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.

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.

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 ß-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.