Psilocybin containing mushrooms: a rapidly developing biotechnology industry in the psychiatry, biomedical and nutraceutical fields

Abstract

Humans have collected and used hallucinogenic mushrooms for ethnic medicinal, recreational, and religious purposes since before recorded history. Currently, the use of these mushrooms is illegal in most countries, but where their use is legal they are applied as self medication. Psilocybin and psilocin, two psychoactive alkaloids, are naturally synthesized by hallucinogenic mushrooms. The chemical structure of these compounds are similar to the neurotransmitter serotonin. Activation of this system by psilocybin and psilocin may produce temporary changes in the brain that induce hallucinations and feelings of euphoria. Adjustment of the serotonin system in this way can moderate symptoms of related mental disorders. This review summarizes relevant and current information regarding the discovery of hallucinogenic mushrooms and their contained psychoactive compounds, the events that lead to their criminalization and decriminilization, and the state of knowledge of psilocybin, psilocin, and derivatives. Last, research on the psychoactive properties of these mushrooms is placed in perspective to possible applications for human dysfunctions.

Introduction

Classic hallucinogenic substances such as lysergic acid diethylamine (LSD), mescaline, peyote, and psilocybin are used by a large number of people, such as in North America where over 30 million adults have used these substances (Krebs and Johansen 2013; Yockey and King 2021). Classic hallucinogenics are either readily found in nature, or synthesized from natural compounds. Psilocybin is a hallucinogenic compound produced by mushrooms. Between 100 (Guzmán 1983) and 1000 (Guzmán et al. 1998) names are used to identify these hallucinogenic mushrooms (Guzmán 2009; Nichols 2016), which are now better known as “magic mushrooms” (Redhead et al. 2007), “shrooms” (Smith et al. 2017), “champinones” (Mayett et al. 2012), “golden teacher”, “liberty cap” (Lassen et al. 1990), and “mushies”, among others (Redhead et al. 2007). Most notably hallucinogenic mushrooms were called “teonanacatl”, meaning “flesh of God”, by the Aztec and Mazatec Mexican tribes (Guzmán 2015), emphasising the mystical or spiritual experiences resulting in meaningful and lasting personal significance (Griffiths et al. 2006).

Mushroom species known to have hallucinogenic properties due to psilocybin include species of Psilocybe, Panaeolus, Pluteus, Inocybe, and Gymnopilus (Christiansen et al. 1984; Allen et al. 1991; Stijve 1995; Guzmán et al. 1998; Musshoff et al. 2000; Guzmán 2008; Awan et al. 2018a, b; Strauss et al. 2022). These species of mushroom synthesizes the hallucinogenic compounds psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine, C₁₂H₁₇N₂O₄P) and psilocin (4-hydroxy-N,N-dimethyltryptamine, C₁₂H₁₆N₂O; de-phosphorylated metabolite of psilocybin). These mushrooms are used for their hallucinogenic properties by numerous cultures (Guzmán 2009), including third world countries across the globe (Allen and Gartz 2009; Guzmán et al. 2014). These mushrooms have only relatively recently been recognized by Western peoples for their therapeutic potential (Tullis 2021; Lowe et al. 2021; Van Amsterdam and Van den Brink 2022; Van Court et al. 2022).

This review explores how, after the discovery and misuse of the hallucinogenic properties of mushrooms, their potential is still vastly undeveloped, yet highly sought after in a rapidly developing industry. Concurrently, it should inform the novice who is interested in these mushrooms about their hallucinogenic and medicinal effects. It will also assist an audience as wide as regulatory authorities, agencies interested in their possible medicinal uses, authorities governing administrative and regulatory legislation, and custodians of biodiversity, to access the most important and recent information and trends.

Terminology

Initially, researchers referred to psychosis-inducing substances as “psychotomimetic” substances, a term suggestive of them fostering a mental state similar to that of an individual with psychosis (Osmond 1957; Nichols 2016). However, “psychotomimetic” was felt to be a negative depiction of how the substance acts in the human body asf “mimicking psychosis”. The alternative term “psychedelics” using the Greek words “psyche”, meaning mind or soul, and “deloun”, meaning “for show” was suggested (Osmond 1957). However, some felt that the term “psychedelic” still painted the salubrious compounds in a negative light (Nichols 2016). A new term “entheogens” was coined because such substances lead to a changed consciousness that could be seen as spiritual (Ruck et al. 1979; Hofmann and Ruck 2004).

The most widely used and preferred collective name for these compounds in scientific writing is hallucinogens (Guzmán 2015) due to their principal effect of producing hallucinations (Nichols 2016). Hallucinogens refer to psychoactive substances (substances that have neurological effects) that produce their effects through manipulation of the 5-hydroxitryptamine (5-HT) or serotonergic system. One drawback of the term hallucinogens is that it is often used to describe a broad category of substances, such as cannabinoids, dissociative agents, LSD, and ecstasy, that each produce unique effects. In a narrower and more descriptive sense, the difficulties in classifying and naming these substances, such as psychedelics and hallucinogens, shows that there is still much to learn of how these substances work and their mechanism of action in the central nervous system, specifically the brain. As the molecular mechanisms of these substances continue to be discovered, this term could change over time.

