Neuroprotective Biomolecules from Hericium erinaceus: A Biochemical Perspective

Abstract

Hericium erinaceus (lion’s mane) is among the most widely marketed natural product preparations for cognitive health and has a long-documented history of use in East Asian traditional medicine alongside botanical drugs. Its principal bioactive constituents, the cyathane diterpenoids, meroterpenoids and structural polysaccharides, belong to the same natural product chemistry, pharmacokinetic and quality control framework applied to plant-derived phytomedicines. Therefore, H. erinaceus faces the same translational questions of batch consistency, bioavailability and mechanistic definition. Scientific evidence remains complicated by the variations in fruiting body and mycelium biochemistry, extraction procedures and pharmacokinetic data. The current work addresses this gap by compiling natural product chemistry, structure–activity relationships, mechanisms of action, preclinical and human studies, along with analytical quality control of H. erinaceus metabolites. It proposes a matrix-specific quality control approach supported by authentication, biochemical makeup and markers for the starting material (fruiting body/mycelium), in addition to critically evaluating the relevant current studies for strengths and weaknesses. Using bioanalytics, mass spectrometry and metabolomics, we also highlight methodologies for compound quantification and novel biomolecule discovery. By linking chemical standardisation to the clinical evidence base, this review provides a quality assessment framework of the kind required for herbal and other natural product remedies. It also identifies the pharmacokinetic and trial design gaps that currently limit integration of these preparations into evidence-based practice.

1. Introduction

The transmission of adaptive memory across generations underpins cultural, scientific, and technological progress [1]. Dementia is projected to affect 153 million people by 2050 and is emerging as the seventh leading cause of death worldwide, intensifying interest in safe, diet-based strategies for protecting brain health [2,3]. This trend urges global research efforts across academia, non-profits and industry to prioritise brain health. Studies highlighting the significance of dietary patterns and micronutrient supplementation on cognitive function and neuroprotection are gaining traction for the same reason [4,5,6]. As the global dietary supplements market is expected to grow to a value of USD 300 billion by 2028, it is essential to have a basic understanding of the biochemistry of the dietary supplements consumed on a regular basis [7]. In this review, we highlight the neuroprotective and neurotrophic bioactive compounds found in the culinary mushroom Hericium erinaceus (lion’s mane (LM)), which is widely consumed in Asian countries such as China, India, Japan, and Korea. LM produces a remarkably diverse metabolome, including >250 secondary metabolites such as polysaccharides, cyathane-type diterpenoids, meroterpenoids, isoindolinone derivatives and other small molecules with neurotrophic, neuroprotective, antioxidant, anti-inflammatory, immunomodulatory, and cytotoxic activities [8]. In parallel, the rapid growth of the LM supplement market is outpacing the development of robust quality standards, as fruiting-body versus mycelium products, extraction conditions and storage all profoundly influence metabolite profiles and stability. This review provides an overview of the neuroprotective and neurotrophic constituents that are abundant in LM and their relevant bioactive roles in neuronal health. The identity of these molecules in LM can be used for quality control, variety selection and cultivation. This review encompasses current knowledge on LM biochemistry, neuroactive mechanisms and preclinical and clinical evidence, identifies critical translational gaps and highlights how LC-HRMS/MS-based untargeted and targeted metabolomics can be used to authenticate LM materials, benchmark neurotrophic metabolites and establish metabolomics-driven quality control frameworks for LM-based functional foods and nutraceuticals targeting brain health.

Earlier reviews established hericenones and erinacines as candidate neuroactive metabolites and summarised the initial NGF studies [9]. Subsequent reviews have broadened our knowledge about the chemical complexity of the fungi, and preclinical research has provided insight into their mechanism of action. Although H. erinaceus is a macrofungus, its therapeutic evaluation shares core challenges with plant-derived phytomedicines, including chemical variability, uncertain bioavailability and limited clinical validation. This review advances research relevant to phytomedicine by integrating the chemistry and proposed mechanisms of its neuroactive constituents with evidence from preclinical and human studies. Attention is given to distinguishing fruiting body and mycelium preparations, selecting appropriate analytical markers and separating chemical standardisation from demonstrated biological potency. This integrated view helps in improving experimental reproducibility, interpreting differences between preparations and designing studies that relate administered bioactive levels to exposure and clinical outcomes. By identifying priorities for analytical validation, pharmacokinetic investigation and adequately powered clinical trials, the review supports the development and evaluation of H. erinaceus-derived preparations for potential neuroprotective applications [10,11,12].

2. Growth Habits and Culinary History of Hericium erinaceus

Hericium erinaceus (Bull.) Persoon, commonly known as yamabushitake, lion’s mane (LM), or monkey’s head mushroom, is a saprophytic fungus in the class Basidiomycetes, subclass Holobasidiomycetidae, order Russulales, and family Hericiaceae. It mainly grows on hardwood species such as Quercus, Acer, and Betula [13,14]. Widely consumed in East Asia, especially China, it is appreciated both as a culinary delicacy and for its medicinal properties [15]. Historical records, such as the History of the Three Kingdoms (280 AD), document its medicinal use, particularly for supporting the health of the five internal organs liver, lung, spleen, heart, kidney, improving digestion, vitality, and strength [16]. More recently, scientific interest has focused on its neuroprotective properties, as the fruiting body and mycelia of LM have been identified as promising materials for neural development and brain health [15,17,18,19]. This dual role in traditional and modern contexts underscores its importance as both a functional food and a potential therapeutic agent.

