Psilocybe cubensis

Not a plant, though indeed magic.

Unfortunately, there is very little peer-reviewed research on cubensis mushroom cultivation due to their illicit nature. However, much internet space has been devoted to the amateur cultivation and study of cubensis and other psychedelic mushrooms for the same reason.

[1]

Propagation

Spore

Mycelia

Cultivation

Almost all cultivation information today is obtained from the illicit market.[1] This includes notable forums Shroomery and Mycotopia as well as several published books.[2][3][4] More recently, Youtube channels have been established providing detailed cultivation information in video format (e.g. PhillyGoldenTeacher). Much of this information was gleaned from amateur experimentation or deduced from other species’ cultivation research, especially Agaricus bisporus[5], and thus should be considered unverified.

[6]

Harvest

Fruit bodies of cubensis tend to occur in semi-consistent “flushes” of mushroom growth: A period of intense growth followed by a refractory period before another growth spurt. Five or six flushes should be considered the typical maximum terminating with contamination of the substrate.[5]

Post-Harvest Processing

Harvest and processing methods should account for the degradation potential of psilocybin and psilocin. Spontaneous, catalytic, and enzymatic pathways contribute to these reactions.[7][8][9][10]

Analytically pure standards of psilocybin and psilocin in water protected from light are only stable for approximately seven days. One-third of psilocybin and one-half of psilocin was found to degrade after 14 days. Exposure to air and especially light increased the degradation rate to the point where immediate consecutive analyses found significant losses. These spontaneously produced degradation products are likely dimers or trimers of the active tryptamines.[7]

Likewise, when psilocybin is incubated with rat brain mitochondria, purified oxidase enzymes, or aqueous ferric ions, blue-colored degradation products are formed. However, only psilocin can produce these products while psilocybin produces colorless byproducts. The ferric ion-catalyzed reactions are not accompanied by oxygen uptake but can be inhibited by the addition of EDTA. The enzymatic reactions, in contrast, do require oxygen uptake and are not inhibited by EDTA.[10]

In the whole Psilocybe spp. carpophores and mycelia, a similar blueing reaction is notable in response to injury. The names P. cyanescens and P. azurescens reflect this reaction. The blue color is not one compound—rather a set of heterogenous quinoid psilocyl oligomers. The phosphate group on psilocybin acts to protect the indole from polymerization.[9]

Chopping fresh cubensis mushrooms into small pieces before drying results in a roughly 30% loss of psilocybin and psilocin.[8]

[8] [11]

Yield

Spawn Run

Fruiting

Sterile water was used to irrigate cased grain, though it is unclear for how long.[5]

[12]

Media

A casing layer of 2 parts peat, 1 part calcium carbonate, and 2 parts perlite/vermiculite can be used as a casing layer of about 2cm depth.[5]

Temperature

Lighting

Pests

Polymeric psilocin, the origin of the injury-induced blue color of cubensis, may serve as a defense mechanism. The irreversible polymerization of psilocyl radicals forms compounds that could hypothetically induce intestinal lesions in insects.[9]

Ecology

Morphology

[12]

Mycelia

Carpophores

Spores

Mycochemistry

Psilocybin and psilocin are considered the main bioactive alkaloids in cubensis mushrooms.[1]

With everything else held constant, psilocybin can vary more than 4-fold in cubensis flushes. This is enough to convolute just about any other experimental effect beyond significance.[5] Similarly, the natural variation casts further doubt on any amateur experimentation without a strong statistical evaluation.

[12] [16]

Infraspecific Variation

[14]

Biosynthesis

[17] [18]

Distribution

Psilocybin is present in equal amount in the cap and stem of cubensis fruit bodies. In contrast, [5]

PsiL (psilocin -> psilocyl radical) and PsiP (psilocybin -> psilocin) are likely localized extracellularly and lysosomally, respectively. The spatial separation prevents psilocybin polymerization until an injury causes cell lysis.[9]

Timecourse

The concentration of psilocybin varies considerably between flushes but does not trend up or down significantly.[5]

Improvement

Identification

Large inconsistencies exist in the Psilocybe spp. genetic and voucher specimen catalogs. For example, several BLAST sequences in the NCBI database labeled Psilocybe cubensis are actually from Fusarium and Amoebozoa. This casts doubt on much of the recent literature.[15]

