A synergistic antifungal agent from Ligusticum chuanxiong: Total synthesis, biological evaluation, and mechanism of ligustrumdiol A.

Abstract

Invasive fungal infections threaten immunocompromised patients, and the toxicity, resistance, and narrow spectrum of current therapies demand new agents. A chemical investigation of the medicinal plant Ligusticum chuanxiong led to the isolation of a rare pentyl-substituted cyclohexene derivative, ligustrumdiol A (LDA). Its structure was fully elucidated by spectroscopic analysis, and an enantioselective total synthesis was accomplished in 12 steps from d-quinic acid with an overall yield of 11.2%. Although LDA alone showed only weak antifungal activity against Aspergillus fumigatus (MIC = 400 μg/mL), it exhibited potent synergistic activity when combined with amphotericin B (AMB). Checkerboard assays revealed that the combination of LDA with AMB reduced the MIC of LDA to 50 μg/mL against A. fumigatus, with fractional inhibitory concentration index (FICI) values below 0.5, indicating strong synergy. Further studies demonstrated that the combination effectively inhibited hyphal growth, disrupted biofilm formation, and impaired preformed mature biofilms. Mechanistically, transcriptomic analysis showed that LDA downregulated genes involved in ribosome function and oxidative phosphorylation, while upregulating MAPK signaling and autophagy pathways. Subsequent experiments confirmed that LDA induced reactive oxygen species (ROS) accumulation, depleted glutathione (GSH), dissipated mitochondrial membrane potential (MMP), reduced ATP levels, and ultimately triggered apoptosis in A. fumigatus. In a murine model of invasive aspergillosis, combination therapy with 0.5 mg/kg AMB and 100 mg/kg LDA achieved a 40% survival rate at day 10 and significantly reduced fungal burden in the heart, liver, lungs, and kidneys, using only one-quarter of the AMB dose required for high-dose monotherapy. Collectively, these findings identify LDA as a novel synergistic partner of AMB and provide a promising strategy to enhance antifungal efficacy while reducing AMB-associated toxicity.