ChapterPDF Available

Abstract and Figures

The aim of our chapter is to review recent developments in a group of medicinally important natural products-alkaloids, with reference to the structure-activity studies in respect of certain diseases. Alkaloids covered by our review come from mushrooms called "hallucinogenic." Hallucinogenic compounds have been chemically identified in mushrooms belonging to various genera, e.g., Agrocybe, Amanita, Conocybe, Galerina, Gymnopilus, Hypholoma, Inocybe, Panaeolus, Psilocybe, Pholiotina, Pluteus, and Weraroa [J.W. Allen, Ethnomycol. J. Sacred Mushroom Stud. 9 (2012) 130-175]. One of the largest classes of alkaloids is indole alkaloids. Indoles are probably the most widely distributed heterocyclic compounds in nature having medicinal importance [K.N. Kumar et al., Molecules 18 (2013) 6620-6662]. Two of simple indole alkaloids: psilocin (3-[2 (dimethylamino)ethyl]-4-indolol) and psilocybin ([3-(2-dimethylaminoethyl)-1H-indol-4-yl] dihydrogen phosphate) are present in most psychedelic mushrooms. Psilocin is a serotonin agonist - psilocybin/psilocin caused effects are thought to be mediated mainly by activation of 5-HT2A receptor. Ligands for the 5-HT2A receptor may be extremely useful tools for future cognitive neuroscience research [D.E. Nichols, Pharmacol. Ther. 101 (2004) 131-181]. They are also other analogs of psilocybin: baeocystin, norbaeocystin, bufotenin, and aeruginascin that are found in hallucinogenic mushrooms. Bufotenin occur also in some animal species (genus Bufo) and plants. Some of the mushroom belonging to genera Gymnopilus and Pholiota were shown to possess bisnoryangonin and hispidin, alkaloids with antimicrobial [K. Shinto et al., J. Home Econ. Jpn. 58 (9) (2007) 563-568] and antioxidant [Lee In-K et al., Mycobiology, 36 (1) (2008) 55-59] activity. Also psychoactive species of genus Amanita, contain the alkaloids (muscimol, ibotenic acid, and muscazone) that react with neurotransmitter receptors in the central nervous system. Isoxazoles, to which these alkaloids belong, have been found to inhibit voltage-gated sodium channels to control pain, enable the construction of tetracycline antibiotic derivatives, and as treatments for depression. The information within this review is intended to serve as a reference tool to better enable future research into important and fascinating area of pharmacognostic science as well as other parts of medical science.
Content may be subject to copyright.
Studies in Natural Products Chemistry, Vol. 46.
Copyright © 2015 Elsevier B.V. All rights reserved.
Chapter 5
Bioactive Alkaloids of
Hallucinogenic Mushrooms
Piotr Paweł Wieczorek,1 Danuta Witkowska, Izabela Jasicka-Misiak,
Anna Poliwoda, Milena Oterman and Katarzyna Zielin´ska
Faculty of Chemistry, Opole University, Pl. Kopernika, Poland
1Corresponding author: E-mail:
Alkaloids are defined as a group of naturally occurring chemical compounds that
mostly contain basic nitrogen atoms [1]. Alkaloid molecules are extremely im-
portant for biomedical science. They have a unique property – an ability to work
as either hydrogen-acceptor or hydrogen-donor for hydrogen bonding, depending
on the type of amine functionality present in alkaloids. This property is critically
important for the interaction (binding) between targets (enzymes, proteins, and
receptors) and drugs (ligands) possessing alkaloid scaffold [2]. Certain drugs with
alkaloid structural features were synthesized by naturally occurring alkaloids, to
which belong indole, indolinone, isoindole, isoxazole, imidazole, indazole, thia-
zole, pyrazole, oxazolidinone, oxadiazole, benzazepine, and many others [2].
Chapter Outline
Introduction 133
Chemical Classes of Alkaloids
Found in Mushrooms 134
Analytical Methods Used for
Alkaloids Isolation and Detection 136
Amanita Mushrooms 140
Psilocybe Mushrooms 142
Biological Samples 145
Biosynthesis of Isoxazoles 151
Biosynthesis of Tryptamine
Alkaloids in Mushroom 152
Biological Activity and
Psychopharmacological Effects 154
Indoleamine Hallucinogenic
Pharmacodynamics 154
Metabolism of Indoleamines
Produced by Mushrooms
and their Psychosomatic
Effects 156
Pharmacodynamics of
Isoxazole Hallucinogens
and Muscarine 160
Biomedical Importance of
Mushrooms’ Isoxazoles and
Indoles 161
Conclusions 162
Acknowledgments 163
References 163
134 Studies in Natural Products Chemistry
From a pharmaceutical and industrial point of view, alkaloids are probably
the most important fungal metabolites. Our work concentrates on two groups of
naturally occurring alkaloids in higher fungi (indoles and isoxazoles). In nature,
indoles are probably the most often occurring heterocyclic compounds, having
medicinal importance [3]. Two simple indole alkaloids: psilocin (3-[2 (dimeth-
ylamino) ethyl]-4-indolol) and psilocybin ([3-(2-dimethylaminoethyl)-1H-indol-
4-yl] dihydrogen phosphate) are present in many mushroom species. These mush-
rooms are called hallucinogenic, psychedelic, entheogenic, magic, medicinal,
neurotropic, psychoactive, sacred, or saint mushrooms [4]. Also other analogs of
psilocybin, known as baeocystin, norbaeocystin, bufotenin, and aeruginascin, were
found in hallucinogenic mushrooms. Hallucinogenic compounds were chemically
identified in mushrooms belonging to various genera, e.g., Agrocybe, Conocybe,
Galerina, Gymnopilus, Hypholoma, Inocybe, Panaeolus, Psilocybe, Pholiotina,
Pluteus, and Weraroa [5]. Allen et al. listed 206 fungal species containing trypt-
amine alkaloids; however, some of them are false positives, which were noted
down at the end of that update [5]. In the case of around 90 species mentioned by
Allen, the presence of psilocin or psilocybin was proven by chemical analyzes.
They are also psychoactive species of genus Amanita, which contain the
alkaloids (muscimol, ibotenic acid, muscazone, and muscarine) reacting with
neurotransmitter receptors in the central nervous system. These alkaloids, ex-
cept muscarine, belong to the chemical group of isoxazoles. Isoxazoles often
exhibit extensive and pharmacologically important biological activities. Musca-
rine is the principal alkaloid toxin in the fungi of genera Inocybe and Clitocybe
[6,7]. Although it does not belong to isoxazoles, in our opinion this alkaloid is
important and is therefore described in our chapter.
Our work analyzes the origin, chemistry, biological activity, and the bio-
medical importance of bioactive alkaloids found in hallucinogenic mushrooms.
In the present review, we aimed to cover the most important scientific papers
within this field of science.
There are four hallucinogenic substances in Amanita sp., which can cause
hallucinogenic effect: muscimol, ibotenic acid, muscazone, and muscarine
(Fig. 5.1a–d). All of these substances except muscarine are isoxazoles [8].
They are quickly absorbed through the digestive system [9]. Ibotenic acid
(a-amino-3-hydroxy-5-isoxazoloacetic acid) is a substance soluble in water
[7]. Pure ibotenic acid is colorless and unstable in solution [8]. The melting
point of ibotenic acid is 150–152°C. The largest amount of this substance in
Amanita mushrooms is located in the red cap and yellow tissue under the cap
(Amanita muscaria) [7]. Muscimol (5-(aminomethyl)-3-hydroxyisoxazole) is
more hydrophobic than ibotenic acid, but it is also soluble in cold water [7,8].
Muscimol is colorless and the melting point is 175°C. It is a product of decar-
boxylation of ibotenic acid [7,10]. Both substances ibotenic acid and muscimol
have structures similar to human neurotransmitters: glutamic acid and GABA
Bioactive Alkaloids of Hallucinogenic Mushrooms Chapter | 5 135
[11]. Muscazone (a-amino-2,3-dihydro-2-oxo-5-oxazoloacetic acid) is a lac-
tam isomer of muscimol [7,11]. The melting point of muscazone is 190°C. It
is also a colorless substance. The hallucinogenic properties of muscazone are
weaker than ibotenic acid or muscimol [7]. Muscarine (2,5-anhydro-1,4,6-tride-
oxy-6-(trimethylammonio)-d-ribo-hexitol) is a substance soluble in water. The
melting point of that compound is 180°C [7]. This alkaloid exhibits less hal-
lucinogenic activity than ibotenic acid and muscimol [12]. Muscarine has three
chiral centers and eight configurational isomers [13].
The main alkaloids tryptamine/indolamine derivatives in mushrooms, exhibit-
ing hallucinogenic properties, are psilocybin and psilocin (Fig. 5.2a, b) [14–18].
In addition to the mentioned alkaloids, mushrooms also contain baeocystin,
which is a derivative of the psilocybin and norbaeocystin (Fig. 5.2c, d) [18,19].
It is also known that there are such psychoactive compounds as aeruginascin and
bufotenine (Fig. 5.2 e, f) [18,20,21]. Psilocybin (O-phosphoryl-4-hydroxy-N,N-
dimethyltryptamine) and its main dephosphorylated metabolite psilocin (N,N-
dimethyltryptamine) belong to tryptamine hallucinogens and are structurally
similar to serotonin [15,19]. It is worth emphasizing that psilocybin is one of the
naturally occurring alkaloids, which contain a phosphorus atom in its structure
[16]. Psilocybin and psilocin in its purest of forms are white crystalline powder
[15]. Psilocybin is soluble in water, while psilocin is a more lipid soluble. Both
substances are soluble in methanol and ethanol [15,18]. The melting point of
psilocybin is 185–195°C, and of psilocin, it is 173–176°C [8,18]. Both of this
indole psychedelic compounds are unstable in light; their stability is good in low
temperatures and in the dark [14,15]. Other compounds belonging to alkaloids
with hallucinogenic properties are baeocystin (4-phosphoryloxy-N-methyltrypt-
amine) and norbaeocystin. These compounds are less explored than psilocybin
FIGURE 5.1 Chemical structures of (a) ibotenic acid, (b) muscimol, (c) muscazone, and (d) muscarine.
136 Studies in Natural Products Chemistry
and psilocin [22]. Baeocystin is a mono-methyl analog of psilocybin, showing a
UV spectrum identical to psilocybin, indicating it to be a 4-substituted indole de-
rivative. The melting point of this substance is 254–258°C [23]. Norbaeocystin is
a demethylated equivalent of psilocybin [18]. The melting point of norbaeocystin
is 188–192°C [24]. Both baeocystin and norbaeocystin are tryptophan derivatives
formed through decarboxylation, indole ring hydroxylation at position 4, and N-
methylation through the activity of S-adenosylmethionine and O-phosphorylation
[8]. Bufotenine (N,N-dimethyl-5-hydroxytryptamine) is a positional isomer of
psilocin [21]. Bufotenine has low lipid solubility [25]. The melting point of bufo-
tenine is 146.5°C. The last hallucinogenic compound mentioned in this chapter –
aeruginascin (Fig. 5.2e) – is a trimethyl analog of psilocybin [22]. It is a quater-
nary ammonium compound N,N,N-trimethyl-4-phosphoryloxytryptamine. Aeru-
ginascin is stable at the room temperature and is more polar than psilocybin [20].
It is difficult to analyze mushrooms extract using available chromatographic
techniques because of the complex composition of mushrooms. In most cases,
before carrying out the proper analysis, it is necessary to use appropriate sample
preparation methods for isolation, purification, and concentration of the mush-
rooms hallucinogens analytes and biological samples.
There are many methods used to isolate substances from samples. These
include liquid–liquid extraction (LLE), Soxhlet extraction, solid-phase extrac-
tion (SPE), or supercritical fluid extraction (SFE). The most popular extrac-
tion methods for isolation hallucinogenic substances from mushrooms are
ultrasonic-assisted extraction and SPE (Table 5.1) [10,26–37]. Ultrasounds are
FIGURE 5.2 Chemical structures of (a) psilocybin, (b) psilocin, (c) baeocystin, (d) norbeaocystin,
(e) aeruginascin, and (f) bufotenine.
Bioactive Alkaloids of Hallucinogenic Mushrooms Chapter | 5 137
TABLE 5.1 The Methods of Extraction and Identification of Ibotenic Acid, Muscimol, Psilocin, and Psilocybin in Mushroom
No. Analyte Matrix
LOD Amounts of
alkaloids References
1. Muscimol A. muscaria
A. pantherina
UAE 96–101.1 HPLC–MS 1.4 mg/L Intraassay
41.3–1988 mg/kg
39.4–1987 mg/kg
4.6 mg/L
2. Ibotenic
A. muscaria
A. pantherina
UAE 99.4–100.9 HPLC–MS 7.8 mg/L Intraassay
52.6–1978 mg/kg
60.7–1901 mg/kg
25.9 mg/L
3. Muscimol A. muscaria
A. pantherina
UAE GC–MS – A. muscaria
284–1052 mg/kg (cap)
A. pantherina
1554–1880 mg/kg (cap)
4. Ibotenic
A. muscaria
A. pantherina
UAE GC–MS – A. muscaria
<10–2845 mg/kg (cap)
A. pantherina
188–269 mg/kg (cap)
5. Muscimol A. muscaria UAE HPLC–UV 30 mg/L 0.38 g/kg (cap),
0.08 g/kg (stem)
6. Ibotenic
A. muscaria UAE HPLC–UV 18 mg/L 0.99 g/kg (cap),
0.23 g/kg (stem)
7. Muscimol A. muscaria
A. pantherina
UAE, SPE Intraday: 101
Interday: 101
HPLC–MS 2.4 mg/kg A. muscaria 133 mg/kg [26]
7.9 mg/kg
138 Studies in Natural Products Chemistry
8. Ibotenic
A. muscaria
A. pantherina
UAE, SPE Intraday: 99.8
Interday: 94.8
HPLC–MS 4.9 mg/kg A. muscaria 146 mg/kg [26]
16 mg/kg
9. Muscimol Amanita
SPE >80 HPLC–MS <10 mg/kg 107 mg/kg [30]
10. Ibotenic
SPE >80 HPLC–MS <10 mg/kg 210 mg/kg [30]
11. Psilocybin P. semilanceata UAE 98 CE–UV 0.045 g/kg [28]
0.225 g/kg
12. Psilocin P. cubensis
P. tampanensis
P. cyanescens
UAE 98.8 HPLC–
60 mg/kg 0.12–2.52 g/kg [29]
220 mg/kg
13. Psilocybin P. cubensis
P. tampanensis
P. cyanescens
UAE 97.3 HPLC–
70 mg/kg 0.38–10.48 g/kg [29]
280 mg/kg
14. Psilocin P. cubensis
P. tampanensis
P. cyanescens
UAE 96.8 HPLC–
6.4 mg/kg 6.42–12.67 mg/kg [31]
15. Psilocybin P. cubensis
P. tampanensis
P. cyanescens
UAE 96.8 HPLC–
6.4 mg/kg [31]
16. Psilocybin Psilocybe
UAE HPLC–FL 4.8–20 mg/kg [33]
TABLE 5.1 The Methods of Extraction and Identification of Ibotenic Acid, Muscimol, Psilocin, and Psilocybin in Mushroom
Samples (cont.)