3. Unique Biochemistry of Hericium erinaceus

Recent studies report more than 250 distinct secondary metabolites from Hericium species, covering a broad range of chemical classes including cyathane diterpenes, alkaloids, benzofurans, chromenes, phenols, pyrones and steroids [20]. Among the cyathane diterpenes, the erinacines represent a particularly significant group, with biosynthetic investigations indicating that these structurally complex metabolites arise through specialised fungal terpene pathways. Recent genomic and transcriptomic analysis have further begun to elucidate the key enzymatic steps involved in their synthesis, providing a clearer framework for understanding their metabolic origin [21]. Recent isolation studies further expand this chemical space through the discovery of cyathane diterpene glycosides like hericinosides A–M with significant α-glucosidase-inhibitory activity [22]. Recently, new alkaloids erinacenone A–L were isolated from LM cultures, with their structures elucidated by spectroscopic techniques, expanding the structural diversity of Hericium metabolites [23]. Similarly, the discovery of a novel isoindolinone derivative, erinacerin W, from mycelia, along with other known erinacines, demonstrates that mycelium culture conditions (e.g., pH, aeration, growth medium) can influence production of bioactive compounds.

Functionally, many of these metabolites show biological activities consistent with neuroprotection, neurotrophic stimulation, immunomodulation, anti-inflammatory, antioxidant and cytotoxic/anticancer effects [8,24,25]. Some of the newly described erinacenones displayed cytotoxicity against MCF-7 cancer cells in vitro, while unusual cyathane and neoverrucosane diterpenoids from LM cultures have been shown to exert anti-neuroinflammatory effects [23,26]. This wide structural and functional diversity highlights the therapeutic and nutraceutical promise of LM metabolites. However, substantial differences between fruiting-body and mycelium metabolite profiles coupled with variability arising from culture and processing conditions indicate that the biochemical composition of supplements made from LM can differ considerably.

The neuroprotective and neurotrophic properties of LM have been linked to a diverse array of over 70 unique secondary metabolites found in its mycelium and fruiting bodies. These neuroactive compounds are broadly categorised into polysaccharides and terpenoids. Advanced analytical techniques, such as liquid chromatography-mass spectrometry (LC-MS) and two-dimensional nuclear magnetic resonance (2D-NMR), have been instrumental in identifying and characterising these metabolites [14,27,28,29]. This section highlights key biomolecules in LM that contribute to brain health and examines their potential applications as therapeutic agents for a variety of medical conditions associated with brain health. By leveraging their neuroactive properties, these compounds offer promising avenues for advancing both neurological health and broader pharmacological therapies.

3.1. Polysaccharides

Polysaccharides are major structural molecules in fungal cell walls and account for roughly 20% of the total biomass of Hericium erinaceus fruiting bodies and mycelium. They have reported antioxidant, immunomodulatory, hypolipidaemic, anticancer and neuroprotective activities [28,30]. However, LM polysaccharides should not be considered as a single pharmacological class. Separation studies coupled with bioanalytics have shown substantial differences in molecular mass, monosaccharide composition, glycosidic linkage, branching and dispersity. All of which influence solubility, gastrointestinal stability, microbial fermentation, receptor recognition and biological activity [31,32].

A recently identified neuroprotective fraction, HEPS, contained two high molecular weight polysaccharides of masses 1.7 × 10⁵ and 1.1 × 10⁵ Da. HEPS at 250 μg/mL improved cell viability, reduced reactive oxygen species (ROS) and mitochondrial damage, enhanced cellular radical scavenging activity and reduced apoptosis in amyloid β-exposed PC12 cells [31,33]. These findings demonstrate efficient neuroprotective activity under in vitro conditions but fail to include oral bioavailability and transport of these molecules across the blood–brain barrier. Similarly, WEP-1 is a 4.01 × 10³ Da water-soluble oligosaccharide in LM composed of mannose, glucose and galactose in a molar ratio of 1.2:16.9:1. Its reported structure includes an α-D-Glc-(1→3) and β-D-Gal-(1→3) backbone with β-D-Glc branches at C-4 and terminal β-D-Man. WEP-1 protected LPS-induced inflammation of intestinal cells by suppressing TLR4/NF-κB signalling and apoptosis [34]. Although TLR4-mediated inflammation is relevant to neurodegeneration, these data primarily support intestinal and peripheral anti-inflammatory activity rather than a direct effect on the central nervous system. HEP-W is another LM polysaccharide with a mass of 15.9 × 10³ Da. This heteropolysaccharide containing rhamnose, fucose, mannose, glucose and galactose activates macrophages through TLR2 and PI3K/Akt/MAPK signalling pathways [35]. This illustrates how linkage and branching patterns influence receptor binding but again supports immunomodulatory effects rather than direct neuronal mechanisms.

Neurotrophic effects have also been reported for an extracellular biopolymer from LM mycelium that has a mass of over 1.0 MDa. Composed of glucose, galactose, xylose, mannose and fructose, this polymer enhanced PC12 cell growth and neurite extension at 0.8 mg/L [36]. However, its direct application to cultured neuronal cells can be questioned without evidence from oral administration and pharmacokinetic studies. In peripheral nerve injury models, oral aqueous extracts and partially purified polysaccharide fractions accelerated sensory recovery, promoting axon regeneration and increased Akt and p38 MAPK signalling after peroneal nerve damage [37]. Nevertheless, the chemical complexity of the administered extracts and dose reporting limits pinpointing these effects to specific polysaccharides [38]. Furthermore, in rat models, an extract containing 20.66% β-(1→3,1→6) glucans from LM preserved cerebellar Purkinje cell integrity and enhanced the ERK-CREB-PSD95 signalling pathway, which is critical for neuronal function and protection [38]. However, since the extract was not pure, it is not possible to pinpoint the bioactivity to a single metabolite.

Overall, LM polysaccharides exhibit antioxidant, immunomodulatory, gut protective and neuroprotective activities, but these effects strongly depend on molecular composition and experimental framework. Direct brain uptake of intact high molecular weight fractions has not been demonstrated. Their neuroprotective activity after oral consumption is therefore more plausibly mediated through intestinal, immune and gut–brain-axis mechanisms. Future studies are needed to provide detailed structural characterisation, standardised dosing, pharmacokinetic data and clear differentiation between direct neuronal and peripheral mechanisms.

