Several mycochemicals are present in mushrooms with different chemical structures and composition such as phenolic compounds, terpenoids, lipids, polysaccharides and proteins, which are easily separated from other constituents by their high molecular weights.
The term 'phenolic compounds' includes a wide range of mycochemicals that are characterized by an aromatic ring bearing one or more hydroxyl groups. Phenolic substances are water-soluble since they most frequently occur in combination with sugar as glycosides, but also as esters and polymers. These compounds belong to different classes based on the number of phenol rings and of the functional groups linked to these moieties. Thus, a classification comprises simple phenols, phenolic acids, phenylpropanoids, flavonoids, flavonols, flavones, stilbenes, and lignans.
Phenolic acids are the main phenolic substances found in mushrooms (Ferreira et al. 2009); they are classified into two groups; hydroxybenzoic acid (HBA) and hydroxycinnamic acid (HCA). Hydroxybenzoic acid derivatives are in the bound form and are part of more complex structures as hydrolyzable tannins, lignins, sugars and organic acids. Hydroxycinnamic acid derivatives are also present mainly in the bound form, attached to cell-wall structural elements, such as lignin, cellulose, proteins or linked to organic acids, through ester bonds, such as quinic or tartaric acids (Manach et al. 2004). The most widespread are the HCAs, which are useful not only as providing the building blocks of lignin but also concerning disease resistance and growth regulation. Five HCAs are common, in fact almost ubiquitous in mushrooms: ferulic, sinapic, caffeic, and p-/o-coumaric acids. HCAs usually are present in mushrooms in combined form as esters; and they are obtained in best yield by mild alkaline hydrolysis, since with hot acid hydrolysis material is lost for the decarboxylation to the corresponding hydroxystirenes.
Caffeic acid occurs in mushrooms regularly as a quinic acid ester (3-o-caffeoylquinic, 4-o-caffeoylquinic, 5-o-caffeoylquinic). Besides, tannic and ellagic acids are observed (Ferreira et al. 2009). In mushrooms, the most prevalent HBAs derivatives are reported to be gallic, protocatechuic, gentisic, homogentisic, p-hydroxybenzoic, 5-sulphosalicylic, syringic, veratric, vanillic (Ferreira et al. 2009) (Table 1).
HBA and HCA compounds are derived biosynthetically from the shikimate pathway. l-phenylalanine and -tyrosine are the crucial amino acids and the building blocks in this pathway.
Flavonoids are another large group of naturally occurring phenolic compounds that are all structurally derived from the parent substance flavone consisting of two benzene rings (A and B) combined with a pyran one (C). Different classes of flavonoids are recognized, such as anthocyanidins, flavonols, flavones, isoflavones, flavanones, and flavonols (Manach et al. 2004). The flavonoids are present in nature as glycosides or aglycones.
It was reported that mushrooms do not synthesize flavonoids, however, the presence of flavonoids was found in various edible mushrooms, e.g. catechin, myricetin, chrysin, hesperetin, naringenin, naringin, formometin, biochanin, resveratrol, quercetin, pyrogallol, rutin, and kaempferol (Gil-Ramirez et al. 2016; Ferreira et al. 2009).
