Purpurascenines A-C, Azepino-Indole Alkaloids from Cortinarius purpurascens: Isolation, Biosynthesis, and Activity Studies on the 5-HT2A Receptor.

Three previously undescribed azepino-indole alkaloids, named purpurascenines A-C (1-3), together with the new-to-nature 7-hydroxytryptophan (4) as well as two known compounds, adenosine (5) and riboflavin (6), were isolated from fruiting bodies of Cortinarius purpurascens Fr. (Cortinariaceae). The structures of 1-3 were elucidated based on spectroscopic analyses and ECD calculations. Furthermore, the biosynthesis of purpurascenine A (1) was investigated by in vivo experiments using 13C-labeled sodium pyruvate, alanine, and sodium acetate incubated with fruiting bodies of C. purpurascens. The incorporation of 13C into 1 was analyzed using 1D NMR and HRESIMS methods. With [3-13C]-pyruvate, a dramatic enrichment of 13C was observed, and hence a biosynthetic route via a direct Pictet-Spengler reaction between α-keto acids and 7-hydroxytryptophan (4) is suggested for the biosynthesis of purpurascenines A-C (1-3). Compound 1 exhibits no antiproliferative or cytotoxic effects against human prostate (PC-3), colorectal (HCT-116), and breast (MCF-7) cancer cells. An in silico docking study confirmed the hypothesis that purpurascenine A (1) could bind to the 5-HT2A serotonin receptor's active site. A new functional 5-HT2A receptor activation assay showed no functional agonistic but some antagonistic effects of 1 against the 5-HT-dependent 5-HT2A activation and likely antagonistic effects on putative constitutive activity of the 5-HT2A receptor.

Photochemical defense as trait of fungi from Cortinarius subgenus Dermocybe.

The photobiological activity of ten colorful species belonging to subgenus Dermocybe of the basidiomycete genus Cortinarius was investigated. Extracts of all species produced singlet oxygen and are thus photoactive. Pigment analysis was performed and showed similarities of the anthraquinone pigments across the species in dependency to their respective pigmentation types. Detailed content analysis of the pigments in the whole agaricoid fruiting body compared to the three different tissue types (pileus, stipe, and lamellae) revealed that the pigments emodin, dermocybin, and dermorubin, as well as their respective glycosides, are enhanced in the gills. In an independent experiment, the gills were shown to be the most photoactive tissues of the fruiting body. Photobiological experiments with invertebrates (i.e., glassworm Chaoborus crystallinus) proved a phototoxic effect of the methanolic extract of the red blood webcap (Cortinarius sanguineus var. aurantiovaginatus). This work adds further evidence to a common photobiological trait in Cortinarius subgenus Dermocybe and underpins the possibility of a photochemical defense mechanism in fungi.

The photosensitizer emodin is concentrated in the gills of the fungus Cortinarius rubrophyllus.

The colorful agaricoid fruiting bodies of dermocyboid Cortinarii owe their magnificent hue to a mixture of anthraquinone (AQ) pigments. Recently, it was discovered that some of these fungal anthraquinones have an impressive photopharmacological effect. The question, therefore, arises as to whether these pigments are also of ecological or functional significance. According to the optimal defense hypothesis, toxic molecules should be enriched in spore-producing structures, such as the gills of agarics. To test this hypothesis, we studied the distribution of fungal AQs in the fruiting body of Cortinarius rubrophyllus. The fungus belongs to the well-studied Cortinarius subgenus Dermocybe but has not been chemically characterized. Here, we report on the pigment profile of this beautiful fungus and focus on the distribution of anthraquinone pigments in the fruiting body for the first time. Here it is statistically confirmed that the potent photosensitizer emodin is significantly enriched in the gills. Furthermore, we show that the extract is photoactive against cancer cells and bacteria.

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

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 (λexc = 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.

Optimized isolation of 7,7'-biphyscion starting from Cortinarius rubrophyllus, a chemically unexplored fungal species rich in photosensitizers.