[19] [20] [14]

Mycology Collections Portal

Inheritance

Methods

The active constituents of cubensis dried carpophores can be extracted using 0.5% (v/v) acetic acid in methanol at a ratio of 1 ml solvent to 10mg powder. This mixture was chosen from tests of 100% methanol, 50% methanol with deionized water or ethanol, and 75% ethanol in isopropanol with 25 mmol/L acetate buffer. Formic and acetic acid added to methanol at various concentrations were also tested for extraction efficiency.² In each case a further 20% of active constituents were recovered with a second extraction with pure methanol. Furthermore, it was found that mechanical agitation of samples increased analyte recovery compared with no agitation. No difference was found in extraction efficiency with a duration range of 20-360 minutes.[8]

[20]

TLC

Visulization

History & Society

The use of Psilocybe mushrooms for medicinal purposes stretches back at least 3000 years.[1]

Albert Hofmann first introduced the medicinal aspects of Psilocybe mushrooms to Western medicine circa 1957. Though the initial experimentation was done with mushroom extracts (from P. mexicana), subsequent research was done with synthetically produced psilocybin.[1]

Psilocybin, and by extension Psilocybe cubensis, was classified as a US Schedule I drug in 1970, halting nearly all domestic research.[1]

Terrance and Dennis McKenna published Psilocybin: Magic Mushroom Grower’s Guide under the psudonyms O. T. Oss and O. N. Oeric in 1976.[22] The book is considered by many to be the origin of the modern psychedelic mushroom cultivation boom.

“Street samples” of psilocybin mushrooms obtained circa 1982 contained up to tenfold variation of psilocybin and psilocin.[5]

[23] [24]

Work Log

17 Apr 2023

Ooof. Gotvaldova et al. is a mess procedurally. All of the experiments used spores on agar as the starting material instead of an isolate.[8]