A brief history of psilocybin-producing mushrooms

Discovery of psilocybin-producing mushrooms

Until scholars began to study hallucinogenic mushrooms more closely in the early 1900s, many considered them a myth (Matsushima et al. 2009; Nichols 2020). It is now theorised that their usage pre-dates that of written history (Nichols 2004), possibly even by millennia (Studerus et al. 2011). The first encounter between humans and hallucinogenic mushrooms could be as early as the times of hunters and gatherers, where sooner or later, man would have ingested certain plants and fungi containing hallucinogenic compounds in search of food (Oss and Oeric 1991). Rock art and fossils provide a few answers to how far back humans have consumed hallucinogenic fungi (Helvenston and Bahn 2003; Franklin and Strecker 2008; Lewis-Williams and Challis 2012; Froese et al. 2016). Some earliest evidence dates back as far as 7000 years with mushroom wall paintings in the Tassili caves in Algeria (Samorini 1992, 2001; Stamets 1999; Metzner and Darling 2005; Matsushima et al. 2009).

The first formal written descriptions of hallucinogenic fungi were from Mexico in the 16 century (Sahagun 1593; Lowe et al. 2021; Van Courte et al. 2022) that described a mushroom from Mexico that produced visions and intoxication during traditional ceremonies. The Aztecs called it “teonanacatl” and “teotlacuilnanácatl” (Sahagun 1593). Weitlaner and Schultes (Schultes 1939, 1940) used the vague descriptions compiled by Sahagun to identify mushrooms gathered by the Mazatec and Aztec Indians in Mexico and identified them as Panaeolus campanulatus var. sphinctrinus, Psilocybe cubensis, and Psilocybe caerulescens in packages collected by Reko (Reko 1919, 1945).

Robert Gordon Wasson, an amateur mycologist and full-time Wall Street banker, and his wife travelled several times to Mexico to meet an elderly “curandera” or healer/shaman (Wasson 1957). The shaman enlightened him about the traditions of Mazatec people, including the use of mushrooms to induce visions and trance states (Wasson 1957). They believed that medicinal powers in the mushrooms allowed consumers to transform their thoughts and views of a disease they needed healing from (Wasson 1957). Heim assisted Wasson with identifications and they described many novel species, including P. cubensis (but reported it as Stropharia cubensis) from several regions in Mexico (Heim 1956, 1958; Heim and Hofmann 1958). Heim (Heim 1958; Heim and Hofmann 1958; Hofmann et al. 1958) sent 100 g of dried Psilocybe mexicana mushrooms for biochemical analysis to Albert Hofmann (Heim 1958; Hofmann et al. 1958), a Swiss chemist, who ingested 2.4 g of the dried mushroom himself to determine whether the active ingredient to be isolated was present (Hofmann 1959; Nichols 2020). He consequently characterized psilocybin as the possible psychoactive compound (Hofmann 1958a, 1958b, 1959; Hofmann et al. 1959).

The first monograph of Psilocybe mushrooms found in Mexico, South America, the United States, Canada, and Java appeared in 1958 (Singer and Smith 1958). Guzmán, an assistant to Singer during his studies in Mexico, continued research on Psilocybe and published a more comprehensive world monograph on the genus (Guzmán 1983), finding that Psilocybe species are widely distributed on all continents, with the majority of the species occurring in Central America (Guzmán 1983). Of these, a relatively small number are currently used outside of their native ranges (Alam Mahmood 2013). The taxonomic status are in dire need of updating and current monographs inclusive of all novel species of these hallucinogenic mushroom genera will be useful (Strauss et al. 2022).

Start of psilocybin research and its criminalisation

During the early discovery and identification of psilocybin, research on other hallucinogenic substances, such as LSD, had already become very popular. The United States Drug Enforcement Administration (DEA) reported that “over 1000 scientific papers, several dozen books, six international conferences, and the treatment of over 40,000 patients with LSD” had been done between 1950 and 1965 (Drug Enforcement Administration 1995). The surge in information on hallucinogenic substances during this period lead to the emergence of many research societies, including the American College of Neuropsychopharmacology, the Substance Abuse Division of the American Psychological Association’s Psychopharmacology, and the Behavioural Pharmacology Society (Belouin and Henningfield 2018).

Hallucinogenic substances, including psilocybin, were entering the public culture at the time as “mind-altering” and “mind-enhancing” compounds (Drug Enforcement Administration 1995; Belouin and Henningfield 2018). These substances were believed to help an individual enter a transpersonal domain, where “individuality” dissipates, enabling one to experience other beings’ consciousness and simultaneously find pain relief and healing (Metzner and Darling 2005). Sandoz Pharmaceuticals began marketing psilocybin under the trade name Indocybin™ (1960) for therapeutic clinical research (Geiger et al. 2018). The results for psilocybin and other hallucinogenic substances were overwhelmingly positive without any reported fatality cases (Metzner and Darling 2005). Between 1960 and 1980, over 100 reports were generated, including analytical and biochemical studies of psilocybin and anecdotal reports of human use (Nichols 2020). For example, Rucker et al. (2016) showed that 79% of patients showed reduction in symptom severity of depressive disorders after treatment with hallunicogenic substances in 19 studies (1949–1973). Krebs and Johansen (2013) evaluated six studies treating alcohol addiction (1966–1970) and found significant recovery up to six months post-treatment. Unfortunately, none of the studies were controlled clinical studies (Nichols 2020), taking away from the value and ethical integrity of these studies, but the results were overwhelmingly positive without any reported fatality cases (Metzner and Darling 2005).