Mushrooms such as the dermocyboid Cortinarius rubrophyllus are characterized by strikingly colorful fruiting bodies. The molecular dyes responsible for such colors recently experienced a comeback as photoactive compounds with remarkable photophysical and photobiological properties. One of them-7,7'-biphyscion-is a dimeric anthraquinone that showed promising anticancer effects in the low nanomolar range under blue-light irradiation. Compared to acidic anthraquinones, 7,7'-biphyscion was more efficiently taken up by cells and induced apoptosis after photoactivation. However, seasonal collection of mushrooms producing this compound, low extraction yields, and tricky fungal identification hamper further developments to the clinics. To bypass these limitations, we demonstrate here an alternative approach utilizing a precursor of 7,7'-biphyscion, i.e., the pre-anthraquinone flavomannin-6,6'-dimethyl ether, which is abundant in many species of the subgenus Dermocybe. Controlled oxidation of the crude extract significantly increased the yield of 7,7'-biphyscion by 100%, which eased the isolation process. We also present the mycochemical and photobiological characterization of the yet chemically undescribed species, i.e. C. rubrophyllus. In total, eight pigments (1-8) were isolated, including two new glycosylated anthraquinones (1 and 2). Light-dependent generation of singlet oxygen was detected for the first time for emodin-1-O-ß-D-glucopyranoside (3) [photophysical measurement: Φ∆ = 0.11 (CD3OD)]. Furthermore, emodin (7) was characterized as promising compound in the photocytotoxicity assay with EC50-values in the low micromolar range under irradiation against cells of the cancer cell lines AGS, A549, and T24.

137Cs and 40K in Cortinarius caperatus mushrooms (1996-2016) in Poland - Bioconcentration and estimated intake: 137Cs in Cortinarius spp. from the Northern Hemisphere from 1974 to 2016.

Cortinarius caperatus grows in the northern regions of Europe, North America and Asia and is widely collected by mushroom foragers across Europe. This study shows that in the last three decades since the Chernobyl nuclear accident, C. caperatus collected across much of Northern Poland exhibited high activity concentrations of radiocaesium (137Cs) - a long-lived radionuclide. The mushroom appears to efficiently bioconcentrate 137Cs from contaminated soil substrata followed by sequestration into its morphological parts such as the cap and stipe which are used as food. The gradual leaching of 137Cs into the lower strata of surface soils in exposed areas are likely to facilitate higher bioavailability to the mycelia of this species which penetrate to relatively greater depths and may account for the continuing high activity levels noticed in Polish samples (e.g. activity within caps in some locations was still at 11,000 Bq kg-1 dw in 2008 relative to a peak of 18,000 in 2002). The associated dietary intake levels of 137Cs have often exceeded the tolerance limits set by the European Union (370 and 600 Bq kg-1 ww for children and adults respectively) during the years 1996-2010. Human dietary exposure to 137Cs is influenced by the method of food preparation and may be mitigated by blanching followed by disposal of the water, rather than direct consumption after stir-frying or stewing. It may be prudent to provide precautionary advice and monitor activity levels, as this mushroom continues to be foraged by casual as well as experienced mushroom hunters.

Nor-guanacastepene pigments from the Chilean mushroom Cortinarius pyromyxa.

For the first time, the pigment composition of basidiocarps from the Chilean mushroom Cortinarius pyromyxa was studied under various aspects like phylogeny, chemistry and antibiotic activity. A molecular biological study supports the monotypic position of C. pyromyxa in subgenus Myxacium, genus Cortinarius. Four undescribed diterpenoids, named pyromyxones A-D, were isolated from fruiting bodies of C. pyromyxa. Their chemical structures were elucidated based on comprehensive one- and two-dimensional NMR spectroscopic analysis, ESI-HRMS measurements, as well as X-ray crystallography. In addition, the absolute configurations of pyromyxones A-D were established with the aid of JH,H, NOESY spectra and quantum chemical CD calculation. The pyromyxones A-D possess the undescribed nor-guanacastane skeleton. Tested pyromyxones A, B, and D exhibit only weak activity against gram-positive Bacillus subtilis and gram-negative Aliivibrio fischeri as well as the phytopathogenic fungi Botrytis cinerea, Septoria tritici and Phytophthora infestans.

Steroidal composition and cytotoxic activity from fruiting body of Cortinarius xiphidipus.

From the fruiting body of ectomycorrhizal fungi Cortinarius xiphidipus, sterols were identified from the crude extract and the cytotoxic effect of ergosta-4, 6, 8(14), 22-tetraen-3-one (ergone) was evaluated. Ten sterols including ergosta-3,5,7,9(11),22-pentaene, (22E)-ergosta-5,7,9(11),22-tetraen-3b-ol, (3ß,22E)-ergosta-5,7,22-trien-3-ol, (22E)-ergosta-7,22-dien-3-ol, neoergosterol, (3ß)-ergosta-5,8-dien-3-ol, (3ß)-ergosta-7-en-3-ol, stigmasterol, stigmasterol 22,23-dihydro and (22E)-ergosta-4,6,8(14),22-tetraen-3-one were identified from the crude extract. The cytotoxic activity of the sterol fraction containing ergosta-4, 6, 8(14), 22-tetraen-3-one was assessed on four tumour cell lines (Neuro-2a, Saos-2, MCF7 and LNCaP-C42). The cytotoxic activity against the four tumour cell lines tested, being Neuro-2a and Saos-2 the most sensitive, with a half-maximal inhibitory concentration (IC50) of 20.8 ± 2.2 and 27.8 ± 1.0 µg/mL, respectively. This is the first report of this Antarctic fungi collected in the Magallanes and Chilean Antarctica Region. This work represents a potential source for the development of anticancer drugs.

