Article

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

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Abstract

The capillary zone electrophoretic (CZE) method for rapid quantitative and qualitative determination of ibotenic acid (IBO) and muscimol (MUS) in Amanita mushrooms naturally grown in Poland was developed. The investigations included the species of Amanita muscaria, Amanita pantherina and Amanita citrina, collected in southern region of Poland. The studied hallucinogenic compounds were effectively extracted with mixture of methanol and 1 mM sodium phosphate buffer at pH 3 (1:1v/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(-1) . The intra-day and inter-day variations of quantitative data were 1.0% and 2.5% RSD, respectively. The recovery values of analyzed compounds were over 87%. The identify of ibotenic acid and muscimol were confirmed by UV spectra, migration time and measurements after addition of external standard. This article is protected by copyright. All rights reserved.

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... As with muscarine, no data for humans have yet been published. A sporophore of Amanita muscaria can contain between 292 and 6570 µg/g of ibotenic acid and between 73 and 2440 µg/g of muscimol [187]. Given the average weight of 60 g and the minimal dose to produce psychotropic effects of 30-60 mg of ibotenic acid and around 6-10 mg of muscimol, a single mushroom is enough to experience hallucinogenic effects [185]. ...
... A sporophore of Amanita muscaria can contain between 292 and 6570 µg/g of ibotenic acid and between 73 and 2440 µg/g of muscimol [187]. Given the average weight of 60 g and the minimal dose to produce psychotropic effects of 30-60 mg of ibotenic acid and around 6-10 mg of muscimol, a single mushroom is enough to experience hallucinogenic effects [185]. ...
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Article
Several families of higher fungi contain mycotoxins that cause serious or even fatal poisoning when consumed by humans. The aim of this review is to inventory, from an analytical point of view, poisoning cases linked with certain significantly toxic mycotoxins: orellanine, α- and β-amanitin, muscarine, ibotenic acid and muscimol, and gyromitrin. Clinicians are calling for the cases to be documented by toxicological analysis. This document is therefore a review of poisoning cases involving these mycotoxins reported in the literature and carries out an inventory of the analytical techniques available for their identification and quantification. It seems indeed that these poisonings are only rarely documented by toxicological analysis, due mainly to a lack of analytical methods in biological matrices. There are many reasons for this issue: the numerous varieties of mushroom involved, mycotoxins with different chemical structures, a lack of knowledge about distribution and metabolism. To sum up, we are faced with (i) obstacles to the documentation and interpretation of fatal (or non-fatal) poisoning cases and (ii) a real need for analytical methods of identifying and quantifying these mycotoxins (and their metabolites) in biological matrices.
... Muscimol is a heterocyclic neurotoxin [1] containing an isoxazole ring. It is produced by mushrooms of the genus Amanita and is a degradation product of ibotenic acid [2]. It was isolated in the 1960s by Japanese scientists [3,4], however, its psychoactive properties were well known long before and attracted great interest. ...
Article
The presented study focuses on the interaction of the well-known neurotoxin muscimol with water. Two approaches for the water solvent are applied - the explicit and the implicit. The muscimol-water clusters were obtained by the molecular dynamics simulations and the first solvation shell was kept for further studies. Implicit water was mimicked via the polarized continuum model (PCM). All three tautomeric forms of the free muscimol molecule are considered in the calculations. The combined theoretical and experimental vibrational IR and Raman studies determined the stability of the prevailing zwitterion form in water. We proved that water molecules in the first solvation shell are crucial for the correct prediction of structural and spectroscopic parameters of muscimol due to its ability to form strong hydrogen bonds. We believe that our findings will shed some light on the binding preferences of muscimol with the γ-aminobutyric acid (GABA) receptor.
... Mushrooms in the genus Amanita are the most well-known psychoactive and poisonous basidiomycete fungi [1]. The Amanita genus consists of cyclopeptide-containing mushrooms, which are responsible for over 90% of all fatal mushroom poisoning instances [2]. ...