Legal action started against hallucinogenic substances after the ban of thalidomide, a medication prescribed for morning sickness that caused severe birth defects in unborn children (Chhabra et al. 2005). As a consequence the “Drug Amendments Act of 1962” was passed, giving the Food and Drug Association (FDA) strengthened regulatory controls on the experimentation of chemical entities in humans, inclusive of psilocybin (Belouin and Henningfield 2018). With the passage of the Controlled Substances Act of 1970, psilocybin, psilocin, LSD, and other hallucinogenic substances were placed under Schedule 1 drugs (Nichols 2004), the most restrictive category defined as having a high potential for abuse, no accepted medical use, and a lack of accepted safety protocols for use under medical supervision (Belouin and Henningfield 2018). This outcome made it virtually impossible to study these substances and ongoing human studies at the time ceased (Belouin and Henningfield 2018). Sandoz immediately ended Indocybin™ production. Ironically, recent research showed that most aspects of recreational use of these mushrooms did not pose significant harm, despite being treated in the same category and with similar punitive measures as serious and addictive hallucinogenic substances such as cocaine (Van Amsterdam et al. 2011; Roberts et al. 2020).

Hallucinogenic mushroom research evolution

The return of psilocybin to modern medicine was with a neuroimaging study showing the effects of psilocybin on the brain through the activation of the agonistic serotonin 2A (HTR_2A) receptor (Vollenweider et al. 1997, 1998; Vollenweider 2001). This re-opened human studies into psilocybin, which considered psilocybin a symptomatic modulator of mental disorders related to the serotonergic system such as depressive disorders, anxiety disorders, schizophrenia, addiction, attention deficit hyperactivity disorder (ADHD), and autism (Lin et al. 2014). Several major institutions began independently researching psilocybin treatment, including the American College of Neuropsychopharmacology, Substance Abuse Division of the American Psychological Association's Psychopharmacology, and the Behavioural Pharmacology Society, to name a few (Belouin and Henningfield 2018).

Pilot studies into the adjustment of the serotonergic system using psilocybin were conducted between 2006 and 2016 (Moreno et al. 2006; Griffiths et al. 2008; Johnson et al. 2008; Grob et al. 2011; Studerus et al. 2011, 2012; Krebs and Johansen 2013; Hanks and González-Maeso 2013; Baumeister et al. 2014; Garcia-Romeu et al. 2014; Tylš et al. 2014; Bogenschutz et al. 2015; Lebedev et al. 2015; Spring et al. 2016; Mithoefer et al. 2016; Ross et al. 2016; Erritzoe et al. 2018). For example, Garcia-Romeu et al. (2014) investigated treating tobacco addiction using psilocybin and showed 80% of participants adhered to smoking cessation after six months. Treatment of alcohol addiction with psilocybin showed improvement in participants' drinking behaviour immediately after treatment (Bogenschutz et al. 2015). Grob et al. (2011) treated terminally ill cancer patients with end-of-life anxiety using psilocybin and showed an improvement in mood lasting at least six months. Carhart-Harris et al. (2016) investigated the use of psilocybin to treat major depressive disorder, and found reduction in depressive symptom ratings in participants from baseline until six-months follow-up.

Modern day psilocybin studies

The FDA granted the “Breakthrough Therapy” designation to Compass Pathways (COMPASS Pathways 2018) to the therapeutic use of psilocybin in the treatment of treatment-resistant major depressive disorder. The FDA typically grants this designation in cases where preliminary studies suggested an enormous improvement of a drug over already available therapies. This designation allowed the fast-tracking of studies. The designation was monumental in the scientific research community and represented a significant shift in how the USA government perceived hallucinogenic substances. It was foreseen that prescriptions by a medical professional could be given for appropriate conditions in future (Hartman 2018).

Following the positive preliminary data and FDA approval, many more studies investigating the same topics in long term aspects and studies on a wider range of disorders were conducted. These include trials involving obsessive–compulsive disorder (OCD) (Reiff et al. 2020; Knopf 2021), cocaine use disorder (ClinicalTrials.gov identifier 04052568), opioid use disorder (ClinicalTrials.gov identifier 04161066), anorexia nervosa (ClinicalTrials.gov identifier 04052568), depression in early Alzheimer’s disease (ClinicalTrials.gov identifier 04123314) (Reiff et al. 2020), post-traumatic stress disorder (Chi and Gold 2020), cluster headaches (Tylš et al. 2016a, b; Castellanos et al. 2020), chronic pain (Castellanos et al. 2020), behaviour disorder and mental health problems (Hanks and González-Maeso 2016; Carhart-Harris et al. 2016; Jungaberle et al. 2018). A number of patents have been obtained or were abandoned in the past (Gerber et al. 2021).