No. Analyte Matrix
LOD Amounts of
alkaloids References
Bioactive Alkaloids of Hallucinogenic Mushrooms Chapter | 5 139
17. Psilocybin UAE 93–98 GC–MS [32]
18. Psilocin P. cubensis
UAE HPLC–UV – [34]
19. Psilocybin P. cubensis
UAE HPLC–UV – [34]
20. Psilocin P. semilanceata
P. cubensis
P. tampanesis
P. cyanescens
UAE HPLC – [36]
21. Psilocybin P. semilanceata
P. cubensis
P. tampanesis
P. cyanescens
UAE HPLC – [36]
22. Psilocin P. subcubensis UAE GC–MS Cap: 0.2
g/kg, stem:
0.2 g/kg
0.2 g/kg (cap),
0.3 g/kg (stem)
23. Psilocybin P. subcubensis UAE GC–MS Cap: 8.6
g/kg, stem:
8.0 g/kg
8.6 g/kg (cap),
8.0 g/kg (stem)
24. Psilocin Mushrooms
LLE GC–MS – [37]
25. Psilocybin Mushrooms
LLE GC–MS – [37]
140 Studies in Natural Products Chemistry
the source of additional energy, which allows separating the analytes from the
sample matrix by destroying mushroom cells. The ultrasonication technique is
faster, inexpensive, and more effective than other traditional methods used for
natural substances isolation. Other extraction methods, which are also used, are
SPE [27,31] and LLE [38]. In later sections, we present brief descriptions of
some works on the isolation and determination of hallucinogenic substances.
The amounts of alkaloids in different mushrooms are shown in Table 5.1 (for
some studies these data are not available).
Amanita Mushrooms
Tsujikawa et al. isolated muscimol and ibotenic acid from A. muscaria and
Amanita pantherina. The dried mushrooms were grounded to a fine powder.
Next, 2 mL of 70% methanol was added to 50 mg of powdered mushrooms.
After shaking (1 min) and ultrasonication (5 min), the sample was centrifuged
(3000 rpm, 3 min), and the supernatant was transferred to another test tube. For
residues, all procedure was repeated in the same way, and 100 mL of combined
extract was evaporated to dryness under the stream of nitrogen. After evapo-
ration, the sample was subjected to dansylation reaction. The dried residue
was dissolved in 100 mL of 25 mM borax solution pH 9.5 and 50 mL of 20 mM
DNS-Cl in acetonitrile. This mixture reacted at room temperature for 90 min.
The reaction was stopped by the addition of 10 mL of ethanoloamine solution
into the aforementioned borax solution. The next step included a conversion of
DNS–IBO to DNS–IBO–Et because DNS–IBO could not be separated from
the matrices. 1 mL of borax solution and 3 mL of ethyl acetate were added to
dansylated solution. After shaking (5 min) and centrifugation (3 min), the ethyl
acetate layer was transferred to another flask. The aqueous layer was extracted
two more times with ethyl acetate (3 mL × 2), and the procedure was conducted
in the same way. The combined ethyl acetate layer (9 mL) was evaporated to
dryness under a stream of nitrogen, and the residue became a derivative with
100 mL of 1.25 M hydrogen chloride in ethanol. The mixture reacted at a tem-
perature of 55°C for 60 min. The reaction was stopped by the evaporation of the
reagent under a stream of nitrogen. The residue was dissolved in 100 mL mix-
ture of ethanol/water (1:1v/v). The sample was filtered before the HPLC analysis.
The obtained recoveries for ibotenic acid and muscimol were 99.4–100.9% and
96.0–101.1%, respectively. The limits of detection were 7.8 mg/L for ibotenic
acid and 1.4 mg/L for muscimol. The limits of quantification were 25.9 mg/L for
ibotenic acid and 4.6 mg/L for muscimol [26].
Tsujikawa et al. also determined hallucinogenic substances in A. panthe-
rina and A. muscaria. The procedure of extraction was conducted in the same
way. After extraction, 200 mL of the sample was evaporated to dryness under a
stream of nitrogen, and the residue was derivatized by a reaction with a mixture
of 50 mL of BSTFA with 10% of TMCS and 50 mL of ethyl acetate containing
20 mg/mL n-pentadecane (IS).
Bioactive Alkaloids of Hallucinogenic Mushrooms Chapter | 5 141
The process of creating a derivative was performed at 80°C for 30 min. Next,
ibotenic acid and muscimol were determined by GC–MS, using a selective ion-
monitoring (SIM) mode [10].
Gennaro et al. extracted the powdered A. muscaria mushroom with ultrapure
water in ultrasonic bath lasting 15 min. After sonification, the supernatant was
transferred to another flask, and the residue was washed twice with ultrapure
water in order to improve the recovery. The combined supernatant was diluted
in ultrapure water and filtered before the HPLC analysis. The limits of detection
were 18 mg/L for ibotenic acid and 30 mg/L for muscimol [28].
Yoshioka et al. extracted ibotenic acid and muscimol, as well as seven other
toxins, from mushrooms as follows: 200 mg of mushrooms was homogenized
in 2.5 mL of 0.5% formic acid in methanol and ultrasonicated for 0.5 and 1 min,
respectively. After centrifugation (1000 rpm, 3 min), the supernatant was trans-
ferred into another flask. The residue was dissolved in 2.5 mL of water, and the
procedure was repeated. The combined extract was adjusted to 5 mL by 50%
aqueous methanol solution. The next step was the SPE. One milliliter of extract
was loaded onto a preconditioned Oasis HLB cartridge. The first 0.5 mL of the
eluate was discarded, and the remaining part of the solution was collected and
diluted 20-fold with 50% aqueous methanol solution. This sample was used
for determination of ibotenic acid and muscimol by the LC–TOF–MS system.
The intraday recoveries were 99.8% for ibotenic acid and 101% for muscimol,
and the interday recoveries were 94.8% for ibotenic acid and 101% for musci-
mol. The limits of detection were 4.9 and 2.4 mg/kg, and the limits of quanti-
fication were 16 and 7.9 mg/kg for ibotenic acid and muscimol [27]. Yoshioka
et al. identified ibotenic acid and muscimol in A. pantherina in concentration
amounts of 146 and 133 mg/g, respectively. The weak point of these results was
that they were based on a single sampling of the mushrooms. Nevertheless, it
is a simple and rapid analytical method, which makes it possible to simultane-
ously identify nine mushroom toxins.
Gonmori et al. also identified ibotenic acid and muscimol in Amanita mush-
rooms using the LC–MS/MS technique [31]. They used acivicin as an inter-
nal standard. One hundred milligrams of mushroom was homogenized with
10 mg of acivicin in 10 mL 50% methanol aqueous solution for 5 min. After
centrifugation (3000 rpm, 10 min), 1 mL of supernatant was mixed with 100 mL
of 0.5% ammonia aqueous solution. This sample was loaded onto Oasis MAX
3 cc (60 mg), which was preconditioned with 1 mL of methanol and 1 mL of 5%
ammonia aqueous solution. The cartridge was washed with a 2 mL mixture
of acetonitryle/water (9:1v/v). Ibotenic acid, muscimol, and internal standard
were eluted with 6 mL of 2% formic acid in methanol. After evaporation to dry-
ness under the stream of nitrogen, the residue was dissolved in a 1 mL mixture
of 0.5% formic acid aqueous solution/methanol (1:4v/v). As a next step, 5 mL of
sample was injected to the LC–MS system. Selected reaction monitoring (SRM)
mode of acquiring LC–MS/MS data was used in this case. The obtained recov-
eries for ibotenic acid and muscimol were above 80%. The limit of detection
142 Studies in Natural Products Chemistry
was lower than 10 mg/kg for both compounds [31]. The use of acivicin as an
internal standard was the novel point of this method, as well as the use of anion
exchange SPE and no requirement for derivatizing before LC–MS/MS analysis.
The authors stressed the fact that this method is much simpler than the method
presented by Tsujikawa et al. [26]. However, the obtained muscimol (MUS) and
ibotenic acid (IBO) recoveries are better in the previous work.
Tsujikawa et al. found that isoxazole compounds tend to be more concen-
trated in the flesh than in the red cuticle of A. muscaria and A. pantherina. They
analyzed Amanita mushrooms that were circulating in the drug market [26].
The total contents of IBO/MUS in the caps were <10–2845/46–1052 ppm
in A. muscaria and 188–269/1554–1880 ppm in A. pantherina. These results
were in general agreement with the data reported in the past [39,40]; however,
the muscimol concentrations were higher than in most of the previous reports.
Authors proposed explanation that A. muscaria sold in the drug market were
dried in the sun or with a heater because drying A. muscaria in the sun or
with a heater causes an increase in MUS in the mushroom by decarboxyl-
ation of IBO [26]. The MUS/IBO contents also depended on the growing
environment and genotype as is described in the review by Michelot and
Melendez-Howell [7].
Psilocybe Mushrooms
Psilocybin and psilocin, the main hallucinogens in Psilocybe mushrooms, are
often extracted using ultrasonic-assisted extraction. Pedersen-Bjergaard et al.
extracted psilocybin from Psilocybe semilanceata before the CE analysis. The
procedure of extraction was as follows: 100 mg of powdered mushrooms were
extracted in an ultrasonic bath lasting 15 min with 3 mL of methanol and with
the addition of 0.5 mg/mL of barbital, which was used as an internal standard.
After extraction, the sample was centrifuged (3200 rpm, 10 min), the superna-
tant was transferred to another flask, and the extraction process was repeated
with 2 mL of methanol containing barbital. The combined extract was diluted
with run buffer (1:1v/v) before CE analysis. The obtained recovery was 98% for
psilocybin. The detection limit was 0.045 g/kg mushrooms for psilocybin and
the quantification limit was 0.225 g/kg mushrooms for psilocybin [29].
Laussmann et al. extracted psilocybin and psilocin from Psilocybe cubensis,
Psilocybe tampanensis, and Panaelous cyanescens. The extraction process was
carried out for fresh, air-dried, and freeze-dried mushrooms. In the case of dried
mushrooms, the samples were homogenized. After the homogenization process,
100 mg of sample was extracted with 100 mL of methanol containing 10 mM
hydrogen chloride and 10 mg/mL of tryptamine as an internal standard in ul-
trasonic bath at 20–25°C lasting 60 min. In the case of fresh mushrooms, 5 g of
mushroom material was extracted in the same solvent, using a laboratory mixer
at 20–25°C for 15 min. After extraction, in both cases the extracts were filtered
before the HPLC analysis. The obtained recoveries were 97.3 and 98.8% for
Bioactive Alkaloids of Hallucinogenic Mushrooms Chapter | 5 143
psilocybin and psilocin, respectively. The limits of detection were 70 mg/kg for
psilocybin and 60 mg/kg for psilocin. The quantification limits were 280 mg/kg
for psilocybin and 220 mg/kg for psilocin [30]. Authors also conducted tests
in order to find optimal sample treatment conditions. They revealed that high-
est alkaloid concentrations are obtained when samples are freeze-dried prior to
extraction. They showed that drying at an elevated temperature (60°C) leads to
decomposition of 90% of psilocin. It is an important piece of information when
comparing results of hallucinogenic substances analyzes content from different
studies. One should take into account not only the growing environment of the
mushrooms, but also the method of extraction and sample treatment.
Saito et al. determined psilocybin and psilocin in the same kind of mush-
rooms used by Lausmann et al. [30]. Twenty miligrams of dried, powdered
mushrooms were added to 1 mL of ethyl acetate containing 5 mg of bufotenin,
which was used as an internal standard. The sample was placed in an ultrasonic
bath for 30 min at a temperature lower than 50°C. After sonification, the sample
was centrifuged (3000 rpm, 5 min), and the supernatant was transferred to an-
other flask. The residue was extracted two more times, using the same method.
The combined extract was evaporated and dissolved in 4% pyridine in 0.2 mL of
acetonitrile, and it was filtered. Then the 50 mL of extract reacted with 50 mL of
10 mM (R)(+)-4-(N, N-dimethyloaminosulfonyl)-7-(2-chloroformylpyrrolidin-
1-yl)-2,1,3-benzoxadiazole in acetonitrile 60°C for 10 min before HPLC–FL
or HPLC–ESI–MS using SIM mode. The recovery was approximately 96.8%
for both compounds. The limit of detection was 0.64 mg/kg for psilocybin and
psilocin [32].
Furthermore, 20 mg of dried, powdered mushroom was added to 1 mL of
methanol containing 4.28 mg/mL 3-indoxyl-phosphate disodium salt as an inter-
nal standard. After 30 min ultrasonification at temperatures lower than 50°C, the
sample was centrifuged (3000 rpm, 5 min), the supernatant was transferred to
another flask and the residue was extracted two more times with 1 mL of metha-
nol, using the same procedure. The combined extract was evaporated, and the
residue was dissolved in 200 mL of 100 mM 1-methylimididazole buffer (pH 7)
and filtered. Next, 10 mL of the extract reacted with 40 mL of 10 mM 5-dimeth-
ylaminonaphthalene-1-[N-(2-aminoethyl)] sulfonamide (DNS-ED) in N,N-
dimethylformamide and 10 mL of 100 mM 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide hydrochloride in pH 7 buffer 60°C for 4 h. After the reaction,
the sample was diluted 20 times with 50 mM mixture of ammonium acetate/
acetonitrile (73:27v/v) and 5 mL was injected to the HPLC–FL [34]. Although
the selectivity increases with the native FL detection after HPLC separation, the
sensitivity seems not to be good enough for a real sample analysis. The detec-
tion sensitivity was about two orders of magnitude higher after derivatizing with
DNS–ED as was shown by Saito et al.
