Bioactive Compounds from Edible Mushrooms as Pharmaceutical Ingredients: A Comprehensive Review of the Developmental Pipeline

Abstract

Edible mushrooms have long been valued as functional foods and traditional remedies, yet a significant developmental gap hinders their transition from nutraceuticals to standardized pharmaceutical ingredients. This narrative review provides a comprehensive and integrative analysis of edible mushroom-derived bioactive compounds as emerging candidates for pharmaceutical development. It examines major chemical classes, including polysaccharides (e.g., β-glucans), proteins (e.g., lectins, FIPs), triterpenoids (e.g., ganoderic acids), nucleosides (e.g., adenosine and cordycepin), and phenolic compounds, which underpin immunomodulatory, anticancer, antioxidant, anti-inflammatory, and metabolic activities. Beyond bioactivity, the review critically examines the downstream processing pipeline required for translation into pharmaceutical ingredients, encompassing controlled biomass production, pre-extraction processing, extraction technologies, isolation and purification strategies, and structural elucidation techniques. Key bottlenecks are identified, including bioavailability limitations of β-glucans (2–5%), lack of standardization, limited human clinical evidence, and regulatory constraints, explaining why robust preclinical evidence has not consistently translated into clinical success. Emerging solutions are also highlighted, including application of multi-omics tools, nano-encapsulation strategies, and synthetic biology approaches to improve scalability and reproducibility. By synthesizing research on natural product chemistry, biotechnology, and pharmacology, this study maps the journey of edible mushrooms from traditional dietary components to pharmaceutical-grade ingredients, providing a focused resource for researchers and industry stakeholders aiming to navigate mushroom-based drug development.

1. Introduction

Edible mushrooms, with an estimated 3000 documented species, represent one of the most widely recognized edible forest products worldwide [1]. Their long-standing culinary relevance across diverse cultures is attributed to their distinctive flavor, aroma, and texture [2]. Historically, mushrooms were valued as both food and therapeutic resources long before their bioactive properties were scientifically understood. Ancient civilizations, including the Romans and Greeks, regularly incorporated mushrooms into their diets, while others revered them as potent medicinal agents. For instance, certain species were referred to as the “elixir of life” in China and the “flesh of the gods” in Egypt [3,4]. Although mycotherapy has been deeply rooted in Asian traditional medicine for millennia, its adoption in Western countries has gained momentum only in recent decades [5].

The intersection of edibility and medicinal value in mushrooms is well established, as many edible species exhibit pharmacological properties, and several renowned medicinal mushrooms are regularly consumed as part of the diet [2]. The health-enhancing effects of edible mushrooms, observed since antiquity, are now attributed to the bioactivity of their secondary metabolites, including polysaccharides, polyphenols, proteins, terpenes, and terpenoids. The literature has documented a wide array of pharmacological activities for these bioactive compounds, including anticancer, antioxidant, hepatoprotective, immunomodulatory and antimicrobial effects [6]. Consequently, edible mushrooms have become a significant source of active ingredients for nutraceutical and pharmaceutical formulations [3]. Notably, the spores and basidiocarps of Ganoderma lucidum, a globally renowned edible and medicinal mushroom, are processed into tablets, injectables, and soft-gel capsules for various therapeutic purposes [7]. Likewise, nutraceuticals derived from edible mushrooms represent an expanding segment of the overall dietary supplement market [8].

Growing recognition of the therapeutic value of edible mushrooms has intensified their demand among consumers and pharmaceutical industries alike. While whole mushroom biomass can be consumed directly, its bioactive constituents can also be isolated and formulated into novel pharmaceutical products. This increased demand has subsequently stimulated the expansion of commercial cultivation and global market of edible mushrooms [9]. Global mushroom production, for instance, increased 15.6-fold between 1990 and 2022 [10]. Also, the global mushroom market is projected to rise from USD 48.8 billion in 2022 to USD 83.5 billion by 2030, at an annual rate of 7% [11]. These commercialization trends highlight the growing opportunity to harness edible mushrooms as sources of natural-based pharmaceutical ingredients.

Although several synthetic drugs exist and are currently in use for disease management, concerns over unfavorable side effects and inconsistent therapeutic outcomes have renewed interest in natural product-based drug discovery [2,12]. Consequently, recent research efforts increasingly target the extraction and purification of mushroom-derived bioactive compounds for pharmaceutical development. For example, quercetin, a flavonol present in some edible mushrooms, is utilized pharmaceutically to regulate blood glucose and enhance insulin sensitivity [13]. Similarly, a biocompatible antioxidant and anti-inflammatory hydrogel developed for diabetic wound healing incorporates G. lucidum polysaccharide (GLP) [14]. Such bioactive molecules provide complementary therapeutic benefits alongside conventional drugs, often enhancing efficacy while lowering side effects [12]. Furthermore, the fruiting bodies, cultured mycelia, and fermentation broths of edible mushrooms are increasingly being recognized as rich reservoirs of novel active compounds. Their attractiveness to pharmaceutical industries is additionally supported by favorable cultivation characteristics, including rapid growth, high biomass yield, and modest space requirements [15].

Despite compelling evidence supporting the bioactivities and pharmacological potential of edible mushrooms, their development pathway into standardized, pharmaceutical-grade raw materials remains poorly integrated. Critical downstream processing stages, such as scalable production, standardized extraction and isolation, and comprehensive molecular characterization, are often addressed in isolation rather than as a unified framework. This knowledge gap may hinder clinical testing and wide-scale therapeutic applications [5]. Accordingly, this comprehensive review elucidates how edible mushrooms are transcending their traditional nutraceutical roles to emerge as viable candidates for modern pharmaceutical development. It synthesizes current knowledge to provide a systematic overview of the multifaceted development pathway, focusing on the major downstream procedures essential for transforming mushroom bioactive compounds into high-purity, reliable pharmaceutical ingredients.

Moreover, unlike previous reviews that have predominantly concentrated on bioactivity cataloging, single mushroom species and/or isolated classes of compounds [16,17,18,19,20], the present review provides a holistic and explicitly translational synthesis of mushroom-derived bioactives. By systematically integrating chemical diversity with extraction and purification strategies, structural elucidation, and pharmaceutical development constraints across multiple edible mushroom taxa, this work bridges natural product chemistry with practical drug development considerations. Importantly, this integrative perspective moves beyond descriptive accounts of bioactivity to critically examine how mushroom bioactives can be rationally prioritized, optimized, and advanced within realistic pharmaceutical development pipelines. To establish their basis for pharmaceutical consideration, the review links the chemical diversity of bioactive constituents in edible mushrooms with their associated biological activities, thereby elucidating the mechanistic basis of their therapeutic potential. It further highlights key translational challenges that connect molecular characterization with the practical requirements for clinical and industrial implementation.

2. Methodology for the Narrative Review

To develop this narrative review, relevant literature was identified through searches of major scientific databases, including Google Scholar, PubMed, Scopus, Web of Science, ScienceDirect, and the Directory of Open Access Journals (DOAJ). Priority was given to studies published between 2020 and 2026 in order to capture recent advances in the development, extraction, and pharmaceutical utilization of bioactive compounds from edible mushrooms. The search strategy employed the core keywords “edible mushrooms” and “bioactive compounds”, combined with other related terms such as cultivation, solid state fermentation, submerged liquid fermentation, extraction, isolation, purification, characterization, standardization and specific pharmacological activities (e.g., anticancer, antimicrobial, antioxidant, neuroprotective, and cardiovascular effects). Moreover, the reference lists of the retrieved articles were manually screened to identify additional relevant studies that were not captured through the electronic database searches. Literature retrieval was completed in 10 January 2026. Following the initial search, duplicate records were removed, and non-English publications were excluded. Studies focusing primarily on other natural product sources, such as medicinal plants, were also excluded. Relevant titles and abstracts were examined to identify studies aligned with the thematic scope of this review, with emphasis placed on studies addressing the biological activities of mushroom-derived bioactive compounds and/or downstream processing aspects pertinent to their pharmaceutical development. Full-text articles were subsequently reviewed and included only if they provided substantive information on edible mushrooms and their bioactive constituents in relation to the predefined thematic scope. The final body of literature (n = 199) comprised original research articles, review papers, institutional/technical reports and relevant book chapters. These sources were qualitatively synthesized to provide an integrated and critical narrative overview of the role of edible mushroom bioactives in pharmaceutical innovation. In this review, the term “bioactive compounds or bioactives” is used as an umbrella descriptor for mushroom-derived molecules exhibiting biological activity, while “secondary metabolites” refers specifically to low-molecular-weight fungal metabolites. The term “pharmaceutical ingredients” is reserved for compounds or standardized extracts considered within a drug-development or regulatory framework.

3. Chemistry and Bioactivity: The Pharmaceutical Basis of Mushroom Compounds

The therapeutic potential of edible mushrooms is primarily attributed to a diverse array of bioactive compounds, which can be broadly categorized into high-molecular-weight macromolecules and low-molecular-weight secondary metabolites, each defined by distinct chemical characteristics and biological functions [21]. High-molecular-weight compounds, including polysaccharides, proteins, and nucleic acid–protein complexes, are predominantly associated with immunomodulatory, antitumor, and antioxidant activities. In contrast, low-molecular-weight metabolites such as terpenoids, sterols, polyphenols, and related secondary compounds largely contribute to antioxidant, anticancer, antiviral, anti-inflammatory, immune-modulating and cardioprotective effects [22].

Importantly, these chemical classes do not act in isolation; rather, their structural features determine molecular mechanisms and pharmacological effects. The abundance and composition of these bioactive constituents vary considerably among mushroom species and are strongly influenced by cultivation substrates, environmental and fruiting conditions (cultivated versus wild), and developmental stage [2,21]. For example, Barros et al. [23] reported markedly higher flavonoid concentrations (16.56 mg/g) in wild-harvested species of Agaricus, Lentinus, and Pleurotus. Conversely, cultivated counterparts of the same genera were found to contain low or undetectable levels of certain phenolic compounds, including flavonoids and lignans [24], underscoring the impact of growth conditions on phytochemical profiles and, by extension, bioactivity.

3.1. High-Molecular-Weight Bioactive Compounds

High-molecular-weight bioactive constituents in edible mushrooms play a central role in mediating many of the health-promoting properties associated with edible mushrooms, particularly those related to immune modulation, tumor suppression, and host defense. Notwithstanding, from a pharmaceutical perspective, the structural complexity and molecular weight of these compounds present challenges related to oral bioavailability, batch consistency, and regulatory definition of an active pharmaceutical ingredient (API). These features necessitate advanced formulation strategies and robust analytical standardization prior to clinical translation.

3.1.1. Polysaccharides

Polysaccharides occur widely in nature, including in plants and microbial extracellular polymeric substances [25]. Among these, mushroom-derived polysaccharides have attracted substantial interest due to their demonstrated relevance in molecular biology, immunology, biotechnology, and pharmaceutical sciences [26]. Compared with polysaccharides from other biological sources, those isolated from mushrooms exhibit remarkable structural diversity, reflected in variations in molecular weight, monosaccharide composition, degree of branching, chain length, and three-dimensional conformation [27]. The strong dependence of polysaccharide bioactivity on these structural features highlights structural elucidation as a prerequisite for reproducible pharmacological outcomes. Mushroom polysaccharides are predominantly composed of α- and/or β-glucans, with β-glucans representing the principal bioactive fraction. These β-glucans constitute one of the two major structural components of the fungal cell wall, alongside chitin [9]. Common glycosidic linkages include (1→3)-β-D-glucan, (1→6)-β-D-glucan, (1→3)-α-D-glucan, and mixed-linkage structures, reflecting substantial molecular heterogeneity [28,29].

Functionally, β-glucans elicit a broad spectrum of biological responses, including immunomodulatory, antitumor, antioxidant, and anti-inflammatory effects, primarily through interactions with pattern-recognition receptors on immune cells [30]. Chan et al. [31], for instance, demonstrated that β-glucans mediate antimicrobial immune responses via specific receptors such as dectin-1, complement receptor 3 (CR3), and Toll-like receptors 2 and 6 (TLR2/6). Sulfated β-glucans from Agaricus brasiliensis have further been shown to prevent HSV-1 and HSV-2 viral entry at low microgram concentrations, highlighting their antiviral potential [32].

Notable examples of bioactive mushroom-derived β-glucans include schizophyllan from Schizophyllum commune, lentinan from Lentinula edodes, pleuran from Pleurotus spp., calocyban from Calocybe indica and the Maitake D-fraction from Grifola frondosa [2,9]. Several of these polysaccharides have demonstrated tumor growth inhibition through activation of macrophages, natural killer (NK) cells, and cytokine (TNF-α, IL-2, and IFN-γ) production, reinforcing their relevance in cancer immunotherapy [33,34]. In addition to homopolysaccharides, mushrooms also contain heteropolysaccharides, such as xylomannans and branched glucans, which further expand their functional repertoire [30].

Polysaccharides may also associate with other biomolecules through covalent or non-covalent interactions to form bioactive complexes, among which polysaccharide–protein complexes (PPCs) are particularly prominent. Well-known examples include Krestin and Coriolan isolated from Trametes versicolor, both comprising β-glucans conjugated to protein moieties [1]. These complexes have been shown to promote macrophage polarization toward tumor-suppressive M1 phenotype, reducing immunosuppressive cytokines and enhancing antitumor immunity [35]. Similarly, Agaricus spp. produces biologically active PPCs characterized by protein-bound β-(1→6)-glucan structures, which have been linked to pronounced immunomodulatory and anticancer effects [31].

3.1.2. Nucleic Acids and Nucleosides

Nucleic acids and their derivatives, including nucleobases, nucleosides, and nucleotides, are essential biomolecules involved in cellular metabolism, gene regulation, and intracellular signaling [36,37]. Edible mushrooms represent a rich dietary source of these compounds, containing purine and pyrimidine nucleobases (e.g., adenine, guanine, uracil, and cytosine), nucleosides (e.g., adenosine, guanosine, uridine, cytidine, thymidine, and inosine), and nucleotides such as adenosine monophosphate (AMP) and guanosine monophosphate (GMP) [37]. Distinct mushroom species exhibit characteristic nucleoside profiles. For example, Agaricus bisporus and Morchella deliciosa are enriched in adenosine, guanosine, xanthosine, and uridine [38], whereas inosine, cytidine, thymidine, and cordycepin have been reported in Antrodia camphorata, Agrocybe aegerita, and Tricholoma matsutake [39,40]. Several mushroom-derived nucleosides, notably adenosine and cordycepin, have been implicated in antidiabetic activities. In particular, cordycepin from Cordyceps spp. has been shown to modulate the expression of diabetes-associated genes by attenuating nitric oxide (NO) production and pro-inflammatory cytokine release through suppression of NF-κB-dependent inflammatory signaling pathways [41]. This modulation is associated with reduced expression of key inflammatory mediators, including IL-1β, IL-6, and TNF-α, as well as the downregulation of upstream regulators such as Toll-like receptor 4 (TLR4) and myeloid differentiation primary response protein 88 (MyD88) [42]. These nucleoside constituents therefore contribute meaningfully to the immunoregulatory, neuroprotective and tissue-repair activities associated with edible mushrooms [37].