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Article
Amanita hemibapha subsp. javanica (Amanitaceae) is an edible Korean wild mushroom. A. hemibapha subsp. javanica is often confused with A. subjunquillea, known as the East Asian death cap, which is potentially fatal when ingested. This study aimed to conduct the first chemical investigation of A. hemibapha subsp. javanica, which resulted in the isolation of seven fatty acid derivatives (1–7) and three steroids (8–10) from the MeOH extract of its fruiting bodies, and their structures were determined by comparing their NMR spectroscopic data with those previously reported, along with the data from LC/MS. Compound 1 was reported previously without the identification of its absolute configuration; its structure, including the absolute configuration was confirmed for the first time, in this study, by using 1H NMR and its fragmentation patterns in MS/MS data, and LC/MS analysis. A recently developed method using competing enantioselective acylation (CEA) coupled with LC/MS analysis was applied for determining the absolute configuration of compound 1, which revealed the 11S-configuration. In the anti-Helicobacter pylori activity test, compound 3 showed antibacterial activity against H. pylori strain 51 with 38.0% inhibition, comparable to that of quercetin (34.4% inhibition) as a positive control. Specifically, compound 4 displayed the most potent antibacterial activity against H. pylori strain 51 with 80.5% inhibition at the final concentration of 100 μm with a MIC50 value of 72 μm. These findings suggested that the active compound 4 is a natural antibiotic that may be used in the development of novel antibiotics against H. pylori. In addition, the first chemical investigation of A. hemibapha subsp. javanica revealed that this mushroom can serve as a promising natural source for the bioactive natural products.
... Several studies have sought to identify and quantify various compounds present in this fungus such as muscarine, bufotenin, muscimol (Figure 1), ibotenic acid [5], muscazone, amatoxins, and phallotoxins [6] by use of analytical techniques including paper chromatography [7], zone capillary electrophoresis, HPLC, and GC/MS [8]. ...
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Article
A simple RP-HPLC method was designed for the quantification of muscimol (5-(aminomethyl)-isoxazol-3-ol) present in five aqueous extracts of Amanita muscaria each from a different developmental stage. Results show that the maximum concentration of muscimol (1,210 mg/ml) was found in the young mushroom stage of development. Moreover, it was also found that this concentration progressively decreases as the fungus ages. The developed method is a simple but effective method for the quantification of muscimol, a widely important metabolite for the pharmaceutical industry as a possible treatment for tardive dyskinesia and Parkinson’s disease. 1. Introduction Amanita muscaria, commonly known as “fly agaric” or “fly amanita”, is one of the best known psychoactive mushrooms in the world due to its psychotropic properties [1]. This fungus is distinguished by a bright red cap featuring small white warty spots, and it is generally found in Europe, Africa, Asia, and the Americas. In Colombia, it was introduced as a symbiont with pine and eucalyptus trees [2]. The main psychoactive constituents of this basidiomycete are neurotoxins ibotenic acid (IBO) and muscimol (MUS), both of which are of interest due to their hallucinogenic and pharmacological properties [3]. Currently, medical research has focused on the use of MUS as GABAA receptor agonists as a possible treatment for tardive dyskinesia and Parkinson’s disease [4]. Several studies have sought to identify and quantify various compounds present in this fungus such as muscarine, bufotenin, muscimol (Figure 1), ibotenic acid [5], muscazone, amatoxins, and phallotoxins [6] by use of analytical techniques including paper chromatography [7], zone capillary electrophoresis, HPLC, and GC/MS [8].
... Hydrophilic interaction column was successfully applied for separation of IBA and MUS when measured by liquid chromatography-triple quadrupole mass spectrometry (LC-MS/ MS) [9,10]. Capillary electrophoresis was reported as another separation technique with high retention capacity for IBA and MUS [11,12]. ...