Bibliography

1. Plotnik, Lauren and Gibbs, Grace and Graham, Thomas, Psilocybin Conspectus: Status, Production Methods, and Considerations, International Journal of Medicinal Mushrooms, vol. 24, no. 1, 2022. doi: 10.1615/IntJMedMushrooms.2021041921. Abstract
   Psilocybin is a psychoactive alkaloid that is produced naturally by approximately 200 species of mushrooms. The potential medical use of this molecule for the t...
2. Nicholas, L. G. and Ogame, Kerry, Psilocybin Mushroom Handbook: Easy Indoor \& Outdoor Cultivation, 2006. Abstract
   Here is a practical step-by-step guide to cultivating four species of psilocybin-containing mushrooms, indoors and outside. Anyone with a clean kitchen, some basic equipment, and a closet shelf or shady flowerbed will be able to grow a bumper crop. This Handbook also includes an introduction to mushroom biology, a guide for supplies, and advice on discreetly integrating psychedelic mushrooms into outdoor gardens. Hand-drawn illustrations and full-color and black-\&-white photographs provide the reader with steps in the cultivation process and exact identification of desired species. The four species detailed include two species that have previously had very little coverage: Psilocybe mexicana (a tiny mushroom used for millennia by indigenous Mexican shamans) and Psilocybe azurescens (a newly described species native to the Pacific Northwest and easily grown outdoors on woodchips). This innovative book also offers a wealth of information about the use of psilocybin-containing mushrooms in both traditional and modern contexts. Contributing ethnobotanist Kathleen Harrison highlights the history, ritual and mythology of sacred Psilocybe mushrooms used in indigenous shamanic settings. The book's authors offer insights into how these principles might be put into practice by the modern voyager, to provide, safe, healing and fruitful journeys.
3. Oss, O. T. and Oeric, O. N., Psilocybin: Magic Mushroom Grower's Guide : A Handbook for Psilocybin Enthusiasts, June 1992. Abstract
   In the 1970s, two of the most influential thinkers of the psychedelic era gathered what was then known about psilocybin botany and culture and presented it in Psilocybin: Magic Mushroom Grower's Guide. Writing under pseudonyms, the McKenna brothers provided simple, reliable, and productive methods for magic mushroom propagation, including black-and-white photographs that showed the techniques of the time.The development of more modern cultivation techniques does not eclipse the cultural contributions of this book. Philosophical asides, whimsical illustrations evoking the mystical nature of mushrooms, and speculations about the relationship of these organisms to humankind provide a lasting legacy. Truly the classic manual on home cultivation, the wisdom of Psilocybin: Magic Mushroom Grower's Guide continues to inspire new students of psycho-mycology--and refreshes psychedelic memories for others.
4. Stamets, Paul and Chilton, J. S., The Mushroom Cultivator: A Practical Guide to Growing Mushrooms at Home, 1983.
5. Bigwood, Jeremy and Beug, Michael W., Variation of Psilocybin and Psilocin Levels with Repeated Flushes (Harvests) of Mature Sporocarps of Psilocybe Cubensis (Earle) Singer, Journal of Ethnopharmacology, vol. 5, no. 3, pp. 287--291, May 1982. doi: 10.1016/0378-8741(82)90014-9. Abstract
   Analysis of Psilocybe cubensis (Earle) Singer grown in controlled culture showed that the level of psilocin was generally zero in the first (or sometimes even the second) fruiting of the mushroom from a given culture and that the level reached a maximum by the fourth flush. The level of psilocybin, which was nearly always at least twice the level of psilocin, showed no upward or downward trend as fruiting progressed, but was variable over a factor of four. Samples obtained from outside sources had psilocybin levels varying by over a factor of ten from one collection to the next.
6. Sommano, Sarana Rose and Suksathan, Ratchuporn and Sombat, Thanarat and Seehanam, Pimjai and Sirilun, Sasithorn and Ruksiriwanich, Warintorn and Wangtueai, Sutee and Leksawasdi, Noppol, Novel Perspective of Medicinal Mushroom Cultivations: A Review Case for ‘Magic’ Mushrooms, Agronomy, vol. 12, no. 12, pp. 3185, December 2022. doi: 10.3390/agronomy12123185. Abstract
