Marek Halama

Do Differences in Chemical Composition of Stem and Cap of Amanita muscaria Fruiting Bodies Correlate with Topsoil Type

Fly agaric (Amanita muscaria) was investigated using a 1H NMR-based metabolomics approach. The caps and stems were studied separately, revealing different metabolic compositions. Additionally, multivariate data analyses of the fungal basidiomata and the type of soil were performed. Compared to the stems, A. muscaria caps exhibited higher concentrations of isoleucine, leucine, valine, alanine, aspartate, asparagine, threonine, lipids (mainly free fatty acids), choline, glycerophosphocholine (GPC), acetate, adenosine, uridine, 4-aminobutyrate, 6-hydroxynicotinate, quinolinate, UDP-carbohydrate and glycerol. Conversely, they exhibited lower concentrations of formate, fumarate, trehalose, α- and β-glucose. Six metabolites, malate, succinate, gluconate, N-acetylated compounds (NAC), tyrosine and phenylalanine, were detected in whole A. muscaria fruiting bodies but did not show significant differences in their levels between caps and stems (P value>0.05 and/or OPLS-DA loading correlation coefficient <0.4). This methodology allowed for the differentiation between the fruiting bodies of A. muscaria from mineral and mineral-organic topsoil. Moreover, the metabolomic approach and multivariate tools enabled to ascribe the basidiomata of fly agaric to the type of topsoil. Obtained results revealed that stems metabolome is more dependent on the topsoil type than caps. The correlation between metabolites and topsoil contents together with its properties exhibited mutual dependences.

Rapid determination of ibotenic acid and muscimol in human urine

Amanita muscaria and Amanita pantherina belong to a group of hallucinogenic mushrooms that contain two psychoactive substances: ibotenic acid (IBO) and muscimol (MUS). These mushrooms have been known for many years and have been used by Siberian tribes in ethnic rituals, where the urine of shaman or deer that consumed and digested the mushrooms was drunk to experience hallucinogenic stimulation. Currently, there are two ways to induce mushroom intoxication: accidentally, by mistaking hallucinogenic mushrooms for eatable fungi, or deliberately, by ingesting mushrooms of the Amanita family for recreation purposes, which has become increasingly popular. The recreational use of hallucinogenic mushrooms has become popular among young people because of easy access to the psychedelic material, which is legally accessible. Mushrooms can be gathered from the forest or purchased on the Internet. Both substances, MUS and IBO, are responsible for ‘mindaltering’ effects, and IBO may be converted to MUS in the human body. These two substances have been found in urine 1 h after consumption. Their properties include the ability to structurally mimic two endogenous neurotransmitters, glutamic acid-IBO, and gamma-aminobutyric acid MUS. A portion of a hallucinogenic compound used for recreation purpose can be rapidly absorbed from the gastrointestinal tract and is excreted through urine. A single active dose of hallucinogens is approximately 6 mg of muscimol (MUS) and 20-60 mg of ibotenic acid (IBO), and the greatest effects occur over 2–3 h after ingestion. After uptake, the concentration of both psychoactive substances is relatively high in urine, which is extremely important for noninvasive and easy collection of diagnostic material. Although Amanita mushrooms are well known for their psychoactive toxic substances, there is no commercial test for either IBO or MUS. Few papers in the literature have described the determination of these compounds in body fluids and mushroom fruiting bodies. In this study, we report for the first time a method for the detection and quantification of MUS and IBO acid in human urine using H NMR spectroscopy. The method was performed by spiking solutions and applying a simple H NMR pulse sequence (1D-NOESY experiment), typically utilized in metabolomics experiments. This method enables the detection of IBO or MUS, as well as the presence of other metabolites or drugs.

Determination of muscimol and ibotenic acid in mushrooms of Amanitaceae by capillary electrophoresis

In this study, the CZE method for rapid quantitative and qualitative determination of ibotenic acid and muscimol in Amanita mushrooms naturally grown in Poland was developed. The investigations included the species of A. muscaria, A. pantherina, and A. citrina, collected in southern region of Poland. The studied hallucinogenic compounds were effectively extracted with a mixture of methanol and 1 mM sodium phosphate buffer at pH 3 (1:1 v/v) using ultrasound‐assisted procedure. The obtained extracts were separated and determined by CZE utilizing a 25 mM sodium phosphate running buffer adjusted to pH 3 with 5% content of acetonitrile v/v. The calibration curves for both analytes were linear in the range of 2.5–7000 μg/mL. The intraday and interday variations of quantitative data were 1.0 and 2.5% RSD, respectively. The recovery values of analyzed compounds were over 87%. The identities of ibotenic acid and muscimol were confirmed by UV spectra, migration time, and measurements after addition of external standard.