Two New Ergosterol Derivatives from the Basidiomycete Cortinarius glaucopus.

Two new sterols 1 and 2 and five known ones 3 - 7 were isolated for the first time from the fruiting bodies of Cortinarius glaucopus. Their structures were established by 1- and 2D-NMR spectra and HR-FABS-MS. The relative configuration of 1 was firmly determined by comparison of the observed 1 H-1 H couplings and NOESY correlations, with those predicted for the computed geometries of the conformers. Calculations were performed by means of DFT with the B3LYP functional at 6-31 + G(d,p) level of theory, in CHCl3 as the solvent. The structures of the new ergosterol derivatives, called glaucoposterol A (1) and B (2), were thus established as (3S,5R,7R,10R,13R,17R,20S,22R,23R,24R)-5,6-epoxy-3,7,23-trihydroxystrophast-8-en-14-one and (22E,3S,5S,9S,10R,13R,17R,20R,24R)-3,5-dihydroxyergosta-6,8(14),22-trien-15-one, respectively. Moreover, the configuration of known strophasterol C (3) was determined as (3S,5R,6S,7R,10R,13R,17R,20S,22S,24R). Glaucoposterol A (1) and strophasterol C (3) represent the second finding in nature of steroids with the rare strophastane skeleton.

Radiocaesium in Cortinarius spp. mushrooms in the regions of the Reggio Emilia in Italy and Pomerania in Poland.

Activity concentrations of 134Cs and 137Cs have been determined in 23 species of mushrooms of the genus Cortinarius (59 individual samples) collected from the Reggio Emilia in Italy 1992-1999 and in 4 species (16 composite samples and 413 individuals) from the Pomerania region in Poland from 1996 to 2015. Across all the Cortinarius species from the Reggio Emilia, the activity concentrations were relatively high in Cortinarius alboviolaceus, Cortinarius duracinus, Cortinarius orellanus, Cortinarius rapaceus, and Cortinarius subannulatus, in which 137Cs was at 10,000 ~ 100,000 Bq kg-1 dry biomass (db) in 1994. Smaller activity concentrations were found in Cortinarius bivelus, Cortinarius bulliardii, Cortinarius cotoneus, Cortinarius largus, Cortinarius lividoviolaceus, Cortinarius purpureus, Cortinarius rufo-olivaceus, Cortinarius torvus, and Cortinarius venetus with levels at 1000 ~ 6000 Bq kg-1 db from 1992 to 1994, and further in Cortinarius anserinus, Cortinarius auroturbinatus, C. largus, Cortinarius praestans, Cortinarius purpurascens, Cortinarius scaurus, Cortinarius sebaceous, Cortinarius talus, and Cortinarius variecolor with activity concentrations at 100 ~ 600 Bq kg-1 db in 1994. All the data were calculated for dehydrated fungal material corrected back to the exact date samples of collection. The greatest activity concentrations of 137Cs both in Italy (1992-1999) and Poland (1996-2010) were found in the popular Cortinarius caperatus, confirming its very high capacity of radiocaesium accumulation. Besides 137Cs, the isotope 134Cs was detected in some species from the Reggio Emilia. An average calculated ratio of activities of 134Cs to 137Cs referenced to 1986 was equal to 0.38 in mushrooms from the Reggio Emilia, and this value slightly differ from that specific for Chernobyl fallout, which was 0.54. It was calculated that 137Cs originating from Chernobyl accident constituted about 68 % of the total activity concentration of the isotope in Reggio Emilia in 1986, while as much as 32 % of 137Cs in mushrooms were from the global fallout from nuclear bomb testing.

A novel orellanine containing mushroom Cortinarius armillatus.

