Targeted isolation of photoactive pigments from mushrooms yielded a highly potent new photosensitizer: 7,7'-biphyscion

Abstract

Pigments of fungi are a fertile ground of inspiration: they spread across various chemical backbones, absorption ranges, and bioactivities. However, basidiomycetes with strikingly colored fruiting bodies have never been explored as agents for photodynamic therapy (PDT), even though known photoactive compound classes (e.g., anthraquinones or alkaloids) are used as chemotaxonomic markers. In this study, we tested the hypothesis that the dyes of skin-heads (dermocyboid Cortinarii) can produce singlet oxygen under irradiation and thus are natural photosensitizers. Three photosensitizers based on anthraquinone structures were isolated and photopharmaceutical tests were conducted. For one of the three, i.e., (-)-7,7'-biphyscion (1), a promising photoyield and photocytotoxicity of EC₅₀ = 0.064 µM against cancer cells (A549) was found under blue light irradiation (λ_exc = 468 nm, 9.3 J/cm²). The results of molecular biological methods, e.g., a viability assay and a cell cycle analysis, demonstrated the harmlessness of 1 in the dark and highlighted the apoptosis-inducing PDT potential under blue light irradiation. These results demonstrate for the first time that pigments of dermocyboid Cortinarii possess a so far undescribed activity, i.e., photoactivity, with significant potential for the field of PDT. The dimeric anthraquinone (-)-7,7'-biphyscion (1) was identified as a promising natural photosensitizer.

Figure 1

Relative singlet oxygen formation efficacy of fungal extracts (PE = petroleum ether extract, MeOH = methanolic extract, 1 mg/mL) under blue light irradiation (468 nm, 24.3 J/cm²). All investigated fungal species were more prone to produce singlet oxygen than an extract containing berberine (λ_{max, EtOH} = 429 nm, ϕ_EtOH = 0.05), i.e., a Berberis ilicifolia root extract. The latter is a plant species commonly known as barberry, which contains the photosensitizer berberine in its roots.

Figure 2

HPLC chromatogram (λ_det = 468 nm) of the EtOAc fraction of C. uliginosus obtained by a liquid–liquid fractionation from the methanol extract. The detection wavelength was fitted to the excitation wavelength of the accompanied assays (i.e., (photo)cytotoxicity and DMA assay). Peaks of interest are indicated by their retention time and by the identified structure. The small structures indicate solely annotated structures, while the normal-sized ones are such which were isolated and are thus structurally verified. Stationary phase: Max RP, Mobile Phase (H₂O/ACN + 0.1%FA), gradient (for details refer SI, Chapter 1.5).

Figure 3

(Left) Dose–response curves of the three cell lines treated with 1 under blue light irradiation (λ_exc = 468 nm blueish plots) and dark conditions (black plots). Error = standard deviation. (Middle) Micrographs (brightfield, 10× objective) of treated (and irradiated) cells. (Right) Results of the (photo)cytotoxicity assay given as effective concentrations (EC₅₀) in µM with confidence interval (95%).

Figure 4

Annexin V/DRAQ7 assay 24 h after (mock) irradiation of A549 cells treated with 1 (c = 0.5 µM). Left) non-irradiated cells showing no sign of induced apoptosis and right) irradiated cell population containing a clear apoptotic cell population.