Phenolic acids and flavonoids identification and quantification from some selected mushrooms [P. ostreatus, P. eryngii (DC.) Quél., Agaricus bisporus (J.E. Lange) Imbach, Cyclocybe aegerita (V. Brig.) Vizzini, Russula cyanoxantha (Schaeff.) Fr., R. virescens (Schaeff.) Fr., Macrolepiota procera (Scop.) Singer, Boletus edulis Bull., Lactarius deliciosus (L.) Gray, Coprinus comatus (O.F. Müll.) Pers., Tuber melanosporum Vittad.] were done by high-performance liquid chromatography coupled with mass spectrometry (HPLC–MS) (Table 1). The compounds identification derives from their retention times, their UV–Vis absorption spectra and mass spectra data and also by comparison with available data (Fogarasi et al. 2018). 4-Hydroxybenzoic acid and 5-feruloylquinic acid were found to be the major compounds in P. ostreatus and A. bisporus with concentrations of 75.042 mg/100 g·fw and 35.040 mg/100 g·fw for P. ostreatus and 79.50 mg/100 g·fw and 71.01 mg/100 g·fw for A. bisporus, respectively. B. edulis extract is characterized by high concentrations of cinnamic acid 168.614 mg/100 g·fw and catechin 145.566 mg/100 g·fw (Fogarasi et al. 2018). Hasnat et al. (2014) reported content of phenolic compounds for R. virescens of 8.74 and 2.21 mg gallic acid/100 g·fw, and flavonoid compounds were 2.83 and 1.02 mg catechin/100 g·fw for the water and ethanol extracts, respectively.
Among phenolic acids, the major amount of protacatechuic acid was found in M. procera (5.19 mg/Kg DW) (Nowacka et al. 2014). Kalogeropoulos et al. evaluated the content of individual phenolic compounds for L. deliciosus; p-OH-benzoic acid (24.5 µg/100 g fw) and p-OH-phenylacetic acid (18.3 µg/100 g fw) were the more abundant among the hydroxyl-benzoic acids, o-coumaric acid (30.2 µg/100 g fw) among the hydroxycinnamic acids, and chrysin (16.5 µg/100 g fw) among the flavonoids.
As concerns C. comatus, among the phenolic compounds, the highest content was detected for quinic acid (14.6 mg/100 g dw) and quercetin (3.01 mg/100 g fw), where the lowest amount was detected for the isoflavonoids genistein (0.023 mg/100 g dw) and daidzein (0.061 mg/100 g dw) (Nowakowski et al. 2020). Besides, Comatin (4, 5-Dihydroxy-2-methoxy-benzaldehyde) isolated and identified from C. comatus has shown hypoglycaemic properties on alloxan-induced-diabetic rats (Ding et al. 2010) (Table1).
In the literature, it is common to find the total phenolic content (TPC) found in mushrooms methanolic extract by the Folin-Ciocalteu assay. However, this assay has some limitations since other readily oxidized compounds such as amino acids, ascorbic acid, and sugars could interfere overestimating the total phenolic content (Arbaayah and Umi 2013).
Phenolic compounds possess antioxidant properties to scavenge free radicals, to prevent lipid peroxidation, and to chelate ferrous ions (Kumar and Pandey 2013).
The general term 'terpenoid' includes all such substances with a common biosynthetic origin. Terpenoids arise from the isoprene molecule CH2=C(CH3)-CH=CH2 and their carbon skeletons originate from the union of two or more of these C5 units. Their classification is according to whether they contain two (C10), three (C15), four (C20), six (C30), or eight (C40) such unit. Essential oils, volatile mono- and sesquiterpenes (C10 and C15), including the less volatile diterpenes (C20), the involatile triterpenoids and sterols (C30), and the carotenoids pigments (C40) are terpenoids. Each of these different classes of terpenoid is of importance in mushroom growth, metabolism, or ecology (Dewick 2009).
Chemically, terpenoids are generally lipid-soluble and are extracted from mushrooms with dichloromethane, light petroleum, or ether and can be separated by flash chromatography on silica gel or alumina using some solvents. Isomerism and the presence of different geometric conformations are common among terpenoids. It depends on the substitution around the cyclohexane ring, twisted in the so-called ‘chair’ form. The stereochemistry of the cyclic terpenoids is highly involved. During purification procedures, structural re-arrangement and isomerization may occur and lead to artifact formation.