Orellanine (3,3',4,4'-tetrahydroxy-2,2'-bipyridine-1,1'-dioxide) is a tetrahydroxylated di-N-oxidized bipyridine compound. The toxin, present in certain species of Cortinarius mushrooms, is structurally similar to herbicides Paraquat and Diquat. Cortinarius orellanus and Cortinarius rubellus are the major orellanine-containing mushrooms. Cortinarius mushrooms are widely reported in Europe where they have caused human poisoning and deaths through accidental ingestion of the poisonous species mistaken for the edible ones. In North America, Cortinarius orellanosus mushroom poisoning was recently reported to cause renal failure in a Michigan patient. Cortinarius mushroom poisoning is characterized by delayed acute renal failure, with some cases progressing to end-stage kidney disease. There is debate whether other Cortinarius mushroom contain orellanine or not, especially in North America. Currently, there are no veterinary diagnostic laboratories in North America with established test methods for detection and quantitation of orellanine. We have developed two diagnostic test methods based on HPLC and LC-MSMS for identification and quantitation of orellanine in mushrooms. Using these methods, we have identified Cortinarius armillatus as a novel orellanine-containing mushroom in North America. The mean toxin concentration of 145 ug/g was <1% of that of the more toxic C. rubellus. The HPLC method can detect orellanine at 17 µg g(-1) while the LC-MSMS method is almost 2000 times more sensitive and can detect orellanine at 30 ng g(-1). Both tests are quantitative, selective and are now available for veterinary diagnostic applications.

Human and experimental toxicology of orellanine.

Orellanine is a nephrotoxic toxin produced by some mushroom species of the Cortinarius genus, typically found in Europe and North America. The nephrotoxicity of Cortinarius orellanus is well known and was first recognized in the 1950s when this mushroom was identified as the cause of a mass poisoning in Poland. Typically, onset of symptoms is delayed for 1-2 weeks after ingestion. Some patients suffer mild gastrointestinal discomfort in the latency period before developing signs of renal impairment due to severe interstitial nephritis, acute focal tubular damage, and interstitial fibrosis. There is no specific antidote to orellanine poisoning. The mainstay of treatment is the prevention of secondary complications of kidney failure, adequate dialysis and, in the case of incomplete recovery, management of chronic renal insufficiency. : In this work, we aim to review about Cortinarius species, including epidemiological studies, chemical structure, toxicokinetics, toxic doses, mechanisms of toxicity, diagnosis, prognosis, and treatment options.

Distribution of mercury in Gypsy Cortinarius caperatus mushrooms from several populations: an efficient accumulator species and estimated intake of element.

Mushroom Cortinarius caperatus is one of the several edible wild-grown species that are widely collected by fanciers. For specimens collected from 20 spatially and distantly distributed sites in Poland the median values of Hg contents of caps ranged from 0.81 to 2.4mgkg(-1) dry matter and in stipes they were 2.5-fold lower. C. caperatus efficiently accumulates Hg and the median values of the bioconcentration factor for caps range from 120 to 18 and for stipes from 47 to 7.3. This mushroom even when collected at background (uncontaminated) forested areas could be a source of elevated intake of Hg. The irregular consumption of the caps or whole fruiting bodies is not considered to pose a risk. Frequent eating of C. caperatus during the fruiting season by fanciers should be avoided because of possible health risk from Hg. Available data on Hg contents of C. caperatus from several places in Europe are also summarized.

Antioxidant polyketide phenolic metabolites from the edible mushroom Cortinarius purpurascens.

Phytochemical investigation of the ethyl acetate extract of the edible macrofungus, Cortinarius purpurascens, led to the isolation of nine anthraquinone-related pigments, citreorosein 6,8-dimethyl ether (1), physcione (2), rufoolivacin (3), rufoolivacin C (4), rufoolivacin D (5), leucorufoolivacin (6), verbindung cr11 (7), verbindung cr60 (8) and 1-Hydroxy-3-methyl-2-isopropanyl-6,8-dimethoxyanthraquinone (9). The structures of these isolated compounds were characterised by spectroscopic methods and comparison with published data. Among the tested compounds, 3-6 exhibited potent DPPH radical-scavenging activity with IC50 values in the range of 3-8 µg/ml. The results indicated that the fungus is a possible source of natural products with potential antioxidant activity.

Synthesis and antimalarial evaluation of a screening library based on a tetrahydroanthraquinone natural product scaffold.