   Fruiting bodies, mycelia, or spores in the form of extracts or powder of various medicinal mushrooms are used to prevent, treat, or cure a range of ailments and balance a healthy diet. Medicinal mushrooms are found in several genera of fungi and their fruit bodies, cultured mycelia, and cultured broth contains phytochemical constituents such as triterpenes, lectins, steroids, phenols, polyphenols, lactones, statins, alkaloids, and antibiotics. Edible mushrooms are considered functional foods that can be used as supplements for complementary and alternative medicines where the markets are growing rapidly. Several species of edible mushrooms possess therapeutic potential and functional characteristics. The psilocybin-containing types, sometimes known as magic mushrooms, have been utilized for generations by indigenous communities due to their hallucinogenic, medicinal, and mind-manifestation properties. Recent clinical research also convinces that these psychedelics have the potential to treat addiction, depression, anxiety, and other mental health concerns. This has escalated the demand for the natural products derived from the mushrooms of these sources, yet the agronomic aspect and biotechnology approaches to produce the active ingredients are not collectively documented. The objectives of this review article are to examine the general type and variation of therapeutic mushrooms, especially those belonging to the Psilocybe. The biotechnology approach for cultivation and the production of secondary metabolites is also appraised. The ultimate purposes are to provide guidance for farmers and companies to pursue sustainable ways to produce natural products for the development of functional food and pharmaceuticals and to support the alteration of the stigmatic drug concerns around psychedelic mushrooms.
7. Anastos, N. and Barnett, N.W. and Pfeffer, F.M. and Lewis, S.W., Investigation into the Temporal Stability of Aqueous Standard Solutions of Psilocin and Psilocybin Using High Performance Liquid Chromatography, Science \& Justice, vol. 46, no. 2, pp. 91--96, April 2006. doi: 10.1016/S1355-0306(06)71579-9.
8. Gotvaldová, Klára and Hájková, Kateřina and Borovička, Jan and Jurok, Radek and Cihlářová, Petra and Kuchař, Martin, Stability of Psilocybin and Its Four Analogs in the Biomass of the Psychotropic Mushroom Psilocybe Cubensis, Drug Testing and Analysis, vol. 13, no. 2, pp. 439--446, 2021. doi: 10.1002/dta.2950. Abstract
   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.
9. Lenz, Claudius and Wick, Jonas and Braga, Daniel and {García-Altares}, María and Lackner, Gerald and Hertweck, Christian and Gressler, Markus and Hoffmeister, Dirk, Injury-Triggered Blueing Reactions of Psilocybe “Magic” Mushrooms, Angewandte Chemie, vol. 132, no. 4, pp. 1466--1470, 2020. doi: 10.1002/ange.201910175. Abstract
   Upon injury, psychotropic psilocybin-producing mushrooms instantly develop an intense blue color, the chemical basis and mode of formation of which has remained elusive. We report two enzymes from Psilocybe cubensis that carry out a two-step cascade to prepare psilocybin for oxidative oligomerization that leads to blue products. The phosphatase PsiP removes the 4-O-phosphate group to yield psilocin, while PsiL oxidizes its 4-hydroxy group. The PsiL reaction was monitored by in situ 13C NMR spectroscopy, which indicated that oxidative coupling of psilocyl residues occurs primarily via C-5. MS and IR spectroscopy indicated the formation of a heterogeneous mixture of preferentially psilocyl 3- to 13-mers and suggest multiple oligomerization routes, depending on oxidative power and substrate concentration. The results also imply that phosphate ester of psilocybin serves a reversible protective function.
10. Levine, Walter G., Formation of Blue Oxidation Product from Psilocybin, Nature, vol. 215, pp. 1292--1293, 1967. url: https://www.nature.com/articles/2151292a0.
11. Laussmann, Tim and {Meier-Giebing}, Sigrid, Forensic Analysis of Hallucinogenic Mushrooms and Khat (Catha edulisForsk) Using Cation-Exchange Liquid Chromatography, Forensic Science International, vol. 195, no. 1, pp. 160--164, February 2010. doi: 10.1016/j.forsciint.2009.12.013. Abstract
    Hallucinogenic mushrooms (e.g. Psilocybe and Panaeolus species) as well as leaves and young shoots of the khat tree (Catha edulisForsk) are illicit drugs in many countries. The exact concentration of the hallucinogenic alkaloids psilocin and psilocybin in mushrooms and the sympathomimetic alkaloids cathinone and cathine in khat is usually essential for jurisdiction. Facing an increasing number of mushroom and khat seizures by German customs authorities, a convenient comprehensive quantitative HPLC method based on cation-exchange liquid chromatography for these rather “exotic” drugs has been developed which avoids time-consuming multi-step sample preparation or chemical derivatization procedures. Using this method a number of different hallucinogenic fungi species and products that are mainly distributed via the internet have been analysed (dried and fresh Psilocybe cubensisSinger as well as P. cubensis collected from “grow boxes”, Panaeolus cyanescensBerkeleyandBroome and so-called “philosopher stones” (sclerotia of Psilocybe species)). Highest total amounts of psilocin have been detected in dried P. cyanescens reaching up to 3.00±0.24mg per 100mg. The distribution of khat alkaloids in different parts of the khat shoots has been studied. High concentrations of cathinone have not only been detected in leaves but also in green parts and barks of stalks. Additionally, the sample treatment for fresh mushroom and khat samples has been optimised. Highest amounts of alkaloids were found when fresh material was freeze-dried.