Kikura-Hanajiri et al. determined 19 hallucinogenic substances, using the
GC–MS and LC–MS. GC–MS was used for qualitative analysis and LC–ESI–
MS – for qualitative and quantitative analysis. Five of these substances were
144 Studies in Natural Products Chemistry
legally controlled tryptamines and phenethylamines originally found in fungi or
plants. In case of plant/mushroom materials, 100 mg of dried mushroom sample
was extracted with 2 mL of methanol containing 50 mL of the internal standard
solution (0.2 mg/mL) in an ultrasonic bath lasting 10 min. The extract was cen-
trifuged (3000 rpm, 5 min) and filtered before the GC-MS analysis. Develop-
ing a LC separation of the 19 compounds in one run was challenging. These
investigations revealed that samples, mostly sold as “mixtures of mushrooms/
plants” via the Internet, possessed the synthesized compounds, 5-MeO-DIPT
(5-methoxy-diisopropyltryptamine) and AMT (a-methyltryptamine) and con-
tained no psilocin or psilocybin.
Tsujikawa et al. identified psilocybin and psilocin in P. cubensis and Cope-
landia genus. The extraction procedure was as follows: 10 mg of dried, pow-
dered mushrooms were double-extracted with 1 mL of methanol in an ultrasonic
bath lasting 30 min. Then the extract was centrifuged (3000 rpm, 2 min), and the
supernatant was transferred to another flask. The combined supernatant was
evaporated under a stream of nitrogen. The residue was dissolved in 100 mL of
mobile phase with 4-hydroxyindole (25 mg/mL), which was used as an internal
standard [35]. Then a 10 mL aliquot was used for the HPLC analysis. Tsujikawa
noted that psilocin and psilocybin have a tendency in P.cubensis to be con-
tained in the cap more than the stem. In Copelandia, this tendency was not so
noticeable. Moreover, it was shown that P. cubensis is psilocybin-rich, whereas
Copelandia is psilocin-rich. However, only two samples of Copelandia were
analyzed within this study, and these samples could be different species because
their appearance and scale of spores were apparently different [35].
Mushoff et al. determined psilocybin and psilocin in P. semilanceata, P.
cubensis, P. tampanensis and P. cyanescens. One hundred milligrams of dried,
powdered mushrooms were extracted with 9 mL of methanol in an ultrason-
ic bath lasting 120 min. After centrifugation, the supernatant was used to the
HPLC analysis [37]. The alkaloids content was determined with <0.003–1.15%
of psilocybin and 0.01–0.90% psilocin. The authors also provided detailed mor-
phological characteristics of investigated hallucinogenic mushrooms, which is
important when analyzing mushrooms.
Keller et al. determined hallucinogenic substances in Psilocybe subcubensis
and Agrocybe praecox, which does not contain hallucinogens such as psilocy-
bin and psilocin. The mushrooms were lyophilized and cut into small pieces.
Fifty milligrams of mushroom was extracted with 1 mL of chloroform in an
ultrasonic bath lasting 60 min. After centrifugation (14,000 rpm, 10 min) and
filtration, 0.5 mL of clear, spiked supernatant was transferred to vial and was
evaporated under a stream of nitrogen. The hallucinogenic mushrooms were ex-
tracted through the same procedure, but only 0.05 mL of supernatant was evap-
orated. The residues were dissolved in 30 mL of N-methyl-N-(trimethylsilyl)-
2,2,2-trifluoroacetamide. The mixtures were heated for 30 min at 70°C. After
heating, the mixtures were cooled and 1 mL of samples were injected into the
GC–MS system. They used SIM mode of data acquiring. The limits of detection
in the cap of P. subcubensis were 8.6 and 0.2 g/kg for psilocybin and psilocin,
Bioactive Alkaloids of Hallucinogenic Mushrooms Chapter | 5 145
respectively. The limits of detection in the stem of P. subcubensis were 8.0 g/kg
for psilocybin and 0.3 g/kg for psilocin [36].
Sarwar et al. determined psilocybin and psilocin in mushrooms. About 200–
500 mg of dried, powdered mushrooms was extracted in 10% solution of acetic
acid and 5 mL of deionized water. After centrifugation (3 min), the supernatant
was transferred into a beaker and neutralized by adding a small volume of so-
dium bicarbonate. This solution was extracted with an equal volume of chloro-
form. The diphasic solution was centrifuged and the chloroform layer was trans-
ferred to another flask and concentrated under air before GC–MS analysis [38].
As we can see in Table 5.1, the high-performance liquid chromatography
is the most widely used analytical technique in the scope of analysis of hallu-
cinogenic substances. Although several other techniques have been applied to
identify these substances in mushrooms, the detection limits are much better for
the HPLC technique than GC or CE methods, especially when using electro-
chemical or MS detection or flow injection analysis [14].
Biological Samples
The leading techniques allowing separation of the main hallucinogenic alka-
loids, i.e., psilocybin, psilocin, bufotenin, ibotenic acid and muscimol, from
biological fluids are LLE [9,41–43] and SPE [16,44–50] (Table 5.2). These
methods allow the satisfactory recovery of trace amounts of the analyte in
physiological samples. The most frequently used analytical methods for the
identification of selected psychoactive compounds from the samples of urine,
plasma, and serum are chromatographic methods, such as high-performance
liquid chromatography (HPLC) with a diode array detector (DAD) [51] or elec-
trochemical detection [14,42,43] gas chromatography (GC) with a MS detec-
tor [16,45,50,52] and electro-migration method-capillary electrophoresis (CE)
with a UV detector [48] (Table 5.2).
In order to detect and identify the analytes in a physiological fluid sample
with suitable sensitivity, the treatment of samples prior to analysis is necessary
for toxicological studies. The extraction of selected hallucinogenic alkaloids
from complicated fluid samples should include the purification of a sample ma-
trix. This step can be taken, using hydrolysis or precipitation of proteins in the
plasma or urine samples. For the urine or blood plasma, which contains a very
large amount of conjugated metabolites, the use of hydrolysis allows satisfac-
tory results to be obtained and allows a lower-level of the analyte detection.
The main hallucinogenic isoxazoles (muscimol and ibotenic acid) in biological
As mentioned above, the main Amanita hallucinogenic alkaloids were ex-
tracted from a biological matrix, such as serum and urine. There are only several
reports that describe the extraction procedure and the process of ibotenic acid
and muscimol identification in such samples. The main extraction procedures
used for isolation of these compounds are LLE and SPE [9,49].
146 Studies in Natural Products Chemistry
TABLE 5.2 The Methods of Extraction and Identification of Ibotenic Acid, Muscimol, Psilocin, Psilocybin, and Bufotenine in
Biological Samples
No. Analyte Matrix
method Recovery (R%) Detection
1. Psilocin Serum LLE LC–MS/MS 0.5 mg/L [41]
2. Psilocin Urine LLE 88 HPLC–ECD [43]
10 mg/L
3. Psilocin Plasma SPE
HPLC–ECD – [42]
10 mg/L
4. Psilocin Urine
SPE 98 GC–MS 5 mg/L [16]
5. Psilocin Serum
SPE 80 LC–MS 2 mg/L [44]
6. Psilocin Urine SPE – GC–MS [45]
10 mg/L
7. Psilocin Urine SPE 88 LC–MS/MS 5 mg/L [46]
10 mg/L
8. Psilocybin Urine SPE 92 LC–MS/MS 5 mg/L [46]
10 mg/L
Bioactive Alkaloids of Hallucinogenic Mushrooms Chapter | 5 147
9. Psilocin Urine SPE 87.6 LC–MS/MS 0.5 mg/L [47]
10 mg/L
10. Psilocin Urine SI–SPE 84 CE–UV 7 mg/L [48]
13 mg/L
11. Psilocin G. spectabilis
Rat plasma
SPE 97.7–104.8 UPLC–PDA 0.05 mg/L [53]
0.12 mg/L
12. Psilocin Urine – – LC–MS/MS 4 mg/L [54]
13. Psilocin Urine SPE 86.6 LC–MS/MS 0.2 mg/L [21]
0.2 mg/L
14. Psilocin Plasma SPE 89.1 LC–MS/MS 0.05 mg/L [21]
0.15 mg/L
15. Psilocin Serum SPE 89.5 LC–MS/MS 0.05 mg/L [21]
0.17 mg/L
16. Bufotenine Urine SPE 88.8 LC–MS/MS 0.10 mg/L [21]
0.14 mg/L
17. Bufotenine Plasma SPE 91.3 LC–MS/MS 0.07 mg/L [21]
0.27 mg/L
18. Bufotenine Serum SPE 91.6 LC–MS/MS 0.05 mg/L [21]
0.11 mg/L
148 Studies in Natural Products Chemistry
19. Psilocin Plasma SPE 86 LC–MS/MS 0.1 mg/L [55]
0.34 mg/L
20. Psilocin Serum
SPE 88 GC–MS 3 ng/g [52]
5 ng/g
21. Ibotenic acid Urine LLE–SPE 74 GC–MS 0.1 mg/L [9]
22. Muscimol Urine LLE–SPE 80 GC–MS 0.1 mg/L [9]
23. Ibotenic acid Serum SPE 87.9–103 LC–MS/MS 1 mg/L [49]
2.5 mg/L
24. Muscimol Serum SPE 89.8–96.4 LC–MS/MS 1 mg/L [49]
2.5 mg/L
TABLE 5.2 The Methods of Extraction and Identification of Ibotenic Acid, Muscimol, Psilocin, Psilocybin, and Bufotenine in
Biological Samples (cont.)
No. Analyte Matrix
method Recovery (R%) Detection
Bioactive Alkaloids of Hallucinogenic Mushrooms Chapter | 5 149
In the first case [9], the authors described the extraction method for ibotenic
acid and muscimol from the urine sample. The extraction procedure included
LLE and SPE. The first step of the proposed procedure was the SPE. For con-
centration and purification of the analytes exchange, the Dowex 50W X8 sor-
bent was used. The urine sample was shaken with an ion exchange sorbent in
a mixture of 0.1 M hydrochloric acid: methanol. Then, it was centrifuged and
again shaken with brine and ethyl chloroformate, i.e., the second step of the
LLE, and the organic layer was finally collected. The sample was then analyzed
using a gas chromatograph with a mass spectrometer (GC–MS). The recov-
ery was 80% for ibotenic acid and 74% for muscimol. The detection limit was
1000 mg/L for both muscimol and ibotenic acid [9].
The SPE was also used in the case of human serum samples. The ion ex-
change Oasis MAX 3cc extraction cartridges were used for extraction of the
ibotenic acid and muscimol from serum [49]. A 100 mL aliquot of human serum
containing ibotenic acid and muscimol was mixed with 100 ng of acivicin (in-
ternal standard, IS) dissolved in methanol, distilled water, and in 0.5% ammo-
nia aqueous solution; the mixture was vortexed. Then the mixture was loaded
on an extraction cartridge (preconditioned with methanol and 0.5% ammonia
aqueous solution). The cartridge was washed with distilled water and then with
methanol. The target compounds and IS were eluted with 0.05% TFA in meth-
anol. The extract was evaporated to dryness. Then, the residue was reconsti-
tuted in methanol and subjected to the LC–MS/MS analysis. The recovery was
87.9–103% (depending on concentration) for ibotenic acid and 89.8–96.4% for
muscimol. These good recoveries were obtained by Haegawa et al. after testing
various compositions for the washing and eluting solutions. The detection limits
were 1.0 and 2.5 mg/L for ibotenic acid and muscimol, respectively [49]. They
are much better than detection limits showed in studies of Stribrny et al. [9].
Very recently, the CE–ESI–MS/MS method used to identify, separate, and
determine the mushroom’s ibotenic acid, muscimol, and muscarine from urine,
has been developed [56]. The obtained LOD values were at the nanomolar con-
centration level showing the usefulness of this method for identification and
quantification of the studied toxins in the human urine.
The main advantages of CE are an extremely small injection volume, high
separation efficiency, and short analysis time [56]. This method is environmen-
tally friendly due to low solvent consumption in comparison with LC.
The main hallucinogenic indoles in biological samples.
Psilocybin and psilocin have free hydroxyl groups, so it is likely that they
are excreted in the urine as conjugated forms. The literature shows that psilo-
cin concentration in urine increases with the process of enzymatic hydrolysis
with b-glucuronidase. This indicates that this substance can conjugate by meta-
bolic pathway to glucuronide conjugate forms [41,43,54,57]. Thus, in this case
hydrolysis forms an important part of proper treatment of the sample prior to
150 Studies in Natural Products Chemistry
Suitable enzyme sources and the power of the enzyme used for enzymatic
hydrolysis is an important factor in the process of obtaining a high concentra-
tion of the analyte free form. Among the conjugates formed in the body with
psilocin, the best proved is a b-glucuronidase derived from Escherichia coli
by enzymatic treatment units from 12500 to 25,000 units per 5 mL of urine
samples [45]. In the first case, of extraction of psilocin from urine, hydrolysis
was used before the LLE extraction. After purification, the resulting supernatant
was used for the LLE. The process of hydrolysis was carried out with b-gluc-
uronidase at a pH of 5, 40°C for 5 h. Then, after the hydrolysis, all samples were
extracted with methanol and membrane filtered and then analyzed by the HPLC
with an electrochemical detector. The recovery of free psilocin was 88–106.2%
(depending on concentration) [43].
The LLE have been also used for the extraction of psilocin and its conju-
gate in serum samples. b-glucuronidase was used for the hydrolysis of gluc-
uronide conjugates of psilocin and the psilocybin analysis of serum samples.
Incubation temperature was 37° C for 2 h. The hydrolysis was carried out at
a pH of 5. The extraction was made on both hydrolyzed and nonhydrolyzed
samples. The serum sample was adjusted to pH 8 and then extracted with
two portions of chloroform. The resulting mixture was centrifuged (10 min,
15,000 g). Then, the organic layer was separated, the solvent was evaporated
and the residue was dissolved in a mobile phase and analyzed, using a liquid
chromatography coupled with a tandem mass spectrometer (LC–MS/MS).
The multiple reaction monitoring (MRM) mode of acquiring LC–MS data
was used. With this method, the satisfactory 0.5 mg/L detection limit was
obtained [41].
