Photoantimicrobial anthraquinones in Australian fungi of the genus Cortinarius

Dermocyboid Cortinarius species of both hemispheres are usually intensely colored and known to contain anthraquinones. Australian dermocyboid fungi have distinct evolutionary histories which are clearly different from Northern Hemisphere species of similar appearance. Allopatric speciation often results in different metabolic adaptations. We were especially interested in the diversity of anthraquinonic pigments and in their photoantimicrobial potential. In this study 33 dried samples from eighteen Australian dermocyboid Cortinarius taxa were extracted with methanol and analyzed via HPLC-DAD-MS. The 9,10-dimethylanthracene (DMA) assay was used to measure the extracts' ability to produce singlet oxygen after irradiation. Furthermore, a photoantimicrobial screening method based on the protocols of Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility (EUCAST), employing the common human pathogens Candida albicans, Escherichia coli, and Staphylococcus aureus was used to identify photoantimicrobials. Based on chromatography, mass spectrometry, and UV/Vis data, peaks were annotated according to literature and in-house data. A chemotaxonomic pigment analysis was established to cluster the species according to their major anthraquinones and to allow identification of the groups by a minimum number of pigments. In agreement with the recorded pigment pattern of the photoactive anthraquinones, nine Cortinarius species (C. alienatus, C. atropurpureus, C. austrovenetus, C. basirubescens, C. canarius, C. clelandii 2, C. globuliformis, C. persplendidus 1, C. sp. "honey pileus 1") showed at least 70 % inhibition of Staphylococcus aureus (Gram-positive bacteria) growth under light irradiation (λ = 428 / 478 nm, H = 30 J/cm², c = 50 μg/mL, pre-illumination time (t_PI) = 60 min) in the photoantimicrobial screening. The extracts of three species (C. alienatus, C. austrovenetus, and C. clelandii 2) showed additional photoactivity against Candida albicans (yeast) under the same conditions. Several relevant (photo)antimicrobials were identified: Emodin, dermocybin, skyrin, physcion (synonym: parietin), 7,7'-biphyscion, and hypericin; From the photoactive extracts, that were not reported to contain photoactive compounds before, austrocortinin and xanthorin were isolated and used for further testing. For austrocortinin a singlet oxygen yield of 0.03 (reference: [Ru(bpy₃)Cl₂], d₄-MeOH) was detected. Accordingly, the anthraquinone did not show activity in the photoantimicrobial assay. Xanthorin, with a singlet oxygen yield of 0.10, led to an inhibition of growth of 78.1 % against Staphylococcus aureus (4.00 μg/mL, λ = 428 nm, 30 J/cm², t_PI = 60 min).