Bioactive Compounds Produced by Macromycetes for Application in the Pharmaceutical Sector: Patents and Products

: It is widely known that mushrooms present several properties with applications in the medicinal and pharmaceutical sectors, including antimicrobial, immunomodulatory, antioxidant, hypotensive, neuroprotective, and anti-inflammatory activities. This article aims to review examples of the bioactive metabolites responsible for those activities, such as polysaccharides, phenols and polyphenols, terpenes, peptides, alkaloids, and steroids, which are produced by several relevant mushroom species. It also discusses their production through solid-state fermentation and submerged fermentation, as well as the processes of obtention of mushroom bioactive extracts and considerations on their stability aiming industrial applications. In addition, the article examines the patent landscape surrounding mushroom-derived bioactives, shedding light on the intellectual property history and innovations driving this field forward. Examples of recently deposited patents in the field are highlighted, as well as the main depositors. China and the United States are the major depositor countries in this field (52% and 35% of patents, respectively), and the principal compounds on the patents are polysaccharides and alkaloids. The article also provides insights into the current market landscape, showcasing mushroom-derived products in the pharmaceutical field available to consumers. From dietary supplements to skincare formulations, the market offerings reflect the growing interest in harnessing the health benefits of mushroom bioactives.


Introduction
Bioactive compounds are beneficial to human health due to their ability to positively influence various physiological processes.The health conditions that mostly affect the world population nowadays, namely cardiovascular disease, cancer, and COVID-19 (FastStats-Leading Causes of Death (cdc.gov)), can be prevented or their effects can be minimized by the use of bioactive molecules along with conventional treatments.Antioxidant compounds, for example, neutralize free radicals in the body and reduce oxidative stress, thus helping to prevent cell damage and lowering the risk of chronic diseases like cancer and neurodegenerative disorders.There are also bioactive molecules that can directly inhibit cancer cell proliferation, induce apoptosis, and modulate signaling pathways involved in cancer development [1,2].Anti-inflammatory compounds can prevent inflammation through different routes, like the inhibition of pro-inflammatory pathways by blocking enzymes such as cyclooxygenase and lipoxygenase, or by the downregulation of the expression of pro-inflammatory cytokines.This can be beneficial to individuals suffering from inflammation of various causes.Inflammation can be a chronic condition caused by environmental stress, inflammatory diets, autoimmune disorders, and infections, among other causes.Even when the causes are multiple, complementary treatments with bioactive compounds can be of great value to improve life quality [3].
Macromycetes, commonly known as mushrooms, are important sources of bioactive compounds used in traditional medicine and nutrition for thousands of years [4].They are low in calories and fat but rich in nutrients, containing essential vitamins and minerals such as vitamin D, vitamin B complex (riboflavin, niacin, and pantothenic acid), potassium, selenium, and copper.Some varieties of mushrooms also provide protein and dietary fiber, making them an important source of alternative proteins for human nutrition [3].They contain antioxidants and immunomodulators such as polyphenols, flavonoids, beta-glucans, and polysaccharides.Compounds like beta-glucans and lovastatin, found especially in oyster mushrooms, may help lower LDL cholesterol levels and improve lipid profiles, reducing the risk of cardiovascular disease.Their fiber content can be a source of prebiotics for the beneficial gut microbiota.Additionally, mushrooms can present antimicrobial activity against various microorganisms, including bacteria, viruses, and fungi [5].This antimicrobial activity is attributed to the presence of bioactive compounds such as polysaccharides, peptides, terpenoids, phenolic compounds, and lectins found in different mushroom species [4].
This manuscript presents an overview of macrofungi as sources of bioactive compounds, focusing on the health benefits of polysaccharides, including glucans, glycans, and exopolysaccharides, peptides, phenols and polyphenols, terpenes, alkaloids, and sterols.Studies demonstrating their in vitro and in vivo effects and mechanisms of action are presented, together with methods to obtain such biomolecules from fungal biomass.A patent search was also carried out in the Derwent Innovations Index database, focusing on the application of macromycetes and their bioactive compounds in the pharmaceutical area.Finally, commercial products derived from mushrooms and their claimed properties are presented, showing the potential of fungal bioactive metabolites in various segments of the pharmaceutical market.

Macromycetes as Sources of Bioactive Compounds
Macromycetes are superior fungi that, under certain conditions, are capable of forming visible structures.They are divided into two phyla-Ascomycota and Basidiomycota [6].The first contains mycelia with septa and generally their cells exist as diploids, while the second is characterized by a dolipore septum with pincer connections.Macromycetes are eukaryotic and heterotrophic organisms with chitin-based cell walls.It is estimated that there are between 2.2 and 3.8 million fungi, of which around 120,000 have been identified and around 2000 have been classified as edible or medicinal [7,8].
The therapeutic properties of mushrooms are mainly due to the wide range of bioactive compounds that they produce or synthesize in their metabolism, either in the fruiting bodies when the biomass is produced by solid fermentation, or in the mycelium or fermentation broth when the biomass is produced via submerged fermentation.Among the metabolites produced by mushrooms are high-molecular-weight compounds, such as carbohydrates, proteins (peptides), and lipids in low proportions.While low-molecular-weight metabolites are terpenes, phenols, sterols, and alkaloids among others (Figure 1).entation 2024, 10, x FOR PEER REVIEW 3 of metabolites produced by mushrooms are high-molecular-weight compounds, such as c bohydrates, proteins (peptides), and lipids in low proportions.While low-molecul weight metabolites are terpenes, phenols, sterols, and alkaloids among others (

Polysaccharides from Mushrooms
Polysaccharides are carbohydrate polymers composed of more than one sug which can be either a single type of monosaccharide (homopolysaccharide) or at least tw different types of monosaccharides (heteropolysaccharide) [11].In macromycete fun polysaccharides play an important role as they can be structural components, as in t case of chitin at the cell wall level; or functional, such as exopolysaccharides (EPS) a glycans, which in turn can form complexes with proteins or lipids [12].

Polysaccharides from Mushrooms
Polysaccharides are carbohydrate polymers composed of more than one sugar, which can be either a single type of monosaccharide (homopolysaccharide) or at least two different types of monosaccharides (heteropolysaccharide) [11].In macromycete fungi, polysaccharides play an important role as they can be structural components, as in the case of chitin at the cell wall level; or functional, such as exopolysaccharides (EPS) and glycans, which in turn can form complexes with proteins or lipids [12].

Glucans
Glucans are the most abundant polysaccharides in macromycete fungi and make up the majority of their cell wall, representing approximately 50-60% of their dry weight mass [16].These compounds are mainly composed of glucosyl residues that can be substituted by a wide variety of glycosyl residues.They can also be classified into α-glucans, β-glucans, and α-β-glucans, presenting a homopolymeric or heteropolymeric structure of various chain lengths, whose structural diversity depends directly on the species of fungus [17].
The biological synthesis of these compounds begins with the synthesis of nucleotide sugars at the cytoplasmic level which are transferred in sequence to a lipid carrier to form assembled repeating units and continue with their subsequent export through the polysaccharide or Wzx/Wzy-dependent pathway [18].
These compounds have several functions, mainly structural, and have been studied as antioxidants, immunoregulators and antitumor agents [19], especially those of the βglucan type, which have been the subject of study because of these bioactive properties.

Glucans
Glucans are the most abundant polysaccharides in macromycete fungi and make up the majority of their cell wall, representing approximately 50-60% of their dry weight mass [16].These compounds are mainly composed of glucosyl residues that can be substituted by a wide variety of glycosyl residues.They can also be classified into α-glucans, β-glucans, and α-β-glucans, presenting a homopolymeric or heteropolymeric structure of various chain lengths, whose structural diversity depends directly on the species of fungus [17].
The biological synthesis of these compounds begins with the synthesis of nucleotide sugars at the cytoplasmic level which are transferred in sequence to a lipid carrier to form assembled repeating units and continue with their subsequent export through the polysaccharide or Wzx/Wzy-dependent pathway [18].
These compounds have several functions, mainly structural, and have been studied as antioxidants, immunoregulators and antitumor agents [19], especially those of the β-glucan type, which have been the subject of study because of these bioactive properties.
Schizophyllan is a water-insoluble nonionic homopolysaccharide composed of a primary β-(1,3)-glucan structure with β-(1,6)-glucopyranosyl side chains on every third or fourth residue of the main chain.This fungal polymer has been noted due to its great capacity as an antitumor and immunobiological agent since [28] first discovered that an aqueous solution of this compound exhibited antitumor activity against sarcoma 180.Schizophyllan was found to activate NK cells, spleen cells, lymphoid cells, and bone marrow cells and increase the production of antitumor cytokines [29].Recent studies such as that of [30] showed a gene silencing effect by the formation of a schizophyllan-mRNA complex, resulting in the suppression of the growth of human lung adenocarcinoma cells.Likewise, the combination of technologies such as the use of nanoparticles for the formation of complexes is being investigated, as in the case of schizophyllan-qutine encapsulated with ellagic acid in the treatment of breast adenocarcinoma in MCF-7 cells, generating a cytotoxic effect [31,32].
Another example of β-glucan is lentinan, which is composed of a main structure of β-(1-3)-glucopyranoside with two branches of β-(1-6)-glucopyranoside for every five positions [33].Extracted from the fungus known as shiitake, this polymer has presented several applications as a bioactive product over the years.Among these, its anticancer and immunomodulatory activity stands out, as evidenced by the activation of macrophages and dendritic cells and increased activity of cytotoxic T lymphocytes and NK cells [34][35][36].In more recent trials, Park et al. [37] confirmed the anticancer potential of lentinan in colon and gastric cancer cells, slowing their proliferation in AGS cells and HT-29 cells by inhibiting the G2/M phase in the cell cycle.In addition to this property, its potential in wound healing, such as those developed in patients affected by diabetes, has also been investigated.Jia et al. [38] developed a hydrogel (GelMA) encapsulated with 10 µM of lentinan, which accelerated wound closure by increasing neovascularization and M2 macrophage proliferation, resulting in a reduction of inflammation.This report strengthens the ability of lentinan to modulate macrophage polarization and angiogenesis through the AMPK/DAF16 pathway, facilitating wound healing.