3.2. Terpenoids

Terpenes, the largest class of natural products, are another type of secondary metabolites of mushrooms which consist of isoprene repeating units, and many terpenes such as cyathane diterpenoids, triterpenoids, and sesquiterpenes have been identified or isolated from various genera of mushrooms including LM [39,40]. Hericenones (meroterpenoids) and erinacines (cyathane diterpenoids) are the two important classes of compounds isolated from LM, proven to induce the biosynthesis of nerve growth factor (NGF) in nerve cells in vitro.

3.2.1. Hericenes, Hericenones and Hericerins

Arnone et al. reported the isolation and characterisation of three geranyl-resorcinols—hericene A, B, and C—from the fruiting bodies of LM [41]. These molecules feature a resorcinol core attached to a geranyl unit along with three distinct fatty acid moieties. Additionally, the fruiting bodies of LM are rich in unique meroterpenes called hericenones (A–J), which are likely oxidation products derived from the geranyl side chains of hericenes via oxidative metabolic pathways. The hericenone content in the fruiting bodies has been reported to be approximately 2.36 mg per gram of dry weight [42,43]. This class of aromatic molecules with an isobenzofuranone skeleton are abundant in Hericium species and is known for their remarkable biological activities [8]. For instance, hericenones A and B have been shown to inhibit the proliferation of HeLa cervical cancer cells at minimum concentrations of 100 μg/mL and 6.3 μg/mL, respectively [44,45].

Structurally, hericenones C–H are benzyl alcohol derivatives featuring simple fatty acid chains. Cyclisation involving the phenolic hydroxyl group and the side chains of hericenones C–E likely results in the formation of hericenones F–H, which exist as racemic mixtures [32]. The biological activity of individual hericenones is influenced by variations in the length and degree of unsaturation of their fatty acid side chains [32]. Another significant class of natural products isolated from LM is the hericerins, which also belong to the geranyl resorcylate family. The chemical structure of hericerin was first elucidated by Kimura and their team, who demonstrated its ability to inhibit pollen germination [46]. In recent decades, these molecules have garnered significant interest in their neurotrophic and neuroprotective properties, with potential applications in managing dementia and related neurological disorders.

Tropomyosin receptor kinase B (TrkB) serves as the receptor for brain-derived neurotrophic factor (BDNF), a critical growth factor involved in neuronal survival, development, proliferation, and synaptic plasticity [47,48]. Recent findings highlight the influence of LM extract and hericerin A on TrkB signalling and neuronal morphology in hippocampal neurons [49]. The study demonstrated that mice orally administered crude LM extract and hericerin A exhibited increased expression of neurotrophins and activation of downstream signalling pathways. This enhancement was associated with significantly improved hippocampal memory, underscoring the therapeutic potential of these compounds for memory improvement and the mitigation of cognitive decline associated with ageing and neurodegenerative disorders [43,49]. Hericenones C–E have been reported to induce nerve growth factor (NGF) biosynthesis, with early studies from the 1990s documenting their activity. These studies also showed that hericenones C–E stimulated NGF synthesis in vitro in mouse astroglial cells at a dose of 33 μg/mL, with bioactivity comparable to that of the mammalian hormone epinephrine [50]. These molecules were among the first compounds of their class to be isolated from a non-animal source [45,50]. In a related study, oral supplementation with LM extracts containing hericenones C and D, along with other natural products in LM, improved recognition memory, hippocampal and cerebellar neurogenesis, and locomotor performance in frail aged mice [51,52].

An intriguing study by Tamrakar and team demonstrated that deacylhericenone, a derivative of hericenone C produced via lipase enzyme treatment, exhibited enhanced neuroprotective properties [53]. This compound showed greater stimulation of BDNF mRNA expression and reduction in oxidative stress, suggesting that the core structure of hericenones may be crucial for neuroprotective activity. Additionally, studies show that hericenone H stimulated NGF synthesis at a dose of 33 μg/mL, achieving a concentration of 45.1 ± 1.1 pg/mL in vitro in mouse astroglial cells [54]. Hericenones F and G also exhibited significant NGF-stimulating activity, though their effects varied based on the batch of cultured cells and cell density used in experiments. In 2008, three additional hericenones were identified, namely 3-hydroxyhericenone F, hericenone I and hericenone J [55]. Among these, 3-hydroxyhericenone F demonstrated significant protective activity against tunicamycin- and thapsigargin (TG)-induced toxicity in a dose-dependent manner, suggesting its potential to mitigate endoplasmic reticulum (ER) stress and prevent apoptosis.

Natural hericenes from LM, specifically hericene B and C, exhibited moderate protective activity against thapsigargin (TG)-induced endoplasmic reticulum (ER) stress at concentrations above 30 μM [56]. However, their protective effects were less pronounced than those of 3-hydroxyhericenone F, which showed significant protection against TG-induced toxicity at concentrations as low as 1.7 μg/mL. Notably, the study also highlighted that regioisomers of hericene B and C demonstrated significant protective activity against tunicamycin (TM)-induced ER stress at a concentration of 10 μM. These findings underscore the importance of the fatty acid chain in determining biological activity; specifically, the binding position of the fatty ester chain plays a critical role in modulating protective effects. This structure–activity relationship (SAR) data provides valuable insights into the molecular determinants of ER stress mitigation by hericenes. Collectively, these studies indicate that geranyl-resorcinols and their derivatives possess significant potential to counteract neuronal apoptosis, a pathological feature detrimental to brain health. The detailed molecular information of hericenes, hericenones and hericerins can be found in Figure 1.

Figure 1. Name, molecular formula, mono-isotopic mass and PubChem IDs of bioactive hericenones, hericerin and hericene isolated from fruit bodies and/or mycelia of H. erinaceus.