Article
Ibotenic acid (IBA) is an amino acid and muscimol (MUS) is the decarboxyl derivative of IBA. They are mushroom neurotoxins with high polarity and low molecular weight. Only one transition (159->113 for IBA and 115 -> 98 for MUS) can be found when directly measured by high performance liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS). Therefore, the identification and quantification of trace amount of the toxins in biomaterial are difficult. A highly sensitive and accurate analytical method for IBA and MUS in plasma was developed by LC-MS/MS with the application of bimolecular dansylation and internal standard calibration. Acetonitrile was used for protein precipitation and for toxin extraction from plasma. The toxins and internal standards (L-tyrosine-¹³C9,¹⁵N for IBA and tyramine-d4 for MUS) were derivatized with dansyl chloride (DNSCl). The reaction conditions of the bimolecular dansylation were optimized and the fragmentation pathways of the derivatives in MS/MS were studied. Method validation was carried out according to the Bioanalytical Method Validation Guidance for Industry (FDA, USA, 2018).The limits of detection for IBA and MUS in plasma were 0.3 ng mL⁻¹ and 0.1 ng mL⁻¹, respectively. The linear ranges in plasma were 1 – 500 ng mL⁻¹ and 1 – 200 ng mL⁻¹ with the correlation coefficients of 0.998 and 0.999 for IBA and MUS, respectively. The recoveries at three spiked levels were 90.7% – 111.4% with relative standard deviations (RSDs) of 6.4% – 10.3% for IBA and the results were 85.1% – 94.2% with RSDs of 5.0% – 8.9% for MUS. The toxin levels in patients’ plasma samples under different poisoning degree were presented.
... At present, only a few analytical procedures exist, including the LC-MS/MS [10], GC-MS [11], or CE-MS [12] techniques, which were developed to determine muscimol and ibotenic acid in serum or urine samples. Generally, the available literature focuses upon the analysis of mushroom extracts in which the content of analyzed analytes is significantly higher [13][14][15][16]. A few more papers describe the methods of psilocybin and psilocin analysis in physiological fluids, the most popular of which are those using HPLC with a mass spectrometry detector (MS) [17][18][19][20][21][22][23] and photodiode array detector (PDA) [24], or using electrochemical detection [9,25]. ...
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Article
The fully automated system of single drop microextraction coupled with capillary electrophoresis (SDME-CE) was developed for in-line preconcentration and determination of muscimol (MUS) and psilocin (PSC) from urine samples. Those two analytes are characteristic active metabolites of Amanita and Psilocybe mushrooms, evoking visual and auditory hallucinations. Study analytes were selectively extracted from the donor phase (urine samples, pH 4) into the organic phase (a drop of octanol layer), and re-extracted to the acidic acceptor (background electrolyte, BGE), consisting of 25 mM phosphate buffer (pH 3). The optimized conditions for the extraction procedure of a 200 µL urine sample allowed us to obtain more than a 170-fold enrichment effect. The calibration curves were linear in the range of 0.05–50 mg L−1, with the correlation coefficients from 0.9911 to 0.9992. The limit of detections was determined by spiking blank urine samples with appropriate standards, i.e., 0.004 mg L−1 for PSC and 0.016 mg L−1 for MUS, respectively. The limits of quantification varied from 0.014 mg L−1 for PSC and 0.045 mg L−1 for MUS. The developed method practically eliminated the sample clean-up step, which was limited only to simple dilution (1:1, v/v) and pH adjustment.
... Efforts to tritiate muscimol were prompted by its pronounced GABA agonist activity [2] but success proved frustratingly elusive for a number of years. However, it was noted in the literature that attempts to dry A. muscaria could result in the decarboxylation of ibotenic acid to afford muscimol [3]. Intriguingly, this curious ibotenic acid decarboxylation pathway allowed colleagues of ours to exploit it in the first and only reported synthesis of [ 3 H] muscimol (3) at high specific activity [4]. ...
Article
Muscimol is a small but structurally unique isoxazole alkaloid isolated from the mushroom Amanita muscaria. Because of its potent gamma aminobutyric acid (GABA) agonist activity, muscimol was the elusive target of tritiation attempts for many years. Our laboratory was the first to solve this radiolabelling challenge by a robust and straightforward high specific activity synthesis of [³H] muscimol involving treatment of related amino acid ibotenic acid with tritiated water in dimethyl sulfoxide at ambient temperature. The surprising simplicity of this method prompts the question of exactly how, when and where tritium is installed in the muscimol framework. Using tritium NMR and mass spectrometry analysis of [³H] muscimol, we now propose a likely answer and sequence of events to explain this mechanistic question.