12. Nagy, László G. and Vonk, Peter Jan and Künzler, Markus and Földi, Csenge and Virágh, Máté and Ohm, Robin A. and Hennicke, Florian and Bálint, Balázs and Csernetics, Árpád and Hegedüs, Botond and Hou, Zhihao and Liu, Xiao-Bin and Nan, Shen and Pareek, Manish and Sahu, Neha and Szathmári, Benedek and Varga, Torda and Wu, Hongli and Yang, Xiao and Merényi, Zsolt, Lessons on Fruiting Body Morphogenesis from Genomes and Transcriptomes of Agaricomycetes, pp. 2021.12.09.471732, April 2022. doi: 10.1101/2021.12.09.471732. Abstract
    Fruiting bodies of mushroom-forming fungi (Agaricomycetes) are among the most complex structures produced by fungi. Unlike vegetative hyphae, fruiting bodies grow determinately and follow a genetically encoded developmental program that orchestrates tissue differentiation, growth and sexual sporulation. In spite of more than a century of research, our understanding of the molecular details of fruiting body morphogenesis is limited and a general synthesis on the genetics of this complex process is lacking. In this paper, we aim to comprehensively identify conserved genes related to fruiting body morphogenesis and distill novel functional hypotheses for functionally poorly characterized genes. As a result of this analysis, we report 921 conserved developmentally expressed gene families, only a few dozens of which have previously been reported in fruiting body development. Based on literature data, conserved expression patterns and functional annotations, we provide informed hypotheses on the potential role of these gene families in fruiting body development, yielding the most complete description of molecular processes in fruiting body morphogenesis to date. We discuss genes related to the initiation of fruiting, differentiation, growth, cell surface and cell wall, defense, transcriptional regulation as well as signal transduction. Based on these data we derive a general model of fruiting body development, which includes an early, proliferative phase that is mostly concerned with laying out the mushroom body plan (via cell division and differentiation), and a second phase of growth via cell expansion as well as meiotic events and sporulation. Altogether, our discussions cover 1480 genes of Coprinopsis cinerea, and their orthologs in Agaricus bisporus, Cyclocybe aegerita, Armillaria ostoyae, Auriculariopsis ampla, Laccaria bicolor, Lentinula edodes, Lentinus tigrinus, Mycena kentingensis, Phanerochaete chrysosporium, Pleurotus ostreatus, and Schizophyllum commune, providing functional hypotheses for ∼10\% of genes in the genomes of these species. Although experimental evidence for the role of these genes will need to be established in the future, our data provide a roadmap for guiding functional analyses of fruiting related genes in the Agaricomycetes. We anticipate that the gene compendium presented here, combined with developments in functional genomics approaches will contribute to uncovering the genetic bases of one of the most spectacular multicellular developmental processes in fungi.
13. Lenz, Claudius and Wick, Jonas and Hoffmeister, Dirk, Identification of ω-N-Methyl-4-Hydroxytryptamine (Norpsilocin) as a Psilocybe Natural Product, Journal of Natural Products, vol. 80, no. 10, pp. 2835--2838, October 2017. doi: 10.1021/acs.jnatprod.7b00407. Abstract
    We report the identification of ω-N-methyl-4-hydroxytryptamine (norpsilocin, 1) from the carpophores of the hallucinogenic mushroom Psilocybe cubensis. The structure was elucidated by 1D and 2D NMR spectroscopy and high-resolution mass spectrometry. Norpsilocin has not previously been reported as a natural product. It likely represents the actual psychotropic agent liberated from its 4-phosphate ester derivative, the known natural product baeocystin. We further present a simple and artifact-free extraction method that prevents dephosphorylation and therefore helps reflect the naturally occurring metabolic profile of Psilocybe mushrooms in subsequent analyses.
14. Gotvaldová, Klára and Borovička, Jan and Hájková, Kateřina and Cihlářová, Petra and Rockefeller, Alan and Kuchař, Martin, Extensive Collection of Psychotropic Mushrooms with Determination of Their Tryptamine Alkaloids, International Journal of Molecular Sciences, vol. 23, no. 22, pp. 14068, January 2022. doi: 10.3390/ijms232214068. Abstract
