The following study references pertain to the conversion of ibotenic acid into muscimol through the preparation of amanita muscaria toadstools.
Key Findings
Drying the amanita mushrooms (until 'cracker dry') only converts up to 30% of the ibotenic acid that was initially present in the fresh samples into muscimol. This leaves a high concentration of ibotenic acid, typically 180 to 1800 ppm in dried amanita mushroom hats.
Fresh A. muscaria typically contains 258 to 471 ppm of ibotenic acid, and a ibotenic acid to muscimol ratio 9:1 or greater.
Ibotenic acid is associated with gastrointestinal distress, nausea, vomiting, diarrhoea, headaches, profuse sweating, hypersalivation, periods of agitation and confusion.
Muscimol is associated with many health benefits including high antioxidant levels, anti-aging properties, supporting the production of growth hormone, diuresis, neuroprotection, anti-hypertensive properties, and the promotion of healing.
In conclusion contributors to these studies suggest a high conversion of ibotenic acid to muscimol is desirable, demanding further preparation beyond drying.
To convert a majority of ibotenic acid that is present in either raw, or dried amanita toadstools, a lemon tek, long-simmered amanita tea is recommend.
Change in Ibotenic Acid and Muscimol Contents in Amanita muscaria during Drying, Storing or Cooking
1993 [1]
Abstract
It is believed by people in mountainous areas that toxins in A. muscaria are reduced by drying, storing or cooking. The main physiologically active substances, ibotenic acid (IBO) and muscimol (MUS), in A. muscaria were therefore investigated. IBO is readily transformed to into MUS through decarboxylation, and MUS is a hallucinogen. Drying A. muscaria in the sun or with a heater caused an increase of MUS in the mushroom, though a lot of precursor IBO was lost. It was suggested that the toxicity of the mushroom would be intensified by processing. IBO and MUS in the mushroom were stable on storage under dry or salt conditions. MUS increased in concentration while IBO decreased during heat-cooking, and the changes were more marked under acidic than alkaline conditions. However, general cooking (within 10 minutes) hardly reduced the toxic substances. Boiling or soaking A. muscaria in water caused most of the IBO and MUS in the mushroom to be released rapidly into the water.
Method for producing muscimol and/or reducing ibotenic acid from amanita tissue
2013 [2]
Abstract
A method for producing muscimol and or/reducing ibotenic acid from Amanita tissue, and or producing a nutritional supplement therefrom.
Key quotes
"0002 Amanita muscaria, and closely related fungi (i.e., Amanita pantherina, Amanita muscaria variant formosa, and others within the Amanita genus) contain Substances that are GABA analogues and antioxidants. For example, according to at least one study, Amanita species were found to have “the highest antioxidant activities” among mushroom species tested." However, when fresh tissue is ingested, even small amounts can cause symptoms of gastrointestinal distress (nausea, vomiting, diarrhea), headaches, profuse Sweating, hypersalivation, periods of agitation and confusion, followed by coma-like sleep. These negative reactions are generally ascribed to the presence of ibotenic acid within fresh tissue, an excitatory neurotoxin. Although ibotenic acid is a neurotoxin with severe adverse effects at high concentrations, its decarboxylated variant, muscimol, is an analogue of gamma aminobutyric acid (GABA). GABA and GABA analogues have many health benefits, including anti-aging properties, supporting the production of growth hormone, diuresis, neuroprotection, anti-hypertensive properties, and the promotion of healing."
"[0003 Fresh A. muscaria typically contains 258 to 471 ppm of ibotenic acid within the entirety of the fungi. Nearly all the ibotenic acid concentrated in the caps, and very little muscimol present. Typically, the ibotenic acid to muscimol ratio of fungal cap tissue would be 9:1 or greater in fresh samples. While drying of the fungal tissue has been reported to convert a portion of the ibotenic acid to muscimol. Such conversion is incomplete and highly variable according to sample variation and conditions. Indeed, a relatively low conversion rate of only 30% is typical by merely drying fungal tissue, leaving an unacceptably high concentration of ibotenic acid, typically 180 to 1800 ppm. A common ibotenic acid to muscimol ratio would be 3:2 in dried specimens, such that the neurotoxin amounts far exceed the GABA analogue. Furthermore, ingesting the dried tissue, which contains the relatively indigestible mushroom cell wall component chitin, would result in adverse physiological effects"
Excitatory Amino Acids: Studies on the Biochemical and Chemical Stability of Ibotenic Acid and Related Compounds[3]
Abstract
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)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and (RS)-α-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 allylglycine, 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°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°C in a pH-dependent manner. In the presence of liver homogenates, ibotenic acid was also shown to decompose. Although muscimol was the only detectable reaction product, mechanisms other than decarboxylation may be involved. Under these conditions, the degradation reaction or reactions were partially dependent on PALP and were inhibited by AOAA and 3MPA but not by allylglycine. The present in vitro studies indicate that ibotenic acid is likely to undergo enzyme-catalyzed decomposition to give muscimol in brain tissue and after systemic administration to animals. These aspects must be taken into consideration in the interpretation of the pharmacological or neurotoxic effects of ibotenic acid after direct application near central neurons, after local injections into animal brains, or after systemic administration to animals.
Questions and Answers with synthetic organic chemist, Dirk Deggler [4]
Q: “May I ask why do you recommend pH 2.9, when the paper you reference worked with pH 2.7?”
Dirk: “I have repeated the decarboxylation experiment numerous times in my lab following the reaction with HPLC or by GCMS in real time and found that reaction proceeds well between pH 2.5 to 3.0.
Most of my experiments were conducted at 2.9 because trying to get to pH 2.7 adding 1 drop of HCl or H3PO4 would push pH down to 2.5. Nielsen et. al. were reacting a maximum of 5 micro moles per millilitre which is 0.79 milligrams per ml. I was reacting between 20-25 grams of IBO per 50mls water after extraction from [the mushrooms] and concentrating on Buchii rotovap. At much higher concentrations of IBO than what Nielsen et. al. had performed in her experiments, I found that 3 hours was required and this was confirmed by US Patent 20140004084A1”
Q: “And why so long boiling time?”
Dirk: “I was reacting 20-25g IBO in 50ml H2O and pH between 2.5-3.0 and I always follow the reaction in real time with by HPLC or GCMS and found that I required 3 hours to fully convert IBO to MUS. At 100’C the rate of reaction, i.e. reaction kinetics proceeds at a given rate governed by the temperature of the reaction. If I wanted the reaction to proceed faster then a pressurised vessel is required to raised the reaction temperature (ie boiling point of water above 100’C if in a pressure vessel) so the reaction kinetics or rate of reaction proceeds faster. Nielsen et al was reacting 5 micro moles per ml I was reacting 0.16 moles in 50 ml water hence 0.16/50 = 0.0032 moles per ml, covert to micro moles u multiply by 1,000,000 = 3200 micro moles per millilitre US patent 201400004084A1 did their decarboxylation's for 3 hours as well.”