... The species diversity and fungi were also reported, Ryvarden and Johansen [5] examined on fungal system of Eastern Africa; however, there have been only three species belonging to Amanita focusing on saprotrophic fungi living on wood in this study. Regarding macro-fungi, there are some families of poisonous fungi, mainly located in the region of South America and the effects of physical factors on Amanitaceae family and isolated the active compounds of Amanitaceae [6,7] Vietnam is located in tropical and subtropical country; therefore, fungi are much diversified. However, very few works have been conducted on the diversity of fungi. ...
... The species diversity and fungi were also reported, Ryvarden and Johansen [5] examined on fungal system of Eastern Africa; however, there have been only three species belonging to Amanita focusing on saprotrophic fungi living on wood in this study. Regarding macro-fungi, there are some families of poisonous fungi, mainly located in the region of South America and the effects of physical factors on Amanitaceae family and isolated the active compounds of Amanitaceae [6,7] Vietnam is located in tropical and subtropical country; therefore, fungi are much diversified. However, very few works have been conducted on the diversity of fungi. ...
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Preprint
The genus Amanita is one of the genera which is diverse in shapes, colors, species and biological characteristics. The species are valuable in medicine and nutritious for human health. However, there are some species belonging to this genus are toxic, especially the species belonging to Amanita Dill. Ex Boehm. The investigation of the species was carried out in Chu Yang Sin national park. The results showed that 15 species of Amanita Dill. Ex Boehm were recorded: (1) Amanita abrupta; (2) Amanita amanitoides; (3) Amanita caesareoides; (4) Amanita caesarea; (5) Amanita cokeri ; (6) Amanita concentrica; (7) Amanita flavoconia; (8) Amanita levistriata; (9) Amanita multisquamosa; (10) Amanita pantherina; (11) Amanita phalloides; (12) Amanita pilosella, (13) Amanita solitaria; (14) Amanita subcokeri; (15) Amanit vaginata .Within 15 species were identified, eight species were newly added to the list of predominant fungi in the Central Highlands of Vietnam included: Amanita abrupta, Amanita amanitoides, Amanita concentrica, Amanita flavoconia, Amanita levistriata, Amanita multisquamosa, Amanita pilosella, Amanita solitaria. Most of the collected Amanita species showed bright colors with a base or fungal rings. They live in areas with high moisture (>85%), at altitude from 800-1200 m above sea level, annually occur from June to November and are saprotrophic on soil, under tree shades, especially coniferous, semi-evergreen trees and on greensward or shrubs.
... Our previous study concerning determination of GHB abuse in saliva has proven the applicability of a CE-coupled conductivity detector for easy and reliable differentiation between the exogenous and endogenous concentration levels of GHB in saliva samples. 14 IBO and PY have also been studied in mushrooms by CE 13,15 and in biological samples with other methods such as NMR 13 and HPLC. 16 All above-mentioned substances have an acidic group with low pK a values (Fig. 1). ...
Article
The aim of the study was to develop a methodology for the determination of γ-hydroxybutyric acid (GHB), ibotenic acid (IBO) and psilocybin (PY) abuse in human saliva. Capillary electrophoresis with a capacitively coupled contactless conductivity detector (CE-C4D) was used with an optimized background electrolyte (BGE) consisting of 17.9 mM L-arginine (Arg), 9.6 mM succinic acid (Suc) and 0.0019% (w/v) hexadimethrine bromide (HDMB) with an adjusted pH of 7.3. Saliva samples were spiked with all three analytes of interest as well as the internal standard, before protein precipitation with acetonitrile (ACN) (1:2, v/v, saliva:ACN). The limits of detection (LOD) and quantification (LOQ) in the saliva were 1.5 and 5.0 mg/L for GHB, 2.1 and 7.9 mg/L for IBO and 3.6 and 12.0 mg/L for PY. The intraday precision varied from 0.7% to 8.6% and interday precision from 3.9% to 11.4% for all substances. The overall accuracy and combined recovery were from 95% to 123% and 98% to 103%, respectively. The present study demonstrates that the use of oral fluid as an alternative sample matrix which is easy and fast to collect and prepare, and in combination with a portable CE instrument built in-house, provides rapid and efficient determination of psychoactive substance intoxication.