α-Glucans
Alpha-glucans are another group of linear or branched polysaccharides, which may be linked by α-D-(1-3), α-D-(1-4) or α-D-(1-6) bonds [19].Pseudonigeran is one of the most studied polymers in this group because it is the most prevalent component of the fungal cell wall; however, there is very limited information about its use as a pharmaceutical biocompound [39].Pseudonigeran is a linear homopolysaccharide linked by α-D-(1-3) bonds.It is a structural component, unlike β-glucans, which are produced by a wide diversity of species.These include Cyclocybe cylindracea, Ganoderma spp., Lentinula edodes, Pleurotus spp.and others.Recent studies have demonstrated that pseudonigeran possesses a high prebiotic potential, as it can stimulate the growth of Lactobacillus and Bifidobacterium, two microbial groups of significant importance to the native intestinal microbiota [40].
Other α-D-glucans isolated from macromycete fungi have been isolated, but their study has been limited.For instance, Patra et al. [41] isolated an α-(1,3)- (1,4) glucan from the edible mushroom Favolus grammocephalus (formerly Polyporus gramocephalus), which demonstrated the activation of macrophages, splenocytes, and thymocytes.This glucan thus has the potential to serve as an immunomodulatory agent in the development of drugs to treat pathological conditions associated with diseases such as AIDS, cancer, and autoimmune disorders.A type of α-(1,4)-D-glucan isolated from the mushroom Albatrellus ovinus has also been reported [42], although it has also been isolated from the mushrooms Pleurotus ostreatus and Agaricus bisporus [43,44].Its properties are poorly understood and have yet to be studied.

Glycans
Glycans are another type of more complex polysaccharides found in macromycetes, which are characterized by being composed of units other than glucose in its main chain and are classified according to the type of sugar that composes it, such as galactans, fucans, xylans, mannans or others [12].
A number of fungal species are known to produce glycans, with α-D-galactan (GAL-Am) being isolated from Amanita muscaria.This presented a structure composed of a main chain of galactan (Galp) linked by α- (1)(2)(3)(4)(5)(6) bonds and branched at O-2 by terminal Galp units.Moreover, it was demonstrated that this compound exhibited a selective reduction in B16-F10 melanoma cells and their clonogenic capacity, without affecting non-tumor cells.This may prove to be a crucial feature in the treatment of cancer [2].Similarly, more complex glycans formed by the combination of multiple sugars have been described.Michorini et al. [45] isolated a fucogalactan from the giant fungus Macrocybe titans.This glycan exhibited a primary α-(1-6)-Galp chain, partially substituted at O-2 by non-reducing terminal units of Fucp residues (Fucan) in the side chain.Additionally, it demonstrated the capacity to reduce melanoma cell migration by 40% (100 µg/mL) through the reduction of cellular activity.
An additional report is that of Chen et al. [48], which presents a GFP-N glycan isolated from the maitake mushroom Grifola frondosa.This glycan is composed of L-arabinose, Dmannose, and D-glucose, with a structure consisting of α-(2,6)-D-Manp, α-(1,4)-L-Araf-C1, and β-(3,6)-D-Glcp.This polymer demonstrated the capacity to activate insulin receptor substrate, phosphatidylinositol-3-kinase, and glucose transporter 4, while inhibiting c-Jun N-terminal kinase 1/2.These findings suggest that this compound may have potential as a hypoglycemic agent at the level of intestinal microflora in vivo in mouse assays.

Exopolysaccharides
Exopolysaccharides (EPSs) represent another type of polysaccharides that are synthesized and secreted to the outside of the cell.These EPSs may be attached to the cell surface or may remain in the extracellular medium.Likewise, their chemical structure can be composed of simple sugars or sugar derivatives [14].
EPSs exhibit a remarkable degree of diversity, varying according to the species of macromycetes.Additionally, their quality and quantity are influenced by external conditions, such as environmental conditions or the micronutrients supplied during their growth [19].In this context, EPSs are presented as a promising alternative for their application in various fields of bioproducts, with a particular emphasis on the pharmaceutical sector and clinical therapy [49,50].This has also led to a surge in research activity over the past decade, as evidenced by a simple search in the ScienceDirect database using the keywords "exopolysaccharides" and "mushrooms".
Sonawane et al. [51] evaluated the hypoglycemic and anticataract activity of a solution of crude EPSs isolated from Phellinotus badius (formerly Phellinus badius) in streptozotocin (STZ)-induced diabetic rats.The EPS solution demonstrated a reduction in blood glucose (37.2%) and body weight gain (47.4%), as well as a reduction in triglycerides, cholesterol and aspartate aminotransferase activities.Additionally, the EPS solution reduced the risk of cataract formation in goat eyes.Other studies have demonstrated a significant hepatoprotective and antioxidant capacity against diethylnitrosamine (DEN)/carbon tetrachloride (CCL 4) induced damage in rats by an exopolysaccharide-peptide complex extracted from Pleurotus ostreatus [52].Similarly, its antioxidant activity has been demonstrated through radical scavenging, reducing capacity and transition metal catalysis chelation in ex-opolysaccharides extracted from the medicinal mushroom Ganoderma sichuanense (formerly Ganoderma lingzhi) [1].