3.1.3. Proteins and Peptides

Proteins and peptides are another major class of bioactive constituents in edible mushrooms, contributing significantly to their nutritional and therapeutic value. Protein content varies widely among species. Notably, Hericium erinaceus (Lion’s Mane), Ophiocordyceps sinensis (Cordyceps), and L. edodes (Shiitake) are particularly protein-rich, often exceeding 20% protein content [30]. Mushroom-derived proteins and peptides have been linked to a broad spectrum of biological activities, including hypotensive and angiotensin-converting enzyme (ACE) inhibitory effects, antioxidant, anticancer, antiviral, and antibacterial properties [43]. The major classes of bioactive fungal proteins include lectins, fungal immunomodulatory proteins (FIPs), and enzymatic proteins such as nucleases, ribonucleases, and laccases [44]. Lectins, also known as glycoproteins, exert their biological effects by interacting with specific sugar residues, leading to agglutination and modulation of immune responses. These interactions have been associated with enhanced insulin secretion, glycemic control, immune stimulation, and chemopreventive effects [1,45]. For instance, investigations into lectins and proteins across Cantharellus cibarius, Imleria badia, Lactarius deliciosus, and Boletus edulis have indicated immunoregulatory activity, including enhanced Th1 lymphocyte responses and suppression of COX-2 and prostaglandin synthesis [46].

Fungal immunomodulatory proteins represent another distinctive group of mushroom proteins with the ability to selectively target immune cells. These proteins have been identified in species such as Flammulina velutipes and several Ganoderma spp. [9]. Notable examples include GMI from Ganoderma microsporum, Fip-gts from G. tsugae, and Ling Zhi-8 from G. lucidum, all of which exhibit antimetastatic and immunoregulatory activities [47,48,49]. Purified and structurally characterized protein fractions from Pleurotus eryngii were shown to suppress tumor cell proliferation in a dose-dependent manner, further highlighting the pharmaceutical relevance of mushroom-derived proteins as natural antitumor agents with additional immunostimulatory properties [50].

In addition, low-molecular-weight peptides such as cordymin, isolated from Cordyceps spp., have demonstrated the ability to reduce blood glucose and glycated hemoglobin levels while alleviating oxidative stress in diabetic animal models [51]. Ribonucleases, which induce apoptosis through RNA degradation, have also been shown to exhibit antibacterial activity against clinically relevant pathogens, including Staphylococcus aureus and Pseudomonas aeruginosa [52]. Edible mushrooms also provide a rich source of essential and non-essential amino acids, including glutamic acid, aspartic acid, arginine, ornithine, and γ-aminobutyric acid (GABA), further enhancing their functional and therapeutic relevance [44].

3.2. Low-Molecular-Weight Bioactive Compounds

Low-molecular-weight bioactive compounds isolated from edible mushrooms primarily function as secondary metabolites and are generally more readily absorbed than macromolecules. Similar to their high-molecular-weight counterparts, these compounds also play pivotal roles in enhancing health, making them attractive candidates for pharmaceutical development [21,22]. These mushroom metabolites align more closely with conventional small-molecule drug development paradigms, owing to their defined chemical structures, improved absorption profiles, and compatibility with established pharmacokinetic and regulatory frameworks [43]. For example, ergothioneine isolated from Pleurotus species exhibits remarkable chemical stability and efficient cellular uptake through the organic cation transporter (OCTN1), resulting in tissue concentrations that are approximately 50–100 times higher than plasma levels [15].

3.2.1. Phenolic Compounds

Phenolic compounds represent one of the most extensively studied classes of mushroom-derived secondary metabolites. Structurally, they are defined by the presence of at least one aromatic ring bearing one or more hydroxyl groups, with molecular complexity ranging from simple phenolic acids to complex flavonoids and tannins [2]. In most edible mushroom species, phenolic acids constitute the dominant phenolic subclass and are commonly grouped into benzoic acid derivatives (C₆–C₁) and cinnamic acid derivatives (C₆–C₃). Frequently reported phenolic acids include gallic, caffeic, p-coumaric, protocatechuic, syringic, vanillic, and trans-cinnamic acids, which have been identified across a wide range of edible mushrooms such as Agaricus, Pleurotus, Russula, Lentinus, and Morchella, often in species-specific profiles and concentrations [53,54].

Functionally, mushroom phenolics exert potent antioxidant activity through free-radical scavenging, metal chelation, and inhibition of lipid peroxidation, thereby mitigating oxidative stress associated with chronic diseases. Experimental evidence supports these roles: phenolic-rich extracts of Pleurotus ostreatus significantly improved oxidative balance in 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma models [55], while methanolic and ethyl acetate extracts of C. cibarius and Pleurotus porrigens demonstrated strong radical-scavenging capacity in vitro [56]. Although generally present at lower levels than phenolic acids, flavonoids have also been detected in several mushroom species. Compounds including quercetin, catechin, rutin, myricetin, and kaempferol contribute to the overall antioxidant capacity of mushroom extracts from species such as B. edulis, P. ostreatus, A. bisporus, L. deliciosus, Craterellus cornucopioides, C. cibarius, Calocybe gambosa, and Melanoleuca spp. [57,58].

Recent applications of untargeted metabolomics can also provide deeper insight into the diversity and functional relevance of phenolic compounds in edible mushrooms. High-resolution LC–MS-based profiling enables the simultaneous detection of known phenolic acids (e.g., gallic, caffeic, p-coumaric acids) alongside previously unannotated derivatives that may contribute to antioxidant or anti-inflammatory activity. During fermentation or developmental transitions, metabolomic analyses can reveal shifts in phenylpropanoid-related pathways, allowing correlation of specific phenolic signatures with enhanced radical-scavenging capacity or enzyme-inhibitory effects [59]. Such integrative metabolomic approaches not only expand the catalog of mushroom-derived phenolics but also support their prioritization, standardization, and potential development as small-molecule pharmaceutical candidates.

3.2.2. Terpenoids and Sterols

Terpenoids constitute a structurally diverse group of natural products derived from repeating isoprene units and are classified into mono-, sesqui-, di-, and triterpenes based on their carbon skeletons. In mushrooms, terpenoids, particularly triterpenoids, represent one of the most prominent classes of anti-inflammatory secondary metabolites [60]. Triterpenoids are composed of six isoprene units and typically exhibit complex polycyclic structures, occurring as alcohols, aldehydes, or carboxylic acids. Several triterpenoids isolated from mushrooms have demonstrated notable pharmacological activities. For instance, ganoderic acids C and F from G. lucidum exhibit hypocholesterolemic effects [60], while triterpenoids from Inonotus obliquus suppress inflammation by reducing the production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and mediators such as prostaglandin E₂ and NO [61]. Similarly, four triterpenoids isolated from G. lucidum: ganodermanontriol, lucidumol A, ganoderic acid C2, and ganosporeric acid A, exhibited protease inhibitory activity against dengue virus (DENV) at micromolar concentrations comparable to the reference inhibitor 1,8-dihydroxy-4,5-dinitroanthraquinone, underscoring their promising antiviral potential [62]. Antrodia cinnamomea is particularly rich in lanostane-type triterpenoids, including antcin A, B, H, and K, as well as eburicoic and dehydroeburicoic acids, many of which exhibit potent bioactivities [63]. These findings highlight the strong structure–activity relationships governing triterpenoid biofunction.

Recent genomic investigations further illuminate interspecies differences in biosynthetic profiles of these secondary metabolites. Whole-genome sequencing of Trametes sanguinea ZHSJ strain revealed an expanded repertoire of genes encoding terpene synthases and cytochrome P450 enzymes, suggesting substantial, yet underexplored, biosynthetic capacity for structurally diverse terpenoids [64]. Such genomic data provide predictive insight into species-specific metabolite production and offer a strategic framework for prioritizing mushroom taxa with high pharmaceutical development potential.

Sterols, a major subclass of triterpenoids, are characterized by the cyclopentane perhydrophenanthrene nucleus and play essential structural and functional roles in fungal cell membranes [65]. Ergosterol (ergosta-5,7,22-trien-3β-ol) is the predominant sterol in mushrooms and serves as a biochemical precursor to vitamin D₂. Beyond its nutritional significance, ergosterol and its derivatives also contribute to therapeutic activities. For example, ergosterol-rich extracts from Auricularia polytricha have demonstrated inhibition of HIV-1 protease activity [66]. Numerous edible mushroom species, including P. ostreatus, P. eryngii, A. bisporus, Russula cyanoxantha, B. edulis, and Coprinus comatus, contain diverse sterol profiles alongside pentacyclic triterpenoids such as oleanolic and ursolic acids, which further contribute to anti-inflammatory and cardioprotective effects [67,68,69,70]. Truffle species (Tuber magnatum, T. melanosporum, T. borchii) further exhibit remarkable sterol diversity, encompassing ergosterol, brassicasterol, campesterol, lanosterol, fungisterol, and dehydroepiandrosterone, emphasizing the chemical and pharmacological richness of fungal sterols [71].

4. From Mycelium to Medicine: Pharmaceutical Development

It is well established that edible mushrooms contain a variety of bioactive compounds with significant therapeutic potential for the development of novel pharmaceutical products. Nevertheless, the quality, safety, and therapeutic efficacy of such products is strongly contingent on the processes involved throughout their development, i.e., from biomass production to the elucidation of bioactive isolates. This section consolidates key information on these procedures, outlining the sequential pathway through which edible mushrooms are transformed into active pharmaceutical ingredients (APIs).

4.1. Sourcing: Origin and Biomass Production

There are presently two possibilities of acquiring edible mushrooms: harvesting from the wild and cultivation (Figure 1). Historically, macrofungal species were sourced exclusively from the wild [2]. However, as their nutritional and therapeutic importance increased, reliance on wild harvesting became unsustainable. Additionally, several factors limit the suitability of wild-sourced mushrooms for pharmaceutical applications, including pest damage, variability in bioactive compound composition, seasonal availability, risk of misidentification with toxic look-alike species, and the potential accumulation of environmental contaminants [72,73,74,75]. Among these concerns, misidentification represents a critical pharmaceutical safety risk, as morphologically similar but toxic mushroom species may be inadvertently harvested and processed, leading to the introduction of toxic compounds into medicinal preparations. Such errors pose serious risks to patient safety and undermine quality control. To overcome these challenges and ensure consistent quality of target bioactive components, controlled cultivation has become essential. Controlled cultivation offers additional advantages, such as rapid biomass production and the ability to generate both fruiting bodies and mycelial biomass under standardized conditions [74]. Mushroom biomass can be produced through solid-state fermentation (SSF) or submerged liquid fermentation (SmF) (Figure 1), both of which aim to produce pure and physiologically active fungal material suitable for downstream pharmaceutical processing [76].

4.1.1. Solid-State Fermentation (SSF)

The production of mushrooms on solid substrate is the most widely applied method for the longest time, owing to its effective utilization of agro-industrial lignocellulosic residues as substrates. This technique supports the conversion of agricultural and industrial waste streams into value-added products, including bioactive compounds relevant to pharmaceutical development (Figure 1) [76]. It remains the most cost-efficient approach for transforming lignocellulosic wastes into a protein-rich edible biomass [2]. For instance, Melanouri et al. [77] demonstrated that ten distinct agro-residues, including wheat straw, poplar sawdust, grape pomace, beech wood shavings, cottonseed cake, corn cobs, coffee residues, olive pulp, barley and oat straw, and rice husk, differentially influenced the cultivation performance of P. ostreatus and P. eryngii. While the magnitude of response varied by substrate type, all residues supported enhanced radial mycelial extension, increased dry biomass accumulation, and elevated extracellular enzyme production, particularly endoglucanase and laccase activities. Similarly, Balenzano et al. [78] reported that grape pomace, used either alone or in combination with wheat straw, effectively supports growth of these mushroom species while improving the nutritional and functional properties of their resulting extracts. This bioconversion process follows a well-defined multistage protocol that includes substrate preparation or composting, inoculation, incubation, fruiting, and harvesting [79]. During the fermentation process, the substrate is incubated under controlled temperature and humidity, enabling mycelial colonization and subsequent fructification over several weeks [76,80]. To prevent biological contamination and resource competition, substrates must undergo high-temperature sterilization prior to inoculation.

Relative to submerged fermentation, SSF offers advantages including lower substrate moisture content and reduced energy requirements [81]. However, a certain level of free water necessary for the optimal development of the mushroom species should be maintained. The low-humidity conditions of the substrate mimic natural fungal habitats, promoting higher fruiting body yields and enhanced concentrations of secondary metabolites important for pharmaceutical applications (Figure 1) [76]. For instance, SSF using the mycelia of P. ostreatus, Hericium erinaceus, and Flammulina velutipes increased the total polyphenol and aglycone isoflavone contents of soybean meal powder by 235.9% and 324.12%, respectively, thereby significantly enhancing its antioxidant activity [81]. Similarly, Nacha et al. [82] reported optimized β-glucan production (54.95% recovery; 79.98% purity) through the SSF of germinated Rice-berry rice with P. ostreatus. Commonly cultivated species using SSF include P. ostreatus and L. edodes [83]. Despite its advantages, the industrial scalability of mushroom production using SSF is constrained by factors such as substrate heterogeneity, species-specific growth variation, heat and mass transfer limitations, pH, and water activity, all of which can influence biomass yield and metabolite profiles [84]. In particular, maintaining the substrate pH within the slightly acidic range (5.0–7.0) optimal for mycelial growth is difficult under SSF conditions, as fungal mycelia continuously secrete organic acids that progressively lower substrate pH during fermentation [84]. This challenge is further exacerbated by the lack of advanced instrumentation for real-time monitoring of internal conditions within solid substrates, especially pH, thereby complicating process control and scale-up [6]. Economically, scale-up requires specialized bioreactor designs and process optimization, increasing production costs and complicating Good Manufacturing Practice (GMP) compliance [8].

4.1.2. Submerged Liquid Fermentation (SmF)

Submerged liquid fermentation (SmF) represents a promising alternative strategy for the controlled production of high-value mycelial biomass and bioactive metabolites from edible mushrooms. This cultivation approach has attracted considerable attention due to its efficiency, scalability, and suitability for producing compounds with broad applications in the nutraceutical and pharmaceutical industries [76,84]. Although SSF remains the predominant method for mushroom cultivation, advances in submerged fermentation technologies continue to improve the feasibility of generating uniform, reproducible, and high-quality mushroom biomass under controlled conditions [85]. The production of nutritionally and therapeutically valuable mushroom biomass via SmF typically involves a series of biotechnological steps that encompasses the preparation of nutrient-rich liquid culture medium, regulation of physicochemical parameters and separation of the resulting mycelial biomass (often in the form of pellets or filamentous structures) from the fermentation broth [76,86].

Unlike solid-state production, SmF offers several advantages, including shorter fermentation cycles (typically 3–7 days), reduced contamination risks, and precise control over cultivation conditions that influence biomass accumulation and metabolite synthesis, such as temperature, pH, and dissolved oxygen levels [6]. For example, Krasnopolskaya et al. [87] demonstrated that F. velutipes exhibited maximal ACE production at 20 °C, whereas optimal biomass accumulation occurred at 30 °C. The influence of such physicochemical factors on mushroom growth and metabolite production in SmF systems has been comprehensively described by Perveen et al. [88].