The modern-day resurgence of research on psilocybin is due to the positive findings in the preliminary data. The number of published review articles and clinical reports (Reiff et al. 2020) is still increasing rapidly, as shown by a current literature search done with the advanced search builder of the NCBI (National Centre for Biotechnology Information) using the search terms “psilocybin” and “psilocin” or “psilocybin AND psilocin” (Fig. 1). Numerous research papers are rapidly appearing on a number of topics, making it difficult to mention all of them. In fact, upon writing this review, new and insightful research papers and reviews continued to appear.

Fig. 1.

Results from an advanced search builder in NCBI (National Centre for Biotechnology Information) using the terms “psilocybin” and “psilocin” or “psilocybin AND psilocin”, to investigate the amount of literature published (source NCBI dated 26/09/2021)

Economic and legal implications

The market cap for hallucinogenic drug companies world wide has been estimated to range from $1.9 billion to $253 billion at the end of November 2021 for the top related industries, which are often also involved with other hallucinogenic compounds (Phelps et al. 2022). This is due to the treatment potential that psilocybin and psilocin have, being non-addictive antidepressant substances that have a low lethality risk with long term benefits (Van Amsterdam et al. 2011; Johnson et al. 2018). Indeed research, and even public access to psilocybin, is increasingly becoming legal in some countries (Phelps et al. 2022). As psilocybin is legally reclassified to enable researchers to investigate its therapeutic potential, it would make a significant impact on treatments available to psychiatric medicine (Rucker 2015; Kargbo 2020; Nichols 2020). It would be important for researchers and industry to collaborate closely, to ensure that what becomes available to health practitioners, patients and the public will be based on solid results and clinical testing, whereas industry could provide funding and unique research questions (Phelps et al. 2022).

Although processes to chemically produce these various compounds exist (Sherwood et al. 2020a; Dörner et al. 2022), the use of the mushrooms and sclerotia (referred to as magic truffles) appeals to the larger, nutraceutical public market. This is because the processes to grow them are completely sterile, specific and organic without the need to add anything such as fungicides. Furthermore, these products also represent a possible synergism of various compounds found in the mushrooms including other hallucinogenic compounds than psilocybin (Dörner et al. 2022), whereas chemically produced compounds will have to be mixed together. Furthermore, the consumed mushroom material may also include other types of bioactive and beneficial compounds (for example Morales et al. 2021; Venturella et al. 2021).

Physiological and psychological activities of psilocybin and psilocin in humans

Absorption

Approximately 20 min after oral consumption of hallucinogenic mushrooms by humans, psilocybin and psilocin can be traced in blood plasma (Dalefield 2017; Puschner 2018). Maximum concentration levels are reached between 80 and 100 min and can be detected for up to six hours (Tylš et al. 2014; Aronson 2016b). Between 90 and 97% of psilocybin is converted into psilocin, of which 80% is found to be in a conjugated form with glucoronic acid or other compounds in the blood plasma (Passie et al. 2002; Tylš et al. 2014). The lipid soluble psilocin crosses the blood brain barrier without mediation and competition (Rautio et al. 2008; Stebelska 2013).

Besides clinical dosing (Passie et al. 2002; Li et al. 2022), microdosing (when amounts small enough not to enduce hallucination are applied intermittently) is shown to also improve depression and anxiety symptoms, social interactions, and cognitive function in clinical settings, and is also used by the public (Cameron et al. 2020). It is, however, important that regulation of dosages applied in settings outside clinical trials, should receive urgent attention (Tullis 2021). Upon legalization, it will be important that the public should be educated on the correct dosages to take, especially based on the types of mushroom species and material that will be consumed. In order to regulate and control the products that will become available to ensure public safety, tracibility of psilocybin and related compounds should be determined, based on strain and species, to provide accreditation to available products.

Physiological and psychological effects

The physiological effects attributed to the spiritual use of these fungi include changes in the sense of time, synaesthesia (alternate ways in how senses are being experienced) and hallucinations (Fricke et al. 2017). For example, psilocybin caused an enhanced responsiveness to music (Wall et al. 2022). Enhanced introspect is achieved and experiences have a mystical feel to it. Both animal and human studies suggest that psilocybin slightly increases an individual’s blood pressure and heart rate (Hasler et al. 2004; Griffiths et al. 2006). The increase was approximately 10–30 mmHg in both systolic and diastolic pressure, and the average heart beat was between 82 and 87 beats per minute (Hasler et al. 2004; Griffiths et al. 2006; Tylš et al. 2014). Psilocybin did not affect an individual’s core body temperature or electrocardiogram (ECG) (Hasler et al. 2004).