The second leading technique used for the hallucinogenic indoles isolation
from the biological matrix is SPE. It is applied mainly to psilocybin, psilocin,
and bufotenine. SPE was applied to the samples of urine and serum, using com-
mercially available columns with different chemical properties of sorbent. The
extraction used, among other sorbents, copolymer (Clean Screen CSDAU206,
ZCDAU020), including ion exchange properties (Oasis MCX, XAD nonionix,
OSP-2a CBA Certifty Varian Bond Elut LRC, Waters MAX), which is widely
used both for polar compounds and nonpolar silica deposits octadecyl modified
with C18 (Bond Elut C18) [16,42,44–48]. In the case of ion exchange sorbents,
the main solution used for the elution had the pH > 7; however, the most com-
monly used one was the ammonium base solution having a concentration from
2% to 5% in methanol or ethyl acetate. Application of the principle as an elu-
ent and ion exchange sorbent gives high-recovery values from 87% to 100%
The example of a procedure based on SPE, using ion exchange columns
Oasis MCX (30 mg, 1 mL), is psilocin extraction from urine samples. In the
first extraction stage, the sorbent was conditioned with methanol, water, sodium
acetate (pH 4). Then, the sample of urine (pH 4) was loaded onto the extrac-
tion cartridge. Next, the cartridge was washed with acetate buffer, methanol,
Bioactive Alkaloids of Hallucinogenic Mushrooms Chapter | 5 151
or a buffer, a mixture of methanol: water (80:20 v/v) or buffer, and a mixture
of methanol: water (50:50 v/v). After the interferents were washed, the sorbent
was dried and then the analytes were eluted with 5% ammonium solution in
methanol. The resulting extract was evaporated to dryness, and the residue was
taken up in a mobile phase and analyzed, using the LC–MS/MS. The obtained
psilocin recovery was 87.6%, the detection limit was 0.5 mg/L, and the limit of
quantification 10 mg/L [47].
Bogusz described the determination of psilocybin and psilocin in serum,
blood, and urine [44]. Before precipitation of the samples, the mixture was cen-
trifuged (5 min, 14,000 g). Then, the ammonium buffer (pH 9.3) was added to
the obtained supernatant, and the mixture was stirred at vortex. The solution
prepared in this way was centrifuged again (10 min, 5000 g). In this case, the
silica sorbent with octadecyl group C18 was used for extraction of psilocybin
and psilocin. The cartridge was conditioned with methanol, water, and ammo-
nium buffer pH 9.3. The supernatant obtained during precipitation was then ap-
plied onto the C18 cartridge and washed with ammonium buffer (pH 9.3). Then
the sorbent was dried and the substances were eluted with methanol: acetic acid
(9:1 v/v). The extract was evaporated to dryness, and then dissolved in a mobile
phase and analyzed by LC-MS with a SIM mode of data acquiring. The recov-
ery of psilocin was 90% and 80% for psilocybin; the LOD for both substances
was 2 mg/L [44].
Definitely higher recoveries, lower limits of detection and quantification are
achieved, using online SPE. This system was used to isolate and concentrate
psilocin from blood plasma. The procedure utilizes a cation exchange extrac-
tion cartridge (OSP-2a CBA). Methanol was used as an eluent. After the extrac-
tion, the sample was analyzed by the HPLC with electrochemical detection.
The recovery achieved by this method was 100%. A satisfactory value limit of
quantification at the level of 10 mg/L was obtained [42]. SPE can be also directly
connected to the analytical system, such as the capillary zone electrophoresis
(CE–UV), in order to improve the selectivity and the rate of extraction. This
system can be based on sequential injection, SI, of the sample and flow control
injection with a thousand concentrations of sample. Sequential injection solid
phase extraction (SI–SPE) was used for extraction and concentration of psilocin
from urine. The C18 extraction cartridge was used for purification, separation,
and concentration of psilocin. The extract was analyzed using the CE–UV. The
recovery was 84–1.2% and a satisfactory detection limit of 7 mg/L was achieved
with this method [48,58].
No precursors to ibotenic acid can be shown without doubts. Ibotenic acid,
muscimol, and muscazone originate probably from the same precursor, b-
hydroxyglutamic acid. Biosynthetic pathway including ring closures and decar-
boxilation determines the structures of these products [7].
152 Studies in Natural Products Chemistry
Psilocybin was demonstrated by many studies to be derived from tryptophan
[59–61]. The precise mechanism of psilocybin biosynthesis is debated by many
authors. In 1959, Hofmann et al. showed that radioactive tryptophan is incor-
porated into psilocin and psilocybin in Psilocibe semperviva [62]. Agurell and
Nilsson investigated the biosynthesis of psilocybin by feeding labeled precursor
to P. cubensis. The study was concerned with the sequence of events, which
leads from tryptophan to psilocybin. They showed that 4-hydroxytryptophan
appears to be a poor precursor of psilocybin and indicated that P. cubensis can
utilize two paths to psilocybin [60]. The first is shown in Fig. 5.3. This sequence
involves chemical modifications as follows: decarboxylation, N-methylation,
4-hydroxylation, and phosphorylation of the 4-hydroxyindole moiety. Agurell
and Nilsson suggested that an alternative route is a conversion of 4-hydroxy-
tryptamine to psilocybin.
Since tryptamine operated as a better precursor for psilocybin synthesis than
tryptophan in cultured P. cubensis, it seems that decarboxylation of tryptophan
to tryptamine is the first step in the psilocybin biosynthesis [64]. Leung and
Paul revealed incompletely methylated psilocybins (these are baeocystin and
norbaeocystin) in Psilocybe baeocystis [65].
These results suggest that methylation might occur as the last step in biosyn-
thesis of psilocybin. Chilton et al. proposed a mechanism by which tryptophan is
transformed to bufotenine and another one showing biotransformation of tryp-
tophan to psilocin (Fig. 5.4) [66]. They revealed that tryptamine-4, 5-epoxide
can be intermediate between tryptamine and psilocin, but it was unknown if it is
also a precursor of serotonin and bufotenine. Another investigation showed that
FIGURE 5.3 The model is based on the data obtained in studies provided by Agurell and
Nilsson [63].
Bioactive Alkaloids of Hallucinogenic Mushrooms Chapter | 5 153
the mushroom efficiently hydroxylated tryptamine to psilocin but N,N-diethyl-
tryptamine was transformed to 4-hydroxy-N,N-diethyltryptamine [67,68].
Baeocystin and norbaeocystin are natural psilocybin analogs first isolated
from P. baeocystis [23,65]. Baeocystin was also detected in some other species
from genera Psilocybe, Conocybe, and Panaeolus [69]. The presence of baeo-
cystin and norbaeocystin in P. baeocystis suggests that the alternative pathway
is probably utilized by some fungi. The possible paths leading to psilocybin and
its analogs are presented in Fig. 5.5.
FIGURE 5.4 Possible paths of biosynthesis of psilocin and bufotenine.
154 Studies in Natural Products Chemistry
Indoleamine Hallucinogenic Pharmacodynamics
Many of the indoleamines have significant biological activity. One of the most
highly studied indoleamines is hormone serotonin (5-hydroxytryptamine or
5-HT). This neuromodulator is mainly synthesized by intestinal cells that regu-
late overall intestinal physiology. Serotonin has been involved in the regulation
of many physiological and pathological events. This neurotransmitter plays an
important role in the central nervous system as well as in the cardiovascular
and gastrointestinal systems [3]. Seven different serotonin receptors and 14 dif-
ferent receptor subclasses were identified [70]. Numerous factors were used
to investigate the 5-HT receptors. Reliable pharmacological investigation on
the hallucinogenic indoleamines are possible since selective antagonists for
relevant serotonergic receptors were discovered and cloned. Indoleamine hal-
lucinogens cause extremely similar experiences in humans. The similarity of
FIGURE 5.5 Alternative pathway for the biosynthesis of psilocybin and its analogs from
Bioactive Alkaloids of Hallucinogenic Mushrooms Chapter | 5 155
their psychopharmacological effects and their ability to produce cross-tolerance
indicate that indolealkylamines (to which psilocin and psilocybin belong) act
through a common receptor mechanism.
It is widely accepted that the unitary mechanism responsible for the effects
of serotonergic hallucinogens is activation of the 5-HT2A receptor; however, it
does not exclude the probability that the interaction of indoleamines with non-
5-HT2 receptors does have also psychopharmacological and behavioral conse-
quences [71].
It was shown that stimulus properties of tryptamines could be blocked by the
5-HT2 receptor antagonists such as pirenperone [72], which formed the basis
for the theory proposed by Glennon et al. claiming that hallucinogenic drugs
act specifically at the 5-HT2 receptor subtypes [73]. Many other works strongly
indicate that the stimulus effects of hallucinogens (mainly LSD–lysergic acid
diethylamide) are mediated by the 5-HT2A receptor [74,75]. Indeed, Vollenwei-
der et al. reported that most of the subjective effects of psilocybin are blocked
by pretreatment with the 5-HT2A antagonist ketanserin [76]. Nevertheless, ket-
anserin had no influence on other drug associated symptoms like reduction of
arousal and vigilance and impairment of multiple-object tracking, suggesting
that psilocin activates several serotonin receptor subtypes [77]. Recently, it has
been noticed that binding affinity of psilocin to the 5-HT receptors is quite dif-
ferent than LSD (see Table 5.3). McKenna et al. investigated the affinities of
21 indolealkylamine derivatives to the 5-HT1A, 5-HT2A, and 5-HT2B receptors,
TABLE 5.3 Binding of Psilocin and (+)-LSD to 5-HT Receptors on the Basis
of Ref. [71]
Binding site
Ki (nM)
Psilocin (+)-LSD
5-HT1A 567.4 (49)*1.1
5-HT1B 219.6 3.9
5-HT1D 36.4 –
5-HT1e – 93.0
5-HT2A 107.2 (25)*3.5
5-HT2B 4.6 30.0
5-HT2C 97.3 (10)*5.5
5-HT583.7 9.0
5-HT657.0 6.9
5-HT73.5 6.6
*Ki in brackets reported by Blair et al. [79].
156 Studies in Natural Products Chemistry
using radioligand competition studies [78]. This study demonstrated that hallu-
cinogenic 4-hydroxy-indolealkylamines, like psilocin, bind potently and selec-
tively to the 5-HT2A receptor. The 5-hydroxylated derivatives (e.g., bufotenine)
displayed approximately equal potency at the 5-HT1A and 5-HT2A sites. Other
serotonin analogs display moderate to high affinity for the 5-HT1 and 5-HT2
subtypes [79–81]. It is worth mentioning that when hallucinogens are tested in
competition with the receptor antagonist, it may profoundly influence apparent
affinity [82]. In spite of the fact that the abuse rate is still growing, especially
among young people, we know relatively little about how the hallucinogenic
tryptamines affect the brain. Most probably, the risk associated with hallucino-
gen administration is commonly known as a “bad trip” and is characterized by
anxiety, fear/panic, dysphoria, and/or paranoia [83]. Visual hallucinations are
caused probably by increasing cortical excitability and altering visual-evoked
cortical responses after activation of the 5-HT2A receptors [84]. Current knowl-
edge about the 5-HT receptor ligands, also in preclinical research and clinical
trials, was reviewed by Filip and Bader [85]. The recent investigations concern-
ing the influence of psilocin and its derivatives on the brain will be described
briefly in subsequent sections.
Metabolism of Indoleamines Produced by Mushrooms
and their Psychosomatic Effects
Psilocybin is a substituted indolealkylamine, which belongs to the group of
hallucinogenic tryptamines. Psilocybin and psilocin are the main psychoactive
compounds of hallucinogenic mushrooms. The toxicity of psilocybin is rela-
tively low (LD50 = 280 mg/kg in rats and LD50 = 285 mg/kg in mice) [86]. This
means that a 60 kg (1 kg = 2.2046 pounds) person must eat 1.7 kg of dried P.
cubensis mushrooms to reach the LD50 value [87]. When administered intrave-
nously in rabbits, psilocybin’s LD50 is around 12.5 mg/kg (TOXNET, National
Library of Medicine, USA). However, nondrug variables can significantly alter
toxic reactions, e.g., diet, physical exertion, expectation, and stress.
Acute adverse effects as well as chronic toxicity of magic mushroom use
has been recently briefly reviewed by van Amsterdam et al. [87]. It should be
noticed that psilocybin is a prodrug for psilocin. This means that whenever a
reference is made to the in vivo effects of psilocybin (e.g., in the following
section), it means that the actual biologically active species is psilocin [88,89].
Indeed, the in vivo experiments on rats showed that psilocybin was rapidly hy-
drolyzed to psilocin. Then psilocin was well taken up by intestinal segments and
transferred to the blood side [90]. The same study revealed that tissue uptake of
intact psilocybin was negligible or absent.
The investigations by the HPLC technique indicated about 50% bioavail-
ability of psilocin after oral administration of psilocybin [89]. Psilocin is de-
tectable in the human plasma within 20–40 min after oral administration, and
the plasma concentration ranges maximum after approximately 80–100 min
Bioactive Alkaloids of Hallucinogenic Mushrooms Chapter | 5 157
[42,89]. After 6 h of drug ingestion, the effects are completely worn out, as was
shown by Hasler et al. [91]. The work by Lindenblatt et al. revealed a large in-
terindividual variation as regards psilocin plasma concentration of healthy vol-
unteers after oral administration of 0.2 mg psilocybin per kilogram of the body
mass [42]. Recent results of pharmacokinetic investigations on rat plasma, after
the oral administration of a Gymnopilus spectabilis extract, showed that psi-
locin was rapidly absorbed into blood and reached maximum concentration at
90 ± 2.1 min [51]. According to another study, the maximum amount of psilocin
concentration in rat plasma, after an intraperitoneal injection of 5 mg/kg, was
obtained after 15 min [92]. Psychopathological effects in humans occur with
plasma levels of 4–6 mg/L [91].
According to another study, the threshold dose of 45 mg psylocybin per kg
of the body weight was assessed clearly as a psychoactive by most of the vol-
unteers. They reported slight amounts of drowsiness, increased sensitivity, and
intensification of preexisting mood states [93]. Hallucinogenic potency of psi-
locin in men is much lower than LSD (10–20 mg for the first and 60–200 mg
for the latter drug) [94]. After the ingestion of higher doses (up to 315 mg/kg of
the body weight), the changes in the mood states, sensory perception (includ-
ing colorful visual illusions, complex scenic hallucinations, and synesthesias)
as well as alterations in perceptions of time and space are produced [93]. The
authors explain that psilocybin is not hazardous with respect to somatic health.
However, some cases are known about where the cause of death was from psi-
locin toxicity.