An additional advantage of SmF lies in the ease of recovering extracellular metabolites, such as bioactive exopolysaccharides, secreted directly into the fermentation broth. Nevertheless, the choice between SSF and SmF ultimately depends on productivity, metabolite yield, and biological efficacy. For instance, laccases produced by P. ostreatus strain ATCC 32783 under SmF and SSF conditions exhibited distinct isoenzyme profiles and levels of enzymatic activity [89]. Bioactive compounds sourced from mycelia produced using SmF tend to exhibit greater compositional consistency, as fermentation parameters can be finely optimized to enhance metabolite quality and bioactivity [76]. Papaspyridi et al. [90] demonstrated the considerable potential of submerged liquid fermentation in batch-operated stirred-tank bioreactors for producing P. ostreatus mycelial biomass with an enhanced metabolic profile. Using this approach, the authors confirmed the biosynthesis of trans-3,4-dihydro-3,4,8-trihydroxynaphthalen-1(2H)-one, indole-3-carboxylic acid, 3-formylpyrrole, and 4-hydroxybenzoic acid—compounds identified as novel metabolites of pharmaceutical relevance. Similarly, SmF has been employed for the cultivation of S. commune, Lentinus polychrous, L. edodes, G. lucidum, and Lentinus squarrosulus, yielding extracts that exhibited significant antioxidant activity, thereby highlighting their potential for further development into pharmaceutical products [91].

For pharmaceutical applications, scaling up SmF processes in bioreactor systems such as bubble columns, stirred tanks, and airlift bioreactors is essential to ensure industrial feasibility. This requires the optimization of bioprocess efficiency and reactor design such that pilot-scale and industrial-scale fermentations achieve yields comparable to or exceeding those obtained at laboratory scale [6,88]. Accordingly, advances in bioreactor engineering and process control are therefore critical for translating submerged mushroom cultivation into large-scale pharmaceutical production platforms. However, high operational costs associated with aeration, agitation, and energy input may limit economic feasibility on the industrial scale. In addition, maintaining genetic stability and ensuring reproducible secondary metabolite production over prolonged fermentation cycles remain key industrial challenges [88].

4.2. Pre-Extraction Processing: Preservation and Disruption

Following the harvest of fruiting bodies or the recovery of cultivated mycelial biomass, immediate preservation is essential to maintain post-harvest quality. Edible mushrooms are highly perishable, with a short shelf life of approximately 1–3 days, and are prone to rapid deterioration due to both intrinsic metabolic activity and extrinsic environmental factors [92,93]. For edible mushrooms intended for pharmaceutical applications, stabilization of the biomass through drying is a critical downstream process, as it prevents degradation and preserves the integrity of bioactive constituents.

A range of drying techniques is employed in mushroom processing, including natural air drying, hot-air drying, freeze-drying, microwave drying, vacuum drying, solar drying, and infrared radiation drying. Each method exerts distinct effects on the chemical composition and stability of mushroom bioactives [8,94]. For instance, Gąsecka et al. [95] reported significant reductions in polysaccharide content in Leccinum scabrum and H. erinaceus following hot-air drying, attributable to the thermal degradation of heat-sensitive polysaccharides into oligosaccharides. Similarly, Wei et al. [96] observed notable differences in polysaccharide extraction yields from Cordyceps militaris depending on the drying method used, with infrared drying (3.61%) outperforming freeze-drying (2.60%) and hot-air drying (2.19%). These findings underscore the importance of selecting an appropriate drying technique during the development of mushroom-based pharmaceutical products. Among available methods, freeze-drying or lyophilization is often preferred by pharmaceutical industries because the low temperatures employed minimize thermal degradation and allow for superior retention of bioactive compounds comparable to those in fresh mushroom material [94].

Beyond preservation, effective disruption of the mushroom cellular structure is necessary to facilitate the extraction of intracellular bioactive compounds. Mushroom cells are protected by a robust chitin-rich cell wall that limits solvent penetration. To overcome this barrier, dried mushroom biomass is typically subjected to mechanical size reduction using mills or homogenizers to produce small fragments or fine powders [97]. Grinding aims to achieve an optimal particle size that enhances solubility and improves extraction efficiency [94]. Extraction yields are strongly influenced by particle size, as finer particles provide increased surface area and greater solvent–matrix interaction [8]. For example, Xiaokang et al. [98] reported that reducing the particle size of L. edodes from 4.75 to 1.75 mm resulted in a corresponding increase in total phenolic content, rising from 4.90 to 6.35 mg GAE/g.

Collectively, drying and grinding represent critical pre-extraction steps that directly influence the stability, accessibility, and recoverability of mushroom-derived bioactive compounds. Their careful optimization is therefore crucial for the successful pharmaceutical application of edible mushroom extracts.

4.3. Extraction: Liberating Compounds from the Fungal Matrix

Extraction constitutes the primary step in isolating pharmaceutical-relevant bioactive compounds from raw mushroom biomass. At its core, the extraction process involves several sequential events: penetration of the solvent into the solid mushroom matrix, dissolution of soluble components, diffusion of the solutes out of the solid phase, and subsequent recovery of the extracted compounds [8,99]. The efficiency of extraction is primarily determined by the dissolution and diffusion dynamics of these steps and is influenced by factors such as the physicochemical properties of the extraction solvent, particle size of the raw material, solvent-to-solid ratio, extraction temperature, and extraction duration [8]. The choice of extraction solvent is a critical determinant of process efficiency and is governed by factors such as solubility, selectivity, safety, and cost. In accordance with the principle of “like dissolves like,” optimal extraction is achieved when the polarity of the solvent closely matches that of the target solutes [99].

A wide range of conventional and advanced extraction technologies is currently employed for mushroom bioactives (Figure 2). Limitations associated with conventional techniques, such as high solvent consumption, long extraction times, and energy inefficiency (

Table 1. Comparative overview of conventional and advanced extraction techniques for mushroom bioactive compounds.

Extraction Method	Typical Extraction Duration	Operating Temperature (°C)	Volume of Organic Solvent Used	Extraction Efficiency	Key Advantages	Major Limitations
Hot-water extraction	1.5–5 h	50–80	None	Moderate	Economical; simple operation; no need for sophisticated equipment	High risk of degradation of thermo-sensitive compounds
Hydro-alcoholic extraction	1–24 h	25–60	Large	Variable	Efficient for extracting both polar and moderately non-polar compounds	High solvent consumption; reliance on costly organic solvents
Enzyme-assisted extraction	Moderate	<50	Moderate	High	Mild operating conditions; environmentally friendly; enhances selectivity and impurity removal	Requires specialized expertise; efficiency depends on enzyme stability and digestion time
Pressurized liquid/subcritical water extraction	Short	>100	Small	High	Automated operation reduces human error; improves extraction efficiency and analyte stability	Thermal degradation of heat-sensitive compounds; co-extraction of impurities under high pressure; expensive specialized equipment
Supercritical fluid extraction	Moderate	Above critical point	None	Very High	Green technology using CO2; highly efficient for non-polar compounds	High operational cost; technical complexity; safety concerns due to high pressure
Ultrasonic-assisted extraction	Short	40–60	Moderate	High	Low energy consumption; compatible with various solvents; enhances mass transfer	Efficiency influenced by ultrasonic attenuation; energy loss in bath systems
Microwave-assisted extraction	Short	Room temperature	None or moderate	High	Rapid heating; reduced solvent use; high selectivity; minimizes thermal degradation when low-polarity solvents are used	Risk of mechanical or thermal damage to bioactive compounds; high equipment cost; requires pressure-resistant and airtight systems

Sources: Refs. [12,18,99].

), have driven the increasing adoption of advanced extraction technologies. A comparative overview of these methods, including their respective advantages and drawbacks, is presented in Table 1. The selection of an appropriate extraction method must account for the chemical nature of the target compounds, the complexity of the fungal matrix, desired yield, and environmental sustainability [100]. As summarized in Table 1, this choice involves balancing extraction efficiency and selectivity against processing time, solvent and energy demands, and the potential degradation of thermolabile compounds, with advanced techniques generally providing higher yields and improved process control compared to conventional solvent-based methods.

4.3.1. Conventional Solvent-Based Methods

Conventional extraction of mushroom bioactive compounds is predominantly based on solid–liquid extraction using water or organic solvents (majorly methanol and ethanol). Among these approaches, hot-water extraction is widely employed, particularly for polar compounds such as water-soluble polysaccharides. This method is valued for its simplicity, cost-effectiveness, safety, and environmental compatibility [18]. However, the elevated temperatures and prolonged extraction times commonly required (Table 1) increase the risk of degrading thermolabile bioactive constituents [3,101]. Sequential extraction strategies, typically involving water followed by alcohol, have also been applied to recover high-value mushroom-derived compounds [101]. Despite improved extraction breadth, this approach often entails the use of high concentrations of organic solvents (30–98.6%) and extended extraction durations ranging from 1 to 24 h, which may limit its industrial and environmental viability (Table 1) [3].

For compounds of moderate to low polarity, such as lipids and certain polyphenols, conventional techniques including Soxhlet extraction and maceration (Figure 2) are commonly employed. In Soxhlet extraction, the solvent is heated to reflux, evaporated, and subsequently condensed onto the mushroom material contained within a cartridge. Continuous percolation of the solvent through the solid matrix enables the solubilization and transfer of target compounds to a collection flask via a siphoning mechanism [13]. In contrast, maceration involves soaking ground mushroom material in organic solvents at ambient temperature, allowing gradual softening of the matrix and diffusion of bioactive constituents into the solvent. Periodic agitation may be applied to enhance extraction efficiency [102].

Table 2. Application of conventional solvent-based methods for extraction of bioactive compounds from edible mushrooms.

Mushroom Material (Pre-Treatment)	Extraction Solvent	Extraction Conditions (Time/Temperature)	Solvent-to-Solid Ratio	Target Bioactive Compound(s)	Extraction Yield	Reference
Lentinula edodes (freeze-dried and ground into 1.75, 3.35, and 4.75 mm)	Hot water	Water bath; 5 h/45 °C	2.5% w/v	Total phenolics	11.29 mg GAE/g	[98]
	Ethanol	Incubated at 90 °C for 1 h	40% v/v		3.68 mg GAE/g
Pleurotus sajor-caju—air dried and knife-milled	Ethanol or hexane	Solvent recycling in a Soxhlet apparatus for 6 h at solvent boiling temperature	1:30 g/mL	Total phenolics	15.6% (ethanol); 1.2% (hexane)	[103]
C. cibarius, Morchella spp., B. edulis—freeze-dried Agaricus bisporus, L. deliciosus, P. ostreatus, L. edodes—tray dried Hericium erinaceus, and G. lucidum—obtained dried	Methanol (M), water (W), and W/M (50:50 v/v)	Extracted with a mechanical shaker at 4 °C for 10 h	1:10 g/mL	Total phenolics	0.65 to 17.11 mg GAE/g d.w., depending on the solvent type and species	[104]
P. ostreatus	Water	Boiled at 100 °C over a period of 3 h	1:20 w/v	Polysaccharides	6.21% yield	[105]
Panus conchatus (fermentation broth)	Water	Bathed at 95 ° C for 1 h	1:1 v/v	Ergothioneine	Varied depending on the carbon and nitrogen sources used for cultivation	[106]
Freeze-dried forms of Aleuria aurantia, Phallus hadriani, and Panus conchatus ground into powder using an electric grinder	Ethanol	Heated on a heating bowl at boiling point for 10 min	1000 g of powdered mushroom fruiting body was poured into 10 mL of absolute ethanol	Polyphenols	0.617–1.501 mg GAE/100 g	[107]
Lycoperdon saccatum, P. ostreatus, Craterellus cornucopioides, Russula cyanoxantha and C. cibarius—air dried and ground in a blender into 0.132 mm, 0.277 mm, 0.433 mm, 0.294 mm and 0.347 mm, respectively	Hexane	Using a Soxhlet apparatus with an attached reflux condenser for 8 h at 40 °C	3.33% w/v	Lipids (fatty acids and sterols)	0.83–3.38%	[108]
Laetiporus sulphureus—lyophilised and reduced to a fine dried powder (20 mesh)	Methanol, ethanol and water	Stirred with solvent at 30 °C for 24 h	10% w/v	Total phenolics	230 (methanol), 65.7 (ethanol), and 47.4 (water) μg GAEs per mg	[109]
Naturally dried T. versicolor ground into fine powder (40 mesh) using a laboratory grinder	Water	4 h at 95 ± 5 °C	1:10 w/v	Total phenolics	433.98 mg GAE/100 g	[110]
H. erinaceus	Ethanol	Soxhlet extraction under optimum conditions (61 °C temperature, 7 h 50 min, and 2 mg/mL)	NA	Phenolic compounds	59.75 mg/g	[111]
Hypsizygus marmoreus—freeze-dried and ground into powder	Water	Mechanically stirred at 10 °C for 6 h	10% w/v	Polysaccharides	NA	[112]
Dried and powdered P. ostreatus, G. lucidum, Fomes fomentarius, Porodaedalea pini, Fuscoporia torulosa, and Phellinus igniarius	Ethanol and water in a sequential order	80% ethanol at room temperature for 24 h; followed by water at 80 °C	NA	Polysaccharides	NA	[113]
Lentinus squarrosulus, Auricularia auricular-judae, Mycetinis copelandii, Baeospora myosura, P. ostreatus, and Volvariella volvacea—air-dried and grind into powder with an electronic blender	Methanol	Heated at 30–40 °C in a Rotary Evaporator	10% w/v	Lipids	NA	[114]
Agaricus campestris, Macrolepiota procera, C. cibarius, Russula vesca, Russula alutacea, B. edulis, P. ostreatus, and A. bisporus	Water and 50% water–ethanol	Stirred at room temperature for 4 h	4% w/v	Polyphenols (flavonoids)	7.61–31.78 mg GAE/g d.w. for water extract 9.28–69.65 mg GAE/g d.w. for hydro-ethanol extract	[115]
Lyophilized basidiocarps of P. ostreatus	Cold and hot water	Cold distilled water (25 °C with continuous magnetic stirring) and boiling water (100 °C under reflux) for 7 h per extraction	16.67% w/v	Polysaccharides	3.40% (hot water)–6.14% (cold water) w/w	[116]
Fruiting bodies of Lentinus velutinus—dried in hot air oven and blended into fine particles	Water and Ethanol	Mushroom biomass was boiled three times with 20 volumes of water for 3 h each, followed by 80% ethanol at 4 °C	NA	Polysaccharides	3.99% yield	[117]
B. edulis and C. cibarius	Acidic water-10% (v/v) acetic acid solution; a mixture of ethanol, water and acetic acid; hexane; and diethyl ether	Orbital shaking at room temperature for 24 h	3.33% w/v	Polyphenols	B. edulis: 0.48–3.73 mg GAE/g C. cibarius: 0.29–0.79 mg GAE/g	[118]
Ganoderma neo-japonicum	Water and ethanol	Boiling for 4 h	NA	Polysaccharides	NA	[119]
Powdered Pleurotus flabellatus	Methanol	Using a Soxhlet extractor for 4–5 h at a temperature below the solvent boiling point	10% w/v	Multiple class of bioactive compounds	NA	[120]
Dried fruiting bodies of Paxillus involutus	Ethyl acetate	Extracted at room temperature for 3 days	16.5% w/v	Phenolics (novel 2,5-diarylcyclopentenone derivatives)	883 g crude extracts	[121]
L. edodes, V. volvacea, Pleurotus eous, P. sajor-caju and A. auricula (freeze-dried and finely milled)	Water	Boiled for 30 min	2% w/v	Total phenolics	2.90–36.19 mg GAE/g dw	[122]
	50% (v/v) ethanol	Room temperature for 24 h	10% w/v		2.75–27.89 mg GAE/g dw
	Diethyl ether	Room temperature for 24 h	10% w/v		1.99–10.46 mg GAE/g dw
Inonotus obliquus (pulverized using a blender)	Hot water	Refluxed for 2 h in a water bath at 100 °C	2.5% w/v	Total glucans, total triterpenoids	3.81% w/w (total glucan) 8.90 mg UAEq/g (total triterpenoids)	[123]
A. auricula judae, Microporus xanthopus, Termitomyces umkowaani, Trametes elegans, and T. versicolor—oven dried and powdered	99.8% Chloroform, 70% ethanol, and hot water	Mushroom material was separately combined with each solvent in an Erlenmeyer flask at 25 °C and shaken in an incubator for 72 h	10% w/v	Multiple compounds, including carboxylic acids, phenols, fatty acids, isoprenoid lipids, and steroids	Varied depending on the species and bioactive compound	[124]
Dictyophora indusiata—air dried and fully ground into powder by a high-speed pulverizer	Hot water	Water bath at 88 °C for 2.5 h	1:40 g/mL	Polysaccharides	Lowest yield compared to other non-conventional extraction methods (UAE, MAE)	[125]
L. edodes—flash-frozen and ground into fine particles using a cryogenic grinder	Ethyl acetate, ethanol, ultrapure water, cyclohexane, and dimethyl sulfoxide (DMSO)	24 h at 4 °C	1:10 g/mL	Secondary metabolites, including indole-3-lactic acid	Water and cyclohexane extracts had the highest number of total compounds, followed by DMSO, ethanol, and ethyl acetate extracts	[126]

NA—not reported in the cited study; UAEq—ursolic acid equivalent; GAE—gallic acid equivalent.

summarizes information on recent studies employing conventional solvent-based methods for the extraction of mushroom bioactives. The collative evidence in Table 2 indicates that conventional solvent-based extraction efficiency in mushrooms is strongly influenced by solvent polarity, temperature, and extraction duration, with aqueous and hydro-alcoholic solvents generally favoring polysaccharide and phenolic recovery, while non-polar solvents such as hexane and diethyl ether preferentially extract lipophilic compounds, albeit often at lower yields.

