Corresponding author: Magdalena Kondeva-Burdina ( magdalenakondeva@gmail.com ) Academic editor: Plamen Peikov
© 2021 Maria Voynova, Aleksandar Shkondrov, Ilina Krasteva, Magdalena Kondeva-Burdina.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Voynova M, Shkondrov A, Krasteva I, Kondeva-Burdina M (2021) In vitro effects of synthetic muscimol and an extract from Amanita muscaria on human recombinant MAOB enzyme. Pharmacia 68(1): 147-150. https://doi.org/10.3897/pharmacia.68.e60705
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The effects of synthetic muscimol and an extract from Amanita muscaria, containing this compound on the activity of human recombinant MAOB enzyme (hMAOB) were studied. Muscimol had statistically significant inducing effect on hMAOB at concentrations 0.25–5 μM, while A. muscaria extract did not influence the enzyme activity at all.
Amanita muscaria, muscimol, MAOB enzyme
Monoamine oxidase (MAO) catalyses the oxidative deamination of monoamines (neurotransmitters, dietary amines, hormones and drugs) in the brain and peripheral tissues, regulating their levels and thus their biological effects. MAO exists in two isoforms, MAOA and MAOB. MAOA selectively deaminates serotonin, whereas MAOB selectively deaminates phenylethylamine and benzylamine. Dopamine is oxidised by both isoforms (
The main psychoactive component in Amanita muscaria (fam. Amanitaceae) (
The aim was to compare the effects of Amanita muscaria extract and synthetic muscimol on human recombinant MAOB enzyme activity.
A. muscaria caps were collected in May 2018 in Vitosha Mountain and identified by Vladimir Vazharov. A voucher specimen was deposited in his personal collection (
Muscimol CRS (Sigma Aldrich, Germany) was dissolved in 50% MeOH (2 mg/ml). Serial dilutions were made as follows: 1 mg/ml; 0.5 mg/ml; 0.1 mg/ml. An aliquot of each standard solution (10 µl) was injected three times in the HPLC. An aliquot of the tincture (10 µl), filtered through a PVDF filter (0.22 µl) was injected three times for HPLC analysis. Muscimol content was in the tincture 2.94 ± 0.03 mg/ml.
Monoamine oxidase activity assay of recombinant human MAOB (hMAOB) was performed using a fluorometric method by Amplex UltraRed reagent (
The hMAOB was incubated for 2 h with A. muscaria extract (at 5 µg/ml, 1 µg/ml, 0.5 µg/ml, 0.25 µg/ml, 0.2 µg/ml, 0.1 µg/ml, 0.05 µg/ml, 0.025 µg/ml, 0.02 µg/ml, and 0.01 µg/ml muscimol) and Muscimol (at 5 µM, 1 µM, 0.5 µM, 0.25 µM, 0.2 µM, 0.1 µM, 0.05 µM, 0.025 µM, 0.02 µM, 0.01 µM) at 37 °C in the dark.
The MAOB activity was expressed as a normalized percent of the untreated control set as 100% and the results were expressed as mean values and standard deviation (± SD) (Graph Pad Prizm, v. 6). Statistical analysis was performed by one-way analysis of variance (ANOVA) with post hoc multiple comparisons procedure (Dunnet’s test) to assess the statistical differences in case of normal distribution. Values of P < 0.05, P < 0.01 and P < 0.001 were considered statistically significant.
Oxidative stress induced by MAO is a potential risk factor for neuronal loss in aging and age-related neurodegenerative disorders, such as Parkinson’s disease (PD). Indeed, oxidative stress generated by increased MAOB activity is known to damage mitochondrial DNA (
It increased statistically significant the hMAOB activity at the highest concentrations: 0.25 – 5 μM, compared to the control (pure hMAOB). Five μM muscimol increased hMAOB activity about five times; 1 μM – with 169 %; 0.5 μM – with 44 % and 0.25 μM – with 27 %.
This paradox of enzyme induction could be explained with the hMAOB protein conformation change induced by high levels of positively-charging molecule such as muscimol. Single-charged and especially double charged small molecules, incl. muscimol could induce changes in the active center and/or activator-binding site of many enzymes (
The classical MAOB inhibitor – selegiline decreased hMAOB activity with 38%, compared to the control (pure hMAOB enzyme).
The extract from A. muscaria had no effect whatsoever on human recombinant MAOB enzyme (Figure
Only the classical MAOB inhibitor – selegiline decreased hMAOB activity with 38 %, compared to the control (pure hMAOB enzyme).
The discovery of novel classes of selective inhibitors of this enzyme is of considerable interest. Identifying MAOB as a potential pathogenic factor in PD stimulated the development of MAOB inhibitors as disease-modifying agents; selegiline and rasagiline, which showed protective role over neuronal cells in both in vitro and in vivo models (
Moreover, preliminary data show that in vitro at concentration higher than physiological, MAO deaminates GABA (
Our findings confirmed the significance of naturally-derived psychoactive alkaloids such as muscimol as leading molecules for possible treatment of neurodegenerative diseases.