An Adventive Panaeolus antillarum in Poland (Basidiomycota, Agaricales) with Notes on Its Taxonomy, Geographical Distribution, and Ecology

Abstract Coprophilous fungus, Panaeolus antillarum rarely recorded in Europe, is reported here for the first time from the Augustów Plane, north-eastern Poland. This thermophilic species was found outdoors in August on horse dung mixed with straw. A chemical analysis did not confirm the presence of the psychoactive alkaloids in collected material. A complete description and illustration of the species based on Polish specimens are presented and notes on its taxonomy, ecology, world distribution and comparison with similar taxa — P. semiovatus var. semiovatus, P. semiovatus var. phalaenarum, and others are also provided.

Research on acute toxicity and the behavioral effects of methanolic extract from psilocybin mushrooms and psilocin in mice.

The pharmacological activities and acute toxicity of the psilocin (PC) and dried residues of the crude extracts of psychotropic mushrooms were investigated in mice. The hallucinogenic substances were effectively isolated, by using methanol, from the species of Psilocybe semilanceata and Pholiotina cyanopus, that were collected in the north-east region of Poland. The chemical analysis of these extracts, which was performed by liquid chromatography with mass spectrometry detection (LC-MS), indicated the presence of psilocin and other hallucinogenic substances, including indolealkylamines and their phosphorylated analogues. When the pure psilocin or fungal extracts were used, slight differences in determined LD50 values were observed. However, the application of PC evoked the highest level of toxicity (293.07 mg/kg) compared to the activity of extracts from Ph. cyanopus and P. semilanceata, where the level of LD50 was 316.87 mg/kg and 324.37 mg/kg, respectively. Furthermore, the behavioral test, which considered the head-twitching response (HTR), was used to assess the effects of the studied psychotropic factors on the serotonergic system. Both, the fungal extracts and psilocin evoked characteristic serotoninergic effects depending on the dose administered to mice, acting as an agonist/partial agonist on the serotonergic system. A dose of 200 mg/kg 5-hydroxytryptophan (5-HTP) induced spontaneous head-twitching in mice (100% effect), as a result of the formation of 5-hydroxytryptamine (5-HT) in the brain. Compared to the activity of 5-HTP, the intraperitoneal administration of 1mg/kg of psilocin or hallucinogenic extracts of studied mushrooms (Ph. cyanopus and P. semilanceata) reduced the number of head-twitch responses of about 46% and 30%, respectively. In contrast, the administration of PC exhibited a reduction of about 60% in HTR numbers.

Phosphorus Profile of Basidiomycetes

GRAPHICAL ABSTRACT Abstract Phosphorus profiles of methanolic extracts of dried basidocarps of edible and toxic mushrooms collected in Poland have been determined. Despite of the presence of standard phosphorus compounds majority of the extracts revealed the presence of phosphonates. Also, the presence of polyphospates was determined in selected cases.

Pholiotina cyanopus, a rare fungus producing psychoactive tryptamines

Abstract Pholiotina cyanopus was collected from wood chips and other woody remnants of undetermined tree species. Its basidiomata were found in June within the area of closed sawmill in the central part of Żywiec city (SW Poland). Description and illustration of Ph. cyanopus based on Polish specimens are provided and its ecology, general distribution and comparison with similar taxa – Pholiotina smithii, Pholiotina sulcatipes, and others are discussed as well. The identity of the active compounds of Ph. cyanopus was additionally determined. Liquid chromatography–mass spectrometry (LC-MS) data sets were obtained to support the occurrence of psilocybin and its analogues – psilocin, baeocystin, norbaeocystin, and aeruginascin in air-dried basidiomata of the species. The content of psilocybin was found to be high (0.90±0.08% of dry weight), besides, analysed samples contained lower concentrations of psilocin (0.17±0.01%), and baeocystin (0.16±0.01%). Additionally, the chemical analysis revealed small amounts of norbaeocystin (0.053±0.004%) and aeruginascin (0.011±0.0007%) for the first time in the species.