4.3.2. Advanced/Green Extraction Techniques

A range of advanced and environmentally benign extraction technologies has been developed for the recovery of pharmaceutical-relevant bioactive compounds from biological resources, including edible mushrooms (Figure 2). These approaches are attracting increasing interest within the pharmaceutical industry because they enable more sustainable processing, shorten extraction time, and often improve extraction efficiency and product quality compared with conventional methods (Table 1).

Enzyme-Assisted Extraction (EAE)

Enzyme-assisted extraction (EAE) exploits the catalytic activity of specific enzymes to degrade structural components of the mushroom cell wall and membranes, thereby facilitating the release of intracellular and bound bioactive compounds. The fungal cell wall is primarily composed of chitin (β-1,4-N-acetylglucosamine) and glucans (β-1,3 and β-1,6 linkages), which can be selectively hydrolyzed using enzymes such as chitinases and glucanases, leading to enhanced extraction efficiency [12,101]. For example, Enman et al. [127] reported the extraction of eritadenine, a hypocholesterolemic compound, from L. edodes using a mixture of α- and β-glucanases. Although the enzymatic pretreatment only marginally increased the yield during subsequent methanolic extraction, it demonstrated the feasibility of enzyme-mediated release of target metabolites. Beyond glycosyl hydrolases, enzymes such as cellulases, pectinases, and proteases are also commonly employed in EAE protocols [99]. Nguyen and Nguyen [128], for instance, obtained a crude extract rich in amino acids, flavonoids, phenolics, proteins, reducing sugars, sucrose, and β-glucans from Pleurotus sajor-caju following pretreatment with exogenous cellulase at 50 °C and pH 5.5 for 8 h.

From an industrial perspective, EAE is particularly attractive because it operates under relatively mild conditions, which helps preserve the structural integrity and bioactivity of thermolabile compounds [129]. Compared with conventional methods, EAE generally requires lower temperatures and shorter processing times while delivering higher extraction yields (Table 3). Zhu et al. [130] demonstrated a 67.72% increase in polysaccharide yield from H. erinaceus using a cellulase:pectinase:trypsin mixture (2:2:1) under optimized conditions (pH 5.71, 52.03 °C, 33.79 min) relative to hot-water extraction. Similarly, Hwang et al. [123] showed that enzymatic extraction using Viscozyme L (a multi-enzymatic complex containing a wide range of carbohydrases) at pH 4.5 and 50 °C for 2 h produced higher levels of β-glucans, triterpenoids, gallic acid, and caffeic acid from I. obliquus than conventional hot-water extraction at 100 °C.

Despite these advantages, EAE presents several practical limitations. The process typically involves multiple downstream steps, including cell lysis, filtration, and centrifugation, which increase technical complexity. In addition, careful optimization and storage conditions are required to maintain enzyme stability and activity [12]. The relatively high cost of commercial enzymes also remains a major constraint for large-scale industrial application of EAE.

Ultrasonic-Assisted Extraction (UAE)

Ultrasonic-assisted extraction (UAE), commonly referred to as sonication, is another emerging green technique for the recovery of bioactive compounds from edible mushrooms. Its effectiveness has been demonstrated in several studies (Table 3); for example, Quintero-Cabello et al. [131] successfully extracted ergosterol, a compound with hypocholesterolemic properties, from Neolentinus lepideus using ultrasound treatment. The underlying mechanism of UAE is acoustic cavitation, which occurs when ultrasonic waves propagate through the solvent medium. These waves are generated by a transducer integrated into either an ultrasonic probe or bath system, converting electrical energy into mechanical vibrations [132]. Cavitation involves the formation, growth, and subsequent implosive collapse of microbubbles, producing localized high shear forces and micro-turbulence. This phenomenon disrupts the mushroom cell wall, enhances solvent penetration, and hastens mass transfer between the solvent and the solid matrix [13,92,133].

As a result, UAE significantly reduces extraction time by promoting faster dissolution and diffusion of solutes. The enhanced heat and mass transfer also allows for an extraction to be performed at relatively lower bulk temperatures, thereby minimizing the degradation of thermolabile compounds while improving overall extraction yield [99,101]. For instance, Hwang et al. [123] reported higher triterpenoid yields from I. obliquus using ultrasound extraction (139 W, 42 kHz, 50 °C) in relation to conventional hot-water extraction (100 °C). Similarly, Cheung et al. [134] observed increased polysaccharide yields from Cordyceps sinensis Cs-HK1 with rising ultrasound intensity (2.44 to 44.1 W/cm²), with UAE outperforming water extraction over treatment times of 10–80 min. Furthermore, ultrasound treatment significantly increased the recovery of polysaccharides from dried and milled A. bisporus by-products relative to untreated samples, with a maximum extraction yield of 4.7% obtained after 15 min at an amplitude of 100 μm [135].

Efficient application of UAE requires careful optimization of processing parameters, including ultrasonic power, extraction time, solvent-to-solid ratio, and temperature. Ma et al. [136] identified optimal conditions for extracting polysaccharides from black fungus mushroom as 350 W ultrasonic power, a fivefold solvent volume, 35 min extraction time, and 90 °C. Likewise, Gogoi et al. [132] demonstrated that ultrasound treatment time, temperature, and solid-to-solvent ratio significantly influenced the recovery of phenolic acids from Pleurotus citrinopileatus, with distinct optimal conditions for aqueous (44 °C, 14 min, and 20 mL/g) and ethanolic (39 °C, 13 min, and 20 mL/g) extracts. From an industrial perspective, ultrasonic probe systems are generally preferred over bath systems because they deliver higher energy density through a localized tip surface, resulting in more efficient cavitation and improved extraction performance [135].

Microwave-Assisted Extraction (MAE)

Microwave-assisted extraction (MAE) is based on the rapid heating of moisture within mushroom tissues, which generates internal pressure sufficient to disrupt cellular structures and promote the release of intracellular bioactive compounds [3]. This process is enabled by the dielectric properties of mushrooms, whereby microwave energy interacts with polar molecules through ionic conduction and dipole rotation, resulting in volumetric heating of the solid matrix [99]. Microwaves are electromagnetic waves composed of oscillating electric and magnetic fields operating within a frequency range of 300 MHz to 300 GHz, with 915, 2450, 5800, and 22,125 MHz being the most commonly applied frequencies in industrial and laboratory-scale heating [3,98,101].

Unlike conventional extraction, where heat is transferred from the external source to the medium via conduction and convection, MAE generates heat directly within the irradiated material. This selective internal heating minimizes energy loss to the surroundings and accelerates mass transfer, thereby enhancing extraction efficiency [101]. Consequently, MAE is recognized for its ability to achieve high extraction yields within short processing times (Table 3) while reducing solvent consumption and overall energy input.

Several studies have demonstrated the effectiveness of MAE for extracting mushroom bioactives (Table 3). Using response surface methodology, Maeng et al. [110] optimized the MAE of total phenolics from T. versicolor, achieving a maximum yield of 470 mg GAE/100 g in 3.8 min with 40% ethanol and a microwave power of 125 W, which exceeded yields obtained by reflux extraction (434 mg GAE/100 g). Similarly, Özyürek et al. [137] successfully extracted polyphenols from Terfezia boudieri, B. edulis, and Lactarius volemus under optimized MAE conditions using methanol–water (80:20, v/v) at 80 °C for 5 min. Comparative studies by Xiaokang et al. [98] further showed that MAE (solid-to-liquid ratio 1:40, 600 W, 15 min) produced significantly higher phenolic contents (11.23 mg GAE/g) from L. edodes than organic solvent-based extraction (3.68 mg GAE/g). In addition, Zhang et al. [138] reported enhanced recovery of gallic acid, protocatechuic acid, catechin, syringic acid, myricetin, and quercetin from Agaricus blazei using MAE (60% ethanol, solvent-to-solid ratio 1:30, 110 °C, 500 W, 5 min) compared with heat reflux extraction and maceration. Despite these advantages, the widespread industrial application of MAE remains constrained by high equipment and operational costs, which currently limit its scalability.

Supercritical Fluid Extraction (SFE)

Supercritical fluid extraction (SFE) is particularly gaining increasing interest from the food and pharmaceutical industries due to its high extraction efficiency and environmentally benign characteristics. In SFE, the extraction medium is a supercritical fluid, defined as a substance maintained above its critical temperature and pressure, where distinct liquid and gaseous phases no longer exist. Under these conditions, the fluid exhibits intermediate physicochemical properties, combining liquid-like solvation capacity with gas-like diffusivity, which enhances mass transfer and penetration into solid matrices [8,13].

Table 3. Application of advanced and non-conventional extraction techniques for the recovery of bioactive compounds from edible mushrooms.