3.2.2. Erinacines and Erinacerins

The mycelium of LM mushrooms contains a class of cyathane-type diterpenoids known as erinacines, characterised by a unique five-to-six-seven tricyclic carbon scaffold. These compounds, considered the primary bioactive agents derived from the mycelium, exhibit distinct structural features, including the presence of a β-configured D-xylose moiety in their scaffold, which makes them detectable using UV detection at 340 nm [57]. A range of erinacines, including A, B, C [58]; D, E, F, G [59]; H, I [60]; P [61]; Q [62]; J, K [63]; R [64]; and erinacol [65] have been isolated from LM mycelium and shown to stimulate NGF biosynthesis. To date, more than 30 erinacines have been identified [43].

Another prominent class of secondary metabolites in the fruiting bodies of the Hericium genus includes isoindoline-1-one derivatives known as erinacerins. Wang et al. [66], during a study of LM from the Tibetan Plateau, isolated ten isoindoline-1-ones and characterised their structural and bioactive properties, including α-glucosidase inhibitory activity. Their work documented the physical and spectroscopic data for erinacerins C–L, demonstrating the potential of solid-state fermentation to enhance secondary metabolite biosynthesis in edible and medicinal mushrooms. Other notable erinacerins include W [67]; A, M and N [68]; as well as Q, S, and T [66]. Erinacerins have been widely recognised for their α-glucosidase inhibitory activity, suggesting their potential as antidiabetic agents [67,68,69,70], while erinacerins M and N have exhibited cytotoxic effects against various human cancer cell lines, including SH-SY5Y, 1321N1, HCT-116, Caco-2, OVK18, and HeLa [68].

In a bioassay using mouse astroglial cells, the amounts of NGF secreted into the medium in the presence of erinacine A–C at 1.0 mM were 250.1 ± 36.2, 129.7 ± 6.5, and 299.1 ± 59.6 pg/mL, respectively. These activities were much stronger than those (69.2 ± 17.2 pg/mL at 1.0 mM) of a known potent stimulator, epinephrine, used as a positive control compound in this assay [58]. In similar studies, the same team reported the NGF synthesis activity of erinacine D (at 1.67 mM NGF secretion was 141.5 ± 18.2 pg/mL), E and F (at 5 mM NGF secretion was 105 ± 5.2 and 175 ± 52 pg/mL, respectively) [59,70]. Lee and team successfully isolated and characterised erinacines H and I from the mycelia of LM [60]. Among these, erinacine H induced NGF secretion, achieving a concentration of 31.5 pg/mL in the culture medium at a treatment concentration of 33.3 µg/mL. In a subsequent study, Chen and team identified erinacine S, a sesterterpene isolated from LM mycelia, which, along with erinacine A, significantly reduced amyloid-beta (Aβ) plaque deposition in the brains of APP/PS1 transgenic mice, a widely used model of AD [71]. More recently, three additional novel cyathane diterpenes, erinacines T, U, and V, were isolated from liquid cultures of LM [72]. These compounds demonstrated pronounced neurite outgrowth-promoting activity in PC12 cells at concentrations ranging from 2.5 to 10 μM. The detailed molecular information of erinacines and erinacerins can be found in Figure 2 and Figure 3, respectively.

Figure 2. Name, molecular formula, mono-isotopic mass and PubChem IDs of bioactive erinacines isolated from fruit bodies and/or mycelia of H. erinaceus.

Figure 3. Name, molecular formula, mono-isotopic mass and PubChem IDs of bioactive erinacerins isolated from fruit bodies and/or mycelia of H. erinaceus.

4. Hericium erinaceus and Brain Health

In vitro studies have demonstrated that bioactive compounds extracted from LM (both fruiting body and mycelia), notably erinacines A and S, promote the development of cerebellar neurons and enhance the process of myelination [73]. These compounds facilitate the maturation of oligodendrocytes and upregulate myelin basic protein (MBP) expression, which is essential for effective axonal conduction [74]. Complementary in vivo studies have shown that oral administration of LM increases neurogenesis in the hippocampus and cerebellum of ageing murine models. This effect is evidenced by elevated expression of doublecortin (DCX), a marker for neuronal precursor cells, and is correlated with improved recognition memory, suggesting potential cognitive benefits [51]. LM extracts demonstrate immunomodulatory properties through the regulation of microglial activity. Erinacines have been reported to promote a neuroprotective microglial phenotype, characterised by decreased pro-inflammatory signalling and enhanced support for neuronal integrity [75].

While preclinical studies are promising, clinical research is still emerging. A pilot study involving patients with mild AD reported that supplementation with erinacine A-enriched LM mycelia led to improvements in cognitive function without significant side effects [76]. The study provided preliminary clinical evidence consistent with a potential cognitive benefit of an erinacine A-enriched preparation, although confirmation requires studies with larger sample sizes. A double-blind, placebo-controlled, parallel-group clinical trial conducted by Mori and team evaluated the efficacy of oral administration of LM in Japanese adults aged 50 to 80 years diagnosed with mild cognitive impairment and attributed the property tentatively to hericenones [77].

Given the pivotal roles of oxidative stress and neuroinflammation in declining brain health, LM has been noted for its robust antioxidant capacity, likely due to its rich content of phenolic and flavonoid compounds. These bioactive compounds enhance endogenous antioxidant defences, thereby reducing the levels of reactive oxygen species (ROS) and inhibiting lipid peroxidation [78]. In addition, LM extracts exert anti-inflammatory effects through the downregulation of the NF-κB signalling pathway, leading to a reduction in the synthesis of pro-inflammatory cytokines. Collectively, these antioxidant and anti-inflammatory properties contribute to the preservation of neuronal integrity. LM has shown antidepressant-like effects by regulating monoamine neurotransmitters and enhancing BDNF expression in the hippocampus. Chronic administration of LM extracts has been found to activate the BDNF/TrkB/PI3K/Akt/GSK-3β signalling pathway, leading to improved neuroplasticity and mood regulation. These effects are further supported by the modulation of inflammatory pathways, including the inhibition of NF-κB signalling, which is often upregulated in depressive states [79].