In 2012, “magic mushroom” ingestion was determined by court-appointed
experts as a cause of death [95]. Plasma toxicology revealed the psilocin level
of 30 mg/L in a 24-year-old heart transplant recipient as a consequence of con-
suming psychedelic mushroom. The fatal ventricular arrhythmia was caused by
excessive sympathetic stimulation of the transplanted heart. Indoleamine hal-
lucinogens in overdose can also produce a psychosis-like syndrome in humans
that resembles first episodes of schizophrenia [76]. The case report presented
by Holger et al. showed a sudden impairment of the left ventricular function,
after P. semilanceata consumption, followed by a rapid recovery. The potential
catecholamine- and serotonin-like characteristics of psilocin was considered as
causative [96]. On the other hand, Beck et al. suggested that adverse reactions to
Psilocybe mushroom intake are not caused by psilocybin but phenylethylamine
(PEA), which was detected for instance in P. semilanceata [97]. This substance,
structurally related to amphetamines, naturally occurs in the nervous system
of mammals and acts probably as a neurotransmitter or neuromodulator. PEA
might be responsible for the cardiovascular effects (tachycardia) and other ad-
verse reactions (nausea and anxiety) of magic mushrooms [97]. However, PEA
is metabolized too rapidly in the digestive system to reach the brain, and it does
not have potential for abuse.
Recently, very interesting findings have been revealed by Carhart-Harris et al.
The authors used functional magnetic resonance imaging (fMRI) to measure the
158 Studies in Natural Products Chemistry
effect of psilocybin on the resting-state brain activity. Their results suggest a
biological mechanism, in which the connectivity of the brain’s connector hubs
is decreased via the 5-HT2A receptor stimulation by psilocybin [98]. Some
preclinical studies show that stimulation of the 5-HT2A receptors increases the
GABAergic transmission and the pyramidal cells (excitatory glutamatergic cells)
inhibition, which may explain the deactivations observed by Carhart-Harris et al.
[99,100]. Their results are in agreement with the hypothesis that the 5-HT2A
receptor-mediated regulation of glutamate release is the mechanism through
which hallucinogens activate the cerebral cortex [99].
The investigations of the fate of psilocin in the rat showed that after 24 h
65% of the dose of 10 mg/kg psilocin is excreted in the urine [101]. A controlled
study in men showed that within 24 h, 3.4 ± 0.9% of the applied dose of psilocy-
bin when excreted with urine was free psilocin. The limited amount (10 mg/L)
was usually reached 24 h after the drug was administered. In this study, eight
volunteers received psilocybin in psychoactive oral doses of 212 ± 25 mg/kg
of the body weight [43]. Terminal elimination half-lives of psilocin calculated
from plasma concentration–time data were estimated at (2.72 ± 1.06 h; n = 6)
[89,91] and from cumulative urinary excretion rates at (3.29 ± 0.57 h; n = 8)
[43]. Three other metabolites of psilocybin were identified: 4-hydroxyindole-
3-yl-acetaldehyde (4H1A); 4-hydroxyindole-3-yl-acetic-acid (4-HIAA); and
4-hydroxytryptophol (4-HTP) [89]. Nevertheless, later pharmacokinetic and
forensic studies revealed that psilocin is mostly eliminated by conjugative
metabolism as psilocin glucuronide [16,45,57]. This is done by glucuronosyl-
transferases: microsomal enzymes originating from liver or intestinal tissues. In
the study provided by Sticht et al., free psilocin was determined in urine at the
concentration of 0.23 mg/L, while the total amount was 1.76 mg/L [16]. These
reports suggest that glucuronidation seems to be an important detoxification
step (Fig. 5.6).
Psilocin has two potential glucuronidation sites, but it was shown, with con-
siderable confidence, that human UDP-glucuronosyltransferases (UGTs) only
catalyze the glucuronidation of psilocin at its hydroxy group [102].
Bufotenine [3-(2-dimethylaminoethyl)-1H-indol-5-ol; 5-hydroxy-N,N-di-
methyltryptamine; 5-HO-DMT] shows activity similar to LSD and other known
hallucinogens. It has the ability to bind and activate hallucinogenic serotonin
(5-HT) receptors, 5-HT2A and 5-HT2C [103]. McBride reported lack of the hal-
lucinogenic response in the case of bufotenine in human experiments due to
poor ability to cross the blood–brain barrier (BBB) [103]. Indeed, the experi-
ments on rats showed that 1 h after injection of bufotenine (1, 30, or 100 mg/kg),
its level was high in lung, heart and blood, and lower in brain and liver [104].
Bufotenine disappeared almost completely within 8 h. These experiments also
indicated that after being injected into rats, bufotenine is rapidly eliminated,
partly by monoamine oxidase A (MAO-A). On the other hand, it was found
that 5-HO-DMT is positive for head-twitch response (rapid side-to-side head
movement that occurs in mice and rats after the serotonin 5-HT2A receptor is
Bioactive Alkaloids of Hallucinogenic Mushrooms Chapter | 5 159
activated), which correlates well with hallucinogenic activity of a variety of
drugs in humans [105].
The head-twitch response is probably the best assay to successively discrim-
inate hallucinogenic drugs from closely related nonpsychoactive compounds
[106]. The work of Fabing and Hawkins showed that 8–16 mg of intravenous in-
jection of bufotenine in human volunteers resulted in primary visual disorders,
permutation of space and time perception, and paresthesias [107]. At the highest
dose, a generalized tingling of the body was observed.
Turner et al. examined the effects of some indolealkylamines on men. When
20 mg of bufotenine was injected into a patient during a 77-min interval, no
psychological changes were shown.
FIGURE 5.6 The psilocybin elimination from the body by glucuronidation process.
160 Studies in Natural Products Chemistry
When 10 mg or more was injected during a 50-s interval, the effects were
extreme (hyperventilation persisting for about 2 min, salivation, and the red-
dish blue color of the face) [108]. It was shown that the elevated levels of en-
dogenous bufotenine (as a product of the serotonin-degradative pathway) may
play a role in autistic spectrum disorders (ASD) and schizophrenia, and can be
correlated with hyperactivity scores in autism [109,110]. Little information ex-
ists about pharmacodynamic properties of baeocystin and norbaeocystin. The
human pharmacology and toxicology of aeruginascin has not been tested yet,
either. Aeruginascin is assumed to undergo a rapid metabolism into its dephos-
phorylation product by analogy to the known Psilocybe alkaloids [20].
Pharmacodynamics of Isoxazole Hallucinogens and Muscarine
The main psychedelic components of some Amanita mushroom species are
muscimol (5-aminomethyl-3-OH-isoxazole) and ibotenic acid [(S)-2-amino-
2-(3-hydroxyisoxazol-5-yl) acetic acid). Muscimol is a product of in vivo ibo-
tenic acid decarboxylation [111]. Both muscimol and ibotenic acid induce a
distinct anorexogenic action on mice (2–3 mg/kg oral) with sedation, hypnosis,
and catalepsy [112,113]. Muscimol is some 6- to 10-fold more toxic than ibo-
tenic acid in animals; its active dose given orally is 7.5–10 mg, and the LD50
(intraperitoneal) is 2.5 mg/kg for mice and 3.5 mg/kg for rats [114]. Chemical
structure of these isoxasoles closely resembles the product of glutamic acid
enzymatic decarboxylation, i.e., g-aminobutyric acid (GABA) [8]. This most
important inhibitory neurotransmitter in the brain plays a significant role in reg-
ulating neuronal excitability throughout the nervous system. There are known
to be three major classes of GABA receptors (GABAA, GABAB, and GABAC).
Gamma-aminobutyric acid regulates the brain excitability mainly via GABAA
receptors, which comprises of five subunits classified into three major groups
(alpha, beta, and gamma) [115]. The subunits of GABA receptors determine
their pharmacological activity. Muscimol binds to both high- and low-affinity
sites of GABAA receptors (Kds of 10 nM and 0.27 mM), as was shown in a radio-
ligand binding study using a bovine brain [116]. It is also a potent partial agonist
at GABAC receptors [117].
Muscimol alters neuronal activity in multiple-brain regions due to wide
distribution of GABAA receptors in the brain. However, brain regional dis-
tribution of muscimol high-affinity-binding sites partially differs from those
of other binding sites of the GABAA receptors. High-affinity muscimol bind-
ing in the brain sections was determined by quantitative autoradiography and
sedative/ataxic effects induced in vivo by muscimol, using a constant speed
rotarod [118]. The results suggest that the behavioral effects of muscimol
are preferentially mediated through high-affinity agonist binding sites of
the forebrain GABAA receptors. The study of muscimol distribution in rats
showed that this drug easily enters the brain [119]. Thirty minutes after intra-
venous administration of [3H]muscimol (1 mg/kg) to rats, this compound was
Bioactive Alkaloids of Hallucinogenic Mushrooms Chapter | 5 161
indicated in brain at the concentration of 200 nmol/kg. The brain regions with
the highest muscimol concentrations were the substantia nigra, the colliculi,
and the hypothalamus [119]. The increase in the levels of radioactive musci-
mol metabolites in plasma was also noticed. Thus, as far as the metabolism
of muscimol is concerned, the transamination appears to be one of its major
pathways [119–122].
Muscazone, the lactam isomer of muscimol, exhibits minor pharmacologi-
cal activities in comparison with the previous substances. The LD50 data for this
compound are not available [123].
As mentioned earlier, many members of the genus Amanita are psychedelic.
The recreational use of them is very risky due to low differences between the
psychedelic and lethal dose of some mushroom species. They can cause annoy-
ing symptoms occurring after the mushroom ingestion like vomiting, hallucina-
tions, restlessness, increased psychomotor drive, and central nervous system
depression. Sometimes, other antycholinergic symptoms, like tachycardia and
increased blood pressure, mydriasis, dry and red skin, can occur, too [124].
Another alkaloid – muscarine, firstly isolated from the mushroom A. mus-
caria in 1868, is highly toxic. It causes profound activation of the peripheral
parasympathetic nervous system by simulating the action of the neurotransmit-
ter acetylcholine at muscarinic acetylcholine receptors [125]. However, mus-
carine is only a trace compound in the fly agaric A. muscaria. As mentioned
earlier, it is the principal toxin in the fungi of genera Inocybe and Clitocybe
(up to 0.43%) [126,127]. Typical muscarinic syndrome within 15–30 min of
ingestion include combinations of nausea, vomiting, diarrhea, abdominal pain,
hypersalivation, diaphoresis, tachycardia, bradycardia, hypotension, lacrima-
tion, blurred vision, miosis, tremor, restlessness, and syncope [126]. Death can
be avoided completely by prompt diagnosis and treatment with atropine [128].
Other highly toxic alkaloids are amanitins. The fatal dose for humans is about
0.1 mg/kg [123]. An excellent review describing the unusual features associated
with A. muscaria and A. pantherina species, their active components and toxins,
was provided by Michelot and Melendez-Howell [7].
Ligands for the 5-HT2A receptor may be extremely useful tools for future cogni-
tive neuroscience research [129]. The indole nucleus is an important element of
many natural and synthetic molecules with significant biological activity. This
double ring system contains seven positions that are open to chemical modifica-
tion. However, the majority of medicinal chemists are focused mainly on modi-
fication of the 4- and 5-positions. It has been shown that modification of either
the 6- or 7-positions significantly reduces the psychoactive effects of the result-
ing substance [130]. Many antimigraine drugs (e.g., almotriptan, zolmitriptan)
are the indolealkylamine derivatives [131].
162 Studies in Natural Products Chemistry
Pure synthetic psilocybin (Indocybin® Sandoz) has already been used and
marketed for experimental and psychotherapeutic purposes in the 1960s [132].
Psilocybin as the 5-HT agonist is useful in studying the neurobiological basis
of cognition and consciousness [93]. It is a valuable tool in the analysis of se-
rotonin–dopamine interactions in acute psychotic states [133]. There are some
works suggesting that psilocybin and other hallucinogens can reduce obsessive-
compulsive disorder (OCD) symptoms in humans [134,135]. Recently, it has
been shown that 1-methylpsilocin has the potential to evoke psilocybin-like ef-
fects on OCD, but is less likely than psilocybin to provoke unwanted halluci-
nogenic effects if administered at equivalent doses [136]. Psilocybin was also
used to study its usefulness in treatment for anxiety in advanced-stage cancer
patients [137] and for cluster headache [138]. In the latter study, 22 of 26 psi-
locybin users reported that psilocybin aborted attacks. However, participants in
this research were not blind to their treatment, which raised the possibility of
the placebo response. The investigators should take into account the fact that
hallucinogen administration involves unique psychological risks like a “bad
trip” or less common, but very harmful for the drug users, prolonged psychoses.
Psilocin and psilocybin are currently regarded as dangerous drugs. The risks
of hallucinogen administration, safeguards for minimizing these risks and new
perspectives were reviewed by Johnson et al. [139] and Tyls et al. [140].
Naturally occurring isoxazoles were efficiently transformed into various
classes of medicinally important molecules. They were found to inhibit voltage-
gated sodium channels to control pain, enable the construction of tetracycline
antibiotic derivatives, and as a treatment for depression [141]. Muscimol was
shown to be a viable candidate for the transmeningeal pharmacotherapy of
intractable focal epilepsy [142,143].
Also, the lipophilic bioisosteres of muscimol and GABA were synthesized
and signed as a therapeutic agent for the treatment of epilepsy [144]. Muscimol is
widely used as a ligand to probe GABA receptors. The development of muscimol
and related compounds as a GABA agonist has been very recently reviewed by
Johnston (2014) [145]. In 2013, the antiinflammatory activity of muscimol in en-
dotoxemia was revealed [146]. This study in mice showed that muscimol (0.1 mg/
kg) significantly decreased lipopolysaccharide-induced placental inflammation.
Ibotenic acid, the second main Amanita alkaloid, is a powerful neurotoxin
used in many investigations as a “brain–lesion” causing agent [147,148]. Intrahip-
pocampal injection of ibotenic acid causes severe neuronal loss, resulting in learn-
ing and memory deficit. Animal models that are relevant for Alzheimer’s disease-
like neurodegeneration are generated by injection of this excitotoxin [149–151].
Research on hallucinogenic alkaloids have gained noticeable importance and
have been given a prominent position in the field of medicine, both with respect
to their biological activity and role played in the introduction of new drugs.
Bioactive Alkaloids of Hallucinogenic Mushrooms Chapter | 5 163
Among the hallucinogenic alkaloids an important role play tryptophan
indole-based alkaloids (psilocybin, psilocin), which have been identified in a
large number of mushrooms, especially of the genus Psilocybe, but are still
waiting for more control studies to ascertain their therapeutic role in some other
conditions, apart from psychiatry.