Mushroom Material (Pre-Treatment)	Extraction Technique	Extraction Solvent/Medium	Operating Conditions	Optimized Extraction Parameters	Target Bioactive Compound(s)	Extraction Yield/Recovery Efficiency	Reference
L. edodes—powdered into 850 μm particle size with an electric mill	UAE combined with autoclaving	Water	P = 550 W, A = 60%, t = 60 min, Solvent/solid ratio = 1:30 (w/v), f = 20 kHz	NA	Polysaccharides	16.3% yield, which was significantly higher than the yield of hot water extract	[139]
Pleurotus citrinopileatus—tray dried and powdered using a household grinder	UAE	Water and Ethanol	Ultrasonic bath, T = 30–55 °C, t = 8–20 min, solvent/solid ratio = 20–50 mL/g, P = 200 W, f = 40 kHz	Water extract = 44 °C, 14 min, and 20 mL/g Ethanol extract = 39 °C, 13 min, and 20 mL/g	Total phenolics and flavonoids	Ultrasonication treatment time, temperature and solid to solvent ratio influenced the yield of phenolic acids	[132]
Flammulina velutipes—freeze-dried and powdered via superfine comminution technique (300 mesh)	UAE	Water	Ultrasonic probe horn of 10 mm diameter, solvent/solid ratio = 1:25 w/v, P = 620 W, T = 45 °C, t = 20 min, f = 20 kHz	NA	Polysaccharides (FVP)	UAE resulted in higher yield (5.59%) of FVP content compared to hot-water extraction (4.45%)	[140]
Armillaria mellea—oven dried, crushed and sieved through a 60 mesh sieve	UAE	Water	Ultrasonic bath, T = 70 °C, P = 280 W, t = 40 min, solvent/solid ratio = 1:20 g/mL	NA	Polysaccharides (AMPs)	3.86% yield	[141]
	EAE	NA	2.3% of cellulase: papain (1:1), material-liquid ratio = 1:20 g/mL, pH = 5, T = 40 °C, t = 140 min			5.21% yield
P. eryngii—dried and powdered	UAE	Water	P = 464 W, f = 80 kHz, T = 60 °C, t = 3.5 h, solid–liquid ratio = 1:36 g/mL	NA	Polysaccharide (PEP)	4.4% yield of crude PEP with a carbohydrate content of 44.4%	[142]
Suillus bovinus—lyophilized and ground by an electric grinder	UAE	93.6% Methanol	Solvent/solid ratio = 1.3% (w/v), f = 20 Hz, P = 70 W, T = 60 °C, t = 5 min, A = 16.86%, 0.71 s−1 cycles	NA	Total phenolics	UAE is an effective method for the recovery of phenolic compounds from wild and cultivated edible mushrooms	[143]
A. auricula-judae—freeze-dried and ground (80-mesh)	UAE	Water, methanol	Solvent-to-sample ratios = 10:1, 20:1, 30:1 mL/g; pulse duty cycles = 0.2, 0.6, 1.0 s−1; T = 10, 40, 70 °C; t = 10 min	1:18 g/mL methanol, T = 59 °C and pulse duty cycle of 0.7 s-1.	Total phenolics	A recovery rate of 94.85% for TPC was achieved using this method	[144]
A. bisporus—air-dried and ground using a blender	MAE	Ethanol/water (80:20, v/v)	Ethanol concentration = 10–90%, t = 1–30 min, solvent/solid ratio = 5–20 mL/0.2 g	t = 16 min; solvent/solid ratio = 12.9 mL/0.2 g; ethanol concentration = 58%	Total phenolics	14.82 mg GAE/g	[145]
P. ostreatus and P. eryngii—air dried and ground	MAE	Water, absolute ethanol, and ethyl acetate	Solid-to-solvent ratio = 1:30 (w/v), T = 80 °C, t = 5 min, P = 30 to 100 W depending on the solvent	NA	Total phenolics	The ethyl acetate extracts of both mushroom species were most biologically effective	[78]
Schizophyllum commune—powdered	MAE	Water	Solvent/solid ratio = 1:4 (w/w); T = 80 °C, 100 °C, and 120 °C; t = 2, 6, and 10 min	T = 80 °C, t = 2 min, solvent/solid ratio = 1:4 (w/w)	Multiple compounds, including amino acids	16.76% to 34.18% crude extract yield (wet basis)	[146]
Multiple Pleurotus spp.—freeze-dried and powdered	MAE	Methanol and water	0–30% methanol in water, solvent/sample ratio = 10:1–20:1 mL/g, T = 40–70 °C, P = 800 W, t = 10 min	Pure water as a solvent, solvent/sample ratio = 17.5:1, T = 44 °C, t = 10 min	Phenolic compounds: p-coumaric acid, t-cinnamic acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, quercetin-3-glucoside, gallic acid, and vanillic acid	The developed UAE method demonstrated high accuracy (>85% recovery) and precision (CV < 10%), confirming its reliability for the profiling of phenolic compounds in oyster mushrooms	[147]
D. indusiata—air dried and pulverized	MAE	Water	Solid/liquid ratio = 1:40, t = 7 min, P = 150 W	NA	Polysaccharides	UAE had a higher yield than MAE; however, the sequential combination of both techniques produced higher yields than both individually	[125]
	UAE		Solid/liquid ratio = 1:40, P = 600 W, T = 80 °C, t = 25 min
L. edodes—dried mushroom bodies were pulverized into a powder using an electric mill	UAE	NA	Ultrasonic probe horn (3 mm-diameter), f = 20 kHz, P = 750 W, A = 60% (0.87 W/mL), t = 40 min	Optimized sequential extraction (EAE followed by UAE): enzyme 0.28% (w/v), A = 62%, and (NH4)2SO4 saturation 69%	Proteins	6.9% yield	[148]
	EAE		0.3% of cellulase (50 U/mg), T = 45 °C, t = 1 h, pH 4.5			4.9% yield
A. bisporus (white strain and brown strain), L. edodes, P. ostreatus (HK35 and Spoppo)—air dried and ground by means of a cross beater mill	UAE	Alkaline medium	Laboratory scale: Ultrasonic probe, P = 400 W, f = 20 kHz, 30 g of sample mixed with 0.1 mol/L NaOH, T ≤ 45 °C Medium scale: P = 1000 W, f = 24 kHz, increase in mass of feedstock and volume of solvent by 10 times	NA	Proteins	7–17% yield	[149]
	EAE	10 mM phosphate buffer	Protease (2.59 AU/G) or papain (1.5 units/mg); enzyme to substrate ratio = 1% (w/w); t = 2 h; protease pH 6.5–8.5 at T = 60 °C; papain pH 6.0–7.0 at T = 65 °C			23–24% yield
A. bisporus—hot air oven dried and ground into fine powder (80 mesh)	EAE	Water	12.36 μL of α-amylase (3000 U/g) and 168 μL of amyloglucosidase (3300 U/g), pH 4.5, T = 50–55 °C, t = 4 h, solvent/solid ratio = 3:25 g/mL	NA	Phenolic acids, flavonoids, and total glucans	26.48–31.30% yield	[150]
L. saccatum, P. ostreatus, C. cornucopioides, R. cyanoxantha and C. cibarius—airdried and ground in a blender into 0.132 mm, 0.277 mm, 0.433 mm, 0.294 mm and 0.347 mm, respectively	SFE	Sc-CO2 (purity 99.9% v/v)	T = 40 °C, p = 30 MPa, CO2 flow rate of 1.94 kg/h, t = 0.5–4 h	t = 4 h	Lipids	0.807–3.316% yield	[108]
A. bisporus—lyophilised and powdered	SFE	CO2 (>99.998% purity) and ethanol (solvent modifier)	Flow rate of 4 mL/min (including carbon dioxide and co-solvent), p = 70–300 bar	p = 244 bar, T = 56 °C, 8% (v/v) co-solvent	Ergosterol	6.23 mg/g d.w	[151]
P. ostreatus—fresh material ground in a blender	SFE	CO2 and water as co-solvent	T = 393.15–433.15 K, p = 15–35 MPa, 10 to 20% H2O, CO2 flow rate of 2.5 L/min−1	p = 25 MPa, T = 433.15 K, 20% H2O	Polysaccharides	2.06–30.73% yield	[105]
Cordyceps sinensis—powder of average particle size 300 μm	SFE	Sc-CO2 and ethanol 1% (v/v) as co-solvent	p = 200, 250, 300 and 350 bar; T = 40, 50 and 60 °C; CO2 flow rate of 0.4 L/h, t = 1.5–2 h	T = 60 °C, p = 300 bar, t = 2 h	Cordycepin and flavonoids	0.53–0.75% (w/w)	[152]
Pleurotus pulmonarius—freeze-dried and ground to a fine powder of pore size 0.12 mm	SFE	Sc-CO2 with and without 5% ethanol as co-solvent	p = 38 MPa, T = 80 °C, t = 2.7 h	NA	Fatty acids, total phenols and ergosterol	0.36% (without co-solvent)–0.93% (with co-solvent) yield	[153]
	UAE	Water	Ultrasound bath, t = 1 h, T = 37 °C			45.14–63.32% yield
Tuber aestivum and Terfezia claveryi—lyophilized and powdered (< 0.5 mm)	PLE	Water, ethanol (100%) and water:ethanol (1:1 v/v)	Accelerated Solvent Extractor; T = 50, 115, and 180 °C; t = 5, 17.5, and 30 min	p = 16.7 MPa, T = 180 °C, t = 30 min	Multiple compounds, including β-glucans and sterols	Varied by species and solvents used	[154]
P. ostreatus (powdered) and G. lucidum (fine pieces)—both lyophilized	PLE (ASE)	Water	T = 50–180 °C, t = 5–30 min, p = 10.2–11.7 MPa	T = 180 °C, t = 26 min for P. ostreatus; and T = 180 °C, t = 22 min for G. lucidum	Polysaccharides (β-d-glucans)	11.9% (G. lucidum); 30.3% (P. ostreatus) (w/w)	[155]
L. edodes—oven dried and ground into fine particles (500 μm) using a blender	PLE (ASE)	Water, ethanol (60% v/v)	T = 40–160 °C, t = 5–30 min, p = 10.3 MPa	T = 142 °C, t = 5 min	Eritadenine	652 mg/100 g d.w	[156]
P. eryngii, H. erinaceus and P. citrinopileatus—dried and powdered using a Moulinex grinder	PLE (SWE)	Water	Liquid to solid ratio = 12 (w/w), T = 160 and 190 °C	T = 190 °C	Total glucans	11.63–47.57% yield, depending on species and temperature	[157]
Agaricus blazei fruiting body	PLE (SWE)	Water	T = 120, 140, 160, or 200 °C; p = 2 and 5 MPa; t = 5–240 min	NA	Polysaccharide (FIII-2-b)	Subcritical water extracts from the fruiting body or mycelium of A. blazei significantly inhibited the expression of immune checkpoint molecules and Axl	[158]
A. blazei mycelium—air dried			T = 160 °C, t = 5 min

NA—not reported in the cited study; t = time; P = power; p = pressure; T = temperature; f = frequency; A = amplitude; GAE—gallic acid equivalent.

Table 3. Application of advanced and non-conventional extraction techniques for the recovery of bioactive compounds from edible mushrooms.

Mushroom Material (Pre-Treatment)	Extraction Technique	Extraction Solvent/Medium	Operating Conditions	Optimized Extraction Parameters	Target Bioactive Compound(s)	Extraction Yield/Recovery Efficiency	Reference
L. edodes—powdered into 850 μm particle size with an electric mill	UAE combined with autoclaving	Water	P = 550 W, A = 60%, t = 60 min, Solvent/solid ratio = 1:30 (w/v), f = 20 kHz	NA	Polysaccharides	16.3% yield, which was significantly higher than the yield of hot water extract	[139]
Pleurotus citrinopileatus—tray dried and powdered using a household grinder	UAE	Water and Ethanol	Ultrasonic bath, T = 30–55 °C, t = 8–20 min, solvent/solid ratio = 20–50 mL/g, P = 200 W, f = 40 kHz	Water extract = 44 °C, 14 min, and 20 mL/g Ethanol extract = 39 °C, 13 min, and 20 mL/g	Total phenolics and flavonoids	Ultrasonication treatment time, temperature and solid to solvent ratio influenced the yield of phenolic acids	[132]
Flammulina velutipes—freeze-dried and powdered via superfine comminution technique (300 mesh)	UAE	Water	Ultrasonic probe horn of 10 mm diameter, solvent/solid ratio = 1:25 w/v, P = 620 W, T = 45 °C, t = 20 min, f = 20 kHz	NA	Polysaccharides (FVP)	UAE resulted in higher yield (5.59%) of FVP content compared to hot-water extraction (4.45%)	[140]
Armillaria mellea—oven dried, crushed and sieved through a 60 mesh sieve	UAE	Water	Ultrasonic bath, T = 70 °C, P = 280 W, t = 40 min, solvent/solid ratio = 1:20 g/mL	NA	Polysaccharides (AMPs)	3.86% yield	[141]
	EAE	NA	2.3% of cellulase: papain (1:1), material-liquid ratio = 1:20 g/mL, pH = 5, T = 40 °C, t = 140 min			5.21% yield
P. eryngii—dried and powdered	UAE	Water	P = 464 W, f = 80 kHz, T = 60 °C, t = 3.5 h, solid–liquid ratio = 1:36 g/mL	NA	Polysaccharide (PEP)	4.4% yield of crude PEP with a carbohydrate content of 44.4%	[142]
Suillus bovinus—lyophilized and ground by an electric grinder	UAE	93.6% Methanol	Solvent/solid ratio = 1.3% (w/v), f = 20 Hz, P = 70 W, T = 60 °C, t = 5 min, A = 16.86%, 0.71 s−1 cycles	NA	Total phenolics	UAE is an effective method for the recovery of phenolic compounds from wild and cultivated edible mushrooms	[143]
A. auricula-judae—freeze-dried and ground (80-mesh)	UAE	Water, methanol	Solvent-to-sample ratios = 10:1, 20:1, 30:1 mL/g; pulse duty cycles = 0.2, 0.6, 1.0 s−1; T = 10, 40, 70 °C; t = 10 min	1:18 g/mL methanol, T = 59 °C and pulse duty cycle of 0.7 s-1.	Total phenolics	A recovery rate of 94.85% for TPC was achieved using this method	[144]
A. bisporus—air-dried and ground using a blender	MAE	Ethanol/water (80:20, v/v)	Ethanol concentration = 10–90%, t = 1–30 min, solvent/solid ratio = 5–20 mL/0.2 g	t = 16 min; solvent/solid ratio = 12.9 mL/0.2 g; ethanol concentration = 58%	Total phenolics	14.82 mg GAE/g	[145]
P. ostreatus and P. eryngii—air dried and ground	MAE	Water, absolute ethanol, and ethyl acetate	Solid-to-solvent ratio = 1:30 (w/v), T = 80 °C, t = 5 min, P = 30 to 100 W depending on the solvent	NA	Total phenolics	The ethyl acetate extracts of both mushroom species were most biologically effective	[78]
Schizophyllum commune—powdered	MAE	Water	Solvent/solid ratio = 1:4 (w/w); T = 80 °C, 100 °C, and 120 °C; t = 2, 6, and 10 min	T = 80 °C, t = 2 min, solvent/solid ratio = 1:4 (w/w)	Multiple compounds, including amino acids	16.76% to 34.18% crude extract yield (wet basis)	[146]
Multiple Pleurotus spp.—freeze-dried and powdered	MAE	Methanol and water	0–30% methanol in water, solvent/sample ratio = 10:1–20:1 mL/g, T = 40–70 °C, P = 800 W, t = 10 min	Pure water as a solvent, solvent/sample ratio = 17.5:1, T = 44 °C, t = 10 min	Phenolic compounds: p-coumaric acid, t-cinnamic acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, quercetin-3-glucoside, gallic acid, and vanillic acid	The developed UAE method demonstrated high accuracy (>85% recovery) and precision (CV < 10%), confirming its reliability for the profiling of phenolic compounds in oyster mushrooms	[147]
D. indusiata—air dried and pulverized	MAE	Water	Solid/liquid ratio = 1:40, t = 7 min, P = 150 W	NA	Polysaccharides	UAE had a higher yield than MAE; however, the sequential combination of both techniques produced higher yields than both individually	[125]
	UAE		Solid/liquid ratio = 1:40, P = 600 W, T = 80 °C, t = 25 min
L. edodes—dried mushroom bodies were pulverized into a powder using an electric mill	UAE	NA	Ultrasonic probe horn (3 mm-diameter), f = 20 kHz, P = 750 W, A = 60% (0.87 W/mL), t = 40 min	Optimized sequential extraction (EAE followed by UAE): enzyme 0.28% (w/v), A = 62%, and (NH4)2SO4 saturation 69%	Proteins	6.9% yield	[148]
	EAE		0.3% of cellulase (50 U/mg), T = 45 °C, t = 1 h, pH 4.5			4.9% yield
A. bisporus (white strain and brown strain), L. edodes, P. ostreatus (HK35 and Spoppo)—air dried and ground by means of a cross beater mill	UAE	Alkaline medium	Laboratory scale: Ultrasonic probe, P = 400 W, f = 20 kHz, 30 g of sample mixed with 0.1 mol/L NaOH, T ≤ 45 °C Medium scale: P = 1000 W, f = 24 kHz, increase in mass of feedstock and volume of solvent by 10 times	NA	Proteins	7–17% yield	[149]
	EAE	10 mM phosphate buffer	Protease (2.59 AU/G) or papain (1.5 units/mg); enzyme to substrate ratio = 1% (w/w); t = 2 h; protease pH 6.5–8.5 at T = 60 °C; papain pH 6.0–7.0 at T = 65 °C			23–24% yield
A. bisporus—hot air oven dried and ground into fine powder (80 mesh)	EAE	Water	12.36 μL of α-amylase (3000 U/g) and 168 μL of amyloglucosidase (3300 U/g), pH 4.5, T = 50–55 °C, t = 4 h, solvent/solid ratio = 3:25 g/mL	NA	Phenolic acids, flavonoids, and total glucans	26.48–31.30% yield	[150]
L. saccatum, P. ostreatus, C. cornucopioides, R. cyanoxantha and C. cibarius—airdried and ground in a blender into 0.132 mm, 0.277 mm, 0.433 mm, 0.294 mm and 0.347 mm, respectively	SFE	Sc-CO2 (purity 99.9% v/v)	T = 40 °C, p = 30 MPa, CO2 flow rate of 1.94 kg/h, t = 0.5–4 h	t = 4 h	Lipids	0.807–3.316% yield	[108]
A. bisporus—lyophilised and powdered	SFE	CO2 (>99.998% purity) and ethanol (solvent modifier)	Flow rate of 4 mL/min (including carbon dioxide and co-solvent), p = 70–300 bar	p = 244 bar, T = 56 °C, 8% (v/v) co-solvent	Ergosterol	6.23 mg/g d.w	[151]
P. ostreatus—fresh material ground in a blender	SFE	CO2 and water as co-solvent	T = 393.15–433.15 K, p = 15–35 MPa, 10 to 20% H2O, CO2 flow rate of 2.5 L/min−1	p = 25 MPa, T = 433.15 K, 20% H2O	Polysaccharides	2.06–30.73% yield	[105]
Cordyceps sinensis—powder of average particle size 300 μm	SFE	Sc-CO2 and ethanol 1% (v/v) as co-solvent	p = 200, 250, 300 and 350 bar; T = 40, 50 and 60 °C; CO2 flow rate of 0.4 L/h, t = 1.5–2 h	T = 60 °C, p = 300 bar, t = 2 h	Cordycepin and flavonoids	0.53–0.75% (w/w)	[152]
Pleurotus pulmonarius—freeze-dried and ground to a fine powder of pore size 0.12 mm	SFE	Sc-CO2 with and without 5% ethanol as co-solvent	p = 38 MPa, T = 80 °C, t = 2.7 h	NA	Fatty acids, total phenols and ergosterol	0.36% (without co-solvent)–0.93% (with co-solvent) yield	[153]
	UAE	Water	Ultrasound bath, t = 1 h, T = 37 °C			45.14–63.32% yield
Tuber aestivum and Terfezia claveryi—lyophilized and powdered (< 0.5 mm)	PLE	Water, ethanol (100%) and water:ethanol (1:1 v/v)	Accelerated Solvent Extractor; T = 50, 115, and 180 °C; t = 5, 17.5, and 30 min	p = 16.7 MPa, T = 180 °C, t = 30 min	Multiple compounds, including β-glucans and sterols	Varied by species and solvents used	[154]
P. ostreatus (powdered) and G. lucidum (fine pieces)—both lyophilized	PLE (ASE)	Water	T = 50–180 °C, t = 5–30 min, p = 10.2–11.7 MPa	T = 180 °C, t = 26 min for P. ostreatus; and T = 180 °C, t = 22 min for G. lucidum	Polysaccharides (β-d-glucans)	11.9% (G. lucidum); 30.3% (P. ostreatus) (w/w)	[155]
L. edodes—oven dried and ground into fine particles (500 μm) using a blender	PLE (ASE)	Water, ethanol (60% v/v)	T = 40–160 °C, t = 5–30 min, p = 10.3 MPa	T = 142 °C, t = 5 min	Eritadenine	652 mg/100 g d.w	[156]
P. eryngii, H. erinaceus and P. citrinopileatus—dried and powdered using a Moulinex grinder	PLE (SWE)	Water	Liquid to solid ratio = 12 (w/w), T = 160 and 190 °C	T = 190 °C	Total glucans	11.63–47.57% yield, depending on species and temperature	[157]
Agaricus blazei fruiting body	PLE (SWE)	Water	T = 120, 140, 160, or 200 °C; p = 2 and 5 MPa; t = 5–240 min	NA	Polysaccharide (FIII-2-b)	Subcritical water extracts from the fruiting body or mycelium of A. blazei significantly inhibited the expression of immune checkpoint molecules and Axl	[158]
A. blazei mycelium—air dried			T = 160 °C, t = 5 min