The mainly terpenoid essential oils include the volatile fraction responsible for the characteristic odor and scent found in many mushrooms. They are commercially important as the basis of skincare in cosmetics and flavorings in the food industry. Fogarasi et al. (2018) reported the presence of α-pinene, β-phellandrene, β-pinene, β-myrcene, and d-limonene in A. bisporus and B. edulis as main terpenoids. The in-tube extraction headspace coupled with gas chromatography-mass spectrometry (HS-ITEX/GC–MS) permits to obtain the volatile profile of selected mushrooms. The volatile constituents strongly influence the aroma profile of each mushroom variety.
Triterpenoids and sterols
Triterpenoids are compounds with a carbon skeleton based on six isoprene units. They biosynthetically derived from squalene, an acyclic C10 hydrocarbon. They have relatively complex cyclic structures, most being either alcohols, aldehydes, or carboxylic acids.
Sterols are triterpenes which are based on the cyclopentane perhydrophenantrene ring system. So, one example is ergosterol, ubiquitous in occurrence in mushrooms. Ergosterol is a component of the fungal cell membrane, which under the influence of UV irradiation is converted to vitamin D2. Besides, ergosterol shows several healthy beneficial properties such as antihyperlipidemic, anti-inflammatory, antioxidant and the effect for inhibiting fungi and bacteria growth (Koutrotsios et al. 2017).
All types of triterpenoids are isolated by very similar procedures, based mainly on column chromatography, GLC and TLC. Identities are confirmed by melting point, rotation, FT-IR, GLC-MS, and NMR experiments.
Table 2 includes the triterpenoids and sterols found in some selected mushroom species.
Different P. ostreatus strains were evaluated for their sterol composition. In all mushroom samples analyzed ergosterol dominated, comprising 51.9–87.4% of sterols, followed by its metabolites ergosta-7-enol (12.7%), ergosta-5,7-dienol (7.6%), and ergosta-7,22-dienol (6%) (Koutrotsios et al. 2017). The ergosterol content in P. eryngii was reported as 20 mg/100 g dw, although a higher value was measured in commercial samples (Souilem et al. 2017). Kikuchi et al. (2017, 2018) reported the isolation and structure elucidation of ergostane type sterols and bisabolane-type sesquiterpenes from P. eryngii with aromatase and nitric oxide production inhibitory effects, respectively (Table 2).
Wang et al. (2013a) reported the identification of novel and rare perhydrobenzannulated 5,5-spiroketal sesquiterpenes, named pleurospiroketals A-E from the edible mushroom P. cornucopiae with inhibitory activity against nitric oxide production in lipopolysaccharide-activated macrophages with IC50 values between 6.8–20.8 µM.
From M. procera were isolated and identified 12 lanostane-type triterpenoids characterized by the presence of a rare '1-en-1,11-epoxy' moiety, namely lepiotaprocerins A-L. Lepiotaprocerins A-F showed significant inhibitions of nitric oxide (NO) production, while lepiotaprocerins G-L, showed cytotoxicity effects against different human cancer cell lines, and lepiotaprocerin I displayed antitubercular activity against Mycobacterium tuberculosis H37Ra with a MIC of 50 µg/mL (Chen et al. 2018).
Three non-isoprenoid botryane sesquiterpenoids, named boledulins A-C were isolated from the cultures of B. edulis Bull. with moderate inhibitory activity against five human cancer cell lines (Feng et al. 2011), while from the edible mushroom L. deliciosus, azulene-type sesquiterpenoids were characterized (Tala et al. 2017).
Many sterols such as campesterol, lanosterol, brassicasterol, β-sitosterol, ergosterol were analyzed in the fruiting bodies of different Tuber species (Table 2). The main sterols found in Tuber magnatum Picco and T. melanosporum fruiting bodies were ergosterol and brassicasterol, which amounted to 63.1–66.7% and 15.7–21.3% of the total sterols, respectively. Also the mycelia of T. borchii Vittad. are a rich source of ergosterol (90.3%). The complex composition profile of the truffle sterols might be taken as the fingerprint for the identification of the truffle species (Yeh et al. 2016).