Much less research has concerned hallucinogenic substances contained in
mushroom Amanita species (muscimol, ibotenic acid). These isoxazole alka-
loids have a long history for their use as sacraments in religious ceremonies
along with medical and recreational purposes and thus need more attention to
explore their therapeutic role. Although hallucinogenic mushrooms have been
used by people for thousands of years, their intriguing features are still being
discovered and their relevance to medical science is growing. The isoxazole
derivatives are associated with important biological activities in different thera-
peutic areas. Some of them are used as antirheumatic drugs, inhibitors for ul-
cers, or anticonvulsant drugs. There are also trials for using these compounds
as anticancer agents.
However, there is also a “dark side” to hallucinogenic mushroom use; they
are most frequently used by young people, predominantly users of other drugs.
Although it has been difficult to demonstrate the toxic effects of hallucinogenic
mushroom use, it is well-established that such use can induce uncontrolled
action in the user.
Collecting hallucinogenic mushrooms require substantial mycological
knowledge as there are many look-a-likes (little brown mushrooms). Some of
these look-a-likes mushrooms are toxic. In rare cases, when the intake of such
mushrooms has been substantial, flashbacks of adverse experiences have been
reported. For these reasons, and perhaps due to the fact that the use of halluci-
nogenic mushrooms is not uncommon for young people, restrictions have been
ordained in many countries over the usage of these mushrooms.
The research is supported by Wroclaw Research Centre EIT+ under the project “Biotech-
nologies and Advanced Medical Technologies” – BioMed (POIG.01.01.02-02-003/08)
financed from the European Regional Development Fund (Operational Programme Innovative
Economy, 1.1.2).
Milena Oterman is a recipient of a Ph.D. scholarship under a project funded by the
European Social Fund.
[1] G. Derosa, P. Maffioli, Curr. Top. Med. Chem. 14 (2014) 200–206.
[2] P. Kittakoop, C. Mahidol, S. Ruchirawat, Curr. Top. Med. Chem. 14 (2014) 239–252.
[3] N.K. Kaushik, N. Kaushik, P. Attri, N. Kumar, C.H. Kim, A.K. Verma, E.H. Choi, Molecules
18 (2013) 6620–6662.
[4] G. Guzman, Econ. Bot. 62 (2008) 404–412.
164 Studies in Natural Products Chemistry
[5] J.W. Allen, Ethnomycol. J. Sacred Mushroom Stud. 9 (2012) 1–195.
[6] Z. Jin, Nat. Prod. Rep. 30 (2013) 869–915.
[7] D. Michelot, L.M. Melendez-Howell, Mycol. Res. 107 (2003) 131–146.
[8] K. Stebelska, Ther. Drug Monit. 35 (2013) 420–442.
[9] J. Støíbrný, M. Sokol, B. Merová, P. Ondra, Int. J. Legal Med. 126 (2012) 519–524.
[10] K. Tsujikawa, H. Mohri, K. Kuwayama, H. Miyaguchi, Y. Iwata, A. Gohda, S. Fukushima,
H. Inoue, T. Kishi, Forensic Sci. Int. 164 (2006) 172–178.
[11] L. Satora, D. Pach, B. Butryn, P. Hydzik, B. Balicka-Slusarczyk, Toxicon 45 (2005)
[12] P. Ginterová, B. Sokolová, P. Ondra, J. Znaleziona, J. Petr, J. Ševcˇík, V. Maier, Talanta 125
(2014) 242–247.
[13] Z. Bikadi, M. Simonyi, Curr. Med. Chem. 10 (2003) 2611–2620.
[14] N. Anastos, S.W. Lewis, N.W. Barnett, D. Sims, J. Forensic Sci. 51 (2006) 45–51.
[15] F. Tylš, T. Pálenícˇek, J. Horácˇek, Eur. Neuropsychopharmacol. 24 (2014) 342–356.
[16] G. Sticht, H. Kaferstein, Forensic Sci. Int. 113 (2000) 403–407.
[17] G. Guzmán, J.W. Allen, J. Gartz, Ann. Mus. Civ. Rovereto. 14 (1998) 189–280.
[18] A.A. Franke, L.J. Custer, L.R. Wilkens, L. Le Marchand, A.M.Y. Nomura, M.T. Goodman,
L.N. Kolonel, J. Chromatogr. B 777 (2002) 45–59.
[19] Z.A. Mahmood, S.W. Ahmed, I. Azhar, M. Sualeh, M.T. Baig, S.M.S. Zoha, Pak. J. Pharm.
Sci. 23 (2010) 349–357.
[20] N. Jensen, J. Gartz, H. Laatsch, Planta Med. 72 (2006) 665–666.
[21] R. Martin, J. Schuerenkamp, A. Gasse, H. Pfeiffer, H. Koehler, Int. J. Legal Med. 127 (2013)
[22] C. Andersson, J. Kristinsson, J. Gry, Occurrence and Use of Hallucinogenic Mushrooms
Containing Psilocybin Alkaloids, Nordic Council of Ministers, Copenhagen (2009).
[23] A.Y. Leung, A.G. Paul, J. Pharm. Sci. 56 (1967) 146.
[24] A.Y. Leung, A.G. Paul, J. Pharm. Sci. 57 (1968) 1667–1671.
[25] R.A. Glennon, P.K. Gessner, D.D. Godse, B.J. Kline, J. Med. Chem. 22 (1979) 1414–1416.
[26] K. Tsujikawa, K. Kuwayama, H. Miyaguchi, T. Kanamori, Y. Iwata, H. Inoue, T. Yoshida, T.
Kishi, J. Chromatogr. B 852 (2007) 430–435.
[27] N. Yoshioka, S. Akamatsu, T. Mitsuhashi, C. Todo, M. Asano, Y. Ueno, Forensic Toxicol. 32
(2014) 89–96.
[28] M.C. Gennaro, D. Giacosa, E. Gioannini, S. Angelino, J. Liq. Chromatogr. Rel. Technol. 20
(1997) 413–424.
[29] S. Pedersen-Bjergaard, E. Sannes, K.E. Rasmussen, F. Tønnesen, J. Chromatogr. B Biomed.
Appl. 694 (1997) 375–381.
[30] T. Laussmann, S. Meier-Giebing, Forensic Sci. Int. 195 (2010) 160–164.
[31] K. Gonmori, K. Hasegawa, H. Fujita, Y. Kamijo, H. Nozawa, I. Yamagishi, K. Minakata, K.
Watanabe, O. Suzuki, Forensic Toxicol. 30 (2012) 168–172.
[32] K. Saito, T. Toyo’oka, T. Fukushima, M. Kato, O. Shirota, Y. Goda, Anal. Chim. Acta 527
(2004) 149–156.
[33] R. Kikura-Hanajiri, M. Hayashi, K. Saisho, Y. Goda, J. Chromatogr. B 825 (2005) 29–37.
[34] K. Saito, T. Toyo’oka, M. Kato, T. Fukushima, O. Shirota, Y. Goda, Talanta 66 (2005)
[35] K. Tsujikawa, T. Kanamori, Y. Iwata, Y. Ohmae, R. Sugita, H. Inoue, T. Kishi, Forensic Sci.
Int. 138 (2003) 85–90.
[36] T. Keller, A. Keller, E. Tutsch-Bauer, F. Monticelli, Forensic Sci. Int. 161 (2006) 130–140.
[37] F. Musshoff, B. Madea, J. Beike, Forensic Sci. Int. 113 (2000) 389–395.
Bioactive Alkaloids of Hallucinogenic Mushrooms Chapter | 5 165
[38] J.L.M. Mohammad Sarwar, Microgram J. 1 (2003) 177–183.
[39] R.G. Benedict, V.E. Tyler, L.R. Brady, Lloydia 29 (1966) 333–342.
[40] K. Tsunoda, N. Inoue, Y. Aoyagi, T. Sugahara, J Food Hyg. Soc. Jpn. 34 (1993) 18–24.
[41] T. Kamata, M. Nishikawa, M. Katagi, H. Tsuchihashi, Forensic Toxicol. 24 (2006)
[42] H. Lindenblatt, E. Kramer, P. Holzmann-Erens, E. Gouzoulis-Mayfrank, K.A. Kovar, J.
Chromatogr. B 709 (1998) 255–263.
[43] F. Hasler, D. Bourquin, R. Brenneisen, F.X. Vollenweider, J. Pharm. Biomed. Anal. 30
(2002) 331–339.
[44] M.J. Bogusz, J. Chromatogr. B 748 (2000) 3–19.
[45] A.F. Grieshaber, K.A. Moore, B. Levine, J. Forensic Sci. 46 (2001) 627–630.
[46] A.A. Elian, J. Hackett, M.J. Telepchak, LC GC North Am. 29 (2011) 854.
[47] M.D.M.R. Fernandez, M. Laloup, M. Wood, G. De Boeck, M. Lopez-Rivadulla, P.
Wallemacq, N. Samyn, J. Anal. Toxicol. 31 (2007) 497–504.
[48] A. Alnajjar, A.M. Idris, M. Multzenberg, B. McCord, J. Chromatogr. B 856 (2007) 62–67.
[49] K. Hasegawa, K. Gonmori, H. Fujita, Y. Kamijo, H. Nozawa, I. Yamagishi, K. Minakata, K.
Watanabe, O. Suzuki, Forensic Toxicol. 31 (2013) 322–327.
[50] J. Karkkainen, M. Raisanen, M.O. Huttunen, E. Kallio, H. Naukkarinen, M. Virkkunen,
Psychiatry Res. 58 (1995) 145–152.
[51] J.B. Chen, M.J. Li, X.T. Yan, E. Wu, H. Zhu, K.J. Lee, V.M. Chu, L.F. Zhan, W. Lee, J.S.
Kang, J. Chromatogr. B 879 (2011) 2669–2672.
[52] C. Albers, H. Kohler, M. Lehr, B. Brinkmann, J. Beike, Int. J. Legal Med. 118 (2004)
[53] J. Chen, M. Li, X. Yan, E. Wu, H. Zhu, K.J. Lee, V.M. Chu, L. Zhan, W. Lee, J.S. Kang, J.
Chromatogr. B Analyt. Technol. Biomed. Life Sci. 879 (2011) 2669–2672.
[54] K. Bjoernstad, O. Beck, A. Helander, J. Chromatogr. B 877 (2009) 1162–1168.
[55] R. Martin, J. Schuerenkamp, H. Pfeiffer, H. Koehler, Int. J. Legal Med. 126 (2012) 845–849.
[56] B.S.P. Ginterová, P. Ondra, J. Znaleziona, J. Petra, J. Ševcˇík, V. Maiera, Talanta 125 (2014)
[57] T. Kamata, M. Katagi, H. Tsuchihashi, Forensic Toxicol. 28 (2010) 1–8.
[58] H.W. Chen, Z.L. Fang, Anal. Chim. Acta 355 (1997) 135–143.
[59] W. Wei-Wei, Aspects of Secondary Metabolism in Basidiomycetes, Department of Botany,
National Taiwan University, Taipei City, Taiwan, 1977.
[60] S. Agurell, J. Lars, G. Nilsson, Acta Chem. Scand. 22 (1968) 1210.
[61] A. Brack, H. Kobel, F. Kalberer, A. Hofmann, J. Rutschmann, Arch. Pharm. Berichte Deut.
Pharm. Gesselschaft 294 (1961) 230.
[62] A. Hofmann, R. Heim, A. Brack, H. Kobel, A. Frey, H. Ott, T. Petrzilka, F. Troxler, Helv.
Chim. Acta 42 (1959) 1557.
[63] S. Agurell, J.L. Nilsson, Acta Chem. Scand. 22 (1968) 1210–1218.
[64] S. Agurell, S. Blomkvis, P. Catalfor, Acta Pharm. Suec. 3 (1966) 37.
[65] A.Y. Leung, A.G. Paul, J. Pharm. Sci. 57 (1968) 1667–1671.
[66] W.S. Chilton, J. Bigwood, R.E. Jensen, J. Psychedelic Drugs 11 (1979) 61–69.
[67] J. Gartz, J. Basic Microbiol. 29 (1989) 347–352.
[68] J. Gartz, Planta Med. 55 (1989) 249–250.
[69] D.B. Repke, D.T. Leslie, G. Guzman, J. Nat. Prod. 40 (1977) 566–578.
[70] W.E. Fantegrossi, K.S. Murnane, C.J. Reissig, Biochem. Pharmacol. 75 (2008) 17–33.
[71] A.L. Halberstadt, M.A. Geyer, Neuropharmacology 61 (2011) 364–381.
[72] F.C. Colpaert, P.A.J. Janssen, Neuropharmacology 22 (1983) 993–1000.
166 Studies in Natural Products Chemistry
[73] R.A. Glennon, R. Young, J.A. Rosecrans, Eur. J. Pharmacol. 91 (1983) 189–196.
[74] M. Titeler, R.A. Lyon, R.A. Glennon, Psychopharmacology 94 (1988) 213–216.
[75] B. Sadzot, J.M. Baraban, R.A. Glennon, R.A. Lyon, S. Leonhardt, C.R. Jan, M. Titeler,
Psychopharmacology 98 (1989) 495–499.
[76] F.X. Vollenweider, M.F.I. Vollenweider-Scherpenhuyzen, A. Babler, H. Vogel, D. Hell, Neu-
roreport 9 (1998) 3897–3902.
[77] O.L. Carter, F. Hasler, J.D. Pettigrew, G.M. Wallis, G.B. Liu, F.X. Vollenweider, Psycho-
pharmacology 195 (2007) 415–424.
[78] D.J. Mckenna, D.B. Repke, L. Lo, S.J. Peroutka, Neuropharmacology 29 (1990) 193–198.
[79] J.B. Blair, D. Kurrasch-Orbaugh, D. Marona-Lewicka, M.G. Cumbay, V.J. Watts, E.L. Bark-
er, D.E. Nichols, J. Med. Chem. 43 (2000) 4701–4710.
[80] A.V. Deliganis, P.A. Pierce, S.J. Peroutka, Biochem. Pharmacol. 41 (1991) 1739–1744.
[81] J.C. Winter, R.A. Filipink, D. Timineri, S.E. Helsley, R.A. Rabin, Pharmacol. Biochem.
Behav. 65 (2000) 75–82.