NA—not reported in the cited study; t = time; P = power; p = pressure; T = temperature; f = frequency; A = amplitude; GAE—gallic acid equivalent.

Among supercritical fluids, carbon dioxide (SC-CO₂) is the most widely used solvent owing to its favorable attributes, including a low critical temperature (31 °C), tunable selectivity, low cost, non-toxicity, high density, low viscosity, and high diffusivity [99,133]. Moreover, the U.S. Food and Drug Administration has designated SC-CO₂ as a “Generally Recognized as Safe” (GRAS) solvent, making it particularly suitable for pharmaceutical and food-related applications.

The solvating power of SC-CO₂ can be precisely modulated by small variations in pressure and temperature near the critical point. Although its inherently low polarity favors the extraction of non-polar compounds such as lipids and sterols, the addition of polar co-solvents (modifiers), such as ethanol, can substantially expand its extraction range to include moderately polar bioactive compounds [101]. For example, Mishra et al. [152] employed 1% (v/v) ethanol as a co-solvent to obtain extracts rich in flavonoids and volatile organic compounds (VOCs) from Cordyceps sinensis. Almeida et al. [151] further reported that the volumetric proportion of ethanol used as a co-solvent was the most critical factor for enhancing ergosterol extraction yield from A. bisporus, albeit at the expense of reduced ergosterol purity in the resulting extract. As with other advanced extraction techniques, optimization of operational parameters, including pressure, temperature, extraction time, and CO₂ flow rate, is essential to maximize SFE efficiency. In an investigation using different pressures and temperatures, for instance, Mazzutti et al. [159] identified optimal conditions for phenolic compounds extraction at 30.0 MPa and 323.15 K. Early studies, such as by Abdullah et al. [160], demonstrated the feasibility of using SC-CO₂ alone to rapidly extract fatty and carboxylic acids from Agaricus spp. More recent investigations have focused on comparative performance and process efficiency relative to conventional methods.

Krivošija et al. [108] reported that a 4 h SC-CO₂ extraction effectively recovered fatty acids and sterols from several mushroom species, including L. saccatum, P. ostreatus, and C. cibarius (Table 3). Although Soxhlet extraction (8 h) yielded slightly higher recoveries, the authors considered the difference negligible in view of the substantial advantages of SC-CO₂, such as reduced solvent use and shorter processing time. Comparable findings were reported by Joradon et al. [161], who observed no significant differences in ergosterol and total polyphenol yields from H. erinaceus obtained via SC-CO₂ (1 h, 46.38 °C, 100 bar) and conventional maceration (72 h). In contrast, Li et al. [162] demonstrated superior sterol recovery from G. lucidum spores using SC-CO₂ at 35 MPa and 48 °C for 3 h with 20% ethanol as a co-solvent compared with Soxhlet extraction. Collectively, these findings highlight the versatility, efficiency, and sustainability of SFE for the recovery of pharmaceutical-grade bioactive compounds from edible mushrooms.

Pressurized Liquid Extraction (PLE)

Pressurized liquid extraction (PLE) is referred to in the literature by several interchangeable terms, including accelerated solvent extraction (ASE), enhanced solvent extraction, pressurized fluid extraction, accelerated fluid extraction, subcritical water extraction (SWE), and high-pressure solvent extraction (HPSE), all of which are based on the same operational principle. In PLE system, the extraction solvent is maintained at elevated temperatures and pressures, typically near its supercritical region. High temperatures enhance solute solubility and diffusion rates, while the applied pressure prevents solvent boiling, thereby preserving the liquid phase above its normal boiling point. This combination promotes disruption of the mushroom matrix and facilitates efficient solvent penetration and mass transfer [99,133].

Similar to other advanced extraction techniques, PLE achieves high extraction efficiencies within shorter processing times and with reduced solvent consumption compared with conventional methods (Table 3). The process is commonly automated (Table 1) and conducted in closed systems that limit exposure to oxygen and light, thereby reducing oxidative degradation of sensitive bioactive compounds [133]. Comparative studies highlight the superior performance of PLE for mushroom bioactives. For example, Hwang et al. [123] evaluated non-conventional (PLE, UAE, EAE) and conventional (hot-water) extraction methods for chaga mushroom (I. obliquus) and reported that PLE at 121 °C and 0.11–0.12 MPa produced the highest yields of β-glucans and total phenolics within 2 h. Similarly, Yang et al. [163] demonstrated that SWE (a specific form of PLE) doubled polysaccharide yields from G. frondosa in 43.65 min compared with hot-water extraction requiring 3 h.

Subcritical water extraction (SWE) employs water as the solvent and enables selective recovery of different classes of bioactive compounds by adjusting temperature. At lower temperatures, SWE preferentially extracts polar compounds, whereas higher temperatures (>100 °C) favor the extraction of less polar constituents due to reduced dielectric constant of water [101]. This means that elevating water above its boiling point under pressurized conditions enhances its ability to solubilize and extract less polar compounds that are otherwise poorly soluble under ambient aqueous conditions. Using this approach, Yang et al. [163] reported an optimal polysaccharide yield of 25.1% from G. frondosa at 210 °C. Extraction conditions strongly influence compound recovery, particularly for phenolic constituents, which generally require elevated temperatures. Krümmel et al. [103] observed a 27% increase in phenolic yield from P. sajor-caju using PLE at 80 °C compared with 40 °C at 10 MPa. Likewise, Sakdasri et al. [164] reported a 25.5% improvement in polyphenol recovery from the same edible mushroom at 140 °C relative to 100 °C using PLE. In addition to temperature, solvent pH can also affect extraction efficiency; Abu-Reidah et al. [165] found that the highest total phenolic content from I. obliquus was obtained at pH 11.5 and 100 °C. These studies collectively underscore the flexibility and effectiveness of PLE as a green and high-performance extraction strategy for pharmaceutical-relevant mushroom bioactives.

These advanced extraction technologies may also be applied in sequential or hybrid configurations to further enhance the recovery of bioactive compounds from edible mushrooms. For example, Zhao et al. [148] showed that EAE followed by UAE produced the highest protein yield (9.4%) from L. edodes, representing approximately a twofold increase compared with EAE alone (4.9%) and a substantial improvement over UAE alone (6.9%). Similarly, Zhang et al. [125] reported that polysaccharide yields from D. indusiata followed the order ultrasound–microwave-assisted extraction > UAE > MAE. The superior performance of the combined approach was attributed to the synergistic effects of acoustic cavitation and microwave irradiation, which collectively enhance cell wall disruption and mass transfer, thereby facilitating more efficient release of target compounds into the extraction solvent. As reflected in the representative studies summarized in Table 3, advanced extraction techniques applied to edible mushrooms demonstrate method- and condition-dependent variations in extraction efficiency, target specificity, and bioactive recovery, underscoring the importance of optimizing process parameters for maximal yield.

4.4. Isolation and Purification: Crude Extract to Defined Ingredient

Crude extracts obtained from edible mushrooms using the extraction methods described above are complex matrices containing a wide range of co-extracted constituents. To obtain defined bioactive ingredients of pharmaceutical relevance, these extracts must undergo further isolation and purification (Figure 3). The presence of impurities can obstruct structural elucidation and compromise the biological activity of target compounds, emphasizing the need for this downstream processing to be effective [19].

High-resolution chromatographic techniques are commonly employed during the final stages of purification. However, because chromatographic media are costly and susceptible to fouling by residual protein and lipid impurities in the mushroom matrix, preliminary fractionation steps are typically applied to simplify the extract and reduce the contaminant load prior to chromatography. Widely used fractionation strategies include liquid–liquid partitioning, precipitation, adsorptive separation, and membrane filtration (Figure 3), owing to their operational simplicity, scalability, and cost-effectiveness [8,99].

Membrane filtration is a size-based separation technique classified into microfiltration, ultrafiltration, and nanofiltration according to membrane pore size [99]. It is extensively used in pharmaceutical processing to clarify extracts, concentrate target molecules, and remove low-molecular-weight impurities. Nevertheless, its selectivity is limited, as effective fractionation of proteins or polysaccharides requires substantial differences in molecular weight [8]. During membrane filtration, a semipermeable membrane selectively retains larger molecules while enabling the passage of smaller molecules. For example, Sari et al. [166] applied cross-flow ultrafiltration to crude L. edodes extracts, generating six fractions of distinct molecular sizes. Subsequent quantitative analysis of the fractions revealed trace levels of β-glucans and decreasing protein content with increasing fraction size. Similarly, Yang et al. [167] purified G. lucidum polysaccharides using a membrane with a 6 kDa molecular weight cut-off (MWCO), achieving a purity of 73.5% and a recovery rate of 69.4%. Using a high-performance ultrafiltration cell with 10, 3 and 1 kDa MWCO, Kaprasob et al. [168] also obtained four molecular size fractions (>10, 3–10, 1–3, and <1 kDa) of bioactive peptides from protein hydrolysate of B. edulis.

Adsorptive separation exploits differences in the affinity of extract components for solid adsorbents. The selection of an appropriate adsorbent as the stationary phase is critical to maximize recovery, ensure effective separation, and prevent irreversible adsorption of target compounds [99]. Common low-cost adsorbents include silica gel, macroporous resins, and alumina. Li et al. [169] demonstrated that ADS-8 resin exhibited superior adsorption and desorption capacities for the isolation of anticancer ganoderic acids GA-Mk and GA-T from G. lucidum mycelia. Concentrations of GA-Mk and GA-T increased from 45 and 22 mg/g in the crude extract to 352 and 141 mg/g, respectively, with recovery yields of 90.1% and 72.2%. In another study, Krüzselyi et al. [170] employed a two-step silica gel-based solid-phase extraction (SPE) approach to isolate minor bioactive constituents from P. citrinopileatus extracts rich in fatty acids. Thin-layer chromatography (TLC) confirmed a marked reduction in linoleic acid content in the eluate compared with the crude methanolic extract.

Graded ethanol precipitation is another widely applied fractionation method, particularly for polysaccharides, and is based on their differential solubility in aqueous ethanol solutions [19]. Increasing ethanol concentration reduces solvent polarity, promoting polysaccharide aggregation and precipitation, thereby yielding fractions enriched in the target compound. Additional deproteinization techniques, including the Sevag method, trichloroacetic acid treatment, and enzymatic digestion, are often employed to remove co-extracted proteins, with the Sevag method being the most commonly used (Figure 3) [18]. Barbosa et al. [105], for instance, precipitated polysaccharides from P. ostreatus crude extracts obtained by SFE–CO₂ (with water as co-solvent) using three volumes of 95% ethanol at 4 °C for 24 h. The polysaccharide-rich fractions were subsequently deproteinized using the Sevag reagent (n-butanol/chloroform, 1:5 v/v) prior to structural analysis. Other edible mushrooms whose crude extracts have been fractionated for polysaccharides using ethanol precipitation and/or deproteinization include Phellinus linteus [171,172], C. versicolor, G. frondosa, G. lucidum and Antrodia cinnamomea [171], Morchella esculenta [173], D. indusiata [125], P. ostreatus and G. lucidum [155], Hypsizygus marmoreus [112], Armillaria mellea [141], P. eryngii [142], Pleurotus cystidiosus [174], L. velutinus [117] and C. sinensis mycelia [175]. Depigmentation of co-extracted colored impurities is another essential step in polysaccharide isolation and is commonly achieved using approaches such as polyamide resin adsorption, activated carbon treatment, or dialysis membrane separation [20]. Although such approaches yield relatively pure carbohydrate fractions, individual fractions may still comprise polysaccharides of varying molecular weights [133].

Salt-based precipitation techniques also play a role in fractionation. Ammonium sulfate [(NH₄)₂SO₄] is frequently used for protein isolation. Mogahid et al. [176] reported the initial separation of a novel hemagglutination-inhibiting lectin from P. eryngii using 80% ammonium sulfate saturation. In addition, Liu et al. [177] combined quaternary ammonium salt precipitation (10% hexadecyltrimethylammonium bromide) with ultrafiltration of 10 kDa MWCO to purify crude polysaccharides (TLH-3) from Tricholoma lobayense. This integrated approach resulted in higher yields, larger processing volumes, and shorter purification times compared with conventional column chromatography.

Fractions obtained from preliminary fractionation often remain chemically complex, containing multiple constituents with closely related physicochemical properties. This complexity complicates the isolation of target bioactive compounds, particularly when structural analogs or isomers are present [8]. Consequently, column chromatography employing finely packed stationary phases (typically <5 μm) is widely adopted as the final step of isolating the target compound. High-performance liquid chromatography (HPLC), which operates under elevated pressure to drive the mobile phase through the column, is especially prevalent at the laboratory scale for the isolation of single, high-purity compounds on the basis of their polarity. For example, Naumoska et al. [178] developed a two-dimensional chromatographic strategy to isolate the neuroprotective compound erinacine A from crude H. erinaceus extract. This approach combined initial fractionation using normal-phase flash chromatography on silica with subsequent purification by semi-preparative reversed-phase HPLC (C₁₈), yielding 19.4 mg of erinacine A with a chromatographic purity of 97.4%. In a similar study, Le et al. [179] repeatedly subjected the ethyl acetate fraction of Phellinus igniarius to silica gel column chromatography (600 g, 160 × 7 cm) to obtain an Inoscavin A-enriched fraction, which was subsequently purified by preparative HPLC using a Luna C₁₈ column to yield Meshimakobnol A. These purified compounds were subsequently employed as reference standards for RP-HPLC–PDA-based quantitative analysis of the same constituents in other Phellinus species. Reversed-phase HPLC (HPLC-RP, C₁₈) was also employed to separate the water-soluble, polar compound muscimol from the aqueous extract of Amanita muscaria [180].