Peptides from Mushrooms
Macrofungi are rich in proteins and can be notable sources of natural bioactive peptides, which can be extracted from their mushrooms or mycelia, whether fresh, dried, frozen and/or freeze-dried.These peptides, called MBPs (Mushroom Bioactive Peptides), are small fragments (2 to ~50 amino acids) produced endogenously or isolated from macrofungal proteins, and which have several beneficial effects on the physiology and health of other living beings, including humans [3].
Peptides are composed of at least two amino acids in which the carboxyl group of the first amino acid connects to the amino group of the next amino acid in the chain through a covalent bond called, in this case, a peptide bond.Sequence-specific polypeptides with more than ~50 residues are called proteins, although there are discrepancies in the literature about when to start adopting this term [53].The bioactive activities of MBPs appear to depend on the composition, structure and sequence of amino acids (especially in relation to their acid-base behavior, determined by free α-amino and α-carboxylic groups and side groups), the length of the amino acid chain (which reflects in its molecular weight), and its electrical charge [54].
Due to their small size, bioactive peptides can be entirely absorbed by the intestine and, consequently, by the bloodstream, being distributed to the body's systems in an intact form, which expands their benefits in various organs and physiological activities [55].Furthermore, bioactive peptides have great morphofunctional stability, tissue-specific affinities, low toxicity and high pharmacological capacity.All these characteristics make these biomolecules excellent candidates for the composition of functional foods and medicines [5].
Proteins can encode and generate a wide variety of bioactive peptides, which can be released without losing their bioactive properties.In fact, peptides from proteins seem to have enhanced bioactivity compared to the properties of whole proteins, mainly because their smaller size increases their ability to cross biological barriers, such as cell membranes, and then be absorbed by cells [56].
There are two main procedures for obtaining MBPs.The first is based on the extraction of endogenous bioactive peptides from macromycetes, which have an intrinsic origin and are synthesized through specific pathways, generally in response to environmental conditions.Their biological structures and functions are generally species-specific and vary depending on the environment in which they live [3].The second method is based on obtaining bioactive peptides that are encoded in the proteins they constitute, forming these macromolecules.These peptides can be released through lysis using peptidases, in processes such as enzymatic hydrolysis, or using basic or acidic solutions, a process called chemical hydrolysis [57][58][59].
The first step in extracting peptides from fungi is to disrupt the cellular structure, especially the cell wall, which is generally accomplished through maceration of the fruiting body and/or mycelium, with an aqueous diluent or lysis buffer.After preliminary bioactivity tests, which can be carried out with enzymatic tests, peptide purification techniques are then carried out via ultrafiltration and/or chromatographic methods based on the physicochemical characteristics of these biomolecules, such as isoelectric point, polarity, molecular mass, solubility, interactions, etc. [60].After purification, identification and characterization of the peptides can be carried out using spectrometric and electrophoretic techniques, for example [61].
The obtaining of MBPs is still largely unexplored and, therefore, knowledge about their bioactive and pharmaceutical properties and obtaining and purification methodologies is still scarce.However, the literature reports provide data supporting several benefits of MBPs that corroborate their pharmaceutical potential, as described below.
For example, there are MBPs that inhibit the angiotensin-converting enzyme (ACE), the central component of the renin-angiotensin system, which makes it possible to control blood pressure by regulating the volume of fluids in the body.ACE converts angiotensin I to angiotensin II, a vessel constrictor.In other words, these ACE-inhibiting peptides can be used to control, prevent and/or reduce high blood pressure, replacing drugs that have several undesirable side effects.The action mechanisms of MPBs in inhibiting ACE are not totally unveiled.They appear to act mainly by inhibiting the synthesis of nucleic acids and proteins involved in ACE activation [62].The fungi Grifola frondosa, Agaricus bisporus, Pleurotus cornucopiae and Pleurotus cystidiosus are macromycetes known to inhibit ACE, with great pharmaceutical potential for the treatment of hypertension [63][64][65][66].
An analysis carried out by Landy et al. [54] regarding data on antihypertensive MBPs suggests that ACE inhibitors have short chains (3 to 17 amino acid residues), with a molecular weight between 301 and 2037.26Da.These data suggest that MBPs could also inhibit ACE in a direct way, by binding themselves to active sites of the enzyme, and that larger peptides may have less capacity to bind to active sites from ACE.Furthermore, most of them present lysine and/or proline in their C-terminal part, which indicates that MBPs with these amino acid residues are more effective in inhibiting ACE.The authors' analysis also suggests that there is a prevalence of hydrophilic characteristics in these peptides and that pentameric and hexameric forms are the most abundant, with their isoelectric points varying from 5.52 to 9.75 [54].
Some MBPs also have antioxidant activity, that is, they help the endogenous antioxidant system to remove free radicals that cause cellular oxidative stress, which can lead to premature aging and severe health problems, such as cancer and degenerative diseases.Most of these peptides can act in limiting the production of reactive species (donating protons, electrons and chelating metals), and in modulating enzymes and pathways of the antioxidant system [67].The majority of this class of MBPs appears to consist of hydrophobic amino acids, which facilitate cellular uptake into different organs through cell membranes, and negatively charged amino acids, which enable the donation of electrons to free radicals, functioning as scavengers and, consequently, inactivating them.Benzene rings of some aromatic amino acids of MBPs (e.g., Phe, Met, Tyr, Lys, Trp, Cys and His) have the ability to stabilize reactive radicals by donating electrons to these molecules and at the same time can maintain their own electrical stability [5].
The basidiomycete Ganoderma lucidum has very efficient antioxidant properties.A bioactive peptide fraction, called GLP (from Ganoderma lucidum peptide), isolated from this fungus appears to act in different ways in the antioxidant system, increasing the levels of glutathione (GSH) and the enzyme superoxide dismutase (SOD), leading to a decrease in lipid peroxidation in the liver, which in turn inhibits the formation of malondialdehyde (MDA), which can trigger a series of inflammatory processes [68][69][70].
Macromycetes also synthesize proteins and peptides with antimicrobial action, endogenously, to combat possible fungal, viral and bacterial infections originating from the environment.Some of these peptides can function as very specific and effective natural antibiotics, with low toxicity compared to synthetic ones.Its antimicrobial properties are possibly due to the amphipathic and cationic characteristics of its structure, which enable it to bind to the negatively charged membranes of microorganisms [71].Antimicrobial MBPs appear to act in different pathways, such as inhibiting the synthesis of nucleic acids, inhibiting protein synthesis, in pathways that cause hyperpermeabilization of biological membranes, inhibiting the activity of some important enzymes and activating apoptotic pathways, processes that lead to death or unviability of microorganisms [72].
Some antibacterial MBPs appear to be positively charged, which, when combined with the negative charges of chemokine receptors on the surface of bacterial cells, cause their depolarization through electrostatic interactions, leading to cell rupture and, consequently, death of the microorganism [56].This mechanism hinders the development of so-called bacterial resistance to antibiotics, which increases their pharmaceutical potential [3].
Plectasin is a bioactive peptide produced by Pseudoplectania nigrella, which has very specific bactericidal properties, without causing toxicity to eukaryotic cells infected by bacteria.Its potential for treating bacterial infections of the nervous system has been studied, especially as it is highly absorbable by cerebrospinal fluid [73].
Some MBPs also have antitumor and immunostimulating properties, causing a significant increase in the production of macrophages and phagocytosis processes that inhibit the growth of various types of cancer (e.g., breast, leukemia) and help fight infections caused by pathogens [74,75].MBPs can increase the cytotoxicity of immune system defense cells, the phagocytic capacity of macrophages and the production of immune cells, and can enhance the activation of immune system pathways and the production of antibodies, thus improving the body's defense against pathogens and inhibiting inflammatory responses [76].Some peptides have been shown to be cytotoxic in treatments against tumor cell lines, reduce cell proliferation rates in mitochondrial pathways and promote expression of tumorigenesis-suppressing caspases in certain types of cancer.These characteristics make them powerful candidates for use as adjuvant agents in conventional treatments (e.g., immunoconjugates or linked to chemotherapeutic agents) or even as alternative medicines to aggressive anti-tumor treatments [54,56].
Pleurotus ostreatus produces two peptides with very promising pharmaceutical characteristics Pleurotus ostreatus peptide (POP) and pleurostrin.POP inhibits protein synthesis and pleurostrin appears to have antifungal properties, and, together, they act to inhibit the growth of fungi, in addition to reducing cell proliferation in some cancer types [77,78].
Furthermore, there are MBPs capable of chelating metals due to residues of some specific amino acids (e.g., Cys, His, Gln), which have the property of chelating divalent mineral metals, such as zinc and calcium.These properties can be exploited to increase the bioavailability of minerals in the body.Other MBPs may also act like analgesics or opioids, improve memory and slow cognitive deterioration in nerve cells [79].
Other MBPs have shown other potential bioactivities, such as antithrombotic activities by inhibiting platelet aggregation, antidiabetic activities by inhibiting α-glucosidase and α-amylase (reducing plasma glucose) and antihypercholesterolemic activities (inhibiting enzymes involved in cholesterol biosynthesis and decreasing the synthesis and secretion of triglycerides and cholesterol through increased bile secretion, modulation of hormone and cholesterol receptors, and increasing hepatic lipid metabolism) [80,81].Some examples of MBPs and their pharmaceutical potential that have demonstrated promising results in studies are shown in Table 1.