... (TLC)[15], fluorometry[16], cell bioassay[17], flow cytometry[18], neurophysiological methods[19], capillary electrophoresis[20,21], hemolysis[22], electrochemical methods[23], electrophoretic method[24], and ELISA[25,26]. An ELISA test kit for detecting saxitoxin in shellfish is also commercially available (RIDASCREEN® Saxitoxin and RIDASCREEN®FAST Saxitoxin; r-Biopharm AG). ...
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Highlights ? We describe the applicability of our previously developed novel enzyme-luminescence method for rapid and sensitive detection of natural neurotoxins (e.g., shellfish and mushroom toxins) using brain model cells (C6 glioma cells) in vitro. ? A racemic mixtures of the gonyautoxins (GTX), including GTX2,3 and GTX1,4 were used for evaluating the inhibition effects of these toxins on glutamate release from the C6 glioma cells. The potency was compared based on IC50 values. ? The activation effect of ibotenic acid (a mushroom toxin) on glutamate release from C6 cells was also evaluated. The potency was compared based on EC50 values. ? We also tested the applicability of our system for real-time detection of glutamate release from primary rat cortical neurons instead of model cells. ? This novel detection technique may be also applicable in determining neuronal differentiation ratio as well finding glutamatergic neurons without immunostaining in situ. ? This sensing tool may also has a great potential for the investigation of the effects of various growth factors and chemicals on neuronal differentiation, neurotransmitter dynamics, neurodegeneration, and synaptogenesis.
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Mushroom poisoning is a deeply concerned food safety problem that affects the public in China every year. Although there are statistics on the number of poisonings and incidents, there is a lack of data on the types of toxic mushrooms, clinical manifestations and toxins. A case of wild mushroom poisoning occurred in Xiamen. Descriptive epidemiological investigation, toxins detection, and morphological and phylogenetic identification were immediately performed. The patients exhibited typical neurotoxic symptoms after consuming wild mushrooms, including chills, vertigo, drowsiness, salivation and coma. The average incubation period was 30 min. Treatments were adopted, including fluid infusion, gastric lavage, catharsis, and liver protection treatment. All patients recovered within 10 days. The species was identified as Amanita pseudosychnopyramis, and its contents of muscarine, muscimol and ibotenic acid were 170.3 ± 5.9 mg/kg, 835.4 ± 43.1 mg/kg and 637.9 ± 54.8 mg/kg in dry weight, respectively, as detected by ultrahigh-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS). To our knowledge, this is the first report of Amanita pseudosychnopyramis poisoning worldwide.
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Four metabolites, hispidin, bisnoryangonin, muscimole, and ibotenic acid, from potentially psychoactive mushrooms were analyzed by GLC--mass spectrometry as their trimethylsilyl derivatives. This method was applied to the first two compounds in Gymnopilus punctifolius (Peck) Singer and to the last two compounds in Amania pantherina (Fr.) Secr.
Article
Forty-seven specimens representing 35 species or varieties of Amanita were examined for the presence of ibotenic acid, muscimol, stizolobic acid, stizolobinic acid, aminohexadienoic acid and chlorocrotylglycine. In addition, crude extracts of A. muscaria were examined chromatographically for the presence of methyltetrahydrocarboline carboxylic acid (MTC). Ibotenic acid and muscimol were found in clearly detectable concentrations in extracts of A. cothurnata, all specimens of A. muscaria, all specimens of A. pantherina and in lower concentrations in A. gemmata. Stizolobic acid and stizobinic acid were found in detectable concentrations in one variety of A. muscaria, in all specimens of A. pantherina and in trace levels after additional purification of the extracts in A. gemmata and in the remaining specimens of A. muscaria. Aminohexadienoic acid and chlorocrotylglycine were detected only in crude extracts of A. smithiana. MTC could not be detected in crude extracts of A. muscaria. Crystalline ibotenic acid (820 mg) was isolated from 17 kg of a specimen of A. pantherina collected in western Washington State.