Among LM biomolecules, extracts enriched in erinacine A appear especially potent and are noted for having favourable properties regarding transportability across biological barriers. This implies better likelihood that the molecule reaches the brain and exerts neuroprotective/neurotrophic effects [80]. Erinacine A, further enhanced neuroprotection by downregulating the expression of inducible nitric oxide synthase (iNOS), reducing nitrotyrosine accumulation, and suppressing the phosphorylation of p38 mitogen-activated protein kinase (MAPK) as well as the expression of C/EBP homologous protein (CHOP). These findings indicate that erinacine A exerts its neuroprotective effects by modulating the iNOS/p38 MAPK/CHOP signalling axis, thereby attenuating neuronal apoptosis and neuroinflammation [81].

The principal mechanisms proposed from predominantly cellular and preclinical studies can be summarised into four categories (Figure 4):

Figure 4. Four major mechanistic pathways through which LM metabolites induce neuroprotective effects.

• Stimulating the synthesis of innate neurotrophins: Many erinacines and some hericenones have been shown to increase synthesis and release of neurotrophins, particularly NGF and, in some studies, BDNF and neurotrophin-3 (NT-3) [51]. These neurotrophins help neurons survive, differentiate, extend axons and dendrites and improve synaptic plasticity through receptor binding processes, which are vital for learning, memory, mood regulation and recovery from injury. Research also shows that hericenones and erinacines stimulate neurite outgrowth in neuronal cell models (often under low NGF levels), suggesting an overall increase in neurotrophic support [82].
• Reduction in oxidative stress: Extracts and isolated molecules from LM relieve oxidative damage through reduction in ROS levels, lipid peroxidation and oxidative damage in neural and glial cells. These molecules also enhance cellular resilience to oxidative stress by upregulating endogenous antioxidant defence pathways through the transcription factor Nrf2, which controls expression of antioxidant enzymes like superoxide dismutase, glutathione peroxidase, etc. [83].
• Activation of anti-apoptotic signalling: LM biomolecules modulate intracellular signalling cascades including p38 MAPK, JNK1/2 and ER-stress response via IRE1α/TRAF2 that regulate apoptosis, cell survival and endoplasmic reticulum (ER) stress responses. This way, LM-derived biomolecules protect neurons against damage arising from oxidative stress, ischemia, neurotoxins, etc., reducing cell death and improving neuronal lifespan [32].
• Modulation of neuroinflammation and neurotrophic homeostasis: Both polysaccharides and low-mass biomolecules from LM suppress inflammatory signalling by downregulating pro-inflammatory cytokines like IL-1β, IL-6, TNF-α and inhibiting iNOS and COX-2 expression, leading to lower nitric oxide (NO) and prostaglandin E₂ (PGE₂) production [83]. Some studies show that LM bioactives may influence metabolic pathways such as insulin-degrading enzyme expression and neurosteroidogenesis pathways. These activities contribute to neuroprotection via regulation of cellular metabolism, neuronal maintenance and myelination [84].

For interpretation of the evidence, in vitro findings were considered primarily hypothesis-generating, animal studies as preclinical evidence of possibility, and controlled human intervention studies as the highest level of evidence considered here. Preclinical studies were interpreted with attention to model relevance, chemical characterisation of the administered material, dose and exposure, controls, replication and correspondence between molecular and behavioural endpoints. Human studies were additionally considered in relation to sample size, blinding/control design, duration, endpoint consistency and chemical standardisation of the intervention. A bibliometric analysis of 839 publications on LM mushrooms published between 1992 and 2023 showed that clinical research represents only a small proportion of the available literature [85]. Consistent with this, the present review identified only eight published human intervention studies relevant to cognitive functions, mood, sleep, stress and/or related neurobiological activity between 2009 and 2025 (Table 1). Table 1 summarises published human intervention studies evaluating the effects of LM on cognition, mood, sleep, stress, and related neurobiological outcomes, while Table 2 presents the major gaps revealed by comparing the in vitro and in vivo evidence and highlights the principal challenges that currently limit the translation of these findings into clinical applications.

Table 1. Critical interpretation of human clinical studies investigating the neuroprotective effects of LM.

Study	Study Design	Preparation and Routine	Results	Interpretation
Mori et al., 2009 [77]	Double-blind, parallel-group RCT involving 30 adults aged 50–80 years with mild cognitive impairment	Approximately 3 g/day of fruiting-body powder for 16 weeks, followed by a 4-week post-treatment observation period	Cognitive scores improved during supplementation but declined after treatment was discontinued	The study was small and relied mainly on a single cognitive screening scale. The active constituents were not quantified, and the persistence of any cognitive benefit after treatment remains uncertain.
Nagano et al., 2010 [86]	Randomised, placebo-controlled trial involving 30 women	2 g/day of fruiting-body material incorporated into cookies for 4 weeks	Improvements were reported in selected measures of depression, anxiety, and non-specific complaints	The short duration, small sample size, and use of multiple outcome measures limit the strength of the conclusions. The findings also cannot be directly extrapolated to patients with clinically diagnosed depression or anxiety disorders.
Saitsu et al., 2019 [87]	Double-blind, parallel-group RCT in adults aged > 50 years; 31 participants included in the final analysis	3.2 g/day of powdered fruiting body for 12 weeks	MMSE scores improved, whereas Benton visual retention and verbal paired-associate test outcomes showed no clear benefit	The cognitive effect was selective rather than consistent across all measures. Near-normal baseline MMSE scores may also have introduced ceiling effects, and the concentrations of potentially active metabolites were not determined.
Vigna et al., 2019 [88]	Controlled dietary intervention involving 77 overweight or obese adults	1.5 g/day for 8 weeks, comprising approximately 1.2 g mycelium and 0.3 g fruiting-body extract	Improvements in mood and sleep parameters were reported, together with changes in pro-BDNF/BDNF-related measures	Interpretation is complicated by the concurrent low-calorie diet, participant attrition, and the absence of quantitative standardisation for erinacines or hericenones.
Li et al., 2020 [76]	Pilot double-blind, placebo-controlled study in patients with mild Alzheimer’s disease; treatment duration of 49 weeks	1.05 g/day of mycelium material standardised to 5 mg/g erinacine A, corresponding to approximately 5.25 mg erinacine A/day	Several cognitive, functional, visual, and biomarker outcomes favoured the intervention group	This was among the longest and most chemically defined clinical studies available, but the sample size remained small. Interpretation is further limited by multiple outcome measures, baseline imaging differences, and four withdrawals associated with gastrointestinal symptoms, nausea, or rash.
Docherty et al., 2023 [89]	Double-blind, parallel-group pilot study involving 41 healthy adults aged 18–45 years	1.8 g/day, assessed both acutely and after 28 days of supplementation	Faster Stroop-task performance was observed 60 min after acute administration, while the reduction in stress approached but did not reach conventional statistical significance	The trial was exploratory and underpowered, with multiple cognitive and mood endpoints. The findings therefore provide preliminary evidence rather than support for broad nootropic effects.
Cernelic Bizjak et al., 2024 [90]	Double-blind pilot study involving 33 adults aged > 55 years over 8 weeks	Erinacine A-enriched preparation providing 3.44 mg erinacine A/day	An improvement in processing speed was observed after statistical adjustment, while changes in microbial diversity correlated with neuropeptide Y	The small sample size limits generalisability. The cognitive finding depended on covariate adjustment, and the microbiome observations should be regarded as exploratory.
Surendran et al., 2025 [91]	Double-blind, crossover acute RCT involving 18 healthy younger adults	Single 3 g dose of a 10:1 fruiting-body extract	No significant effect was observed for composite global cognition or mood, although an isolated improvement in pegboard performance was reported	The principal outcomes were negative, and the improvement in one psychomotor task requires independent replication before biological significance can be established.