Phenols and Polyphenols from Mushrooms
Phenols and polyphenols are organic compounds found abundantly in nature, particularly in plants and certain macromycetes.In those organisms, phenols and polyphenols act on various biological activities and physiological functions.They are involved in processes such as defense mechanisms against pathogens and predators, regulation of growth and development, and adaptation to environmental stressors.In macromycetes, those compounds can inhibit the growth of bacteria and other microorganisms, thus protecting the mushroom from diseases, predation and decay.However, the action of phenols and polyphenols is not only limited to those benefits for the fungi but is also extended to biological activities of interest in human health [91][92][93].
Phenolic compounds are characterized by the presence of at least one aromatic ring (C 6 ) and one or more hydroxyl (OH) groups in the molecule.The structures of these compounds can extend from simple molecules to complicated polymers [92,94].The total phenolic content of medicinal mushrooms is around 6.0 mg per gram of dried matter, with phenolic acids, flavonoid compounds, tannins, and tocopherols being the principal classes.Even if many different compounds are identified inside those classes, the content of phenolic compounds in mushroom species has not been as precisely characterized as in the case of plants, especially concerning phenolic acids [92].
The most prevalent phenolic acids discovered in mushroom species include p-coumaric, cinnamic, p-hydroxybenzoic, benzoic, ferulic, and gallic acids.Phenolic acids are divided into two principal classes, benzoic acid derivatives (with a C1-C6 backbone) and cinnamic acid derivatives (with a C3-C6 backbone).In the study by Çayan et al., 26 fungal species were analyzed concerning the phenolic acid compounds, and fumaric acid was found to be the most abundant in 16 out of 26 mushrooms.Catechin hydrate, a polyphenol, was identified as the major phenolic compound in the other species.Higher phenol content was identified on the mushroom species Suillus granulatus (71.79 µg/g) and Lepista nuda (currently Collybia nuda) (68.38 µg/g) [95].
The class of polyphenols known as flavonoids can also be present on mushrooms, at quantities of up to 3.0 mg per gram of dried matter.Homogentisic acid, benzoic acid (4-OH-benzoic, vanillic, protocatechuic, salicylic) and cinnamic acid derivatives (caffeic, ferulic, p-coumaric, myricetin and catechin) are some of the principal flavonoids reported in mushroom species [96].However, some researchers affirm that edible mushrooms lack the enzymes necessary for synthesizing flavonoids, and studies have not found significant flavonoid absorption in cultivated fruiting bodies or mycelia grown in flavonoid-rich environments.Flavonoids detected in mushrooms could arise from sample contaminations or procedural issues, and at the moment there is still no consensus about this topic.Employing appropriate analytical methods for flavonoid detection can further help the understanding of flavonoid synthesis in mushroom cells [97,98].
Tannins are other significant secondary mushroom metabolites, and are categorized into hydrolyzable and condensed types.Hydrolyzable tannins consist of esters of gallic acid or ellagic acid with glucose, while condensed tannins are polymers of flavan-3-ols or flavan-3,4-diols.The structures of hydrolyzable tannins vary due to the different oxidative bonds formed through intermolecular oxidation reactions, typically appearing as oligomers with a molecular weight of 2000-5000 Daltons.Condensed tannins, also referred to as procyanidins, can be converted into anthocyanins through acid-catalyzed reactions.Tannin contents from mushroom samples from the Pleurotus genus are highly reported, being in the range of 36 to 40 mg/g.Those substances can accelerate wound healing, aid in cancer prevention, and assist in treating inflamed mucous membranes and ulcerated tissues [99,100].
The biological activities of mushroom phenolic compounds are wide ranging, including antioxidant, antitumor, anti-inflammatory, antihyperglycemic, antimicrobial, antiosteoporotic and anti-tyrosinase activities [92].Reactive oxygen species (ROS) are key catalysts for oxidation in vivo and in vitro, associated with numerous diseases and aging processes.Excessive ROS accumulation damages lipids, proteins, carbohydrates, and DNA, inducing oxidative stress, cell dysfunction, and cell death.These processes are prevalent in various diseases like cancer, inflammation, atherosclerosis, and cardiovascular issues.Phenols and polyphenols have been demonstrated to have antioxidant potentials, neutralizing ROS, through different pathways, including the inactivation of metals, acting as oxygen scavengers, or inhibiting free radicals [101].
In a study by Guo et al., a positive linear correlation between total antioxidant capacities and total phenolic contents was observed in 49 edible macro-fungi.Macro-fungi like Thelephora ganbajun, Boletus edulis, Volvariella volvacea, Butyriboletus regius (formerly Boletus regius), Suillus bovinus and Agaricus blazei display high antioxidant capacities and total phenolic contents, being important sources of natural antioxidants for the prevention of diseases caused by oxidative stress [102].
Numerous terpenes and terpenoids (terpenes with the addition of functional groups, usually oxygen-containing) can be obtained from medicinal mushrooms, and are responsible for their antioxidant, anti-inflammatory, anticancer activities, and immunomodulatory [96].The mushroom extracts from Hypsizygus tessullatus (brown and white varieties) and Flammulina velutipes, for example, showed good antiproliferative activities against human breast cancer cell lines (CF-7 and MDA-MB-231 cell lines).This anticancer activity was attributed principally to the phenolic compounds quantified on the sample, which included significant flavonoid compounds [103].
Furthermore, the ability of natural polyphenols to scavenge reactive oxygen species (ROS) positions these compounds as valuable candidates for the production of anti-aging lotions or creams in the cosmetic industry [92].In the study by Ziemlewska et al., water extracts from the mushrooms Grifola frondosa (Maitake), Hericium erinaceus (Lion's Mane) and Ganoderma lucidum (Reishi) were assessed for antioxidant properties, bioactive content, and skin effects.Maitake extract exhibited the highest levels of flavonoids and phenols, along with efficient scavenging of radicals.Importantly, all extracts mitigated skin irritation, increased skin hydration, and reduced transepidermal water loss.Furthermore, the incorporation of mushroom extracts into washing gels decreased skin irritation and intracellular production of free radicals compared to a cosmetic base.Overall, these findings highlight the potential of mushroom extracts in enhancing cosmetic formulations [104].
Phenolic compounds have also been shown to be useful in the treatment of COVID-19.Ganoderma lucidum, a common mushroom in Chinese medicine, produces luteolin and quercetin, phenolic compounds that were screened for anti-COVID-19 action [105,106].Ganoderma lucidum extract has also been applied in skincare products, as well as Pleurotus ostreatus extract, showing promising cosmeceutical potential [107].
Concerning the process of extraction of phenol compounds from the mushrooms, this depends strongly on the effectiveness and efficiency of the selected extraction method.Conventional methods for phenol compound extraction from mushrooms include maceration and Soxhlet method of lipid extraction.Those methodologies are simple and depend principally on the solvent and external factors such as temperature, time, and agitation, which are adjusted to improve the solubility of the compounds.Nevertheless, those techniques demand long extraction periods and high solvent volumes and may lead to low extraction yields.Novel methods of extraction, which are green, economical, and faster, have been researched, however.One example is Ultrasound-Assisted Extraction (UAE), applied in the article by Machado-Carvalho et al. for the extraction of phenolic compounds from the mushroom Inonotus hispidus.A total phenolic content of 219.01 µg/g (dry weight) was identified on the extract, with hispidin as the main polyphenol.The extract demonstrated potential as a source of antioxidant compounds, with possible industrial, pharmaceutical, and food applications [108].
After extraction, the application of those mushroom phenols and polyphenols in industry faces some challenges, especially related to their stability, solubility, bioavailability, and bioactivity, especially during storage and processing.These challenges restrict their utilization in both food and medicinal contexts.Sensitivity to storage conditions like temperature, oxidation, and light, along with gastrointestinal conditions such as pH and enzyme interactions, complicates their incorporation into dietary supplements.Furthermore, exposure to alkaline environments can prompt the formation of undesirable derivatives, while metabolites with a bitter taste present additional hurdles for integration into food products.However, research and technologies have been developed to encounter those challenges and expand the application of those compounds, utilizing strategies that include novel drug delivery systems and encapsulation or nanoencapsulation of the mushroom extracts with success.

Terpenes from Mushrooms
Terpenes and terpenoids are a class of fungal secondary metabolites that possess chemical structure diversity and a broad range of biological activities.The terpenoids are classified into different classes based on the number of linked isoprene units as monoterpenes (C 10 ), sesquiterpenes (C 15 ), diterpenes (C 20 ), sesterterpenes (C 25 ), triterpenes (C 30 ), and polyterpenes (longer chains of C 5 ) [109].
The biosynthesis of terpene originates from the mevalonate pathway, and mevalonic acid is the main precursor from isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP).The monoterpenes are formed from geranyl diphosphate (GPP, C 10 ), resulting in the phosphorylation of mevalonic acid.The GPP combined with IPP produces the farnesyl diphosphate (FPP, C 15 ), a precursor molecule from sesquiterpenes.The diterpenes are produced by condensation of the FFP and IPP to form geranylgeranyl diphosphate (GGPP, C 20 ).FPP and GGPP are molecules precursors from triterpenoids (C 30 ) and tetraterpenoids (C 40 ) [110,111].
Macromycete fungal species contain a variety of bioactive terpenes that have potential pharmacological applications.These terpenes include sesquiterpenes, diterpenes, triterpenes, monoterpenes, and polyketides (Table 2).Terpene biosynthesis for higher fungi depends on the type of terpene synthase produced, which is a peculiar characteristic of each fungal species and its bioactivity [110].
The monoterpenes are rarely isolated from filamentous fungi, such as Aspergillus.Among the mushrooms, the production of monoterpenes was reported by only three species, including Agaricus bisporus, Pleurotus cornucopiae, and Pleurotus ostreatus.Many of these monoterpenes possess antioxidant and anti-inflammatory activities [117,135].

Alkaloids from Mushrooms
Different groups of mushrooms are known to be potential producers of high-molecularweight bioactive compounds, such as alkaloids [140].Alkaloids are secondary metabolites, some of which are produced by macrofungi; these compounds exert protective activities on the organism, for example, to combat pathogen attacks.They also act to mediate interspecies communication, acquisition of nutrients against competitors, and act in symbiotic interactions [141].
Among the main alkaloids produced by macrofungi are indoles.These compounds are some of the main nitrogen-containing secondary metabolites, characterized by a structure containing the indole nucleus or derivatives (Figure 3A).
type of compound has psychotropic activity by modulating serotonin receptors [152]; ex-amples of this group of alkaloids are psilocybin, aeruginasin, and bufotenine [145].
The β-carbolines are organic amines with multi-target pharmacological activities by modulating enzymes and receptors involved in depressive disorders monoamine oxidases.Examples of these compounds are β-carboline-1-propanoic acid, brunnein A, etc.These compounds also intercalate into DNA, inhibit CDK, topoisomerase and monoamine oxidase and interact with benzodiazepine receptors and 5-hydroxyserotonin receptors.Furthermore, these biometabolites also have sedative, anxiolytic, hypnotic, anticonvulsant, antitumor, antiviral, antiparasitic and antimicrobial properties [153,154].
Carbazoles have a tricyclic structure, consisting of two six-membered benzene rings fused on each side of a ring.One side of a ring contains five-membered nitrogen [155].Examples of this group are carquinostatin, koenimbin, and koenigicine, among others.Some of these compounds have biological properties against diseases such as ischemiareperfusion, atherosclerosis, inflammation, autoimmune diseases and cancer [151,156].Finally, ergot is an indole alkaloid that shares a common tetracyclic system formed biosynthetically by condensation of tryptophan and dimethylallyl pyrophosphate (Figure 3B); lysergol and ergonovine are part of this group [145].

