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Enhancement of Indole Alkaloids Produced by Psilocybe cubensis (Earle)

Singer (Agaricomycetideae) in Controlled Harvesting Light Conditions

Article  in  International Journal of Medicinal Mushrooms · January 2009

DOI: 10.1615/IntJMedMushr.v11.i4.80

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 International Journal of Medicinal Mushrooms
DOI: 10.1615/IntJMedMushr.v11.i4.80
pages 419-426

Enhancement of Indole Alkaloids Produced by Psilocybe

cubensis (Earle) Singer (Agaricomycetideae) in Controlled
Harvesting Light Conditions1
Hussein Riahi (1), Hasan Rafati (2), Ali Mohammadi (1)
1) Department of Biology, Shahid Beheshti University, Evin, Tehran
2) Medicinal Plants and Drugs Research Institute (MPDRI), Shahid Beheshti University, Evin, Tehran

Abstract
Different variables including species, strain, glucose and ammonium succinate concentration in
the growth medium, pH, temperature, timing and oxidation have been accounted for the Psilocin
(PC) and Psilocybin (PB) content in the "Magic Mushrooms"(MMs). These are but some of the
variables in a constellation of factors that complicate consistency in the production of PC and PB.
In an attempt to study the effect of light on chemical constitutions, some samples were kept in
dark, some samples were kept in dim lighting, whereas others were exposed to natural but indirect
light. After picking and drying Mushrooms, a simple one-step extraction involving
homogenization of the dried fruit bodies of fungi in chloroform and derivatization with MSTFA
was performed. The samples were then analyzed by gas chromatography–mass spectrometry. This
investigation shows that the relative PC and PB content of the mushrooms is highly dependent on
the light condition. This variation could amount to 100 fold of active components (PB and PC) in
samples harvested in dark condition compared to the samples harvested in indirect light condition.
Therefore it can be concluded that UV light may have destructive effect on the active components,
which would readily explain why sun-struck collections are less potent

Keywords: Psilocybe cubensis; psilocybin; psilocin; gas chromatography–mass spectrometry

(GC–MS); MSTFA; Psychedelic fungi;

1. Introduction
Psilocybe cubensis is a species of psychedelic mushroom whose primary, pharmacologically active
constituents are Psilocybin (PB) and Psilocin (PC). It belongs to the Strophariaceae family with a
grey to violet-gray color, and bruise bluish/purplish when crushed or dried (Bluing Reaction). The
caps are planar when fully mature, and their gills are adnate (horizontally attached to the stem) to
adnexed (slightly indented at the attachment point) depending on the subspecies. The gills are
closely spaced and drop dark purple spores (1)(Rafati et al., 2009).
The psychedelic effects of some species of the genus Psilocybe were first described by Wasson in
1957 (2). Two hallucinogenic components of the tryptamine type, PB (Psilocybin or 4-
phosphoryloxy-N, N-dimethyltryptamine), the main psychotropic compound, and PC (Psilocin or
4-hydroxy-N, N-dimethyltryptamine) were then isolated by Hofmann and his colleagues (3).They
have structural similarity to the neurotransmitter serotonin, and their highly hallucinogenic potency
is thought to occur from their influence on the serotoninergic nervous system. (4)
The content of active components of the mushrooms have been reported nearly the same in few
references (i.e. 0.75% (14), 0.74% (5)). Different strains have shown a noticeable change in the

1
.Citation: Rafati, H., Riahi, H., Mohammadi, A., 2009. Enhancement of Indole Alkaloids Produced by Psilocybe
cubensis(Earle) Singer (Agaricomycetideae) in Controlled Harvesting Light Conditions. International Journal of
Medicinal Mushrooms 11.

1
content (from 0.48% in Amazonian strains to 0.65% in Mexican strain (1)). Bigwood and Beug
found a fourfold variation in potency in cultivated specimens and up to a tenfold variation
in potency from wild specimens (6). Mushrooms grown indoors seem consistently more
potent than field-collected specimens, probably due to nutritional factors (precursors) and
protection from the damaging effects of ultraviolate radiation (1). Gartz found that raising
tryptamine concentrations of cow dung and rice media by 25 millimolars directly increases
affected the potency of P. cubensis mycelia, specifically in PC content: from 0.09% to
3.3% of the dried mass. (PB content was actually depressed, but not nearly on the same
order of magnitude).