    Since not only psilocybin (PSB) but also PSB-containing mushrooms are used for psychedelic therapy and microdosing, it is necessary to know their concentration variability in wild-grown mushrooms. This article aimed to determine the PSB, psilocin (PS), baeocystin (BA), norbaeocystin (NB), and aeruginascin (AE) concentrations in a large sample set of mushrooms belonging to genera previously reported to contain psychotropic tryptamines. Ultra-high performance liquid chromatography coupled with tandem mass spectrometry was used to quantify tryptamine alkaloids in the mushroom samples. Most mushroom collections were documented by fungarium specimens and/or ITS rDNA/LSU/EF1-α sequencing. Concentrations of five tryptamine alkaloids were determined in a large sample set of 226 fruiting bodies of 82 individual collections from seven mushroom genera. For many mushroom species, concentrations of BA, NB, and AE are reported for the first time. The highest PSB/PS concentrations were found in Psilocybe species, but no tryptamines were detected in the P. fuscofulva and P. fimetaria collections. The tryptamine concentrations in mushrooms are extremely variable, representing a problem for mushroom consumers due to the apparent risk of overdose. The varied cocktail of tryptamines in wild mushrooms could influence the medicinal effect compared to therapy with chemically pure PSB, posing a serious problem for data interpretation.
15. Bradshaw, Alexander J. and Backman, Talia A. and {Ramírez-Cruz}, Virginia and Forrister, Dale L. and Winter, Jaclyn M. and {Guzmán-Dávalos}, Laura and Furci, Giuliana and Stamets, Paul and Dentinger, Bryn T. M., DNA Authentication and Chemical Analysis of Psilocybe Mushrooms Reveal Widespread Misdeterminations in Fungaria and Inconsistencies in Metabolites, Applied and Environmental Microbiology, vol. INVALID\_SCITE\_VALUE, no. INVALID\_SCITE\_VALUE, pp. e01498-22, November 2022. doi: 10.1128/aem.01498-22. Abstract
    The mushroom genus Psilocybe is best known as the core group of psychoactive mushrooms, yet basic information on their diversity, taxonomy, chemistry, and general biology is still largely lacking. In this study, we reexamined 94 Psilocybe fungarium specimens, representing 18 species, by DNA barcoding, evaluated the stability of psilocybin, psilocin, and their related tryptamine alkaloids in 25 specimens across the most commonly vouchered species (Psilocybe cubensis, Psilocybe cyanescens, and Psilocybe semilanceata), and explored the metabolome of cultivated P. cubensis. Our data show that, apart from a few well-known species, the taxonomic accuracy of specimen determinations is largely unreliable, even at the genus level. A substantial quantity of poor-quality and mislabeled sequence data in public repositories, as well as a paucity of sequences derived from types, further exacerbates the problem. Our data also support taxon- and time-dependent decay of psilocybin and psilocin, with some specimens having no detectable quantities of them. We also show that the P. cubensis metabolome possibly contains thousands of uncharacterized compounds, at least some of which may be bioactive. Taken together, our study undermines commonly held assumptions about the accuracy of names and presence of controlled substances in fungarium specimens identified as Psilocybe spp. and reveals that our understanding of the chemical diversity of these mushrooms is largely incomplete. These results have broader implications for regulatory policies pertaining to the storage and sharing of fungarium specimens as well as the use of psychoactive mushrooms for recreation and therapy. IMPORTANCE The therapeutic use of psilocybin, the active ingredient in “magic mushrooms,” is revolutionizing mental health care for a number of conditions, including depression, posttraumatic stress disorder (PTSD), and end-of-life care. This has spotlighted the current state of knowledge of psilocybin, including the organisms that endogenously produce it. However, because of international regulation of psilocybin as a controlled substance (often included on the same list as cocaine and heroin), basic research has lagged far behind. Our study highlights how the poor state of knowledge of even the most fundamental scientific information can impact the use of psilocybin-containing mushrooms for recreational or therapeutic applications and undermines critical assumptions that underpin their regulation by legal authorities. Our study shows that currently available chemical studies are mainly inaccurate, irreproducible, and inconsistent, that there exists a high rate of misidentification in museum collections and public databases rendering even names unreliable, and that the concentration of psilocybin and its tryptamine derivatives in three of the most commonly collected Psilocybe species (P. cubensis, P. cyanescens, and P. semilanceata) is highly variable and unstable in museum specimens spanning multiple decades, and our study generates the first-ever insight into the highly complex and largely uncharacterized metabolomic profile for the most commonly cultivated magic mushroom, P. cubensis.