Sterols from Mushrooms
Steroids are fat-soluble compounds that have a basic structure of 17 carbon atoms arranged in rings linked together.One of the most important steroids is ergosterol, which is a sterol that makes up the cell membranes of fungi and some protists [157].This sterol is a precursor to vitamin D2 [158].Ergosterol is transformed into viosterol by the action of ultraviolet light and is subsequently converted into ergocalciferol, which is a form of vitamin D [159].
Steroids are essential eukaryotic constituents, are secondary metabolites of mushroom metabolism and are biosynthesized through triterpenoids [157].According to Rangsinth et al. and Baker et al. [159,160], the main sterol in mushrooms is ergosterol.These compounds are increasingly relevant in the pharmacological sphere due to their health benefits, mainly due to their antioxidant properties, immunological activity, and effects against diabetes and cancer.Among the ergosterol-producing mushrooms, the following stand out: Agaricus bisporus, Tuber melanosporum, Lentinula edodes, and Agaricus blazei [161].
The ergosterol content in mushrooms varies between species and between different parts of the mushroom, for example, in button and morel mushrooms, the ergosterol content can reach concentrations of 3.52 mg/g and 0.45 mg/g, respectively [162].Regarding Indole alkaloids are derived from the amino acid tryptophan, which is obtained via the shikimate route [141,142].The synthesis of indole alkaloids begins with chorismate in the shikimic acid pathway and involves the intermediates anthranilate (AT) and indole-3-glycerol-phosphate (IGF).The IGF intermediate is transformed into indole, which can couple with serine to form tryptophan (Trp).The Trp can be decarboxylated and converted to tryptamine (TPT), or prenylated at C4 to produce 4-dimethylallyl tryptophan (4-DMAT).The compounds Trp, IGF, TPT and 4-DMAT can serve as precursors for the synthesis of fungal indole alkaloids [141][142][143].
Indole alkaloids from mushrooms have a wide range of biological properties, which is why the pharmaceutical sector has shown great interest in these compounds as a more efficient alternative to synthetic drugs currently used for neuropsychiatric diseases, such as depression, anxiety, post-mortem stress, traumatic stress, obsessive-compulsive disorders, depressive disorders, bipolarity and schizophrenia, among others [144,145].
According The use of macrofungi with hallucinogenic effects constitutes a ceremonial tradition of Mesoamerican indigenous cultures.These fungi have compounds characterized by the ability to induce neurotrophic effects, mental changes, and even hallucinations after being ingested [148,149].One of the first indole alkaloids consumed by man was psilocin (500-200 BC), obtained mainly from Psilocybe mexicana.This compound is a substance analogous to serotonin and acts as an agonist of serotonin 5-HT receptors, which can cause psychic effects (altered states of consciousness, synesthesia, increased introspection) [150].
Indole alkaloids can be classified into two groups: (i) non-isoprenoids, among which are tryptamines, β-carbolines, carbazoles; and (ii) isoprenoids, such as ergot and terpeneindoles (Figure 3B).Non-isoprenoids are phenolic lipids derived from the polyketide pathway and can have varied and diverse structures that do not follow the isoprene unit pattern.Differently, the isoprenoids are molecules synthesized through condensations of the five-carbon compound isopentenyl diphosphate (IPP) and dimethylallyldiphosphate derived from the mevalonate pathway [141,148].Tryptamines are known as simple alkaloids derived from indole formed by a benzopyrrole with an ethylamine-type side chain at C-3 and substituents at positions C-4 and C-5 on the indole nucleus [141,148,151].This type of compound has psychotropic activity by modulating serotonin receptors [152]; examples of this group of alkaloids are psilocybin, aeruginasin, and bufotenine [145].
The β-carbolines are organic amines with multi-target pharmacological activities by modulating enzymes and receptors involved in depressive disorders monoamine oxidases.Examples of these compounds are β-carboline-1-propanoic acid, brunnein A, etc.These compounds also intercalate into DNA, inhibit CDK, topoisomerase and monoamine oxidase and interact with benzodiazepine receptors and 5-hydroxyserotonin receptors.Furthermore, these biometabolites also have sedative, anxiolytic, hypnotic, anticonvulsant, antitumor, antiviral, antiparasitic and antimicrobial properties [153,154].
Carbazoles have a tricyclic structure, consisting of two six-membered benzene rings fused on each side of a ring.One side of a ring contains five-membered nitrogen [155].Examples of this group are carquinostatin, koenimbin, and koenigicine, among others.Some of these compounds have biological properties against diseases such as ischemiareperfusion, atherosclerosis, inflammation, autoimmune diseases and cancer [151,156].Finally, ergot is an indole alkaloid that shares a common tetracyclic system formed biosynthetically by condensation of tryptophan and dimethylallyl pyrophosphate (Figure 3B); lysergol and ergonovine are part of this group [145].

Sterols from Mushrooms
Steroids are fat-soluble compounds that have a basic structure of 17 carbon atoms arranged in rings linked together.One of the most important steroids is ergosterol, which is a sterol that makes up the cell membranes of fungi and some protists [157].This sterol is a precursor to vitamin D 2 [158].Ergosterol is transformed into viosterol by the action of ultraviolet light and is subsequently converted into ergocalciferol, which is a form of vitamin D [159].
Steroids are essential eukaryotic constituents, are secondary metabolites of mushroom metabolism and are biosynthesized through triterpenoids [157].According to Rangsinth et al. and Baker et al. [159,160], the main sterol in mushrooms is ergosterol.These compounds are increasingly relevant in the pharmacological sphere due to their health benefits, mainly due to their antioxidant properties, immunological activity, and effects against diabetes and cancer.Among the ergosterol-producing mushrooms, the following stand out: Agaricus bisporus, Tuber melanosporum, Lentinula edodes, and Agaricus blazei [161].
The ergosterol content in mushrooms varies between species and between different parts of the mushroom, for example, in button and morel mushrooms, the ergosterol content can reach concentrations of 3.52 mg/g and 0.45 mg/g, respectively [162].Regarding the ergosterol content in mushroom capsules, this can reach concentrations of 3.76 mg/g and 4.56 mg/g for white and brown mushrooms [163].
Ergosterol has properties similar to some medicines.However, the main advantage of this biometabolite is that it does not cause hepatotoxicity or skin allergy.Its average lethal dose in rats is extremely high-2.05mol/kg.In addition, ergosterol was considered a non-mutagenic and non-carcinogenic compound [159,164].
Ergosterol is an effective antioxidant agent that acts in different ways depending on the type of free radical.According to [165], this biometabolite is capable of neutralizing free radicals induced by terbutyl hydroperoxide.Furthermore, ergosterol also reacts with the hydrogen peroxide radical (H 2 O 2 ), whereby it inhibits or prevents lipid oxidation.The authors in [166] demonstrated that ergosterol-rich extracts obtained from Auricularia nigricans (formerly Auricularia polytricha) present a significant defense against neurotoxicity induced by oxidative stress.
On the other hand, ergosterol also has anti-inflammatory properties.According to Yoo et al. [167] this metabolite inhibits inflammation induced by lipopolysaccharides in conjunction with the phosphorylation of p38, extracellular signal-regulated protein kinase and mitogen-activated proteins.According to Huan et al. [168], ergosterol suppresses the expression of inflammatory mediators through different signaling pathways, including the Janus kinase signal transducer and the JAK-STAT interaction chain, routes that are related to inflammation, cell division and death.Nallathamby et al. [169] found that Cordyceps militaris extract enriched with ergosterol exerts neuroprotective effects by attenuating radical production activated by lipopolysaccharide.
Ergosterol and its derivatives also have biological activity against cancer.This disease still remains one of the most common causes of morbidity and mortality in the world [170].Compounds of natural origin with chemopreventive and chemotherapeutic properties have led to a new era in the research of medicines to combat cancer and its lethal malignancies, due to which current treatments are insufficient especially when this disease is in advanced stages [159].Chen et al. found that ergosterol fractions obtained from Ganoderma lucidum exhibited potent cytotoxicity against MDA-MD-231 and HepG2 cell lines, while for normal cells, the biometabolite had no effect.Rangsinth et al. [159] reported that ergosterol derivatives such as 5,6-dehydroergosterol and ergosterol peroxide also possess anticancer activity specifically in breast and cancer-related cells.Martínez-Montemayor et al. [171] showed that ergosterol also inhibits the expression of the enzymes ATK1, ATK2, etc., which attenuates the invasion of cancer into organisms.Li et al. [172] found that the Sanguinoderma rude (formerly Amauroderma rude) stratum rich in ergosterol inhibits melanoma B16 cancer cells.According to researchers, the inhibition of cancer by ergosterol occurs mainly because ergosterol positively regulates multiple tumor suppressors such as pro-apoptotic genes, including FasL, Bad, Bim and Trail.Yazawa et al. [173] found that a diet with an ergosterol content between 0.01 and 0.1% inhibits bladder carcinogenesis and that the inhibition of cancer cells occurs by blocking androgenic signaling.Other examples of the anticancer properties of ergosterol are summarized in Table 3.
Other biologically important properties of ergosterol are its antidiabetic and antimicrobial activity.Regarding the first, Xiong et al. [174] demonstrated that treatment with ergosterol obtained from Pleurotus ostreatus in mice reduced blood glucose with type 2 diabetes, in addition, ergosterol stimulated the expression of GLUT4 modulated by the PI3K pathway.Furthermore, according to Rangsinth et al. [159], ergosterol also increases glucose uptake due to which it positively regulates the activity of glucose transporter 4 and the phosphorylation of Akt and PKC in muscle cells as well as tissues.
Regarding antimicrobial activity, according to [175], steroids such as stigmasterol, sitosterol and ergosterol exhibit potent antimicrobial activity against a broad spectrum of microorganisms.For example, ergosterol has antifungal properties demonstrated against filamentous fungi such as Aspergillus flavus, Penicillium digitatum, among others [159].In fact, Li et al. [176] showed that ergosterol inhibits the growth of Helicobacter pylori, the main etiological agent associated with chronic gastritis and duodenal ulcers [177].Ergosterol also affects the growth of Trypanosoma cruzi, the etiological agent that causes Chagas disease, as ergosterol permeabilizes the plasma and mitochondrial membranes, causing the death of the parasite.Finally, ergosterol also has antiviral properties against the COVID-19 virus.

