were quantified using Reverse Phase High Performance Liquid Chromatography. Ergosterol concentration declined in a dose dependent manner for both pathogenic yeasts similar to the positive control terbinafine, while there was a slight accumulation of squalene in C. gattii only. 2,3-oxidosqualene levels accumulated in both fungi relative to the untreated control. Lanosterol production showed an oscillatory trend for the two microorganisms. Together, these findings indicate that the rhizome extract is capable of inhibiting squalene epoxidase and 2,3-oxidosqualene/ lanosterol cyclase causing a decrease in ergosterol production