Table 2. Summary of the key research gaps in clinical trials of LM metabolites identified through comparative analysis of reported in vitro and in vivo findings.

Metabolite/Preparation	In Vitro Evidence (Models and Key Effects)	In Vivo and or Clinical Evidence	Major Research Gaps	References
Erinacine A	Promotes NGF and BDNF expression. Activates NRF2/HO-1 pathway. Reduces NO, TNF-A, IL-1Β in microglia. Enhances neurite outgrowth in PC12, BV-2 and hippocampal neuron cultures.	Rodents: improved neuronal survival and reduced apoptosis in models of optic nerve injury, ischemia, and neurodegeneration. Oral dosing increases brain NGF. Clinical: pilot RCTs (n = 20–40) show modest cognitive improvements and elevated BDNF/NGF.	Scarce pharmacokinetic and BBB penetration data. Human trials are short-term and small. Erinacine content varies across preparations.	[90,92,93]
Hericenones	Stimulate NGF secretion. Enhance neurite outgrowth. Structure–activity studies show fatty-acid chain length influences potency. Effects shown at µM levels in PC12 and cortical neuron lines.	Limited in vivo data. Most studies use crude fruiting-body extracts. Clinical studies using powdered fruiting bodies suggest mild cognitive benefits in MCI and ageing, but hericenone concentrations are rarely quantified.	Lack of pharmacokinetics and BBB permeability data. No in vivo dose response for individual hericenones. Clinical studies confounded by complex mixtures.	[77,93]
Standardised extracts (Mixture of fruiting body and mycelia)	Synergistic induction of neurotrophins (NGF/BDNF). Antioxidant and anti-inflammatory effects greater than isolated compounds.	Mouse models: improved locomotor and cognitive frailty. Reduced neuroinflammation. Early human observational data only. Composition varies widely.	In vitro synergy difficult to interpret. Limited dose–exposure–response data. No large controlled clinical trials with fully characterised extract chemistry.	[51,52]
Polysaccharide-rich extracts (β-glucans)	Reduce oxidative stress, inhibit apoptosis, modulate macrophage and microglial inflammatory signalling. Promote neurite outgrowth in neuronal systems at high concentrations.	Rodent models: improvements in cognition and inflammation in metabolic/inflammatory conditions. No polysaccharide-focused neurocognitive RCTs in humans.	Very poor BBB penetration suggests indirect action via immune/gut–brain pathways. Effective in vitro doses not physiologically achievable. Extract biochemistry poorly standardised.	[93,94]
Whole fruiting-body powder	Limited direct in vitro evaluation. Contains low levels of hericenones, phenolics, and antioxidants.	RCT in MCI (n = 30): improved cognitive scores over 16 weeks. Effects dissipate after discontinuation. Small studies suggest mood/sleep effects but inconsistent methodology.	Chemical composition highly variable. Lack of biomarkers. Cognitive endpoints subjective. No long-term safety or durability data.	[32,77,86]
Erinacine-enriched mycelium supplements	Similar neurotrophic and anti-inflammatory effects as purified erinacine A. Induce NGF, BDNF, and antioxidant genes.	Small pilot human studies report improved cognitive performance and changes in BDNF/NPY signalling. Ongoing early-phase trials exploring dose and formulation.	Wide variability in erinacine A/S/C content. Lacking pharmacokinetics and dose optimisation. No large independent RCTs.	[82,90,94]

Bioavailability and Gut–Brain Interactions of LM Metabolites

In rats, oral administration of an erinacine A-containing mycelium extract at 2.381 g/kg (roughly 50 mg/kg erinacine A) resulted in bioavailability of 24.39% [95]. Erinacine S has likewise shown measurable BBB permeability in rats, with an estimated oral bioavailability of 15.13% following an equivalent 50 mg/kg exposure [12]. These findings support in vivo blood–brain barrier penetration of selected erinacines in rodents, although corresponding human pharmacokinetic data and therapeutically relevant brain concentrations remain to be established, particularly for lower-dose and commercial preparations. Similar in vivo evidence for individual hericenones and hericerins remains limited, while gastrointestinal absorption and CNS penetration of polysaccharides has not been documented.