Patents: Technological Development and Innovation
Patent applications related to macrofungi and bioactive compounds have been increasing worldwide in recent years.A patent search in the Derwent Innovations Index database identified patent applications in many areas, whether for human or animal pharmaceuticals, agriculture, polymer extraction methods, and biochemical compounds.Products such as encapsulates, tablets, extracts, liquid compositions, alternative supplements, probiotic foods and nutraceuticals, supplementary human and animal nutrition ingredients, etc., were identified in the patent documents.
With the aim of finding patent documents in the field of pharmacology, an International Patent Classification (IPC) code group A61K-31/* was selected, where class A61 (medical or veterinary science), subclass A61K (preparations for medical, dental, or hygienic purposes), and group A61K-31 (medicinal preparations containing organic active ingredients) were chosen.Along with this IPC, the names "macromycet*", "macro$fung*", and "mushroom" were added.This resulted in the final search algorithm: A61K-031* AND (macromycet* OR macro$fung* OR mushroom*).
With this algorithm, 1237 patent documents were retrieved from the Derwent Innovations Index database.These documents were considered in the analysis of the historical technological development of the segment and in the evaluation of the number of patent publications per year (Figure 4b).By refining the search, selecting documents related to the areas "Pharmacology Pharmacy" and "Biotechnology Applied Microbiology", excluding the areas "Food Science Technology" and "Agriculture", and restricting to patents filed in the last five years, 176 patent documents were found.By reading the titles and abstracts of these documents, 174 were selected for being related to the pharmaceutical or therapeutic field (Figure 4a,c).
The first patent was filed in Japan in 1982 (JP57047482-A).The patent was assigned by the General Electric Company and Zh Biseibutsu Kagaku Ken.The invention refers to producing an antibiotic using a macromycete fungus species, Leucoagaricus leucothites (currently Leucocoprinus leucothites).This bioproduct, a carcinostatic substance, has the therapeutic potential to help treat leukemia.Patented by the assignee, the cultivated macromycetes of the genus Leucoagaricus had their metabolic liquors of interest extracted using solvents such as (m)ethanol, butanol, ethyl acetate, butyl acetate, ether, diozane and acetone.The carcinostatic compound acts to inhibit cancer cells from spreading [181].
In the Derwent Innovations Index database, Japanese patents were the most numerous and prominent until the early 2000s, with important contributions of the assignee Kagome Co Ltd.According to the company's website, its mission is to provide quality food to solve social issues [182].
gel, prevents inflammation and skin aging.Its mechanism of action focuses on improving the skin's water retention properties, increasing collagen content in the cortex, and improving skin elasticity [200].
As can be seen in Figure 4c, the patents filed in the last five years have been divided into applications for disorders of the nervous system; therapy against multiple types of cancer; treatments against dermatological problems and anti-aging; and treatment for problems of the digestive and blood systems and numerous others, such as treatment against respiratory diseases, inflammation, immune diseases, obesity, influenza, COVID-19, cardiovascular diseases, etc.Of the six patents filed by Kagome Co., Ltd. up to the 2000s, two, JPH09235224A and JPH09235226A, were designed to help treat ischemic brain diseases linked to memory disorders.The bioactive compounds (phenols, palmitic acid, stearic acid, and linoleic acid) are extracted from the Hericium erinaceus mushroom as reported in both patent documents.The compounds of interest are extracted from Hericium erinaceus with a homogeneous solvent system consisting of water and an organic solvent.The dried solid is subsequently treated by fractional chromatography and then fractionated to isolate each of the three compounds.The final product can be formulated into an oral agent, such as a tablet, granules, powder preparation, or capsule.The only difference between one patent and the other concerns the derivatives used for the formulation.While the first (JPH09235224A) contains benzyl alcohol derivatives, the second (JPH09235226A) has chroman derivatives [183].
Over time, in the 1980s and 1990s, the focus of patent applications continued to be on the anti-tumor properties of bioactive fungal compounds.In 1983, Noda Shokukin Kogyo Co., Ltd.patented an anti-cancer agent derived from a glycoprotein of xylose produced by a mycelium belonging to Basidiomycetes (Lentinula edodes).Using membrane filtration and freeze-drying methods, the powder obtained from the mushroom was subjected to ethanol extraction, and the precipitate was separated by fractionation chromatography.When the patent was filed, they did not know which bioactive compound was responsible for the anti-cancer effect.Still, they knew that the action of the extracted anti-cancer substance was to increase immunity [184].
In 1989, Nichirei Corp., using ergosterol (Agaricus sp.), patented a method of extracting this compound with organic solvents to treat uterine cancer.An organic solvent, such as methanol, ethanol, acetone, ethyl acetate, chloroform, etc., extracts the compound.The extract obtained is subjected to solid-liquid separation by filtration, centrifugation, or a combination.Finally, the extract is put under adsorption chromatography for further purification.This produces three ergosterol derivatives with a remarkable cytocidal effect on cancer cells.The patent states that the destructive impact acts on the membranes of cancer cells, breaking them down through a biochemical reaction [185].
In 1995, New Oji Paper Co., Ltd.patented a method for hydrolyzing proteins from the mycelium of Basidiomycetes (Pleurotus sajor-caju, (currently Lentinus sajor-caju)) to prevent or treat hypertension.Its mechanism of action is a substance that acts as an angiotensinconverting enzyme inhibitor.Through extraction with a water-soluble solvent such as acetone or ethyl acetate, a protein extract is obtained from the mycelium of the fungus.By freeze-drying and then precipitating again with acetone, a mixture of converting enzymeinhibiting peptides is obtained, which can be administered orally or parenterally [186].
In the 2000s, several patents with applications against cancer, neurological and psychological problems, dermatological problems, and many others gained focus in the Western market.As an example of applications against cancer, the Japanese patent JP2000159682A can be cited, the aim of which is to strengthen the anti-tumor activity contained in the β-glucan polysaccharides of mushrooms (Agaricus sp.) by irradiating them with infrared rays, which increase the amount of lipoperoxides in their composition, increasing their antitumor activity proportionally [187].Another good example, filed by Nagatomo Yakuhin KK, another Japanese company, concerns a method for extracting the anti-tumor polysaccharide β-glucan from Pachyma hoelen (currently Wolfiporia hoelen) mushrooms.By boiling the sample at 100 • C for 5 to 6 h, the macropolysaccharide β-glucan is extracted efficiently [188].
Regarding innovative applications of neurological treatments, the international patent WO2010124089A2 of Synosia Therapeutics Inc. can be mentioned, the aim of which is to help treat people with an addiction to illicit drugs such as cocaine.One of the active alkaloid ingredients (ketamine) comes from the Ergot fungus (Claviceps purpurea).The invention relates to the use of imidazol-alkylamine inhibitors of dopamine β-hydroxylase.Their mechanism of operation consists of inhibiting plasma esterases to slow down the elimination of cocaine, which can result in elevations in plasma cocaine levels, increasing at least one negative subjective symptom of cocaine abuse and dependence, such as irritability, agitation, and extreme suspicion [189].A more recent example is the patent CA3186405A1, granted by Optimi Health Corp., the aim of which is to provide a drug composed of indole-alkylamines for the treatment of depression and anxiety through their psychoactive properties.The alkaloid compounds (baeocystine, norbaeocystine, N, Ndimethyltryptamine, 5-hydroxytryptamine (serotonin), 5-hydroxytryptophan, psilocybin and psilocin) found in mushrooms such as Psilocybe cyanescens can be extracted using a deep eutectic solvent composed of choline chloride, sugar, an allylene glycol, a diol and an acid, xylitol or proline [190].
Another field of activity extensively explored in patent documents was dermatological treatments.The patent JP2005089391A, granted to Kanebo Cosmetics Inc., created a pharmaceutical composition with an active ingredient obtained from the mushroom Tricholoma orirubens to treat dry skin caused by the degradation of hyaluronic acid.The active compound acts on fibroblasts, inhibiting the degradation of hyaluronic acid.The compound's name was not mentioned, but it is known to have phenols in its composition [191].Another example, patent JP2005068061A, filed by Kao Corp., developed a pharmaceutical product capable of inhibiting the apoptosis of melanocytes, making it effective in preventing the appearance of gray hair and vitiligo.By extracting a lanostane-type terpenoid from the fungus Ganoderma lucidum with organic solvents such as methanol, ethanol, propanol, 1,3-butanediol, acetone, tetrahydrofuran, it is possible to obtain a product in the form of injections, inhalants, suppositories, oral preparations, ointments, creams, lotions, emulsions, gels, etc. [192].
In addition to the conventional applications listed above, there are also diverse and pioneering fields of application.Patent US20020127243A1, filed by Sun Farm Corp., created a dietary supplement with the therapeutic functions of helping against viral infections and low immunity.The active compounds (phytoestrogens, β-glucans, saponins, inositol hexaphosphate, and lectins) extracted from mushrooms, such as Lentinula edodes, help by increasing immune activity, such as increasing the production of lymphocytes, immunoaccelerators, immunopotentiators and immunomodulators [193].Another diverse application example is patent US20080200429A1, filed by ImmuDyne Inc., where β-glucan compounds are extracted from Agaricus blazei mushrooms to treat bone loss or low bone density conditions.This happens through the inhibition of osteoclasts and the maturation of osteoblasts by β-glucans, leading to decreased bone resorption and increased bone formation, making β glucans ideal agents for treating diseases such as osteoporosis.The method of obtaining such compounds mainly involves enzymatic hydrolysis of fungal cells or biomass [194].
The growth of this sector in the 21st century shows that the market is awakening to this branch of biotechnology, especially about the therapeutic applications of bioactive fungal compounds.As can be seen in Figure 4a, the main countries with technology for producing or obtaining pharmaceutical products from mushrooms are China, the United States, South Korea and Japan, with 52%, 35%, 6.0% and 2% of patents, respectively.Therefore, it is clear that the most prominent researchers and producers of products with bioactive compounds derived from fungi in the last five years have been in North America and Asia, according to the Derwent Innovations Index database.This phenomenon can be explained by a linear increase in the global market trend, mainly in emerging countries such as China, to transform abundant and cheap substrates and natural resources into value-added products through bioprocesses.In this way, a resource that is easy, quick, and cheap to grow, such as mushrooms, becomes a raw material for high-value products in the pharmaceutical natural products market [195].
On the other hand, moving forward to the 21st century, the peak of publications related to the topic was more specifically in the last five years, with the highest peak of patent filings identified in the year 2022, post-pandemic.However, in the last two years, the number of patents applied to the pharmaceutical sector has decreased (Figure 4b).This could be due to the post-pandemic period, where concerns about collective health have reduced, and the trend has advanced towards environmental, climate, and sustainable development concerns [196].
The main patent depositors in the last five years were Hunan Ansen Biotechnology Co., Ltd.(Zhuzhou, China), Changde Jizhi Biotechnology Ltd., Co. (Hunan, China), and Shanghai Cheermore Biotechnology Co., Ltd.(Shanghai, China).Both companies develop food products or medicines linked to pharmaceutical and medicine manufacturing, dietetic foods adapted for medical purposes, or high-quality international food raw materials and related application technologies [197].
The patent CN114887052A assigned to the company Changde Jizhi Biotechnology Ltd., Co. claims several components to assist in the treatment of cardiovascular diseases through a multivitamin complex composed of several compounds, including Cordyceps sinensis (currently Ophiocordyceps sinensis) leaves and Agaricus sp.peptides.The multivitamin can improve the medicinal effect of cardiovascular disease treatments by increasing immunity, quickly balancing nutrition, and supplementing energy [198].
Hunan Hanzhen Biotechnology Co., Ltd.'s patent CN112315973A refers to developing anticancer drugs with the selenoside-III polysaccharide specific to the colon cancer-resistant mushroom Pleurotus ostreatus.The polysaccharide selenoside-III can kill cancer cells and simultaneously does not damage normal cells by specifically promoting apoptosis of HCT116 cells [199].
Shanghai Cheermore Biotechnology Co., Ltd.'s patent CN110638687A concerns polysaccharides from the mushroom Lentinula edodes.Such a composition, a paste or aqueous gel, prevents inflammation and skin aging.Its mechanism of action focuses on improving the skin's water retention properties, increasing collagen content in the cortex, and improving skin elasticity [200].
As can be seen in Figure 4c, the patents filed in the last five years have been divided into applications for disorders of the nervous system; therapy against multiple types of cancer; treatments against dermatological problems and anti-aging; and treatment for problems of the digestive and blood systems and numerous others, such as treatment against respiratory diseases, inflammation, immune diseases, obesity, influenza, COVID-19, cardiovascular diseases, etc.
In addition to the pharmacological applications, the presence of bioactive compounds in each patent was also analyzed, making it possible to see the predominance of certain compounds in relation to a specific pharmacological application.For example, bioproducts used to treat neurological and psychological disorders predominantly use alkaloids, like indole-alkylamines, in the form of pharmacological compositions in paste, encapsulated, liquid, or tablet form.Meanwhile, for treating cancerous disorders, polysaccharides, like β-glucan, make up the vast majority in the same formats as above or fungal extracts.For dermatological disorders, polysaccharides and alkaloids are used the most in gels, ointments, or fungal extracts.
Another point worth highlighting is that some patents, precisely 56, include more than one bioactive compound in their formulations, showing that joint action can be more efficient and effective than just one active ingredient.Patent US2023293610-A1, whose field of action is to treat a person with an inflammatory disease, has alkaloids (indolealkylamines), phenols (mycophenolic acid), and terpenes (isopulegol) in its formulation.Of the most diverse mushrooms used, Agaricus sp. was the most prominent.The patent comprises methods of administering doses with at least one edible or medicinal mushroom or extract thereof, a cannabinoid, a terpene, a curcuminoid, and a flavonoid.Treating inflammation in this way involves the application of combined corticosteroids to suppress the immune response and slow the inflammation process [201].
Another great example is patent US2022331344-A1, deposited by Resurgent Biosciences Inc.The patent comprises a botanical extract of a psychotropic macrofungus in the case of several species and genera, such as Agrocybe, Amanita, Conocybe, Panaeolus (formerly Copelandia), Coprinus, Entoloma, and Galerina.The aim is to create a soluble tablet with a combination of psychoactive compounds such as psilocybin, psilocin, norpsilocin, baeocystin, norbaeocystin, aeruginascin, serotonin, bufotenine, β-carboline alkaloids, muscarine, muscimol, muscazone and ibotenic acid extracted from one or more psychotropic fungi.This pharmaceutical formulation can be used to treat drug addiction, obsessive-compulsive disorder, intermittent explosive disorder, kleptomania, pyromania, trichotillomania, etc.Its active ingredient consists of cholinesterase inhibitors [202].
Another point that deserves attention is the most cited patent documents.Patent US2022304980-A1, which has 22 citations, relates to a measured dosage composition for treating neurological conditions such as anxiety, post-traumatic stress disorder, attention deficit disorders, depression, memory loss, dementia, cognitive dysfunction, hearing loss, vision loss, neurological pain, insomnia, erectile dysfunction, physiological pain or discomfort, or a combination thereof.Its active compounds include psilocybin, psilocin, baeocystin, norbaeocystin, salts and isomers, and combinations.Patented by Sw Holdings Inc., the active compounds were extracted from mushrooms of the genera Panaeolus, Copelandia, and Psilocybe.Its operating mechanism includes alkaloids acting on the central nervous system, altering brain function, and resulting in temporary changes in perception, mood, consciousness, and behavior [203].
The patent US2022273680-A1, which has ten citations, also refers to a method through a liquid mixture with serotonin for preventing or treating various psychological disorders, such as depression, psychotic disorders, schizophrenia, and bipolar I disorder, among others.Its active biocompounds are psilocybine, psilocin, baeocystine, norbaeocystine, lysergide (LSD), dimethyltryptamine, carboxamidotryptamine, ibogaine, and 3,4methylenedioxy-methamphetamine.Patented by the University of Maryland in Baltimore, psychoactive substances can be extracted from numerous species of mushrooms, including the genera Psilocybe, Gymnopilus, Panaeolus, Copelandia, Hypholoma, Pluteus, Inocybe, Conocybe, Panaeolina, Gerronema, Agrocybe, Galerina, and Mycena.Its mechanism of action consists of acting on serotonin receptors in the nervous system of the person being treated and can be ingested via pill, tablet, or liquid [204].
Finally, the patent US2023021957-A1 has nine citations.It has a scope of functions for treating compulsive eating disorders, aiding or causing weight loss, or treating obesity or a complication associated with obesity.Its active ingredients are indole alkylamines with their psychoactive effects.Patented by Neonmind Biosciences Inc., its alkaloid compounds come from mushrooms of the genus Psilocybe, where the active ingredients such as psilocin, psilocybin, and their analogs are manufactured through dry mushroom powder and an analog of psilocin or purified psilocybin is manufactured synthetically, 4-thiol-dimethyltryptamine, 1methylpsilocin, 4-fluoro-N, N-dimethyltryptamine, O-acetylpsilocin, 4-hydroxy-N-methyl-N-isopropyltryptamine or 4-hydroxy-N-methyl-N-ethyltryptamine.This alkaloid will act on selected serotonin receptors [205].