Ion-exchange chromatography (IEC) and gel filtration chromatography, also referred to as size-exclusion chromatography (SEC), are among the most commonly applied chromatographic techniques, particularly for the purification of mushroom polysaccharides. IEC separates compounds based on differences in net surface charge, incorporating ion-exchange mechanisms with liquid chromatographic separation [181]. Bioactive components of edible mushrooms bearing ionizable functional groups can be selectively retained on charged resin beads and subsequently eluted by modifying the mobile-phase pH or ionic strength. Anion-exchange resins such as DEAE-agarose or DEAE-cellulose are typically used for neutral or acidic polysaccharides and phenolic compounds, whereas cation-exchange resins (e.g., sulfonate or carboxylate matrices) are suitable for alkaloids [99,181]. In contrast, gel filtration chromatography relies on the molecular-sieving properties of porous beads to separate constituents according to their hydrodynamic size or molecular weight [181]. Unlike membrane filtration, smaller molecules are retained longer within the pores of the stationary phase, while larger molecules elute earlier. Common stationary phases include Sephadex (cross-linked dextran), agarose gels, and polyacrylamide-based media (Bio-Gel P) [99].

These charge- and size-based chromatographic approaches have been effectively combined for polysaccharide purification across different species. Liu et al. [177] initially fractionated T. lobayense polysaccharides by DEAE ion-exchange chromatography to obtain TLH-1 and TLH-S fractions, followed by purification on a Superdex-75 gel filtration column (Cytiva, Wilmington, DE, UAS) (2.6 × 50 cm) to isolate the potent antioxidant polysaccharide TLH-3. However, when compared with a salt-precipitation–ultrafiltration strategy, the authors highlighted key limitations of column chromatography, including high separation-media (gel) costs, prolonged processing times (up to 8 days), and restricted sample loading capacity. These limitations emphasize the need to transition towards faster, more efficient and usually automated chromatographic processes, such as flash chromatography. Similarly, Luo et al. [182] purified a white mycelial polysaccharide (PBMP1) from P. baumii through sequential separation on a DEAE-cellulose-52 column (2.6 × 30 cm) followed by a Sephadex G-100 column (2.6 × 60 cm). Applying the same chromatographic sequence, Yang et al. [173] isolated a new polysaccharide (MEP-1) from hot-water extracts of M. esculenta using a DEAE-Cellulose A52 column (2.6 × 30 cm) and a Sephadex G-100 column (1.6 × 100 cm), achieving a yield of 1.58%. In a comparable study, Wang et al. [142] sequentially applied crude P. eryngii polysaccharides (PEP) to a DE-52 cellulose column (1.0 mL/min elution velocity) and a Sephadex G-100 column (0.25 mL/min), yielding a highly purified fraction (PEP-0.1-1) with a sugar content of 99.42%. Polysaccharides isolated from A. mellea and subsequently shown to exhibit significant antidiabetic activity in streptozotocin (STZ)-induced diabetic mice were purified using a DE-52 cellulose column (3.5 cm × 20 cm) [141]. Water-soluble polysaccharides from P. ostreatus were first separated from co-extracted proteins by exploiting electrostatic interactions on a DEAE-Sepharose Fast Flow column (43 × 1.5 cm), followed by further purification using Sephadex G-75 gel filtration chromatography (93 × 1.5 cm) [116].

Beyond polysaccharides, analogous multistep fractionation workflows have been employed for protein isolation from edible mushroom matrices. A bioactive protein (PEP) exhibiting anti-inflammatory activity was isolated from P. eryngii extracts through initial ammonium sulfate precipitation (60–80% saturation), succeeded by purification on a DEAE-Sepharose Fast Flow column, resulting in a product with >95% purity [183]. In another study, Lau et al. [184] employed graded ammonium sulfate precipitation (10–100% saturation) to concentrate proteins from a crude aqueous extract of A. bisporus. Protein precipitates that exhibited the highest ACE inhibitory activity were further fractionated by reversed-phase HPLC (HPLC-RP), and the active fractions were subsequently purified by gel filtration (Biosep SEC-S2000 column (Phenomenex, Torrance, CA, USA); 300 × 7.8 mm), yielding seven fractions with recovery rates ranging from 0.7% to 5.1%. Furthermore, Zhou et al. [185] fractionated Ramaria botrytis extracts by precipitation with 80% (NH₄)₂SO₄, followed by sequential chromatographic purification of the crude protein on a DEAE–Sepharose Fast Flow column (2.0 × 40 cm) and a Superdex 75 10/300 size-exclusion column. This combined fractionation and chromatographic strategy led to the isolation of a ubiquitin-like antitumor protein (RBUP) with a yield of 9.43%.

In parallel with macromolecular purification, similar chromatographic principles have been extended to low-molecular-weight secondary metabolites. For instance, the ethyl acetate extract of Paxillus involutus was subjected to successive chromatographic separations, including silica gel column chromatography (200–300 mesh), octadecylsilane (ODS)-based semi-preparative reversed-phase HPLC (250 × 10 mm), and Sephadex LH-20 column chromatography. These techniques were applied iteratively to isolate rare 2,5-diarylcyclopentenone derivatives and to identify novel compounds designated as involutenones [121]. Similarly, Imam et al. [126] isolated anticancer metabolites, including indole-3-lactic acid, from aqueous L. edodes extracts through sequential purification involving silica gel column chromatography (230–400 mesh) followed by semi-preparative reversed-phase HPLC (C₁₈). In a related study, Béni et al. [186] recovered four secondary metabolites, osmundalactone, 5-hydroxy-hex-2-en-4-olide (lactones), and the terphenyl quinones spiromentins C and B, from Tapinella atrotomentosa by subjecting its chloroform extract to flash silica gel column chromatography and subsequent normal-phase HPLC purification.

Collectively, these studies underscore that sequential fractionation and chromatographic purification are indispensable for obtaining mushroom-derived bioactives at high purity. Such purification workflows (Figure 3) constitute a controlled pipeline that inherently enables standardization, as each step is systematically monitored (e.g., by TLC) and analytically validated to ensure that the isolated compounds meet defined criteria for identity, purity, and bioactivity, thereby enabling the generation of reproducible, pharmaceutical-grade materials.

4.5. Structural Elucidation and Characterization

Following isolation and purification, rigorous structural elucidation and physicochemical characterization are essential for identification and purity validation of bioactive compounds derived from edible mushrooms. Structural characterization not only confirms molecular composition and architecture but also underpins structure–activity relationships (SAR), quality control, and regulatory acceptance in pharmaceutical development [17]. For example, Guo et al. [20] highlighted that the glycosidic linkages, branching degree, and molecular weight of Volvariella polysaccharides critically influence their physicochemical properties and biological activities. Similarly, the biological activities of proteins derived from edible mushrooms are strongly influenced by their molecular weight, amino acid composition, structural conformation, and peptide sequence [187].

Fully elucidated compounds further serve as molecular templates for the rational design or modification of related analogs aimed at optimizing biological performance [171]. Given the extensive chemical diversity of mushroom metabolites, including polysaccharides, proteins, terpenoids, phenolics, alkaloids, and sterols, no single analytical technique is sufficient for comprehensive structural resolution. Consequently, contemporary studies adopt integrated, multi-platform analytical strategies that combine chromatographic, spectroscopic, and spectrometric approaches to achieve unambiguous structural identification (

Table 4. Structural elucidation and analytical characterization of bioactive compounds isolated from edible mushrooms.

Edible Mushroom Species	Identified Bioactive Compound(s)	Chemical Class of Compound	Molecular Weight/Formula	Analytical Techniques Employed	Monosaccharide/Amino Acid Composition	Key Structural Features	Reported Bioactivity	Reference
D. indusiata	NA	Polysaccharides	1534–24,110 kDa	FT-IR, NMR (1H), HPSEC-MALLS	Fuc, Ara, Gal, Glc, Xyl, Man, Fru, GlcA	NMR analysis indicated strong interactions between bound water molecules and polar functional groups on the polysaccharide surface, suggesting a highly hydrated macromolecular structure	Antioxidant activity	[125]
P. involutus	2,5-diarylcyclopentenone derivatives (involutenones A–H)	Phenolic compounds	C18H14O6 (A), C18H14O7 (B), C27H24N2O7 (C), C21H17NO5 (D), C22H19NO7 (E), C19H16O8 (F), C18H14O7 (G), C18H16O7 (H)	NMR (1H and 13C), HRESI-MS	NA	Absolute configurations of selected involutenones were established through comparison of experimental and calculated electronic circular dichroism (ECD) spectra	Antioxidant activities	[121]
B. edulis	KBMPHF 1-4	Peptides	KBMPHF1 (>10 kDa), KBMPHF2 (3–10 kDa), KBMPHF3 (1–3 kDa), and KBMPHF4 (<1 kDa)	LC-MS/MS	Alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine	ACE-inhibitory activity of low-molecular-weight peptide fractions was associated with hydrogen bonding interactions and the presence of hydrophobic cavities	Antioxidant activity, ACE inhibition	[168]
P. ostreatus and G. lucidum	NA	Polysaccharides	NA	NMR (1H and HSQC) and GC–MS	Glc (86%), Man (4.9–9.4%) and Gal (1.1–3.5%)	NMR (HSQC) analyses revealed that all extracts of both mushrooms contained mainly α- and β-glucans and heteropolysaccharides with branched and linear (1→3) and (1→6) linkages	NA	[155]
H. marmoreus	Fucomannogalactan (FMG-Hm)	Hetero-polysaccharide	17.1 kDa	HPSEC-RI, GC–MS, NMR (1H, HSQC-edit, HSQC-TOCSY, HSQC-NOESY)	Fuc, Man, and Gal (1:1.08:3.17)	Structural analyses confirmed a branched fucomannogalactan composed of an α-(1→6)-linked galactopyranosyl backbone partially substituted at O-2 with terminal α-L-fucopyranose and β-D-mannopyranose residues	Antimelanoma property	[112]
A. mellea	AMP	Polysaccharides	NA	FT-IR, HPLC	Man, Rha, Glc, Gal, and Fuc, with varying ratios depending on the extraction method	FT-IR revealed presence of O-H bending vibration, C-H stretching vibration, C = O stretching vibration, and C-H variable angle vibration, as well as presence of pyranose	Antidiabetic activity	[141]
P. eryngii	PEP-0.1-1, PEP-0-1 and PEP-0-2	Polysaccharides	3235 kDa, 2041 kDa, and 23.933 kDa for PEP-0.1-1, PEP-0-1, and PEP-0-2 respectively	UV-vis, FT-IR, HPLC, NMR (1H, 13C NMR, COSY, HSQC, HMBC, NOESY)	PEP-0.1-1 contained Gal and Glc (6.63:93.37); PEP-0-1 contained Fru, Gal, Glc, Xyl, Man, and Fru (0.14:0.73:94.2:0.61:2.79:1.53); PEP-0-2 contained Fru, Gal, Glc, Xyl and Man (0.64:16.62:68.56:0.51:13.67)	FT-IR and multidimensional NMR analyses revealed pyranose ring structures with C–O–C glycosidic linkages; PEP-0.1-1 exhibited a linear (1→4)-linked glucopyranosyl backbone, whereas PEP-0-1 and PEP-0-2 displayed branched (1→4)-Glcp and (1→6)-Galp linkages	Anti-inflammatory activity	[142]
	PEP	Protein	40 kDa	MALDI-TOF-MS	NA	PEP shared partial amino acid sequence homology with the known protein Pleery1 (48 kDa); however, its distinct molecular weight led the authors to classify PEP as a novel protein isolated from P. eryngii	Anti-inflammatory activity	[183]
P. baumii	PBMP1	Polysaccharide	2.95 × 103 kDa	HPLC, GC-MS, FT-IR, NMR (1H, 13C)	Fuc (1%), Glc (24.54%), and Gal (0.29%)	FT-IR and NMR analysis confirmed that PBMP1 is a hetero-polysaccharide with an α-glycosidic linkage and highly branched 1,3,4-Glc and 1,4,6-Glc branches with Fuc as the non-reducing terminus	NA	[182]
P. ostreatus	NA	Polysaccharides	NA	FT-IR, XRD, NMR (1H)	NA	The anomeric bonds identified using FT-IR and NMR analyses indicate that the extracts are a mixture of heteropolysaccharides, β-glucans, α-glucans, and oligosaccharide	Antioxidant activity	[105]
				GC-MS, FT-IR, NMR (1H, 1H COSY and 1H, 13C HMQC)	Glc, Fuc, Rha, Gal, Xyl, Man, Ara	Cold- and hot-water-soluble fractions contained a branched partially methoxylated mannogalactan and slightly branched (1→6)-β -D-glucan, respectively; the DMSO-insoluble polysaccharide was identified as linear (1→3)-α-D-glucan, while the DMSO-soluble one was a branched (1→3)(1→6)- β-D-glucan	NA	[116]
A. campestris, M. procera, C. cibarius, R. vesca, R. alutace, B. edulis, P. ostreatus, and A. bisporus	NA	Phenolic compounds	NA	FT-IR	NA	Spectral data for extracts confirmed the presence of bioactive functional groups including, –OH, >NH, –CHO, –COOH, and –COOR; all extracts exhibited the presence of a broad peak for hydrogen bonded –OH stretching	Antioxidant activity	[115]
C. sinensis	CSF1, CSF2 and CSF3	Nucleosides, polyphenols	NA	HPTLC, GC-MS	NA	HPTLC confirmed the presence of flavonoids (ascorbic acid, gallic acid and quercetin) and nucleobases while GC-MS analysis revealed VOCs (e.g., α-linoleic acid, 9,12-octadecadienoic acid (Z,Z)-, trimethylsilylester)	Antibacterial and hypoxia-protective activities	[152]
L. edodes	LE331	Indole derivatives	C11H11NO3	HPLC, LC-MS, NMR (1H and 13C)	NA	Identification of the most cytotoxic isolate by LC-MS and 1D NMR revealed it to be 2- hydroxy-3-(1H-indol-3-yl) propanoic acid, also known as indole-3-lactic acid (ILA)	Anticancer activity	[126]
T. lobayense	TLH-3 and TLH-3′	Polysaccharides	4.24 kDa and 4.23 kDa for TLH-3 and TLH-3′, respectively	FT-IR, HPLC	Rha, Man, GlcA, GalA, Glc, Gal, Ara	Molecular weights, FT-IR, and monosaccharide composition analysis concludes that TLH-3 and TLH-3 are the same polysaccharide	Antioxidant activity	[177]
R. botrytis	RBUP	Protein	18.5 kDa	ESI-MS/MS	NA	ESI–MS/MS sequencing revealed that RBUP shares 69% amino acid sequence similarity with ubiquitin from Coprinellus congregatus	Hemagglutinating and antitumor activities	[185]
A. cinnamomea, C. versicolor, G. frondosa, G. lucidum, and P. Linteus	NA	Polysaccharides	10.2–722.7 kDa	HPSEC, FT-IR	Fuc, Gal, Glc, Man, Rha, Rib, Xyl, GalA, GlcA	All mushroom extracts contained (1→3;1→6)-β-D-glucans in varying proportions; the degree of branching of (1→3;1→6)-β-D-glucans in all polysaccharides ranged from 0.21 to 0.26; the authors similarly reported the existence of triple-helix structures in the samples	Immunomodulatory activity	[171]
P. pulmonarius	NA	Lipids	NA	GC-FID, GC-MS, HPLC	NA	GC analysis detected 136 chemical constituents, with 9-oxononanoic acid, linoleic acid, oleic acid, stearic acid, and ethyl octadecanoate being the most abundant in all of the extracts; HPLC also confirmed the presence of ergosterol	Antioxidant activity	[153]
L. velutinus	LVP	Polysaccharide	336 kDa	FT-IR, SEC, TLC	Only glucose	FT-IR revealed that LVP is a polysaccharide with sugar ring structures	Anticancer and antioxidant activities	[117]
P. sajor-caju	NA	Phenolic compounds and lipids	NA	GC-MS, LC-ESI-MS/MS	NA	GC-MS and LC-ESI-MS/MS analyses identified compounds belonging to phenols and lipids, with linoleic, chlorogenic and vanillic acids being the major compounds	Antioxidant and antimicrobial activities	[103]