16. Lenz, Claudius and Sherwood, Alexander and Kargbo, Robert and Hoffmeister, Dirk, Taking Different Roads: L-Tryptophan as the Origin of Psilocybe Natural Products, ChemPlusChem, vol. 86, no. 1, pp. 28--35, 2021. doi: 10.1002/cplu.202000581. Abstract
    Psychotropic fungi of the genus Psilocybe, colloquially referred to as „magic mushrooms”, are best known for their l-tryptophan-derived major natural product, psilocybin. Yet, recent research has revealed a more diverse secondary metabolism that originates from this amino acid. In this minireview, the focus is laid on l-tryptophan and the various Psilocybe natural products and their metabolic routes are highlighted. Psilocybin and its congeners, the heterogeneous blue-colored psilocyl oligomers, alongside β-carbolines and N,N-dimethyl-l-tryptophan, are presented as well as current knowledge on their biosynthesis is provided. The multidisciplinary character of natural product research is demonstrated, and pharmacological, medicinal, ecological, biochemical, and evolutionary aspects are included.
17. Schäfer, Tim and Kramer, Kristina and Werten, Sebastiaan and Rupp, Bernhard and Hoffmeister, Dirk, Characterization of the Gateway Decarboxylase for Psilocybin Biosynthesis, ChemBioChem, vol. n/a, no. n/a, pp. e202200551, 2022. doi: 10.1002/cbic.202200551. Abstract
    The l-tryptophan decarboxylase PsiD catalyzes the initial step of the metabolic cascade to psilocybin, the major indoleethylamine natural product of the “magic” mushrooms and a candidate drug against major depressive disorder. Unlike numerous pyridoxal phosphate (PLP)-dependent decarboxylases for natural product biosyntheses, PsiD is PLP-independent and resembles type II phosphatidylserine decarboxylases. Here, we report on the in vitro biochemical characterization of Psilocybe cubensis PsiD along with in silico modeling of the PsiD structure. A non-canonical serine protease triad for autocatalytic cleavage of the pro-protein was predicted and experimentally verified by site-directed mutagenesis.
18. {Watkins-Dulaney}, Ella and Straathof, Sabine and Arnold, Frances, Tryptophan Synthase: Biocatalyst Extraordinaire, ChemBioChem, vol. 22, no. 1, pp. 5--16, 2021. doi: 10.1002/cbic.202000379. Abstract
    Tryptophan synthase (TrpS) has emerged as a paragon of noncanonical amino acid (ncAA) synthesis and is an ideal biocatalyst for synthetic and biological applications. TrpS catalyzes an irreversible, C−C bond-forming reaction between indole and serine to make l-tryptophan; native TrpS complexes possess fairly broad specificity for indole analogues, but are difficult to engineer to extend substrate scope or to confer other useful properties due to allosteric constraints and their heterodimeric structure. Directed evolution freed the catalytically relevant TrpS β-subunit (TrpB) from allosteric regulation by its TrpA partner and has enabled dramatic expansion of the enzyme's substrate scope. This review examines the long and storied career of TrpS from the perspective of its application in ncAA synthesis and biocatalytic cascades.
19. Zhang, Xiaochun and Yu, Huan and Wang, Ziwei and Yang, Qi and Xia, Ruocheng and Qu, Yiling and Tao, Ruiyang and Shi, Yan and Xiang, Ping and Zhang, Suhua and Li, Chengtao, Multi-Locus Identification of Psilocybe Cubensis by High-Resolution Melting (HRM), Forensic Sciences Research, vol. 7, no. 3, pp. 490--497, July 2022. doi: 10.1080/20961790.2021.1875580. Abstract
    Hallucinogenic mushroom is a kind of toxic strain containing psychoactive tryptamine substances such as psilocybin, psilocin and ibotenic acid, etc. The mushrooms containing hallucinogenic components are various, widely distributed and lack of standard to define, which made a great challenge to identification. Traditional identification methods, such as morphology and toxicology analysis, showed shortcomings in old or processed samples, while the DNA-based identification of hallucinogenic mushrooms would allow to identify these samples due to the stability of DNA. In this paper, four primer sets are designed to target Psilocybe cubensis DNA for increasing resolution of present identification method, and the target markers include largest subunit of RNA polymerase II (marked as PC-R1), psilocybin-related phosphotransferase gene (marked as PC-PT), glyceraldehyde 3-phosphate dehydrogenase (marked as PC-3) and translation EF1α (marked as PC-EF). Real-time PCR with high-resolution melting (HRM) assay were used for the differentiation of the fragments amplified by these primer sets, which were tested for specificity, reproducibility, sensitivity, mixture analysis and multiplex PCR. It was shown that the melting temperatures of PC-R1, PC-PT, PC-3 and PC-EF of P. cubensis were (87.93 ± 0.12) °C, (82.21 ± 0.14) °C, (79.72 ± 0.12) °C and (80.11 ± 0.19) °C in our kinds of independent experiments. Significant HRM characteristic can be shown with a low concentration of 62.5 pg/µL DNA sample, and P. cubensis could be detected in mixtures with Homo sapiens or Cannabis sativa. In summary, the method of HRM analysis can quickly and specifically distinguish P. cubensis from other species, which could be utilized for forensic science, medical diagnosis and drug trafficking cases. Supplemental data for this article are available online at https://doi.org/10.1080/20961790.2021.1875580.