[82] A.J. Sleight, N.J. Stam, V. Mutel, P.M.L. Vanderheyden, Biochem. Pharmacol. 51 (1996)
[83] M.W. Johnson, W.A. Richards, R.R. Griffiths, J. Psychopharm. 22 (2008) 603–620.
[84] M. Kometer, A. Schmidt, L. Jancke, F.X. Vollenweider, J. Neurosci. 33 (2013) 10544–
[85] M. Filip, M. Bader, Pharmacol. Rep. 61 (2009) 761–777.
[86] R.S. Gable, Addiction 99 (2004) 686–696.
[87] J. van Amsterdam, A. Opperhuizen, W. van den Brink, Regul. Toxicol. Pharm. 59 (2011)
[88] A. Horita, L.J. Weber, Biochem. Pharmacol. 7 (1961) 47.
[89] F. Hasler, D. Bourquin, R. Brenneisen, T. Bar, F.X. Vollenweider, Pharm. Acta Helv. 72
(1997) 175–184.
[90] K. Eivindvik, K.E. Rasmussen, R.B. Sund, Acta Pharm. Nord. 1 (1989) 295–302.
[91] F. Hasler, Untersuchungen zur Humanpharmako- kinetik von Psilocybin, University of
Bern, Bern, 1997.
[92] K. Saito, T. Toyo’oka, T. Fukushima, M. Kato, O. Shirota, Y. Goda, Anal. Chim. Acta 527
(2004) 149–156.
[93] F. Hasler, U. Grimberg, M.A. Benz, T. Huber, F.X. Vollenweider, Psychopharmacology
(Berl.) 172 (2004) 145–156.
[94] A. Shulgin, Ann. Shulgin, TiHKAL: The Continuation, Transform Press, Berkeley, 1997.
[95] T.H. Lim, C.A. Wasywich, P.N. Ruygrok, Intern. Med. J. 42 (2012) 1268–1269.
[96] H.M. Nef, H. Mollmann, P. Hilpert, N. Krause, C. Troidl, M. Weber, A. Rolf, T. Dill, C.
Hamm, A. Elsasser, Int. J. Cardiol. 134 (2009) E39–E41.
[97] O. Beck, A. Helander, C. Karlson-Stiber, N. Stephansson, J. Anal. Toxicol. 22 (1998) 45–49.
[98] R.L. Carhart-Harris, D. Erritzoe, T. Williams, J.M. Stone, L.J. Reed, A. Colasanti, R.J. Ty-
acke, R. Leech, A.L. Malizia, K. Murphy, P. Hobden, J. Evans, A. Feilding, R.G. Wise, D.J.
Nutt, Proc. Natl. Acad. Sci. USA 109 (2012) 2138–2143.
[99] J.L. Scruggs, D. Schmidt, A.Y. Deutch, Neurosci. Lett. 346 (2003) 137–140.
[100] Q.J. Zhang, S. Wang, J. Liu, U. Ali, Z.H. Gui, Z.H. Wu, Y.P. Hui, Y. Wang, L. Chen, Brain
Res. 1312 (2010) 127–137.
[101] K.W. Kalberger, F. Rutschmann, J. Biochem. Pharmacol. 11 (1962) 261–269.
[102] N. Manevski, M. Kurkela, C. Hoglund, T. Mauriala, M.H. Court, J. Yli-Kauhaluoma, M.
Finel, Drug Metab. Disposition 38 (2010) 386–395.
[103] M.C. McBride, J. Psychoactive Drugs 32 (2000) 321–331.
Bioactive Alkaloids of Hallucinogenic Mushrooms Chapter | 5 167
[104] R.W. Fuller, H.D. Snoddy, K.W. Perry, Neuropharmacology 34 (1995) 799–804.
[105] S.J. Corne, W. Pickerin, Psychopharmacologia 11 (1967) 65–65.
[106] R.M. Bilder, Arch. Clin. Neuropsychol. 28 (2013) 511–512.
[107] H.D. Fabing, J.R. Hawkins, Science 123 (1956) 886–887.
[108] W.J. Turner, S. Merlis, Arch. Neurol. Psychiatry 81 (1959) 121–129.
[109] E. Emanuele, R. Colombo, V. Martinelli, N. Brondino, M. Marini, M. Boso, F. Barale, P.
Politi, Neuroendocrinol. Lett. 31 (2010) 117–121.
[110] N. Takeda, R. Ikeda, K. Ohba, M. Kondo, Neuroreport 6 (1995) 2378–2380.
[111] D.R. Curtis, D. Lodge, H. McLennan, J. Physiol. 291 (1979) 19–28.
[112] P.G. Waser, Psychopharmacol. Bull. 4 (1967) 19–20.
[113] Y. Matsui, T. Kamioka, J. Pharm. Pharmacol. 31 (1979) 427–428.
[114] W. Theobald, O. Buch, H.A. Kunz, P. Krupp, E.G. Stenger, H. Heimann, Arzneimittelforsc-
hung 18 (1968) 311–315.
[115] T.A. Smith, Br. J. Biomed. Sci. 58 (2001) 111–121.
[116] S.M.J. Dunn, R.P. Thuynsma, Biochemistry 33 (1994) 755–763.
[117] M.C. Graham, A.R. Johnston, Jane R. Hanrahan, Kenneth N. Mewett, Curr. Drug Targets –
CNS Neurol. Disord. 2 (2003) 260–268.
[118] D. Chandra, L.M. Halonen, A.M. Linden, C. Procaccini, K. Hellsten, G.E. Homanics, E.R.
Korpi, Neuropsychopharmacology 35 (2010) 999–1007.
[119] M. Baraldi, L. Grandison, A. Guidotti, Neuropharmacology 18 (1979) 57–62.
[120] P. Krogsgaardlarsen, G.A.R. Johnston, J. Neurochem. 25 (1975) 797–802.
[121] P. Krogsgaard-Larsen, B. Frolund, K. Frydenvang, Curr. Pharm. Des. 6 (2000) 1193–1209.
[122] L.J. Fowler, D.H. Lovell, R.A. John, J. Neurochem. 41 (1983) 1751–1754.
[123] P. Patnaik, A Comprehensive Guide to the Hazardous Properties of Chemical Substances,
Wiley and Sons, New York, (2007).
[124] M. Lukasik-Glebocka, A. Druzdz, M. Naskret, Przegl. Lek. 68 (2011) 449–452.
[125] A.M. Watanabe, B.B. Katzung B.G. Katzung (Ed.), Základní a klinická farmakologie: H and
H, Brno and 1992, p. 83.
[126] Y. Lurie, S.P. Wasser, M. Taha, H. Shehade, J. Nijim, Y. Hoffmann, F. Basis, M. Vardi, O.
Lavon, S. Suaed, B. Bisharat, Y. Bentur, Clin. Toxicol. (Phila.) 47 (2009) 562–565.
[127] K. Genest, D.W. Hughes, W.B. Rice, J. Pharm. Sci. 57 (1968) 331–333.
[128] D.R. Benjamin, Mushrooms, Poisons, and Panaceas: A Handbook for Naturalists, Mycolo-
gists, and Physicians, W. H. Freeman and Co, (1995).
[129] D.E. Nichols, Pharmacol. Ther. 101 (2004) 131–181.
[130] W.E. Fantegrossi, K.S. Murnane, C.J. Reissig, Biochem. Pharmacol. 75 (2008) 17–33.
[131] A.M. Yu, AAPS J. 10 (2008) 242–253.
[132] T. Passie, J. Seifert, U. Schneider, H.M. Emrich, Addict. Biol. 7 (2002) 357–364.
[133] F.X. Vollenweider, P. Vontobel, D. Hell, K.L. Leenders, Neuropsychopharmacology 20
(1999) 424–433.
[134] F.A. Moreno, P.L. Delgado, Am. J. Psychiatry 154 (1997) 1037–1038.
[135] F.A. Moreno, C.B. Wiegand, E.K. Taitano, P.L. Delgado, J. Clin. Psychiatry 67 (2006)
[136] A.L. Halberstadt, L. Koedood, S.B. Powell, M.A. Geyer, J. Psychopharmacol. 25 (2011)
[137] C.S. Grob, A.L. Danforth, G.S. Chopra, M. Hagerty, C.R. McKay, A.L. Halberstadt, G.R.
Greer, Arch. Gen. Psychiatry 68 (2011) 71–78.
[138] R.A. Sewell, J.H. Halpern, H.G. Pope Jr., Neurology 66 (2006) 1920–1922.
[139] M. Johnson, W. Richards, R. Griffiths, J. Psychopharmacol. 22 (2008) 603–620.
168 Studies in Natural Products Chemistry
[140] F. Tyls, T. Palenicek, J. Horacek, Eur. Neuropsychopharmacol. 24 (2014) 342–356.
[141] P. Jayaroopa, K.A. Kumar, IJPCBS 3 (2013) 294–304.
[142] R.C. Collins, Neurology 30 (1980) 575–581.
[143] N. Ludvig, S.L. Baptiste, H.M. Tang, G. Medveczky, H. von Gizycki, J. Charchaflieh, O.
Devinsky, R.I. Kuzniecky, Epilepsia 50 (2009) 678–693.
[144] P. Krogsgaard-Larsen, B. Frolund, K. Frydenvang, Curr. Pharm. Des. 6 (2000) 1193–1209.
[145] G.A. Johnston, Neurochem. Res. 39 (2014) 1942–1947.
[146] M.H. Gharedaghi, M. Javadi-Paydar, Y. Yousefzadeh-Fard, M. Salehi-Sadaghiani, P. Java-
dian, N. Fakhraei, S.M. Tavangar, A.R. Dehpour, J. Matern. Fetal Neonatal Med. 26 (2013)
[147] J.S. Durmer, A.C. Rosenquist, Neuroscience 106 (2001) 765–781.
[148] R. Faipoux, O. Rampin, N. Darcel, S. Gougis, D.W. Gietzen, D. Tome, G. Fromentin,
FASEB J. 20 (2006) A178-A178.
[149] S. Eleuteri, B. Monti, S. Brignani, A. Contestabile, Neurotox. Res. 15 (2009) 127–132.
[150] H.J. Lee, I.J. Lim, S.W. Park, Y.B. Kim, Y. Ko, S.U. Kim, Cell Transplant. 21 (2012)
[151] J.Y. Zhang, P. Li, Y.P. Wang, J.X. Liu, Z.J. Zhang, W.D. Cheng, Y.Y. Wang, PLoS One 8
... Undoubtedly, indole-type alkaloids, such as psilocybin, harmine (i.e., ayahuasca alkaloids), and dimethyltryptamine (DMT) among others, have become the epicenter of this recent psychedelic-medicinal wave, as it is discernible in the rising number of publications, research programs, and clinical trials involving these metabolites [16,18,19]. In fact, mushroom-derived indole alkaloids have demonstrated an extraordinary therapeutic potential for neuropsychiatric conditions [20,21]. Unfortunately, the chemical and pharmacological studies of these alkaloids have been overshadowed for decades due to their illegal and psychotropic connotation, hampering the discovery of potential bioactive natural alkaloids derived from magic fungi. ...
... Hence, the legislative actions adopted by the governments truncated the progress accomplished in the elucidation of the therapeutical potential of natural psychedelics, and most of the research in this field conducted during the 70s and 80s was kept hidden [34]. Despite the diplomatic conflict and after decades of latency, the research in psychedelics entered a stage of a renaissance in the early 21st century as a result of the persistent interest in their pharmacological potential by some medical-psychiatric scientists and private research institutions [20,21]. In this context, one of the flagship projects that has driven this renewal is the magic mushrooms study at the Johns Hopkins Center, showing the high safety and efficiency of psilocybin for challenging mental disorders [36]. ...
... Psilocin is the active metabolite, responsible for the hallucinogenic effects whereas psilocybin can act as a prodrug and it is converted into the active form after enzymatic dephosphorylation in vivo [55]. Both psilocybin and psilocin are chemically unstable; however, it is believed that the phosphate group on psilocybin also improves the stability of the active metabolite, preventing degradation in the mushrooms, biomass, and extracts [20,56]. Some procedures and solvents favor the phosphate ester hydrolyzation in psilocybin, and the subsequent enzymatic oxidation of psilocin leading the develop of the characteristic blue colorations in the mushroom extracts by formation of quinoid oligomers [57,58]. ...
Full-text available
Neuropsychiatric diseases such as depression, anxiety, and post-traumatic stress represent a substantial long-term challenge for the global health systems because of their rising prevalence, uncertain neuropathology, and lack of effective pharmacological treatments. The approved existing studies constitute a piece of strong evidence whereby psychiatric drugs have shown to have unpleasant side effects and reduction of sustained tolerability, impacting patients’ quality of life. Thus, the implementation of innovative strategies and alternative sources of bioactive molecules for the search for neuropsychiatric agents are required to guarantee the success of more effective drug candidates. Psychotherapeutic use of indole alkaloids derived from magic mushrooms has shown great interest and potential as an alternative to the synthetic drugs currently used on the market. The focus on indole alkaloids is linked to their rich history, their use as pharmaceuticals, and their broad range of biological properties, collectively underscoring the indole heterocycle as significant in drug discovery. In this review, we aim to report the physicochemical and pharmacological characteristics of indole alkaloids, particularly those derived from magic mushrooms, highlighting the promising application of such active ingredients as safe and effective therapeutic agents for the treatment of neuropsychiatric disorders.
... Generally, 100 g of dried Amanita muscaria contains about 150 mg of MUS and 30 mg of IBO; therefore, 10 g of dried mushrooms can evoke a psychedelic effect. The toxicity of MUS-LD 50 values in mice is 2.5 mg/kg [4]. Their psychotropic properties result from their ability to activate the N-methyl-d-aspartate glutamate and GABA receptors [5]. ...
... Muscimol (pKa: 4.8 and 8.4 [1,43]) was purchased from Abcam (Cambridge, UK). Psilocin (pKa 9.41 [4]) was from Sigma-Aldrich (Poznań, Poland). The chemical structures of the investigated analytes are shown in Figure 5. ...
Full-text available
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.
... Numerous studies have been performed on the isolation and chemical and biological characterization, and these studies are still increasing. These investigations have also confirmed that families such as alkaloids [1], peptides [2], phenoxazines [3], amines [4], or nitrogenous sesquiterpenoids [5] could show outstanding activities of pharmacological or agronomic interest. The alkaloid family is one of the most relevant of these, given its production by a wide range of living beings, its structural variety, as well as the biological activities that have been discovered a long time ago. ...