Article
The complex pharmacological profile (excitation/inhibition) of ibotenic acid on single neurons in the mammalian CNS prompted studies on the stability of ibotenic acid and a number of structurally related excitatory amino acids under different in vitro conditions in the presence or absence of enzymes. Ibotenic acid, (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-7-carboxylic acid (7-HPCA), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and (RS)-alpha-amino-3-hydroxy-4-bromo-5-isoxazolepropionic acid (4-Br-homoibotenic acid) were all inhibitors of (S)-glutamic acid decarboxylase (GAD) in mouse brain homogenates, but only ibotenic acid was shown to undergo decarboxylation during incubation with brain homogenates. The formation of the decarboxylated product, muscimol, which primarily occurred in a synaptosomal fraction, was dependent on the presence of pyridoxal-5-phosphate (PALP) and was inhibited by (S)-glutamic acid, 3-mercaptopropionic acid (3MPA), aminooxyacetic acid (AOAA), and allyglycine, suggesting that ibotenic acid is a substrate for GAD. The overall decomposition rate for ibotenic acid (8.7 nmol min-1 mg-1 of protein), which apparently embraces other reactions in addition to decarboxylation to muscimol, was higher than the rate of decarboxylation of (S)-glutamic acid (3.2 nmol min-1 mg-1 of protein). At pH 7.4 and 37 degrees C, but in the absence of enzymes, none of the excitatory amino acids under study underwent any detectable decomposition, whereas ibotenic acid and 7-HPCA, but not AMPA and 4-Br-homoibotenic acid, decomposed, partially by decarboxylation, at 100 degrees C in a pH-dependent manner. In the presence of liver homogenates, ibotenic acid was also shown to decompose.(ABSTRACT TRUNCATED AT 250 WORDS)
A capillary zone electrophoretic (CZE) method was developed for the rapid determination of psilocybin in Psilocybe semilanceata. Following a simple two step extraction with 3.0+2.0 ml methanol, the hallucinogenic compound was effectively separated from matrix components by CZE utilizing a 10 mM borate-phosphate running buffer adjusted to pH 11.5. The identity of psilocybin was confirmed by migration time information and by UV spectra, while quantitation was accomplished utilizing barbital as internal standard. The calibration curve for psilocybin was linear within 0.01-1 mg/ml, while intra-day and inter-day variations of quantitative data were 0.5 and 2.5% R.S.D., respectively. In addition to psilocybin, the method was also suitable for the determination of the structurally related compound baeocystin.
Article
Central pontine myelinolysis (CPM) is a demyelinating disease of the pons often associated with demyelination of other areas of the central nervous system (CNS). The term 'osmotic demyelinization syndrome' is used for pontine and extrapontine myelinolysis. In this paper, we are concerned with CPM although the extrapontine one is based on the same pathogenesis. Both share the diagnostic methods, and their prevention and therapy are the same. The etiology and pathogenesis of this disorder are unclear and will be discussed. However, almost all cases of CPM are related to severe diseases. Chronic alcoholism is still the most common underlying condition of CPM patients. In the literature, 174 cases of CPM have been reported in alcoholics since 1986, which is equivalent to an incidence of 39.4%. Likewise, 95 cases of CPM following the correction of hyponatremia have been documented since 1986 (21.5%). The role of hyponatremia and its correction will be outlined in the discussion of the pathogenesis of CPM. The third largest group of CPM cases are liver transplant patients (17.4%), with the development of CPM being attributed to the immunosuppressive agent cyclosporine in particular. Depending on the involvement of other CNS structures, the clinical picture can vary considerably. The large-scale introduction of magnetic resonance imaging has increasingly facilitated the antemortem diagnosis of CPM, although the radiological findings lag behind and do not necessarily correlate with the clinical picture. As yet, there is no specific therapy of choice. A number of therapeutic approaches have been tested and although they have not been compared with regard to their rate of success, they have all led to a substantial improvement in the prognosis of CPM.
Article
The review lists natural sources,i.e. strains and species of fungi producing predominantly psychoactive tryptamines (indolealkylamines), their chemical structure and properties, toxic effects on the man and psychic symptoms of intoxication. It describes the biosynthesis and production of some tryptamines by the mycelial culture ofPsilocybe bohemica Šebek, a survey of methods for their analysis and isolation. It evaluates the worldwide use and abuse of psychoactive fungi as sources of drugs in general and in the Czechia in particular during the last two and a half decades.