20. Dörner, Sebastian and Rogge, Kai and Fricke, Janis and Schäfer, Tim and Wurlitzer, Jacob M. and Gressler, Markus and Pham, Duyen N. K. and Manke, David R. and Chadeayne, Andrew R. and Hoffmeister, Dirk, Genetic Survey of Psilocybe Natural Products, ChemBioChem, vol. 23, no. 14, pp. e202200249, 2022. doi: 10.1002/cbic.202200249. Abstract
    Psilocybe magic mushrooms are best known for their main natural product, psilocybin, and its dephosphorylated congener, the psychedelic metabolite psilocin. Beyond tryptamines, the secondary metabolome of these fungi is poorly understood. The genomes of five species (P. azurescens, P. cubensis, P. cyanescens, P. mexicana, and P. serbica) were browsed to understand more profoundly common and species-specific metabolic capacities. The genomic analyses revealed a much greater and yet unexplored metabolic diversity than evident from parallel chemical analyses. P. cyanescens and P. mexicana were identified as aeruginascin producers. Lumichrome and verpacamide A were also detected as Psilocybe metabolites. The observations concerning the potential secondary metabolome of this fungal genus support pharmacological and toxicological efforts to find a rational basis for yet elusive phenomena, such as paralytic effects, attributed to consumption of some magic mushrooms.
21. Stebelska, Katarzyna, Chapter 84 - Assays for Detection of Fungal Hallucinogens Such as Psilocybin and Psilocin, pp. 909--926, January 2016. doi: 10.1016/B978-0-12-800212-4.00084-4. Abstract
    Methods applicable to the detection of hallucinogenic indole compounds, namely psilocybin, psilocin, and related molecules, that have been developed so far for purposes of mycological research, toxicological studies, or forensic investigations are described in this chapter. The most current literature as well as some interesting older publications are cited, providing a historical overview of the subject. Methods of qualitative and quantitative analysis are presented. Additionally, preliminary purification steps and sample treatment before the exact quantitative determination are proposed. The methods described utilized various analytical techniques for the measurement of psilocybin/psilocin content in the analyzed material, including capillary electrophoresis, chemiluminescence detection systems, high-performance liquid chromatography with electrochemical, ultraviolet, or fluorescence detection systems, gas chromatography, and liquid chromatography coupled with mass spectrometry. The methods developed for simultaneous determination of several commonly abused psychoactive substances along with psilocin/psilocybin are also mentioned.
22. Oss, O. T. and Oeric, O. N. and the Obscure, Irimias, Psilocybin, Magic Mushroom Grower's Guide: A Handbook for Psilocybin Enthusiasts, January 1976. Abstract
    Pp. 63; 8 full page color plates, 54 text figures (black-and-white photos and line-drawings). Color pictorial stiff wrappers, sm 8vo. Psilocybin is one of the most active, and least toxic of all psychedelics. It is very well be the most perfect psychedelic! No complex chemicals, special equipment or knowledge of chemistry is needed to grow Psilocybin mushrooms at home. Given the spores, all that is needed to grow them is a little bit of grain, some chalk, a pressure cooker and a few mason jars. Signature of Nancy Weber on the title page. No other ownership marks.
23. Roberts, Aaron, An Overview of Decriminalization Efforts in Regard to Psychedelic Plants in the United States, 2019-2020, no. 3944724, October 2021. doi: 10.2139/ssrn.3944724. Abstract
    This paper examines the recent developments made in psychedelic-related drug policy in the United States. The paper gives an overview of the decriminalization efforts made at the state and local levels. The paper also looks at the historical, cultural, political, and public health factors that have shaped psychedelic policy throughout American history and into the current day. Lastly, the paper shares some concerns about discrimination and unequal access present in psychedelic-assisted psychotherapy.
24. Sinclair, Andrea, High Times in Ancient Egypt: The Use and Abuse of Psychoactive Plant Identifications in Alternative Egyptology, 14.04.21. url: https://hcommons.org/deposits/item/hc:41627/. Abstract
    Text to a presentation on the misrepresentation of ancient Egyptian psychoactive consumption in academic publications and public media that was given by me at the Alternative Egyptology Symposium, hosted by the Allard Pierson Museum, Amsterdam, 14-04-2021. There is an academic paper in preparation.