Commercial Products with Pharmaceutical Properties Obtained from Macromycetes
Mushrooms have a long history of use in traditional medicine, particularly in Asian countries.They have been found to possess a wide range of medicinal properties.Mushrooms are a rich source of bioactive compounds, with over 100 medicinal functions identified.Further research is needed to explore the full range of applications of mushrooms as a source of pharmaceuticals [206].
A number of products have been developed for therapeutic and commercial use, primarily comprising polysaccharides, with β-glucans being a particularly prominent example.These include polysaccharide peptide (PSP) and krestin (PSK) from Trametes versicolor; lentinan isolated from Lentinula edodes; schizophyllan (also known as Sonifilan, Sizof), and so on.Iran, or SPG, is derived from Schizophyllum commune; Befungin is extracted from Inonotus obliquus; D-fraction is derived from Grifola frondosa; GLPS polysaccharide fraction is derived from Ganoderma lucidum; and active hexose correlated compound and other substances are also present [207].
Pandey et al. [208] presented the results of their investigation into commercial mushroomderived pharmaceutical products.These include Ganoderma lucidum, which has been produced in three different forms, including a product with wound-healing and antiinflammatory activity, produced by CV Skinlabs.The first product is Abs, U.S., which contains anti-oxidative properties.It is produced by La Bella Figura Gentle Enzyme Cleanser, Italia.The second product is called California Gold, and the last is called Immune Duo.Both products contain immune-system-strengthening activity and are derived from Agaricus bisporus.
Another mushroom utilized in the production of the product known as "Krestin" (β-glucan derived) is Coriolus versicolor (currently Trametes versicolor).This product is prescribed as an adjuvant in cancer and antitumor treatment and is produced by the Japanese company Sankio, Kureha.A product derived from L. edodes, produced by the Chinese company ShenYuan ChemPharm Co., Ltd. C. (Xiaogan, China), with the commercial brand XiaoGan [209], is a multifunctional anti-cancer, anti-oxidative, wound-healing, and immunomodulating agent.
Once more, two commercial products are presented as examples of immune-strengthening activity.The first is a product called Mushroom 8-Plex Powder, produced by Zhou Nutrition and containing fermented and organic mushrooms.The second is a product produced by Source Naturals, called Mushroom Immune Defense, which contains a combination of 16 mushroom mycelia biomass.Finally, we consider a product with antioxidant properties that contains beta glucan derived from Schizophyllum commune, a fungus native (cosmopolitan) to the Czech Republic.This product is manufactured by Contipro A.S. [208].
The beneficial compounds found in mushroom types such as Reishi, Shiitake, Turkey tail, Chaga, and Maitake are causing a stir in the food world because they help regulate blood sugar and heart health.Because of their umami flavor and potential health advantages, these mushrooms are utilized in a variety of products, including capsules, powders, tinctures, and infused goods like coffee and tea.In order to satisfy customer needs and adhere to product registration and clearance procedures for medicinal or supplement goods in the food and beverage, dietary supplement, and pharmaceutical applications, the sector is also concentrating on sustainability and organic practices [210,211].
Another area of pharmacological interest is the use of mushroom-derived bioactive compounds.These include neurotrophic substances, which are synthesized from tryptophan as a secondary metabolite in some mushroom species.The most well-known and researched example is psilocybin.There has been a recent resurgence of interest in this molecule as a potential treatment for a range of common psychiatric disorders, including major depression and obsessive-compulsive disorder [212].Currently, several developed countries, including the United States and Australia, have approved the use of psilocybin as an alternative treatment for mental illnesses [213].Table 4 provides a summary of other products, their active ingredients, characteristics, and the companies that produce them.
The market for medicinal mushrooms is expected to expand by USD 5.94 billion at a compound yearly growth rate of 9.96% between 2022 and 2027.Several key factors contribute to the growth of the medicinal mushroom market [214].One such factor is the growing need for goods produced from mushrooms, which is being driven by factors such as changing customer preferences and growing awareness of their health benefits [215].The industry is also benefiting from the rising acceptance of online purchasing, which has made these products more accessible to a wider clientele.The industry is growing faster as a result of its development into uncharted territory.A significant proportion of therapeutic mushroom claims are derived from the extrapolation of cell cultures.
A significant number of businesses had previously received warnings for using the term "medical mushrooms" and making health claims for their products that contain mushrooms [214].Despite the existence of compelling evidence for the health benefits of certain mushroom products, the industry is constrained by a number of factors: (a) the extensive array of nutraceuticals currently available on the market; (b) the absence of standardized production and testing protocols that are necessary to guarantee product quality; and (c) the fact that the majority of medicinal products derived from mushrooms do not contain a single chemically defined molecule that is employed in conventional pharmacological treatments, rendering them neither pharmaceuticals nor nutraceuticals (food/functional food).
While some mushrooms, such as Ganoderma lucidum, are believed to hold the key to curing all human diseases, such assertions are exaggerated [214].No known treatment has this ability, not even conventional or alternative medicine.While it is inaccurate to view products made from mushrooms as a replacement for contemporary medications, they do occupy a unique category.In certain instances, they enhance overall health and act in conjunction with other therapies [216,217].
A significant number of nations have conducted research into the medicinal properties of mushrooms.However, most of these studies have focused on pre-clinical investigations, which involve the use of chemicals, enzymes, and animal models to screen for bioactivity.A considerable number of these investigations have been conducted using human cells in culture, but many are still in the pre-clinical phase or have not yet reached clinical trial stages I, II, or III [214].To demonstrate the safety and efficacy of medicinal mushrooms, high-quality, long-term, randomized, double-blind, placebo-controlled clinical studies-including sizable population studies-are essential for producing statistically significant results.