Fatty acids and lipids
Mushrooms are an essential source of fatty acids that occur mainly in bound form, esterified to glycerol, as fats or lipids. They are crucial as membrane constituents in the mitochondria and chloroplasts and provide mushrooms with a storage form of energy. The content of total lipids ranges mostly from 1 to 4% of the dry weight. Besides, mushroom fats are rich in unsaturated fatty acids (PUFA) and particularly in linoleic acid (Koutrotsios et al. 2017).
Lipids are known by their distinct solubility properties and are extracted with alcohol, ether or dichloromethane from mushrooms.
The general structures for the three main classes of mushrooms lipids are reported in Fig. 1.
Structural variation within each class is due to the different fatty acid residues that may be present. The identification of lipids mainly requires the determination of their fatty acid components. Fatty acids are determined as methyl esters (FAMEs) after hot saponification of the sample, followed by reaction with BF3/MeOH. The resulting FAMEs are analyzed by GC–MS by comparison with standard FAMEs and confirmed utilizing mass spectra library (Helrich 1990).
In some selected mushrooms species, the fatty acid composition is characterized by a prevalence of polyunsaturated linoleic acid (C18:2ω6), monounsaturated oleic acid (C18:1ω9), and saturated palmitic acid (C16:0) (Table 3). The fatty acids are divided into saturated (SFA), monounsaturated (MUFA), and polyunsaturated (PUFA). In particular, the ratio between the single components of PUFA is fundamental in preventing cardiovascular diseases. PUFAs are a family of so-called 'essential' fatty acids that are converted to tissue hormones useful to prevent blood clotting and hypertension (Pietrzak-Fiećko et al. 2016).
Koutrotsios et al. (2017) evaluated the fatty acid profile of different P. ostreatus strains, collected in Greece, including saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), ω3 and ω6 fatty acids. PUFA was the major fatty acid class detected; linoleic acid (C18:2ω6) dominated in all samples (56.8–80.5%) followed by oleic (C18:1ω9) and palmitic (C16:0) (6.3–19.5 and 7.5–12.1%, respectively) (Table 3).
Jing et al. (2012) reported a selective method where fatty acids from cultivated mushrooms P. eryngii, C. aegerita and C. comatus were derivatized with BAETS as the labeling reagent and identified by high-performance liquid chromatography with fluorescence detection and online mass spectrometry (HPLC-FLD-MS/MS).
Total fatty acids (TFAs) values for P. eryngii, C. aegerita and C. comatus (dw) were 42.60, 48.95, and 79.21 mg 10 g−1, respectively, while UFA:SFA ratio were 3.23, 3.29, and 3.03, respectively. Linoleic (C18:2ω6) and oleic (C18:1ω9) acids were the main FA found and their content was between 27.17–49.34 mg 10 g−1 and 4.08–22.15 mg 10 g−1, respectively.
Also for P. cornucopiae the linoleic acid (C18:2ω9) was the main FA, with a composition characterized by a higher content of mono (MUFA) and polyunsaturated FA (PUFA) than of saturated FA (SFA) (Rodrigues et al. 2015).
The lipids analyzed for A. bisporus showed a high content of unsaturated acids with linoleic acid (C18:2ω6) as the main constituent of fruiting bodies (33.3%) and stems (39.4%). The total saturated fatty acid (SFA) content was between 22.1 and 26.5% of total lipids, palmitic acid (C16:0) was the major SFA at about 14% followed by stearic acid (C18:0) at about 4%. Oleic acid (C18:1ω9) was the major monounsaturated fatty acid (MUFA) present at about 1.5% of total lipids (Sande et al. 2019).
As concerns R. cyanoxantha the major fatty acid found was linoleic acid (C18:2ω6) (43.65%) followed by oleic acid (C18:1ω9) (28.39%) and palmitic acid (C16:0) (12.95%) (Grangeia et al. 2011).