Psilocybin Psilocin

Figure 1. Chemical structures of Psilocin and Psilocybin

Gartz also made the interesting observation in the culturing of P. cubensis mycelium that
the raising of malt sugars to more than 10% resulted in the complete suppression of PB
production (7). These observations underscore that the nutritional content of the substrate
significantly affects potency. Additionally, Gartz found that younger specimens are generally
more potent than mature ones, an observation many users have also made. These are but some of
the variables in a constellation of factors that complicate consistency in the production of PC and
PB.Despite the suggestion by P. Stamets (1) that ultraviolet radiation from the Sun markedly
lessens the potency of PB-containing mushrooms, but we have not found any reporting the effect
of light on the PC/PB content.
The determination of PC and PB has often been studied by thin-layer chromatography (1,5,7,12-13
, 15-17), high performance liquid chromatography (HPLC) with ultraviolet detection (1,5,7,12-
13,18-19), paper chromatography (14) ,electrochemical detection (13,18-19) and fluorescence
detection (19), gas chromatography (GC) with flame ionization detection and infrared
spectrometry (20), ion-mobility spectrometry (21), and gas chromatography-mass spectrometry
(GC-MS) with/without trimethylsilyl derivatization (7-8,15-17 20-21). In this study we have
evaluated the effect of cultivation light on the relative amount of PB/PC using GC-MS technique.

2. Sample Preparation:

The mycelium was grown on pasteurized compost consisted of wheat straw, chicken manure and
gypsum. During the mycelium growth, temperature and relative humidity were maintained at 25 °C
and 95%Rh respectively. After colonization of compost with mycelium, substrate was covered
with casing soil. After 10 days case-run the ambient conditions were changed to initiate
fructification. A temperature of 18-20'C was maintained during harvest period. In an attempt to
study the effect of light on chemical constitutions, some samples were kept in dark, some samples
were kept in dim lighting, where as others were exposed to natural but indirect light. Mushrooms
were picked and dried for further studies.

2
3. Sample Analysis:

3.1. Reagents: Chloroform (A.R.). (N Methyl- N-(trimethylsilyl)-2, 2, 2-trifluoroacetamide

(MSTFA)) / obtained from E. Merck (Darmstadt, Germany).

3.2. Equipment: GC/MS (HP 6890/5973), centrifuge, pestle and mortar.

3.3. Procedure for Pure Mushrooms:

Parts of fungal fruit bodies (cap and stem) were placed in oven overnight at 50 °C and grounded to
a fine powder in a mortar. 70 mg of powdered samples were accurately weighed and extracted
with 1 ml chloroform in an ultrasonic bath for 1 hr. The samples were then Centrifuged at 10,000
rpm for 10 min and filtrated through a cotton filter, resulting a clear solution. The resulting
solution was pipetted into a GC vial and evaporated to dryness at 50°C under a gentle stream of
nitrogen. Each residue was dissolved in 30 µl MSTFA and heated for 30 min at 70°C. After
cooling, 1 µl of the sample was directly used for GC–MS analysis. (21)

4. Results

A mass spectrum of MSTFA derivative of PC/PB is shown in Figs. 3. The drift time for the
PC/PB was 17.60 minutes. The dephosphorylation of PB to PC in vivo has been well
documented (9,22-23) and is thought to account for most or all of its central nervous
system activity (22).Casale described the rapid formation of PC after complete
dephosphorylation of PB by heating the dilute acetic acid extract (8). In the present study,
reaction of MSTFA with PC and PB results in the same silonized derivative (Bis
Trimethylsilyl Psilocin) following complete dephosphorylation of PB , probably due to the
heating (Fig 3).
Relative content of PC/PB in dark and light cultivated mushrooms (Fig.2,4-5)can then be
calculated using the AUC as an indirect index of the content (Table 1). The statistical analysis
(ANOVA) of the results show that content of PC/PB was significantly (p<0.001) different among
the samples (Table 2 ).The results of the LSD analysis shows that The maximum PC/PB content
was observed in the Dark sample while minimum was observed in the Light sample. (Table 3).

5. Discussion

PB/ PC -containing mushrooms are commonly called "magic mushrooms"(MMs) or simply

"shrooms". They are naturally occurring and have been used as a god-like traditional medicine for
centuries in the religious ceremonies by shamans in Central and South America. Currently, they
have been used extensively for recreational purposes as hallucinogenic substances in various
countries in Europe, America, Japan and elsewhere. (10). Convention on Psychotropic Substances
has defined the PB and PC, and the mushrooms containing these substances as the Schedule I
drugs, which includes dangerous drugs claimed to create a serious risk to public health, and whose
therapeutic value is doubtful.They act on the central nervous system to produce changes in
perception, mood, and thinking ability. The effects produced by PC and PB are similar to those
produced by LSD and mescaline (11)
In the US, an FDA-approved study supported by "Multidisciplinary Association for Psychedelic
Studies" (MAPS) began in 2001 to study the effects of PB on patients with obsessive-compulsive
disorder (OCD). MAPS has also proposed studying PB's potential application for the treatment of
cluster headaches based on anecdotal evidence presented to them by a cluster headache sufferer.

Different variables including species, strain, glucose and ammonium succinate concentration in the
growth medium, pH, temperature, timing and oxidation have been accounted for the PC and PB
content in the "Magic Mushrooms"(MMs). For example oxidation, absence of glucose and low

3
levels of ammonium succinate will all give poor yields of PC/PB production. Whereas, adjustment
of pH, temperature and timing reported to increase the PC/PB content (8). These are but some of
the variables in a constellation of factors that complicate consistency in the production of PC and
PB.To the best of our knowledge, no data on the effect of the light on the relative PC/PB content in
Psilocybe Cubensis mushroom has been published so far. From the results presented here, the
ultraviolet radiation from the Sun could be considered to markedly lessen the potency of MMs.