Common somatic symptoms after consuming psilocybin and psilocin that could be perceived negatively such as dizziness, weakness, tremor, nausea, vomiting, drowsiness, yawning, paresthesia, blurred vision, and increased tendon reflexes (Tylš et al. 2014; Aronson 2016b; Puschner 2018; Van Courte et al. 2022). Muscle relaxation is also experienced with intoxication. In fact, recently wood lover’s paralysis (WLP) start to receive more attention as a serious side effect, albeit temporary, when ingesting certain wood inhabiting hallucinogenic species (Dörner et al. 2022; Van Court et al. 2022). Besides the negative symptoms mentioned, some individuals are also reported to become fearful, especially when having a prior depressive episode (Hasler et al. 2004; Van Courte et al. 2022). However, it is unclear exactly what is meant by a prior depressive epidode, for example if the individual have in fact been clinically diagnosed by major depressive disorder. Two case studies where patients with previous psychiatric comorbidities showed increased aggression, depression, and psychosis, respectively, upon consumption of hallucinogenic mushrooms or different types (Nielen et al. 2004), highlights the importance that although these hallucinogenic mushrooms may appear as miracle self-help drugs, proper clinical diagnoses, and oligogenic profiles are crucial.

Psilocybin and psilocin in the brain

When psilocybin is ingested by the body, it is readily converted into psilocin (Arora 1986). Psilocybin and psilocin are structurally similar and functionally related to serotonin (Tylš et al. 2014) (Fig. 2). Hence they can affect the serotonergic system in both humans (Chi and Gold 2020) and animals (Horita and Weber 1962), and possibly even insects with serotonergic pathways (Awan et al. 2018a, b). The serotonergic system regulates various physiological processes that are vital to healthy body function, some of which include circadian rhythm, appetite, pain, vascular tone, platelet formation, motor activity, and cognitive function (Kenna et al. 2012; Mann 2013). In the brain, serotonin functions as a neurotransmitter to modulate anxiety and stress as well as promote patience and coping (Carhart-Harris and Nutt 2017). Sex differences in the behavioural effects of psilocybin have also been noted (Tylš et al. 2016a, b).

Fig. 2.

Chemical structures of psilocybin and structurally related compounds. The illustrations was created by ChemDraw Ultra (https://chemistrydocs.com/chemdraw-ultra-12-0/)

Psilocin is a high-affinity agonist of serotonin HTR_2A (HT: serotonin; R: receptor; 2A: family linked to function) receptors (Puschner 2018; Madsen et al. 2019) in the brain, particularly in the prefrontal cortex (Tylš et al. 2014). The overall result of HTR_2A activation is increased cortical activity secondary to down-stream postsynaptic glutamate effects (Puschner 2018). Psilocin is also active at the HTR_1A, HTR_1D, and HTR_2A receptors, but with lesser effects (Aronson 2016a). Psilocin binds to these receptors in the following order: HTR_2B > HTR_1D > DRD₁ > HTR_1E > HTR_1A > HTR_5A > HTR₇ > HTR₆ > DRD₃ > HTR_2C > HTR_1B > HTR_2A (Ray 2010; Tylš et al. 2014)., where DRD₁ and DRD_{3 are} different receptors subtypes of dopamine (D: dopamine; R: receptor; D: family). More receptor subtypes where weak bonds occur include the receptors for adrenergic α2_A, Imidazoline₁ and HTT transporters (Ray 2010). While the HTR_2A receptor activation is considered necessary for hallucinogenic effects (Nichols 2004), the role of other receptor subtypes is much less understood.

Psilocybin and psilocin are detectable in human urine (at 3–10%, and 90–97%, respectively), showing that psilocybin is readily converted to psilocin after consumption (Passie et al. 2002). The elimination half-life of psilocybin is 50 min and the elimination constant is 0.307/h (Lindenblatt et al. 1998). Approximately 70% of psilocybin is excreted through urine within three hours after oral administration and is completely eliminated from the body within 24 h (Aronson 2016a). Metabolism of both psilocybin and psilocin is facilitated by Monamine Oxidase A, which is also responsible for metabolism of serotonin (Lenz et al. 2020).

Chemical characteristics and production of psilocybin, psilocin, and related compounds

Chemical characteristics

Psilocybin and psilocin are tryptamine molecules (Fig. 2) that have an indole ring with attached amine side chains (Metzner and Darling 2005). The tryptamine chemical backbone serves as a basic structural unit for indolealkylamine as well as serotonin (Fig. 2) (Snook 2016; Thukral et al. 2020). Psilocybin and psilocin appear to be volatile compounds, and can be vaporized at normal temperatures into surrounding air. Wasson noted that he hallucinated inside a house that contained a significant amount of fresh hallucinogenic mushrooms (Wasson 1957).

Psilocybin and psilocin present as a white crystalline powder in their pure forms (Tylš et al. 2014) and with molecular weights and melting points of 284.252 g mol⁻¹ and 204.273 g mol⁻¹, and 220–228 °C and 173–176 °C, respectively. Psilocybin has been found to be water soluble due to the presence of bulky phosphate groups, while psilocin is considered to be more lipid-soluble (Tylš et al. 2014). However, both psilocybin and psilocin have been found to be soluble in methanol and ethanol (Dinis-Oliveira 2017). Notably, both of the compounds are insoluble in petroleum, ether, and chloroform (Barceloux 2012). Psilocin can be diluted in an acidified aqueous solution as well as in dimethylsulfoxide (Barceloux 2012). Both substances are unstable in light, particularly when dissolved into a solution, but show good stability at low temperatures in dark areas with chemically inactive atmospheres (Anastos et al. 2006; Gotvaldová et al. 2020). They are found in the highest concentrations in the cap of the mushroom (Gotvaldová et al. 2020).