Article
The fly agaric is a remarkable mushroom in many respects; these are its bearing, history, chemical components and the poisoning that it provokes when consumed. The 'pantherina' poisoning syndrome is characterized by central nervous system dysfunction. The main species responsible are Amanita muscaria and A. pantherina (Amanitaceae); however, some other species of the genus have been suspected for similar actions. Ibotenic acid and muscimol are the active components, and probably, some other substances detected in the latter species participate in the psychotropic effects. The use of the mushroom started in ancient times and is connected with mysticism. Current knowledge on the chemistry, toxicology, and biology relating to this mushroom is reviewed, together with distinctive features concerning this unique species.
Article
It is usually believed that drugs of abuse are smuggled into the United States or are clandestinely produced for illicit distribution. Less well known is that many hallucinogens and dissociative agents can be obtained from plants and fungi growing wild or in gardens. Some of these botanical sources can be located throughout the United States; others have a more narrow distribution. This article reviews plants containing N,N-dimethyltryptamine, reversible type A monoamine oxidase inhibitors (MAOI), lysergic acid amide, the anticholinergic drugs atropine and scopolamine, or the diterpene salvinorin-A (Salvia divinorum). Also reviewed are mescaline-containing cacti, psilocybin/psilocin-containing mushrooms, and the Amanita muscaria and Amanita pantherina mushrooms that contain muscimol and ibotenic acid. Dangerous misidentification is most common with the mushrooms, but even a novice forager can quickly learn how to properly identify and prepare for ingestion many of these plants. Moreover, through the ever-expanding dissemination of information via the Internet, this knowledge is being obtained and acted upon by more and more individuals. This general overview includes information on the geographical range, drug content, preparation, intoxication, and the special health risks associated with some of these plants. Information is also offered on the unique issue of when bona fide religions use such plants as sacraments in the United States. In addition to the Native American Church's (NAC) longstanding right to peyote, two religions of Brazilian origin, the Santo Daime and the Uniao do Vegetal (UDV), are seeking legal protection in the United States for their use of sacramental dimethyltryptamine-containing "ayahuasca."
Article
The constituents of seven mushrooms sold as Amanita muscaria or Amanita pantherina (five A. muscaria and two A. pantherina) and four "extracts purported to contain A. muscaria" products that are currently circulated in Japan were determined. All mushroom samples were identified as A. muscaria or A. pantherina by macroscopic and microscopic observation. The dissociative constituents, ibotenic acid (IBO) and muscimol (MUS), were extracted with 70% methanol twice and determined by gas chromatography/mass spectrometry. The IBO (as the hydrate)/MUS contents were in the range of <10-2845ppm/46-1052ppm in the cap of A. muscaria and 188-269ppm/1554-1880ppm in the cap of A. pantherina. In the caps, these compounds had a tendency to be more concentrated in the flesh than in the cuticle. On the other hand, the IBO/MUS contents in the stem were far lower than in the caps. In the "extracts purported to contain A. muscaria" products, IBO/MUS were detected below the lower limit of calibration curve (<10ppm/<25ppm) or not detected. However, these samples contained other psychoactive compounds, such as psychoactive tryptamines (5-methoxy-N,N-diisopropyltryptamine and 5-methoxy-N,N-dimethyltryptamine), reversible monoamine oxidase inhibitors (harmine and harmaline) and tropane alkaloids (atropine and scopolamine), which were not quantified. This is the first report of the chemical analysis of Amanita mushrooms that are circulated in the drug market.
Article
Amanita muscaria has a bright red or orange cap covered with small white plaques. It contains the isoxazole derivatives ibotenic acid, muscimol and muscazone and other toxins such as muscarine. The duration of clinical manifestations after A. muscaria ingestion does not usually exceed 24 hours; we report on a 5-day paranoid psychosis after A. muscaria ingestion. A 48-year-old man, with no previous medical history, gathered and ate mushrooms he presumed to be A. caesarea. Half an hour later he started to vomit and fell asleep. He was found comatose having a seizure-like episode. On admission four hours after ingestion he was comatose, but the remaining physical and neurological examinations were unremarkable. Creatine kinase was 8.33 microkat/l. Other laboratory results and brain CT scan were normal. Toxicology analysis did not find any drugs in his blood or urine. The mycologist identified A. muscaria among the remaining mushrooms. The patient was given activated charcoal. Ten hours after ingestion, he awoke and was completely orientated; 18 hours after ingestion his condition deteriorated again and he became confused and uncooperative. Afterwards paranoid psychosis with visual and auditory hallucinations appeared and persisted for five days. On the sixth day all symptoms of psychosis gradually disappeared. One year later he is not undergoing any therapy and has no symptoms of psychiatric disease. We conclude that paranoid psychosis with visual and auditory hallucinations can appear 18 hours after ingestion of A. muscaria and can last for up to five days.