For poorly absorbed macromolecules such as polysaccharides, an indirect mechanism of neurobiological action seems more plausible than direct CNS BBB permeability. Following oral consumption, these molecules may interact with the intestinal epithelium and respective receptors, modulating gut-barrier and peripheral immune responses. Microbial fermentation in the gut could also generate bio-transformed products capable of signalling the brain through circulatory, endocrine, immune or vagal pathways. This concept is supported by studies on mice administered standardised LM extract, resulting in reduced hippocampal inflammatory and cellular-senescence markers without reshaping the gut microbiome [11]. Additionally, a human pilot study using erinacine A-enriched supplement increased gut microbial diversity that correlated with circulating neuropeptide Y [90]. Collectively, these findings support the gut–brain interaction as a plausible pathway through which poorly absorbed LM constituents may influence neuronal health. However, direct links to this mediation and causality are yet to be established through studies.

5. Hericium erinaceus as Food and Dietary Supplements

The use of LM as a natural product remedy draws on its history as an edible mushroom and its chemically diverse composition. Mushrooms are recognised as dietary sources of nutrients [96,97], and LM contains protein, dietary fibre, vitamins and minerals [98], with alanine and leucine among the abundant amino acids reported in its fruiting body [99]. Glutathione and ergothioneine have also been reported [29], having antioxidant and anti-inflammatory properties [100]. These constituents provide a compositional chemical profile, although their presence alone does not establish the neuroprotective activity of an LM preparation [101,102,103,104].

Human studies have evaluated LM in several formulations. A pilot trial in healthy young adults receiving 1.8 g/day of a proprietary LM blend reported preliminary benefits in performance speed and stress-related outcomes [89]. Supplementation with 3.2 g/day of powdered fruiting body for 12 weeks was associated with improved cognitive function in another study [87]. A four-week trial using LM-containing cookies, providing 2 g/day of fruiting body powder, reported reductions in depression and anxiety scores [86]. Improvements in depression, anxiety and sleep-related scores were also reported in overweight and obese adults receiving a mycelium/fruiting body preparation alongside a low-calorie diet for eight weeks [88].

These findings remain preliminary and arise from preparations that differ in fungal material, processing and administered dose. The contribution of specific neuroactive constituents therefore remains difficult to resolve. Larger studies using chemically characterised preparations are needed to establish reproducibility and support meaningful comparisons between experimental findings and commercial products. Quality assurance is consequently central to evaluating LM as a natural product remedy with potential neuroprotective applications.

5.1. Biochemical Markers, Quality Control and Bioactivity

Quality assurance of LM preparations should distinguish species authentication, chemical standardisation and assessment of biological activity. Authentication should draw on voucher-verified reference material, supported by documentation of fruiting body or mycelial origin, strain, cultivation substrate, production conditions and processing history. Internal transcribed spacer (ITS) barcoding can support species identification but should be interpreted alongside morphological and chemical evidence and, where necessary, additional molecular markers (Figure 1, Figure 2 and Figure 3).

Chemical standardisation should reflect the production matrix, since fruiting body powders, extracts, mycelial biomass and fermented or mixed products are not analytically interchangeable. Selected hericenones and hericenes can be used to assess fruiting body preparations, whereas erinacine A and additional erinacines can be used for mycelial products. Mixed formulations require markers representing both components and documentation of their dry-weight contributions. Ergosterol can indicate fungal biomass but is neither specific to LM nor a measure of neuroactive potency. Marker presence and abundance should therefore be established analytically and interpreted against authenticated materials representing the same production matrix.

A single-laboratory validated UHPLC–UV method provides a practical basis for simultaneous quantification of selected hericenones, hericenes, erinacine A and ergosterol [10]. Where UV response or co-elution limits selectivity, complementary detection methods can strengthen confidence in marker assignment and quantification. Such measurements support compositional specifications, while their relationship to biological potency requires separate evaluation.

This distinction is reinforced by the incompletely defined structure–activity relationships of LM metabolites. Purified hericenones C–E did not consistently reproduce the NGF-inducing activity observed with crude extracts in a human astrocytoma model [105]. In another cellular study, deacylhericenone, a lipase-derived metabolite of hericenone C, showed greater BDNF mRNA induction and protection against oxidative stress than the parent compound [53]. These observations highlight the importance of evaluating individual compounds and extract composition when interpreting reported neuroactivity.

For erinacines, structural variation around the cyathane diterpenoid scaffold likewise warrants systematic evaluation under comparable assay conditions. Studies using purified, stereochemically verified compounds at relevant concentrations would strengthen relationships between chemical structure and neurobiological activity. This helps determine whether selected quality control markers predict biological effects in the long run.

5.2. Mass Spectrometric Approaches to Ensure Quality of LM Supplements

As public health nutrition increasingly emphasises strategies for promoting healthier eating patterns without compromising the enjoyment of food, mushrooms have emerged as a promising component for enhancing dietary quality [106]. In a market offering foods and drinks containing LM as well as products containing 100% unprocessed LM, it is essential to implement measures that preserve the mushroom’s biochemistry, which underlies its bioactivity. Critical factors such as strain selection, optimal growth conditions, and appropriate harvest timing must be carefully considered to maintain the functional properties of the mushroom. Advanced bioanalytical techniques, particularly LC-HRMS/MS metabolomics-based high-throughput screening, can serve as valuable tools to monitor and ensure the stability and integrity of bioactive compounds throughout the production, processing, and distribution stages [107,108,109,110]. This approach is crucial for delivering consistent and effective fungal-based nutritional products. Figure 5 provides an overview of the proposed metabolomic workflow for the evaluation of bioactives in LM and its supplements.

Figure 5. LC-HRMS/MS-based metabolomics workflow for the biochemical evaluation of neuroprotective and neurotrophic molecules in LM and its dietary supplements.

5.2.1. Extraction and Stability of LM Neuroactives

Reliable assessment of LM preparations depends on the recovery and preservation of constituents associated with their reported neurotrophic and neuroprotective activities. Polysaccharides, proteins, lipids and other components of LM supplement matrices can interfere with the detection and quantification of these constituents [111,112,113]. Consequently, extraction and analytical procedures should be validated within the relevant product matrix.