Other alkaloids than psilocybin and psilocin, such as aeruginasin, baeocystine, norbaeocystine, norpsilocin, lumichrome, and verpacamide A, were observed to be structurally related to psilocybin and psilocin and could be isolated from hallucinogenic species (Sherwood et al. 2020a, b; Dörner et al. 2022). Aeruginascin is N-trimethyl analogue of psilocybin, possessing a molecular formulae of 4-hydroxy-N,N,N-trimethyltryptamine (4-HO-TMT) generated due to hydrolysis of psilocybin (Jensen et al. 2006; Sherwood et al. 2020a, b). It was originally discovered in Inocybe aeruginascens (Chadeayne et al. 2020). Human exposure to this compound also cause hallucinations with euphoric experiences, similar to psilocybin (Gartz 1989). Notably, aeruginascin is closely related to bufotenidine, an analogue of serotonin present in the toad venom (Glennon et al. 1991; Glennon 2006).

Baeocystine is a tryptamine natural product present in psilocybin-producing mushrooms such as Inocybe corydalina, Psilocybe baeocystis, and P. cubensis (Leung and Paul 1967, 1968; Sherwood et al. 2020a, b). It is a tryptamine alkaloid with a chemical formula of [3-[2-(methylamino)ethyl]-1H-indol-4-yl] dihydrogen phosphate, and observed to possess hallucinogenic properties. Notably, in animal studies baeocystin alone was not evident to exhibit hallucinogenic properties (Sherwood et al. 2020a, b). Norbaeocystin [3-(2-aminoethyl)-1H-indol-4-yl] dihydrogen phosphate, is a tryptamine derived alkaloid where the tryptamine carries an additional phosphoryloxy substituent at position 4 (Leung and Paul 1968).

Norpsilin was extracted from P. cubensis and has a formulae of 3-[2-(methylamino)ethyl]-1H-indol-4-ol (Lenz et al. 2017). The most recently discovered alkaloids from Psilocybe species are Lumichrome and Verpacamide A (Dörner et al. 2022). Lumichrome has a formula of 7,8-dimethyl-1H-benzo[g]pteridine-2,4-dione. It is derived from an alloxazine in being a tautomer of a 7,8-dimethylisoalloxazine. It shows blue fluorescence due to photolysis of riboflavin in acidic or neutral solutions. Lumichrome is a natural product found in the bacteria Streptomyces, Nocardia alba, and plants (Rajniak et al. 2018). Verpacamide A is written as 2-[3-[(3S,8aS)-1,4-dioxo-2,3,6,7,8,8a-hexahydropyrrolo[1,2-a]pyrazin-3-yl]propyl] guanidine and is a cyclo-(L-arginine-L-proline) inhibitor. It is a natural product also found in the bacteria Streptomyces nigra, Pseudomonas, and the sponge Axinella vaceleti (Dörner et al. 2022).

Harmane and harmine are tremorogenic β-carboline alkaloids with a structure of 9H-beta-carboline carrying a methyl substituent at C-1 (Herraiz and Chaparro 2006), and a harman skeleton with methoxy- substituted at C-7 (Djamshidian et al. 2016), respectively. These β-carboline alkaloids have been reputed to be hallucinogenic (Mamedov et al. 2018; Dörner et al. 2022). They also inhibit the monoamine oxidase (MAO) enzyme family that breaks down serotonin, psilocybin and similar compounds (Lenz et al. 2020). These compounds have been extracted from various plant-derived food commodities such as vinegar, grapes, rye, soybeans, barley, corn, wheat, wine, beer, whisky, brandy, sake, and inhaled substance (tobacco) (Poindexter and Carpenter 1962; Adachi et al. 1991; Zheng et al. 2000). Additionally, they are endogenous to animal tissues and have been extracted from beef and sardines (Poindexter and Carpenter 1962; Rommelspacher et al. 1982; Adachi et al. 1991). Recent studies have found them to also be present in Psilocybe species, including two new forms, namely norharmane and cordysinin C/D (Dörner et al. 2022). Norharmane, 9H-Pyrido[3,4-B]indole, is a natural product found in, for example, the plants Ophiopogon and Strychnos johnsonii. β-carboline is the parent compound, which is a tricyclic structure comprising of an indole ring system where the ortho- is fused to C-3 and C-4 of a pyridine ring and a mancude organic heterotricyclic parent (Aiypo et al. 2021). Cordysinin, molecular formula (3S,6R,8aS)-6-hydroxy-3-(2-methylpropyl)-2,3,6,7,8,8a-hexahydropyrrolo[1,2-a]pyrazine-1,4-dione, is a pyrrolopyrazine where hexahydropyrrolo[1,2-a]pyrazine-1,4-dione is substituted by a hydroxy group at position 6, and a 2-methylpropyl group at position 3. It is a natural product and has been isolated from the mycelia of Cordyceps sinensis (Yang et al. 2011).