RT: 5.51 - 41.39

17.60 NL:
100 2.68E6
95 TIC MS
ps ilocybin
90 m

85
18.90
80

75

70

65
Relative Abundance

60

55

50

45 26.70

40 37.33

35
22.70
30

25
16.69
20 16.02
28.14
15 19.32 22.83
24.40
21.75 28.25 37.95 39.52
10 13.65 19.97 31.00 32.48 34.92 35.84
6.27 8.70
15.02
5 8.98 12.40 12.68

6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
Tim e (m in)

Psilocin
RT: 16.71 - 18.74
17.60 NL:
100 2.68E6
95 TIC MS
ps ilocybin
90 m

85

80

75

70

65
Relative Abundance

60

55

50

45

40

35

30

25

20

15

10
16.78 16.89 16.99 17.10 17.19 17.25 17.44 17.53 17.71 17.90 18.03 18.10 18.20 18.25 18.46 18.52 18.63 18.72
5
16.8 17.0 17.2 17.4 17.6 17.8 18.0 18.2 18.4 18.6
Tim e (min)

Figure 2: Dark sample Plasmogram of Psilocybe Cubensis ( DTime =17.60 ms (Pc) ) : (A) Total (B) Expanded

4
Figure 3. Mass Spectrum of MSTFA derivative of PC/PB

Psilocin
RT: 17.38 - 17.68
17.58 NL:
100 3.13E5
TIC MS
95 ps ilocybin
d
90

85

80

75

70
Relative Abundance

65

60 17.51

55

50

45

40

35
17.67

17.41 17.42
30 17.39
17.47

17.38 17.40 17.42 17.44 17.46 17.48 17.50 17.52 17.54 17.56 17.58 17.60 17.62 17.64 17.66 17.68
Tim e (m in)

Figure 4: Dim lighting sample Plasmogram of Psilocybe Cubensis ( DTime =17.60 ms (Pc) )

Psilocin
RT: 16.96 - 17.94
17.52 NL:
100 1.62E5
TIC MS
psilocybin
95 n

90

85

17.59
80
Relative Abundance

17.23
75

70

65

60 17.18

17.67
55 17.02 17.05

17.42
50 17.44 17.87 17.88
17.08 17.36 17.39 17.77
17.11 17.81
17.34
45

17.0 17.1 17.2 17.3 17.4 17.5 17.6 17.7 17.8 17.9
Time (min)

Figure 5: Light sample Plasmogram of Psilocybe Cubensis ( DTime =17.60 ms (Pc) )

5
 6000000
5000000
4000000
3000000
2000000 Series1
1000000 Series2
S2
0
S1
rk
Da t in
g
t
gh gh
Li Li
m
Di

Mean Difference
Treat (I) Treat (J) (I-J) Std. Error Sig.

Groups Replica 1 Replica 2

Dark 5083237.00 5740283.00
Dim Lighting 505846.40 103905.50
Light 133300.20 17296.11

Table 1. Results of the analysis

SS DF MS F P
x * treat Between Groups 4E+013 2 1.820 E+013 180.019 .001
Within Groups 3E+011 3 1.011 E+011
Total 4E+013 5

Table 2 : ANOVA showing comparison of samples

6
 Dim Lighting
5106884.09650(*) 317994.49658 .001
* The mean difference is Dark
significant at the .001 level.
Light
5336461.88750(*) 317994.49658 .000
Table 3: Multiple
Comparisons of samples Dark
(LSD) -5106884.09650(*) 317994.49658 .001
Dim Lighting
Light
References: 229577.79100 317994.49658 .523
1) Stamets , Paul. Psilocybin
Mushrooms of the World, Dark
Homestead Book Company. , -5336461.88750(*) 317994.49658 .000
Light
(1978, P.109) ISBN:
0930180038 Dim Lighting
-229577.79100 317994.49658 .523
2) Wasson, V.P.Wasson, R.G.
Mushrooms, Russia and History, Pantheon, New York, 1957.

3) Hofmann, A. Frey, A. Ott, H. Petrzilka, Th. Troxler, F. Konstitutionsaufklarung und Synthese von
Psilocybin, Experienta 15 (1958) 397–399.

4) Kamata,T. Nishikawa, M and et al,"Liquid Chromatography-Mass Spectrometric and Liquid

Chromatography-Tandem Mass Spectrometric Determination of Hallucinogenic Indoles Psilocin and
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7
 15) Timmons JE. The identification of psilocin and psilocybin using gas chromatography-mass
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Rafati, H., Riahi, H., Mohammadi, A., 2009. Enhancement of Indole Alkaloids Produced
by Psilocybe cubensis(Earle) Singer (Agaricomycetideae) in Controlled Harvesting Light
Conditions. International Journal of Medicinal Mushrooms 11.

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