Full-text available
Alkaloids are a wide family of basic N-containing natural products, whose research has revealed bioactive compounds of pharmacological interest. Studies on these compounds have focused more attention on those produced by plants, although other types of organisms have also been proven to synthesize bioactive alkaloids, such as animals, marine organisms, bacteria, and fungi. This review covers the findings of the last 20 years (2002–2022) related to the isolation, structures, and biological activities of the alkaloids produced by mushrooms, a fungal subgroup, and their potential to develop drugs and agrochemicals. In some cases, the synthesis of the reviewed compounds and structure−activity relationship studies have been described.
... The sample was pink oyster mushroom combined with two drops of H 2 SO 4 , Wagner's reagent was added into the sample. The presence of alkaloids is characterized by the formation of white deposits by Mayer's reagent, red deposits by Dragendorff's reagent, and brown deposits by Wagner's reagents (Wieczorek et al., 2015). ...
Full-text available
The quality of mother culture is one of Oyster Mushroom cultivation's essential factors as it determines the quality and quantity of the crop. Potato Dextrose Agar (PDA) is the most common media for making the mother cultures of edible mushrooms; therefore, it is essential to find alternative media to increase crop yields. This research aims to find out the most effective alternative media of mother of pink oyster mushroom (Pleurotus flabellatus) and the effect on the phytochemical characteristic. The research method used was experimental research, a completely randomized single factor experiment, with four treatments and six repetitions: PDA (Potato Dextrose Agar), CDA (Cron dextrose Agar), GBDA (Green bean dextrose Agar), SDA (Soy dextrose Agar). The Agar medium was used to measure pink oyster growth, while broth media were used to determine the biomass weight and the phytochemical compound. The research results showed that the diameter on the CDA medium had the most extensive results among all. Biomass test results showed that the most effective media to obtain biomass were the CDB and GBDB medium. The phytochemical results shows were alkaloids and saponins, while steroids and tannins were not detected in all samples.
... In past few years, mushroom bioactive components and its health effects were reviewed. For instance, Mingyi et al. (2019) reviewed mushroom polysaccharides and its health effects, Wieczorek et al. (2015) reviewed mushroom alkaloids and its health effects. However, realizing the role of mushroom polyphenols as an active health ingredient, a comprehensive discussion on the functional activities of mushroom polyphenols is lacking. ...
Phenolic compounds are minor metabolites usually present in mushroom species. Because of their potential advantages for human health, such as antioxidant and other biological activities, these bioactive components have been gaining more interest as functional foods, nutraceutical agents for providing better health conditions. This review aims to comprehensively discuss the recent advances in mushroom phenolic compounds, including new sources, structural characteristics, biological activities, potential uses and its industrial applications as well as the future perspectives. Phenolic acids as well as flavonoids are considered the most common phenolics occurring in mushroom species. These are responsible for its bioactivities, including antioxidant, anti-inflammatory, antitumor, antihyperglycaemic, antiosteoporotic, anti-tyrosinase and antimicrobial activities. Several edible mushroom species with good phenolic content and show higher biological activity were highlighted, in a way for its futuristic applications. Trends on mushroom research highlighting new research areas, such as nanoformulation were discussed. Furthermore, the use of phenolic compounds as nutraceutical and cosmeceutical agents as well as the future perspectives and recommendations were made.
... However, gas chromatography is not recommended for the quantification of tryptamines because of their poor volatility and heat lability. [28][29][30] In our study, ultra-high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) was employed to investigate the stability of five tryptamine alkaloids in the biomass of cultivated fruiting bodies of the psychoactive species Psilocybe cubensis. [31][32][33] 2 | MATERIALS AND METHODS ...
Psilocybin, psilocin, baeocystin, norbaeocystin, and aeruginascin are tryptamines structurally similar to the neurotransmitter serotonin. Psilocybin and its pharmacologically active metabolite psilocin in particular are known for their psychoactive effects. These substances typically occur in most species of the genus Psilocybe (Fungi, Strophariaceae). Even the sclerotia of some of these fungi known as “magic truffles” are of growing interest in microdosing due to them improving cognitive function studies. In addition to microdosing studies, psilocybin has also been applied in clinical studies, but only its pure form has been administrated so far. Moreover, the determination of tryptamine alkaloids is used in forensic analysis. In this study, freshly cultivated fruit bodies of Psilocybe cubensis were used for monitoring stability (including storage and processing conditions of fruiting bodies). Furthermore, mycelium and the individual parts of the fruiting bodies (caps, stipes, and basidiospores) were also examined. The concentration of tryptamines in final extracts was analyzed using ultra-high-performance liquid chromatography coupled with mass spectrometry. No tryptamines were detected in the basidiospores, and only psilocin was present at 0.47 wt.% in the mycelium. The stipes contained approximately half the amount of tryptamine alkaloids (0.52 wt.%) than the caps (1.03 wt.%); however, these results were not statistically significant, as the concentration of tryptamines in individual fruiting bodies is highly variable. The storage conditions showed that the highest degradation of tryptamines was seen in fresh mushrooms stored at −80°C, and the lowest decay was seen in dried biomass stored in the dark at room temperature.
... In recent years, a number of alkaloids have been discovered in Basidiomycota; fungi produce natural alkaloids, the most studied groups are the indoles and isoxazoles, two simple indole alkaloids: psilocin (3-[2 (dimethylamino) ethyl] -4-indolol) and psilocybin ([3-(2-dimethylaminoethyl) -1H-indol4-yl] dihydrogen phosphate) are present in many mushroom species (Wieczorek et al., 2015;Sandargo, 2019). Other isolated alkaloids are the Erinacerines commonly isolated from both the cultures and fruiting bodies of Hericium erinaceus (Bull.) ...
Full-text available
The discharge of industrial and urban wastewater without prior treatment is a growing problem in Paraguay. The search of technologies to reduce the environmental impacts generated by this problem, is an area of interest in recent times. Phytoremediation is a remediation alternative that proposes the application of artificial or constructed wetlands where a complex system of plants, microorganisms, and substrate act together to remove contaminants. The objective of this work was to describe the performance and efficiency of two macrophytes in constructed wetlands of horizontal subsurface flow for the treatment of urban wastewater in the city of San Lorenzo. A septic tank was used as primary treatment, followed by two wetlands, one with Cyperus giganteus and other with Typha dominguensis. The results of water analyzes show that both wetlands built at pilot scale proved to be effective for the treatment used, demonstrating a significant reduction for each parameter studied with respect tothe entrance to the system. The removal (%) of ammoniacal nitrogen was 42% (CG) and 65% (TD); for total phosphorus it was 45% (CG) and 58% (TD), the biochemical oxygen demand was 64% (CG) and 81% (TD), while for chemical oxygen demand efficiency it was 40% (GC) and 61% (TD), respectively.
The prospection of unusual sources and undiscovered habitats is valuable in natural product research. Indeed, the fungi kingdom has received special attention since its ability to produce novel and intriguing secondary metabolites with various biological uses. Among secondary metabolites, alkaloid-derived structures present a wide range of bioactivities, including antineurodegenerative, antidepressive, anxiolytic, anti-inflammatory, cytotoxic, and insecticidal properties. Furthermore, various studies showed particular properties of those alkaloids in reducing nicotine addiction and alcohol dependence. Alkaloids are categorized into several groups based on their heterocyclic ring system and biosynthetic precursor, such as indole, isoxazoles, and muscarine. Therefore, this chapter focuses on those fungi’s bioactive alkaloids with emphasis on pharmacokinetics as well as the current analytical approaches for extraction and compound identification. Furthermore, the main biological activities and action mechanisms of these fungus alkaloids will also be discussed.
Fungi are eukaryotic organisms that can produce a wide range of secondary metabolites with a significant impact on society. Some metabolites are exploited for their activity as antioxidant, anti-inflammatory, antitumor, and anti-microbial agents, and in the production of cancer vaccines, among other pharmaceutical applications. Since the discovery of penicillin, the pharmaceutical industry has been greatly interested in fungi as sources of natural bioactive compounds, and fungi metabolites have made an indispensable contribution to improving human and animal health throughout the last decades. Starting with the development of antibiotics, the pharmaceutical industry has increasingly turned to these compounds for a variety of applications. The increase in the number of patents registered worldwide is a strong indicator that the market realizes the great potential of fungi secondary metabolites. In general, the pharmaceutical industry trend is centered on adopting different strategies to discover new drugs, and fungi secondary metabolites are viewed as having significant potential. This chapter explores the current pharmaceutical applications of secondary metabolites found in fungi. Initially, the most recent mushroom studies and their commercial pharmaceutical and cosmeceutical applications are explored. An overview of the different classes of fungi secondary metabolites with biologically relevant activities is then presented. Recently marine fungi were found to be a rich source of secondary metabolites. Due to the recent relevancy of marine fungi, an overview of marine fungi secondary metabolites with relevant pharmaceutical-related activities is also presented. Finally, the potential of fungi metabolites as a source of natural pigments and the methodologies used to characterize and explore fungi secondary metabolites are also analyzed.
Development of rapid and reliable immunochemical methods for monitoring psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine; Pyb) and psilocin (dephosporylated metabolite; Psi), the psychoactive compounds contained within hallucinogenic mushrooms (magic mushrooms), is desirable in order to identify these mushrooms and regulate their illicit use. Because no antibody was publicly available for this purpose, we generated two independent monoclonal antibodies (mAbs) against Pyb or Psi, and then developed enzyme-linked immunosorbent assays (ELISAs) by using them. To generate the specific antibodies, novel immunogenic conjugates were prepared by linking Pyb or Psi molecules to carrier proteins by modifying their 2-(N,N-dimethylamino)ethyl side chains. Spleen cells from mice immunized with these conjugates were fused with P3/NS1/1-Ag4-1 myeloma cells, and hybridoma clones secreting anti-Pyb and anti-Psi mAbs were established. These mAbs were characterized for their biochemical features and then applied to competitive ELISAs, which used microplates coated with Pyb or Psi linked with albumin. These ELISAs enabled the determination of Pyb or Psi with measurable ranges of ca. 0.20−20 or 0.040−2.0 μg/assay (limit of detection was 0.14 or 0.029 μg/assay), respectively. The related tryptamines were satisfactorily discriminated as exemplified by the cross-reactivity of the ELISA to determine Pyb (or Psi) with Psi (or Pyb) that were found to be 2.8% (or <0.5%), respectively. The Pyb and Psi contents in a dried powder of the hallucinogenic mushroom, Psilocybe cubensis, were determined to be 0.39 and 0.32 (w/w)%, respectively. The ELISAs developed using the current mAbs are promising tools for identifying illegal hallucinogenic mushrooms.
Full-text available
Psilocybin, a psychoactive alkaloid contained in hallucinogenic mushrooms, is nowadays given a lot of attention in the scientific community as a research tool for modeling psychosis as well as due to its potential therapeutic effects. However, it is also a very popular and frequently abused natural hallucinogen. This review summarizes all the past and recent knowledge on psilocybin. It briefly deals with its history, discusses the pharmacokinetics and pharmacodynamics, and compares its action in humans and animals. It attempts to describe the mechanism of psychedelic effects and objectify its action using modern imaging and psychometric methods. Finally, it describes its therapeutic and abuse potential.
Ibotenic acid (IBO) and muscimol (MUS) in the fruit body of Amanita muscaria during the reproduction stage were investigated. The mean levels of IBO and MUS throughout the fruit were x: 343ppm and x: 22ppm, respectively; most was detected in the cap of the fruit (IBO x: 519ppm, MUS x: 30ppm), then in the base (IBO x: 290ppm, MUS x: 20ppm), with the smallest amount in the stalk (IBO x: 253ppm, MUS x: 17ppm). The concentrations of IBO and MUS in the cap decreased gradually after increasing early on, and those in the stalk decreased gradually, where as in the base there was an increase; the levels in the whole body were nearly constant during maturation. Since the changes were similar in lone and colonial mushrooms, difference of mushroom-growing location had no influence on the concentrations of IBO and MUS. Also, difference of size of the fruit body had no influence on the concentrations of IBO and MUS. The large variations of the IBO and MUS contents may depend on individual differences of growth circumstances. Although the fruit body grew to about 6 times the weight of the base during maturation, the concentration of IBO remained nearly constant. Detection of MUS may reflect the enzymatic decarboxylation of IBO before the analysis, since the changes in the concentration of MUS paralleled those of IBO at a lower level.
Throughout an arc described by the islands of the Caribbean, the shores of the Amazon, and as far as the western ridges of the Andes, in Colombia and Peru, there has been used since pre-Columbian times a snuff variously known as cohoba, niopo, parica, and yopo, among other designations.1-11 With minor variations, this snuff is prepared from seeds of Piptadenia peregrina by grinding with calcined clam shells or wood ashes. It is inhaled or is forcibly blown into the nostrils of the person to be intoxicated by it. Descriptions of the action of the snuff vary somewhat, but the one we have previously reported,12 given us by Dr. Jacques Fourcand, is typical. The inhalation of the snuff is quickly followed by rigidity and staring and, at times, a convulsion. This gives way to an excited state, which may last an hour more or less and in which there
A simple and rapid method for the determination of nine mushroom toxins, ibotenic acid, propargylglycine, choline, muscimol, muscarine, α-amanitin, β-amanitin, phalloidin, and phallacidin, in mushroom samples has been developed. Mushroom toxins were extracted with 0.5 % formic acid in methanol/water, purified with Oasis HLB cartridges, and analyzed by liquid chromatography–time-of-flight mass spectrometry. The separation was performed on a pentafluorophenylpropyl column, and the mass spectrometer was operated in positive ion mode with electrospray ionization. Calibration curves were linear over the range of 0.01–1 μg/ml (R 2 = 0.999). The detection limits for the toxins in mushroom samples were 0.0098–4.9 μg/g, which were low enough for the investigation of food poisoning cases. The intraday and interday recoveries for blank mushroom extracts fortified with mushroom toxins were 72.5–107 % and 75.9–108 %, and relative standard deviations were <7.9 and 8.2 %, respectively.
Muscimol, a psychoactive isoxazole from Amanita muscaria and related mushrooms, has proved to be a remarkably selective agonist at ionotropic receptors for the inhibitory neurotransmitter GABA. This historic overview highlights the discovery and development of muscimol and related compounds as a GABA agonist by Danish and Australian neurochemists. Muscimol is widely used as a ligand to probe GABA receptors and was the lead compound in the development of a range of GABAergic agents including nipecotic acid, tiagabine, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, (Gaboxadol(®)) and 4-PIOL.