Conclusions
Mushrooms have been considered as true natural pharmaceutical factories since ancient times.Many recent studies over the last 50 years have proven the production and characterization of these compounds produced by macromycetes cells.Many medicinal effects of these bioactive compounds have been proven by research at a preliminary, preclinical level in animal models and in in vivo and ex vivo studies.Some clinical studies have already been carried out, but modern science cannot yet categorically state that these effects are similar to modern medicine and its treatments.Despite this, there has been a growing interest in mushrooms as medicines, nutraceuticals and cosmeceuticals in recent years, both due to the marketing carried out by certain segments and the dissemination of information via the internet.This has generated a significant number of patents in the field, as well as a market worth billions of dollars around the world, which will only grow in the coming years.If we take into account the number of classes of compounds, their plurality of described molecules, and the immense diversity of mushroom species not yet explored, we can conclude that this market is still only in its infancy.Robust studies in clinical and (meta)population trials, with effective and safe methods, as well as modern, cheap and effective in silico molecular analysis tools will allow for exponential growth of discoveries and their diverse applications as new drugs of interest.

Figure 1 .
Figure 1.Biometabolites with biological activity produced by mushrooms.

Figure 1 .
Figure 1.Biometabolites with biological activity produced by mushrooms.

Figure 2 .
Figure 2. Variety of polysaccharides produced by macromycete fungi with pharmaceutical bioactive potential.

Figure 2 .
Figure 2. Variety of polysaccharides produced by macromycete fungi with pharmaceutical bioactive potential.
to Plazas et al., Charlson et al., and Leichsenring et al. [145-147], neuropsychiatric diseases currently represent a challenge for global health systems due to their constant growth in number of cases and the lack of medicine and effective treatments.Furthermore, currently used psychiatric medications have unpleasant side effects and reduced tolerability, impacting patients' quality of life [145].

Figure 4 .
Figure 4. (a) The map with the countries' respective patent numbers over the last five years.(b) The graph showing the number of published patents per year, retrieved from the Derwent Innovations Index database, related to macromycete fungi in pharmacology and applied microbiological biotechnology.(c) The number of patents registered in the last five years is classified according to their applications and bioactive compounds.The numbers of the bioactive compounds may overlap, as some patents have more than one compound in their formulation.Phe-Phenol and Polyphenols; Pol-Polysaccharides; Ter-Terpenes and terpenoids; Pep-Peptides; Alk-Alkaloids; Erg-Ergosterol.

Figure 4 .
Figure 4. (a) The map with the countries' respective patent numbers over the last five years.(b) The graph showing the number of published patents per year, retrieved from the Derwent Innovations Index database, related to macromycete fungi in pharmacology and applied microbiological biotechnology.(c) The number of patents registered in the last five years is classified according to their applications and bioactive compounds.The numbers of the bioactive compounds may overlap, as some patents have more than one compound in their formulation.Phe-Phenol and Polyphenols; Pol-Polysaccharides; Ter-Terpenes and terpenoids; Pep-Peptides; Alk-Alkaloids; Erg-Ergosterol.

Table 1 .
General characteristics and pharmaceutical potential of some MBPs previously reported.

Table 2 .
Biological properties of terpenes obtained from mushrooms.

Table 3 .
Biological properties of ergosterol.

Table 4 .
Biological properties of mushrooms.