Studies on the genomes of some psilocybin-producing species indicated that more diverse metabolites similar to the analogues of psilocybin most likely will be discovered (Dörner et al. 2022). The genes generaly form a cluster with its evolutionary origin possibly tied to insect relationships, which are thought to be the result of convergent evolution (Awan et al. 2018a, b) or horizontal gene transfer (Reynolds et al. 2018; McKernan et al. 2021). It is also thought that there could be interactions between these compounds when ingested and that the different compounds have varying effects (Sherwood et al. 2020a, b). Some of the adverse effects experienced following digestion of mushrooms could also be attributed to these other compounds. For example, aeruginascin in some species could be responsible to cause temporary muscle weakness (WLP) (Sherwood et al. 2020a, b).

Understanding the genomic and transcriptomic structures related to genes of the psilocybin pathway and those of similar compounds, are still at the initial stages (Van Courte et al. 2022). Furthermore, refining the annotations and comparisons with more strains and species, and species in other genera than Psilocybe, are still much needed, besides the fact that the species identities of all of the available strains should actually all be verified. This is hampered by the fact that these strains are still illegal in many countries, and reputed biobank collections other than commercial collections representing the substantial genetic variation present, are scarce (McKernan et al. 2021). A better understanding of exactly what compounds are produced by each species and strain, linked to morphology as well as DNA sequence based identifications and genome data, will greatly help to better understand these fascinating compounds.

Production in vivo (biosynthesis)

Numerous strains of Psilocybe species, different strains of P. cubensis, and hallucinogenic species from other genera, are commonly used by the public to produce mushrooms and sclerotia, as can be seen in a basic internet search. These various strains are reputed to have differing potencies and other abilities, such as ease and speed of growth, and flushing. There is also variation in content between different parts of the fruiting bodies (Gotvaldová et al. 2020). Continued development, and even hybridization between strains, are being done to increase yield and psilocybin content. The available strains have not yet been scrutinized adequately in scientific literature due to the legal restrictions that have only been lifted recently in some countries, although their properties are most likely well known by commercial companies using them for the production of psilocybin (Harris 2022). A great need thus exists for publically available and reputed scientific output on the genetic identities, stability, production properties, and cultivation techniques of these strains.

Since the discovery of psilocybin, three biosynthetic pathways have been proposed for psilocybin production in vivo (Fig. 3). In the first pathway (Fig. 3a) L-tryptophan is the pathway precursor, with five reactions leading to the biosynthesis of psilocybin (Hofmann et al. 1958; Agurell et al. 1968). The first biosynthetic pathway was determined by radiotracer labelling using ¹⁴C- and ³H- in Psilocybe semperviva (Hofmann 1958; Agurell et al. 1966, 1968). The pathway originated with L-tryptophan that initially undergoes decarboxylation to yield tryptamine, followed by a successive N,N-dimethylation, C-4 hydroxylation, and 4-O-phosphorylation to yield psilocin and psilocybin (Agurell et al. 1966). A second pathway (Fig. 3b) included the hydroxylation of tryptamine to psilocin through the transformation of N,N-dimethyl-tryptamine to 4-hydroxy-N,N-dimethyltrypatmine (Gartz and Moller 1989). Psilocybin is produced through phosphorylation of the oxygen group. Psilocin is formed through methylation of the nitrogen containing side chain.

Fig. 3.

Multiple pathways for the biosynthesis of psilocybin in vivo. A The first biosynthesis pathway from tryptophan that is decarboxylated and methylated to N,N-dimethyltryptamine (DMT) (indicated with black arrows), that is oxidatively phosphorylated to psilocybin. B The second pathway from 4-hydroxytryptamine to 4-hydroxy-N-methyltryptamine to psilocin through methylation (indicated by dark grey arrows), and last psilocybin through phosphorylation (indicated by dark grey arrows). C The third biosynthesis pathway from tryptophan to norbaeocystin, and methylation of norbaeocystin and baeocystin resulting in psilocybin (indicated by light grey arrows). In the pathways the gene PsiD encodes for tryptophan decarboxylase, PsiH for a P₄₅₀ monooxygenase, PsiK for a kinase, and PsiM for a methyltransferase. The rectangle dotted box demarcated the three distinguishable pathways as indicated in the subsections of A, B and C. The illustrations were created by ChemDraw Ultra (https://chemistrydocs.com/chemdraw-ultra-12-0/)

Incompletely methylated psilocybin compounds, named baeocystin and norbaeocystin, found in specimens of Psilocybe baeocystis and Psilocybe semilanceata (Leung and Paul 1968; Repke and Leslie 1977; Repke et al. 1977), could be precursors to a third pathway (Fig. 3c). This was confirmed when baeocystin and norbaeocystin from P. cubensis, Psilocybe cyanescens, and Psilocybe serbica were used in an in vitro enzymatic route for psilocybin production in transformed Escherichia coli cells (Fricke et al. 2017, 2019a). In this pathway L-tryptophan is decarboxylated by PsiD (tryptophan decarboxylase) to produce tryptamine, which is oxidised by PsiH (P₄₅₀ monooxygenase) to produce 4-hydroxytryptamine (Fricke et al. 2017, 2019a). PsiK (a kinase) catalyses the phosphotransfer step to produce norbaeocystin, and then a methyltransferase, named PsiM, catalyses a methyl transfer to produce baeocystin as precursor for psilocybin. In vivo production of psilocybin can also be done using Sacharomyces cerevisae (Milne et al. 2020; Wang et al. 2021), representing large biotechnological potential for production.