As part of a research program aimed at discovering new antimalarial leads from Australian macrofungi a unique fungi-derived prefractionated library was screened against a chloroquine-sensitive Plasmodium falciparum line (3D7) using a radiometric growth inhibition assay. A library fraction derived from a Cortinarius species displayed promising antimalarial activity. UV-guided fractionation on the CH(2)Cl(2)/MeOH extract from this fungus resulted in the isolation of four known compounds: (1S,3R)-austrocortirubin (1), (1S,3S)-austrocortirubin (2), 1-deoxyaustrocortirubin (3), and austrocortinin (4). Compound 2 was used as a natural product scaffold in the parallel solution-phase synthesis of a small library of N-substituted tetrahydroanthraquinones (5-15). All compounds (1-15) were tested in vitro against P. falciparum 3D7 parasites and (1S,3S)-austrocortirubin (2), the major fungal constituent, was shown to be the most active compound with an IC(50) of 1.9 µM. This compound displayed moderate cytotoxicity against neonatal foreskin fibroblast (NFF) cells with an IC(50) of 15.6 µM.

Analysis of the mushroom nephrotoxin orellanine and its glucosides.

Orellanine is a nephrotoxin found in various Cortinaceae mushroom species. Unintentional consumption after these species were confused with edible mushrooms such as Cantharellus tubaeformis has caused several casualties. In this work, a quantitative HPLC-ESI-MS/MS method for total orellanine in Cortinarius rubellus, spiked blood plasma, and a mushroom stew prepared from C. tubaeformis with the addition of a single specimen of C. rubellus is presented. The existence of mono- and diglucosylated orellanine in C. rubellus was also proven, although quantitative analysis could not be obtained for the glucosides due to rapid hydrolyzation to orellanine in the extract. Extraction with 3 M HCl or water mainly yielded orellanine, while MeOH or acidified MeOH mainly extracted mono- and diglucosylated orellanine. The highest recovery of total orellanine was obtained with 3 M HCl, which was subsequently used for quantitative analysis. A C18 HPLC column and low pH in the eluents retained all these toxins. Orellanine could be detected at a 4.9 ng/mL level in all extracts, which is well below the threshold for acute toxic effects. Additionally, the fragmentation pattern of orellanine upon electrospray MS/MS was probed. The method described is useful for two important applications. First, it allows quantitative analysis of processed food products that may be contaminated by orellanine from Cortinaceae mushrooms. Second, orellanine is currently being evaluated as a potential cure of metastatic renal cancer, and this work provides a method for monitoring orellanine at low concentrations within the therapeutic interval in blood serum.

Toward the antioxidant and chemical characterization of mycorrhizal mushrooms from northeast Portugal.

UNLABELLED: Mushrooms are widely appreciated all over the world for their nutritional properties and pharmacological value as sources of important bioactive compounds. Mycorrhizal macrofungi associate with plant roots constituting a symbiotic relationship. This symbiosis could influence the production of secondary metabolites, including bioactive compounds. We focused on the evaluation of antioxidant potential and chemical composition of mycorrhizal mushrooms species from Northeast Portugal: Amanita caesarea, Amanita muscaria, Amanita pantherina, Chroogomphus fulmineus, Cortinarius anomalus, Cortinarius collinitus, Cortinarius violaceus, Lactarius quietus, Lactarius volemus, Russula sardonia, Suillus luteus, and Tricholoma ustale. A similar profile of metabolites was observed in the studied species with the order sugars > fat > ascorbic acid > phenolic compounds > tocopherols. Nevertheless, the samples revealed different compositions: prevalence of sugars in L. volemus, fat and ascorbic acid in A. muscaria, phenolic compounds in C. anomalus and tocopherols, and antioxidant activity in S. luteus. PRACTICAL APPLICATION: Chemical characterization of 12 mycorrhizal mushrooms was achieved. They are sources of nutraceuticals, such as sugars and fatty acids, and contain bioactive compounds, such as vitamins and phenolic acids. Edible species can be incorporated in diets as sources of antioxidants, while nonedible species can be explored as sources of bioactive metabolites.

Structure and absolute configuration of toxic polyketide pigments from the fruiting bodies of the fungus Cortinarius rufo-olivaceus.

Two new polyketide-derived pigments, named rufoolivacins B (), and D (), with a 4',10-coupled aryl linkage between polysubstituted 1-naphthol and 1,4- or 1,2-anthraquinone, together with nine known metabolites including rufoolivacins A () and C (), have been isolated from the fruiting bodies of the Chinese toadstool Cortinarius rufo-olivaceus (basidiomycetes). Their structures were characterized on the basis of spectroscopic methods, including 2D-NMR experiments (COSY, NOESY, HSQC, and HMBC). The axial chirality of and was assigned through analysis of their CD spectra and ZINDO and TDDFT calculations. Compounds and were found to be unusual natural products incorporating an ortho-anthraquinone chromophore. All the metabolites were shown to be toxic toward the brine shrimp.