Article
A reliable analytical method was developed for the quantification and identification of muscimol (MUS) and ibotenic acid (IBO), the toxic constituents of Amanita muscaria and Amanita pantherina. MUS and IBO were extracted from mushrooms by aqueous methanol and derivatized with dansyl chloride (DNS-Cl). After extraction with ethyl acetate and evaporation of the solvent, the residue was ethylated with 1.25 M hydrogen chloride in ethanol. The resulting derivatives were quantified by high-performance liquid chromatography with UV detection and identified by liquid chromatography electrospray ionization tandem mass spectrometry. Calibration curves were linear in the range of 25-2500 ppm for MUS and 40-2500 ppm for IBO, respectively. This method was successfully applied to identify and quantify MUS and IBO in Amanita mushrooms naturally grown and circulated in the drug market.
  • D Michelot
  • L M Melendez-Howell
Michelot, D., Melendez-Howell, L. M., Mycol. Res. 2003, 107, 131–146.
  • K Tsujikawa
  • K Kuwayama
  • H Miyaguchi
  • T Kanamori
  • Y Iwata
  • H Inoue
  • T Yoshida
  • T Kishi
Tsujikawa, K., Kuwayama, K., Miyaguchi, H., Kanamori, T., Iwata, Y., Inoue, H., Yoshida, T., Kishi, T., J. Chromatogr. B 2007, 852, 430–435.
  • M Brvar
  • M Mozina
  • M Bunc
Brvar, M., Mozina, M., Bunc M., Wien. Klin. Wochenschr 2006, 118, 294–297.
  • D B Repke
  • D T Leslie
  • N G Kish
Repke, D. B., Leslie D. T., Kish N. G., J. Pharm. Sci. 1978, 67, 485-487.
  • K Tsujikawa
  • H Mohri
  • K Kuwayama
  • H Miyaguchi
  • Y Iwata
  • A Gohda
  • S Fukushima
  • H Inoue
  • T Kishi
Tsujikawa, K., Mohri, H., Kuwayama, K., Miyaguchi, H., Iwata, Y., Gohda, A., Fukushima, S., Inoue, H., Kishi, T., Forensic Sci. Int. 2006, 164, 172–178.
  • R G Benedict
  • Tyler Jr
  • V E Brady
Benedict, R. G., Tyler Jr., V. E., Brady, L. R., Lloydia 1966, 29, 333–342.
  • S Pedersen-Bjergaard
  • E Sannes
  • K E Rasmussen
  • F Tønnesen
Pedersen-Bjergaard, S., Sannes, E., Rasmussen, K. E., Tønnesen, F., J. Chromatogr. B 1997, 694, 375-381.
  • B Marciniak
  • T Ferenc
  • J Kusowska
  • J Ciecwierz
  • E Kowalczyk
Marciniak, B., Ferenc, T., Kusowska, J., Ciecwierz, J., Kowalczyk, E., Med. Pr. 2010, 61, p. 583–595.
  • T Takemoto
  • T Nakajima
  • T Yokobe
Takemoto, T., Nakajima, T., Yokobe, T., Yakugaku zasshi 1964, 84, 1232-1233.
  • M F M Tavares
Tavares, M. F. M., Forensic Sci. Int. 2007, 173, 130-136.
  • J L Da Costa
  • A Y Wang
  • G A Micke
  • A O Maldaner
  • R L Romano
  • H A Martins-Júnior
  • O N Neto
  • M F M Tavares
da Costa, J. L., Wang, A. Y., Micke, G. A., Maldaner, A. O., Romano, R. L., Martins-Júnior, H. A., Neto, O. N., Tavares, M. F. M., Forensic Sci. Int. 2007, 173, 130-136.