Given the lipophilic to moderately polar nature of many LM terpenoids, ultrasound-assisted extraction with aqueous ethanol, commonly at 60–80%, has been employed to recover these constituents [114,115,116]. Further purification by counter-current or silica chromatography can support the isolation of neuroactive diterpenoids for characterisation and reference standard preparation [113,115]. For routine quality control, the priority is reproducible analyte recovery with sufficient removal of interfering matrix components.

The chemical stability of hericenones and erinacines remains incompletely characterised in finished LM products. Studies on whole fruiting bodies have demonstrated losses of selected bioactive compounds under elevated temperature and humidity [117], whereas systematic stability and shelf-life data for capsules, powders and extracts remain limited [118]. Accordingly, drying, extraction and storage conditions require evaluation for their effects on the retention of neuroactive constituents. Controlled processing, protective packaging and stability testing should therefore form part of product quality assurance [20]. Matrix-specific validation of recovery, accuracy, precision and analyte stability is essential to distinguish genuine compositional variation from analytical losses.

5.2.2. Untargeted LC-HRMS/MS for Authentication

The accurate mass and fragmentation data provided by LC–HRMS/MS [119] can support quality assurance by comparing the chemical profiles of LM preparations with those of taxonomically authenticated reference materials. Comparisons should account for the fungal material used, processing conditions and matrix effects [113]. Appropriate blanks, pooled quality control samples and internal standards are therefore needed to distinguish reproducible product differences from analytical variation.

Statistical comparisons can prioritise differences in metabolite signals for further investigation [120,121], while multivariate analysis can identify constituents contributing to variation between preparations and reference materials [122,123,124]. Such differences may channel investigations into depleted neuroactive constituents or unexpected components that require chemical confirmation before being attributed to adulteration or poor product quality.

Feature-based molecular networking and ion-identity molecular networking can further support the grouping of structurally related metabolites and the association of different adducts with the same constituent [125,126]. Applied to LM, these approaches can guide the selection of compounds for confirmatory analysis and assessment of batch consistency. Their principal value is therefore to support authentication and compositional assessment, as chemical similarity does not always demonstrate neuroprotective efficacy.

5.2.3. Targeted Analysis for Standardisation of LM Preparations

Targeted LC–HRMS/MS complements chemical fingerprinting by selectively measuring predefined LM constituents associated with reported neurotrophic and neuroprotective activities, including hericenones and erinacines [116,127]. Isolation followed by NMR characterisation can support structural confirmation [83,128,129], and compounds with known identity and purity can serve as reference standards for absolute quantification [127,130].

Matrix-matched calibration can help address ion suppression or enhancement [131], while extraction recovery requires separate evaluation [132,133,134]. Expected molecular formulae and adducts can guide precursor selection [135,136,137,138], with retention time and diagnostic tandem mass spectra providing complementary evidence for the identification of structurally related LM metabolites [116,139]. For example, fragments associated with fatty acid side chains in hericenone-related compounds can provide supporting structural information [20,42,50,57]. These features are most useful when incorporated into validated methods for defined quality control markers.

Targeted acquisition methods, including parallel reaction monitoring or selected/multiple reaction monitoring on suitable instruments, can support selective measurement of these markers [140,141,142]. Software such as Skyline assists in data processing and quantitative evaluation [143]. Following matrix-specific validation, these measurements can support batch specifications, verification of declared constituent content and monitoring changes during storage. Marker selection should reflect the preparation and the compounds implicated for reported biological activity.

Together, authenticated reference materials, reproducible chemical profiles and validated marker measurements provide a foundation for standardising LM preparations. Reporting measured constituent content alongside the administered dose would strengthen comparisons between experimental studies and commercial products, while keeping chemical quality assurance distinct from the biological evidence required to establish neuroprotection.

6. Conclusions

Hericium erinaceus (LM) occupies a distinctive position at the intersection of nutrition, ethnomedicine and neuropharmacology. Preclinical evidence supports biologically plausible neuroprotective effects through modulation of neurotrophin signalling, neurite growth, oxidative and ER stress, inflammatory pathways and microglial activity. However, these effects vary between extracts and isolated compounds. Moreover, activity seen in crude preparations cannot always be attributed to individual metabolites.

Human evidence remains limited. The eight intervention studies reviewed between 2009 and 2025 used chemically different preparations, including fruiting body, erinacine-enriched mycelium and mixed fruiting body/mycelium formulations. These materials should not be treated the same chemically, particularly because many earlier trials lacked quantitative characterisation of proposed neuroactive molecule/molecules. Pharmacokinetic evidence from humans is also limited: oral bioavailability and BBB permeability have been demonstrated mainly for erinacines A and S in rodents, while comparable human CNS permeability data are unavailable. High molecular weight polysaccharides are more plausibly associated with intestinal, immune and gut–brain mechanisms unless direct systemic or brain uptake is observed.

Expanding LM supplement market further highlights the need for robust quality standards. Variability arising from strain, cultivation, material type, extraction, processing and storage can substantially alter product chemistry. Quality control should therefore distinguish authentication from potency and apply matrix-specific markers for fruiting body and mycelium products, supported by appropriate polysaccharide characterisation and stability assessment. LC-HRMS/MS-based targeted and untargeted metabolomics provide useful tools for authentication, chemical profiling and quantitative assessment of key metabolites. Future research should prioritise standardised preparations, NMR-purified reference standards, validated analytical methods, reliable human pharmacokinetic studies and adequately powered clinical trials. Overall, current evidence supports the biological plausibility of neuroactive effects from selected LM constituents but does not yet completely establish their mechanisms or clinical efficacy in humans. Addressing the analytical, pharmacokinetic and clinical evidence gaps identified here will be essential for reliable translation into its untapped use in modern phytomedicine.