NA—not reported in the cited study; HRESI-MS—High-Resolution Electrospray Ionization Mass Spectrometry; HPSEC-MALLS—High-Performance Size-Exclusion Chromatography coupled with Multi-Angle Laser Light Scattering; HSQC—Heteronuclear Single Quantum Coherence; RI—refractive index detector; TOCSY—Total Correlation Spectroscopy; NOESY—Nuclear Overhauser Effect Spectroscopy; UV-vis—ultraviolet-visible spectroscopy; HMBC—heteronuclear multiple bond correlation; HPTLC—high-performance thin layer chromatography; ESI–MS/MS—electrospray ionization-tandem mass spectrometry; GC-FID—gas chromatography flame ionization detector; kDa—Kilodalton. Monosaccharide notation: Glucose → Glc; Galactose → Gal; Mannose → Man; Fucose → Fuc; Rhamnose → Rha; Xylose → Xyl; Arabinose → Ara; Fructose → Fru; Ribose → Rib; Glucuronic acid → GlcA; Galacturonic acid → GalA.

). As summarized in Table 4, representative studies illustrate the structural features of bioactive compounds isolated from diverse edible mushroom species using these analytical tools.

Chromatographic techniques, particularly HPLC, are routinely employed to assess compound purity and homogeneity, especially for low-molecular-weight constituents such as triterpenoids. Mass spectrometry (MS), often coupled with liquid or gas chromatography (LC–MS, GC–MS), provides accurate molecular mass determination, elemental composition, fragmentation patterns, and insights into substitution and functional group arrangements. This technique operates by ionizing chemical species, followed by separating the resulting ions according to their mass-to-charge (m/z) ratios under vacuum conditions [113]. Coupling MS with chromatographic separation enhances its applicability to complex extracts and enables sensitive detection of bioactive compounds present at low concentrations [188]. Nuclear magnetic resonance (NMR) spectroscopy remains the cornerstone of definitive structural elucidation. By exploiting the magnetic properties of specific atomic nuclei, NMR provides detailed information on molecular connectivity, stereochemistry, and conformational features through one- and two-dimensional experiments. Its capacity to identify functional groups and distinguish between structural and stereochemical isomers makes NMR indispensable for natural product characterization. Nevertheless, its application is constrained by relatively low sensitivity and the requirement for highly purified, homogeneous samples [17,133]. In addition, NMR is resource-intensive, demanding both high investment and dedicated, specialized expertise.

For macromolecular bioactives such as polysaccharides and proteins, which exhibit hierarchical structural organization (primary, secondary, tertiary, and quaternary levels), additional analytical tools are required. These include size-exclusion chromatography (SEC), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), Fourier-transform infrared spectroscopy (FT-IR), circular dichroism (CD), X-ray diffraction (XRD), and amino acid sequencing (Table 4) [19]. For instance, Luo et al. [189] employed Size-Exclusion Chromatography with Multi-Angle Light Scattering and Refractive Index Detection (SEC–MALLS–RI) to identify a G. lucidum polysaccharide (GL-PWQ3) as highly branched with a compact, spherical conformation formed by stacked molecular chains. Similarly, Liu et al. [16], using scanning electron microscopy (SEM) and atomic force microscopy (AFM), demonstrated that branched polysaccharide chains from the same species (RGLP-1) intertwine to form circular aggregates. FT-IR spectroscopy is particularly valuable for rapid functional group identification and for probing molecular interactions and conformational changes relevant to biological activity [188]. Characteristic absorption bands at defined wavenumbers provide insights into structural motifs and bonding patterns [142].

Complementary techniques, including UV–visible (UV-Vis) spectroscopy, and optical rotation measurements, further support compound characterization, stability evaluation, and formulation development. These advanced analytical tools constitute a robust framework for the unambiguous identification and characterization of mushroom-derived bioactives, facilitating their translation from complex natural extracts into chemically defined, reproducible ingredients suitable for pharmaceutical innovation. A critical insight emerging from Table 4 is the recurrent lack of certain information across key characterization parameters, including molecular weight, precise monosaccharide or amino acid composition, definitive structural features, and, in some cases, bioactivity attribution. This pattern highlights substantial knowledge gaps in the comprehensive structural and functional elucidation of many mushroom-derived bioactives, despite their demonstrated biological potential. Notably, several studies report bioactivity without complete molecular definition, while others characterize chemical features without linking them to specific biological functions. These gaps present valuable research opportunities to apply these advanced analytical platforms in a more integrated manner to achieve full structure–activity relationships, standardize bioactive entities, and uncover novel mechanisms of action.

5. Critical Bottlenecks in Pharmaceutical Translation and Innovative Solutions

Despite substantial progress in the extraction, isolation, purification, and structural elucidation of mushroom-derived bioactives, significant hurdles remain before these compounds can achieve clinical and commercial success. Comprehensive molecular characterization in itself does not ensure biological reproducibility, scalable production, or clinical viability. Consequently, the translation of chemically well-defined mushroom bioactives into market-ready pharmaceutical ingredients is constrained by interconnected biological, technological, regulatory, and industrial challenges. This section therefore critically examines the principal bottlenecks limiting pharmaceutical translation and discusses emerging strategies aimed at bridging the gap between molecular characterization and real-world therapeutic application.

A major translational challenge is the poor oral bioavailability of many promising mushroom-derived compounds, particularly high-molecular-weight polysaccharides such as β-glucans. Comparative oral absorption studies indicate that β-glucans (>100 kDa) exhibit low systemic absorption, with reported bioavailability of only 2–5% [190], in contrast to triterpenoids from G. lucidum, which demonstrate moderate absorption levels of approximately 15–25% [191]. Nonetheless, triterpenoids, despite being lipophilic, face the complementary challenge of being rapidly metabolized via cytochrome P450 enzymes-mediated (CYP3A4) pathways and extensively bind to plasma proteins, leading to short effective half-lives of 2–4 h [192]. These pharmacokinetic limitations significantly restrict the efficacy of conventional oral formulations (e.g., tablets and capsules), which remain the preferred route of administration. From an industrial perspective, this necessitates either complex dosing strategies that may compromise patient compliance or substantial investment in advanced drug delivery systems, such as nano-encapsulation [192]. Nano-encapsulation has shown promise in enhancing compound stability and bioavailability; for instance, silver-based nanoparticles (AgNPs/He) synthesized using Hebeloma excedens extracts exhibited improved stability and bioaccessibility of phenolic compounds compared with crude mushroom extracts [193].

Another critical barrier to pharmaceutical translation is the lack of standardization and robust quality control frameworks, which are essential for the development of pharmaceutical-grade therapeutics. The absence of standardized practices severely limits reproducibility and impedes regulatory approval [192]. These challenges span the entire production pipeline, from variability in mushroom raw materials to inconsistencies in extraction and purification protocols. Biological heterogeneity in mushroom sourcing results in variable starting materials, while non-standardized extraction processes can lead to multi-fold differences in commercial products’ bioactive content. Furthermore, the high cost and limited availability of validated analytical methods constrain effective batch-to-batch consistency assessment for complex mushroom biomolecules [192,194]. Consequently, mushroom-based pharmaceutical products lacking rigorous quality control are prone to inconsistent therapeutic performance.

An additional complexity arises in defining the “active pharmaceutical ingredient” (API) for mushroom-derived preparations, particularly when bioactivity results from synergistic interactions among multiple constituents rather than a single compound [195]. Addressing this challenge requires the implementation of controlled cultivation systems compliant with Good Manufacturing Practice (GMP), coupled with genetic authentication strategies such as DNA barcoding to ensure species-level accuracy [196]. The implementation of GMP is complicated by biological variability arising from species differences, cultivation substrates, growth conditions, and post-harvest processing. These factors directly affect metabolite profiles and bioactivity, making batch-to-batch consistency particularly challenging for polysaccharides and crude extracts [194]. Given that toxicological evaluation in accordance with the guidelines of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) is a critical step for pushing lead candidates toward pharmaceutical registration, impurity profiling constitutes a major regulatory hurdle [192]. Mushroom-derived products may contain residual proteins, endotoxins, heavy metals, pesticides, mycotoxins, or co-extracted secondary metabolites, all of which complicate toxicological characterization and safety assessment. Advanced analytical platforms, including LC–MS/MS fingerprinting combined with chemometric analyses, offer robust solutions for quality assurance and standardization; however, their widespread industrial adoption remains limited by high operational costs [192]. Another major challenge lies in the limited understanding of the mechanisms of action of many mushroom-derived compounds. Clear elucidation of the molecular pathways and signaling targets, through which these bioactive compounds interact with biological systems, is essential for rational drug design, dose optimization, and eventual regulatory approval [52].

More critically, the evidence base is limited by a scarcity of well-designed, randomized controlled human trials capable of defining safe and effective dosage ranges. Current knowledge therefore relies heavily on simplified in vitro systems and preclinical animal models, which often fail to adequately capture human pharmacokinetics, immune system complexity, and disease heterogeneity [5,192]. This translational gap arises from a convergence of aforementioned factors, including intrinsic pharmacokinetic constraints, poor bioavailability, model-dependent overestimation of efficacy, and the inherent difficulty of translating complex, multi-component mushroom extracts into standardized, regulatorily acceptable pharmaceutical entities. Collectively, these limitations help explain why robust preclinical findings have not consistently yielded clinical success. Nonetheless, the translational landscape is evolving, and mushroom-derived therapeutics are increasingly progressing toward clinical evaluation, with several edible mushroom-based formulations already assessed for bioactivity in phase I–III clinical trials (Table S1).

Finally, regulatory ambiguity remains a major barrier to the pharmaceutical development of mushroom-derived bioactives. Compounds intended for drug development must satisfy stringent and costly requirements, including extensive toxicological testing and human clinical evaluation, before qualification as Investigational New Drugs (INDs), in contrast to the less demanding regulatory pathways applied to dietary supplements. When marketed as dietary supplements, mushroom products are subject to relatively permissive regulatory frameworks that emphasize safety and labeling rather than demonstrated clinical efficacy. This burden, in combination with limited intellectual property protection for minimally modified natural compounds, frequently deters sustained industrial investment [196]. This challenge is further amplified by marked differences between European and American regulatory frameworks. In the European Union, the European Food Safety Authority (EFSA) applies a precautionary regulatory approach, particularly under the novel food and botanicals framework, which demands comprehensive compositional, toxicological, and human safety data, even for materials with a documented history of dietary use [197]. This process is often protracted and inflexible, especially for complex mushroom extracts whose bioactivity may arise from synergistic interactions rather than a single, well-defined API. By contrast, the United States Food and Drug Administration (FDA) provides a more clearly defined translational route through its Botanical Drug Development Guidance. This framework explicitly accommodates complex natural products, allowing progression as INDs provided batch consistency, chemical fingerprinting, and clinical safety are adequately demonstrated [197,198]. Although the FDA pathway still imposes rigorous quality and efficacy standards, its explicit recognition of “botanical drugs” category offers greater regulatory clarity and predictability [196].

Collectively, this regulatory asymmetry contributes to translational uncertainty and may help explain why mushroom-derived therapeutics more frequently advance through clinical development pipelines in the United States than in Europe. For instance, a comparative analysis by Calin et al. [199] showed that the US has conducted a markedly greater number of psilocybin (a tryptamine alkaloid derived from various mushroom genera) clinical trials, spanning both early- and late-stage development, than the EU. Addressing this imbalance will require greater harmonization and cross-recognition between regulatory frameworks, particularly through the development of clearer, internationally aligned pathways for complex natural products [197]. Establishing shared standards for quality control, safety evaluation, and characterization of multi-component extracts could reduce translational uncertainty, enhance global investment confidence, and facilitate more equitable advancement of mushroom-derived therapeutics across jurisdictions while maintaining rigorous safety and efficacy benchmarks.

6. Conclusions and Future Perspectives

Edible mushrooms represent a chemically rich and biologically versatile resource with considerable promise for pharmaceutical innovation. This review emphasizes that their therapeutic potential extends well beyond traditional nutraceutical applications, supported by a diverse repertoire of bioactive compounds with immunomodulatory, anticancer, antioxidant, anti-inflammatory, and cardioprotective properties. However, successful pharmaceutical translation requires a paradigm shift from bioactivity-driven discovery toward process-integrated development. A central conclusion of this review is that molecular characterization alone is insufficient to ensure clinical and industrial viability. Instead, reproducible biomass production, optimized extraction and purification workflows, rigorous structural elucidation, and robust quality control must be addressed as interconnected components of a unified development pipeline. The profound disconnect between strong preclinical evidence and limited clinical success does not reflect a lack of therapeutic promise, but rather unresolved challenges related to bioavailability, pharmacokinetics, model predictivity, standardization, and regulatory alignment. These constraints highlight the need for rational prioritization of mushroom-derived compounds based on criteria that extend beyond biological activity to include chemical definability, manufacturability, safety margins, and regulatory compatibility. Without such criteria, promising candidates remain difficult to advance as pharmaceutical-grade entities. The future of mushroom-based pharmaceuticals hinges on embracing interdisciplinary, innovative strategies. In addition to already outlined approaches, key research and development priorities include:

• Integration of novel drug delivery strategies, including nano-formulations and encapsulation technologies, to protect compounds from degradation and enhance their targeted delivery.
• Application of synthetic biology and metabolic engineering to sustainably produce high-value mushroom bioactives such as cordycepin, ergothioneine, and specific triterpenoids in yeasts or other microbial hosts using recombinant plasmids, thereby directly addressing sourcing and standardization challenges.
• Deployment of omics technologies (genomics, transcriptomics, metabolomics) to identify novel compounds, refine structure–activity relationships and better unravel intricate molecular mechanisms associated with biosynthesis.

Overall, edible mushrooms occupy a strategic intersection between natural product chemistry and modern pharmaceutical science. With coordinated advances in biotechnology, analytical chemistry, and regulatory science, mushroom-derived bioactives are well positioned to contribute meaningfully to the next generation of natural-based pharmaceutical therapeutics.