The genes encoding the enzymes of the third pathway were annotated from genome sequences of P. cubensis and P. cyanescens (Fricke et al. 2017, 2019b), and refined consequently from genomes of more species (Awan et al. 2018a, b; Reynolds et al. 2018; McKernan et al. 2021). Besides the genes for psiD, psiH, psiK and psiM, genes were also found for two major facilitator-type transporters (psiT1 and psiT2) and a putative transcriptional regulator (psiR) (Fricke et al. 2017, 2019b). Degenerate primers have been designed for psiD and psiK (Reynolds et al. 2018).

Chemical production

Chemical production of psilocybin and the other related alkaloids are possible (for example Nichols and Frescas 1999; Lenz et al. 2017; Shirota et al. 2003; Kargbo et al. 2020; Sherwood et al. 2020a, b; Dörner et al. 2022) unless consumed in the form of dried or powdered fruiting bodies or slerotia. Crystaline forms (Nichols and Frescas 1999; Sherwood et al. 2020a) are usuable unstable even though preferably used in the for-profit drug industry and for commercial-scale production by large pharmaceutical companies, each usually with their intellectual property applied (Phelps et al. 2022). Usage of forms of another molecule, namely psilacetin (C₁₄H₁₈N₂O₂) (Fig. 2), as a prodrug for psilocin, appear to be easier, more stable, and cheaper (Chadeayen et al. 2019).

Conclusion

Research on hallucingenic substances have come a long way since the criminalization of most hallucinogenic chemical compounds with a large and growing number of research papers appearing. Hallucinogenic substances, such as psilocybin and psilocin, have great research and therapeutic potential in psychiatric and neurological disorders and could serve as a model to better understand the serotonergic system. Self medication makes these fungi important as nutraceauticals. With legalization starting to occur in some countries across the world, and responsible research these fungi could result in breakthroughs in the pharmaceutical arena serving medical, psychiatric, and nutraceutical fields, easing the lives of many.

The potential of hallucinogenic mushrooms is especially relevant in third world countries, where western medicines, such as antidepressants, are not readily available, or trusted. For example, in South Africa depression is prevalent in up to 34.7% of individuals across all sociological groups (Igboeli et al. 2021), yet most of these people may not have access to supportive medicines. Depression can lead to fatal outcomes such as suicide, with South Africa having roughly 8000 reported suicides each year and at least 23 suicides successfully completed each day. However, in the Americas and elsewhere hallucinogenic mushrooms have a long history of traditional use (Allen and Gartz 2009; Gerber et al. 2021). Traditional healers, for example in Tanzania (Tibuhwa 2018), have accepted medicinal mushrooms as a traditional therapeutic practice and species are sold in traditional markets. Traditional healers are acknowledged as health professionals in numerous cultures, and are legally practicing in many countries, such as South Africa where these healers are used by more than 80% of the population (Liverpool et al. 2004).

Care should be taken to give credit to the peoples who discovered the uses of these fungi, and to follow proper ethical clearance and benefit sharing according to international treaties protecting their rights (Gerber et al. 2021; Van Courte et al. 2022). Furthermore, illegal movement of strains are happening across the world from countries where these fungi could still be undescribed, or unstudied. In their new locations these strains are cultivated and even hybridized with other strains. This sharing of material is uncontrolled, unregulated and against the Convention for Biological Diversity. This is particularly concerning since the true species identities, genetic diversity (Van Courte et al. 2022), geographical origins of these species, and even commonly used strains, are still largely uncharacterized.

Large gaps in knowledge still exist on exactly what compounds are present in specific species, their individual or possible synergistic effects, taxonomic and biodiversity gaps, and the degree to which species have been sequenced to ensure proper identification (Dörmer et al. 2022; Strauss et al. 2022; Van Court et al. 2022). Lack of natural product accredidation, and responsible use by the public upon legalization without aggrevating other possible psychiatric conditions, should be seriously considered. The potential to consume similar looking toxic species (Strauss et al. 2022; Van Courte et al. 2022) or contaminated mushroom products, are realistic dangers. These gaps, however, represent exciting research opportunities in a very relevant field, besides the clinical and commercial aspects that perhaps receive more attention at the moment.

In conclusion, the natural availability, easy and organic cultivation, and low-risk pharmacological profile of psilocybin producing mushrooms make them a worth-while investment in health sectors where there are not always financial resources to use expensive phramaceuticals. Furthermore, the use of extractions from mushrooms, which then also includes other compounds beneficial to the experience of using these products, are often more desireable (Harris 2022). The treatment efficacy of psilocybin and psilocin are not dependent on any of the 5-HT precursor compounents, making them an ideal replacement therapy. Due to the organic nature of psilocybin and psilocin, the unexpected chemical side effects experienced with synthetic anti-depressants should be minimal. These mushrooms could thus positively impact on the quality of life for numerous people, especially those who have exhausted all other possibilities. More so, because psilocybin is a compound sought after by large corporate drug companies, but also used as self medication by the public.

Author contributions

DS: literature search and writing; SG, ZM, MG: editing and supervision.

Funding

This work did not received any funding.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.