Abstract
Visceral leishmaniasis (VL), caused by Leishmania donovani, is one of the most lethal parasitic diseases, causing 30000 deaths annually. Pentavalent antimonials, with limited effectiveness, cytotoxicity, drug resistance, and therapeutic limitations, underscore the urgent need for the development of novel, selective, and cost-effective antileishmanial agents. The Indian pharmaceutical industry, a major global economic sector, is showing growing interest in mushrooms in developing alternative drugs against several diseases. Thus, this study focuses on the antileishmanial potential of the hydroalcoholic extract of Amanita princeps, a wild edible mushroom, with detailed mechanistic analysis. In our research, the cell-inhibitory activity of the extract was evaluated by the MTT assay against promastigotes, axenic amastigotes, and intracellular amastigotes, yielding IC₅₀ values of 21.20, 30.26, and 38.93 μg/mL, respectively, with high selectivity indices (>10). A detailed mechanistic study revealed that the extract can disrupt the NO-arginase axis, suppress antioxidant enzymes (SOD and catalase), and elevate lipid peroxidation, ultimately causing intracellular oxidative stress. Further, flow cytometric and fluorescence microscopic study confirmed elevated reactive oxygen species, mitochondrial membrane depolarization, nuclear fragmentation, sub-G0/G1 cell cycle arrest, leading towards apoptosis as evidenced by AO/EtBr dual staining. Furthermore, the extract was able to restore host immunological balance by upregulating pro-inflammatory cytokines (IL-12β, TNF-α) and suppressing anti-inflammatory mediators (IL-10, TGF-β). Finally, GC-MS, network pharmacological and molecular docking study identified Urs-12-en-28-ol, a ursane triterpenoid with the highest affinity against DHFR (-9.7 kcal/mol) and demonstrated significant in vitro antileishmanial activity. Conclusively, our findings establish Amanita princeps as a promising candidate for next-generation antileishmanial drug development.