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Chemical investigation of a dichloromethane extract of the aerial parts of Waltheria indica led to the isolation and characterization of five polyhydroxymethoxyflavonoids, namely, oxyanin A (1), vitexicarpin (3), chrysosplenol E (4), flindulatin (5), 5-hydroxy-3,7,4'-trimethoxyflavone (6), and six quinolone alkaloids, waltheriones M-Q (2, 7, 8, 10, 11) and 5(R)-vanessine (9). Among these, compounds 2, 7, 8, 10, and 11 have not yet been described in the literature. Their chemical structures were established by means of spectroscopic data interpretation including (1)H and (13)C, HSQC, HMBC, COSY, and NOESY NMR experiments and UV, IR, and HRESIMS. The absolute configurations of the compounds were established by ECD. The isolated constituents and 10 additional quinoline alkaloids previously isolated from the roots of the plant were evaluated for their in vitro antifungal activity against the human fungal pathogen Candida albicans, and 10 compounds (7, 9, 11-16, 18, 21) showed growth inhibitory activity on both planktonic cells and biofilms (MIC ≤ 32 μg/mL). Their spectrum of activity against other pathogenic Candida species and their cytotoxicity against human HeLa cells were also determined. In addition, the cytological effect of the antifungal isolated compounds on the ultrastructure of C. albicans was evaluated by transmission electron microscopy.

Candida albicans is a major cause of fungal diseases in humans, and its resistance to available drugs is of concern. In an attempt to identify novel antifungal agents, we initiated a small-scale screening of a library of 199 natural plant compounds (i.e., natural products [NPs]). In vitro susceptibility profiling experiments identified 33 NPs with activity against C. albicans (MIC50s ≤ 32 μg/ml). Among the selected NPs, the sterol alkaloid tomatidine was further investigated. Tomatidine originates from the tomato (Solanum lycopersicum) and exhibited high levels of fungistatic activity against Candida species (MIC50s ≤ 1 μg/ml) but no cytotoxicity against mammalian cells. Genome-wide transcriptional analysis of tomatidine-treated C. albicans cells revealed a major alteration (upregulation) in the expression of ergosterol genes, suggesting that the ergosterol pathway is targeted by this NP. Consistent with this transcriptional response, analysis of the sterol content of tomatidine-treated cells showed not only inhibition of Erg6 (C-24 sterol methyltransferase) activity but also of Erg4 (C-24 sterol reductase) activity. A forward genetic approach in Saccharomyces cerevisiae coupled with whole-genome sequencing identified 2 nonsynonymous mutations in ERG6 (amino acids D249G and G132D) responsible for tomatidine resistance. Our results therefore unambiguously identified Erg6, a C-24 sterol methyltransferase absent in mammals, to be the main direct target of tomatidine. We tested the in vivo efficacy of tomatidine in a mouse model of C. albicans systemic infection. Treatment with a nanocrystal pharmacological formulation successfully decreased the fungal burden in infected kidneys compared to the fungal burden achieved by the use of placebo and thus confirmed the potential of tomatidine as a therapeutic agent.

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This paper reviews the use of TLC-bioautography in the search for antifungal compounds from natural sources. The main methods used for antifungal screening are presented, with special emphasis on bioautography. Different aspects of the technique, including the latest chromatographic developments such as HPTLC and HPLC microfractionation are presented. The present status and recent advances made in antifungal bioautography are discussed, and a comprehensive review of the applications over the last 6 years is presented. Various strategies applied in the search for antifungal compounds from natural sources are discussed, with a highlight on the challenges faced when screening complex crude mixtures. The activities of approximately 100 antifungal compounds of natural origin are presented with their minimum inhibitory quantity. The most active natural source compounds against Candida, Cladosporium, Colletotrichum, and Fusarium species are highlighted, and the compound activities discussed. In addition, perspectives concerning future improvements in bioautography sensitivity and reproducibility are noted.

A dichloromethane extract of the roots from the Panamanian plant Swartzia simplex exhibited a strong antifungal activity in a bioautography assay against a genetically modified hypersusceptible strain of Candida albicans. At-line HPLC activity based profiling of the crude extract enabled a precise localization of the antifungal compounds, and dereplication by UHPLC-HRESIMS indicated the presence of potentially new metabolites. Transposition of the HPLC reversed-phase analytical conditions to medium-pressure liquid chromatography (MPLC) allowed an efficient isolation of the major constituents. Minor compounds of interest were isolated from the MPLC fractions using semipreparative HPLC. Using this strategy, 14 diterpenes (1-14) were isolated, with seven (5-10, 14) being new antifungal natural products. The new structures were elucidated using NMR spectroscopy and HRESIMS analysis. The absolute configurations of some of the compounds were elucidated by electronic circular dichroism spectroscopy. The antifungal properties of these compounds were evaluated as their minimum inhibitory concentrations in a dilution assay against both hypersusceptible and wild-type strains of C. albicans and by assessment of their antibiofilm activities. The potential cytological effects on the ultrastructure of C. albicans of the antifungal compounds isolated were evaluated on thin sections by transmission electron microscopy.

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