The fatty acid composition of different wild Boletus species collected in Portugal was reported by Heleno et al. (2011). (Table 3). The major fatty acid found in B. edulis was oleic acid (C18:1ω9) (42.5%) followed by linoleic acid (C18:2ω6) (41.32%) and palmitic acid (C16:0) (9.57%). A very similar profile of fatty acid composition was reported for 33 samples of wild B. edulis in the form of caps and stems, collected from selected regions of Poland. The dominant fatty acids in all samples analyzed were C18:2ω6, C18:1ω9, and C16:0 (Pietrzak-Fiećko et al. 2016).
Kalogeropoulos et al. (2013) reported the fatty acid composition of wild L. deliciosus from Greece. The prevalent fatty acids were linoleic acid (C18:2ω6) (31.78%), followed by stearic acid (C18:0) (29.83%) and oleic acid (C18:1ω9) (21.82%) (Table 3).
Another class of lipids found in mushrooms is glycosphingolipids (GLSs) and the cerebrosides in particular. A polar head (usually a monosaccharide or a carbohydrate chain) and a fatty acyl group are linked to a long-chain aminoalcohol called a long-chain base (LCB). The fatty acyl chain is amide-linked to the LCB and together they make up the ceramide; the monosaccharide or oligosaccharide group is linked to the primary alcoholic function of the ceramide (Fig. 2).
GLSs are ubiquitous membrane constituents of mushrooms and are believed to possess a wide range of biological activities, including modulation of growth and regulation of differentiation. They are involved in membrane phenomena, such as cell–cell recognition, cell–cell adhesion, antigenic specificity, and other kinds of transmembrane signaling.
β-Glucosylceramide is by far the most common GLS from mushrooms. A peculiarity of glucosylceramides from mushrooms is the frequent occurrence of a di-unsaturated C18 sphingosine with a methyl branching at C-9. Structure determination was based on carbohydrate analysis, methylation analysis, chemical degradation, and extensive use of FAB-MS (Itonori et al. 2004). Three cerebrosides with different lengths of the fatty acid portion have been isolated and identified from Pleurotus cornucopiae (Paulet) Rolland (Lee et al. 2017). Furthermore, purified acidic glycosphingolipids (AGLs) from P. eryngii were reported to induce interleukin-2 (IL-2) release from invariant natural killer T (iNKT) cells inducing prolonged retention of IL-4 in serum in vitro and in vivo (Fu et al. 2016). So through iNKT cell activation AGLs isolated from P. eryngii might be involved in the maintenance of immunohomeostasis.
An important secondary metabolite from mushrooms is lovastatin, a polyketide employed as a cholesterol-lowering drug that inhibits (3S)-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. This is a key enzyme in the synthesis of mevalonate, since it is the immediate precursor of cholesterol and lovastatin is the lead compound of all of the drugs classified as statins. Lovastatin was discovered from Aspergillus terreus and Monascus ruber in the 1970s and is a natural product in oyster mushrooms (Chen et al. 2012) (Fig. 3).
Mushrooms are a significative source of polysaccharides. The structural complexity of polysaccharides is ascribed to the linkage between two sugar units, through an ether linkage, in several different ways. The reducing end of one sugar (C1) can condense with any hydroxyl group of a second sugar (at C2, C3, C4, or C6) so that during polymerization some sugars may be substituted in two positions, leading to branched chains structures. Besides, the ether linkage can have either a α- or β-configuration, due to the stereochemistry of simple sugars, and both kinds of linkage can co-exist in some molecule.
Generally, the polysaccharides are present in the mushroom cell wall and include α-glucans and β-glucans. These macromolecules are composed of glucopyranose units linked with glycosidic bonds of the type (1 → 6)-β, (1 → 3)-β, or (1 → 3)-α. Mushrooms are characterized by different kinds of polysaccharides that include not only glucans but also heteroglycans and proteoglycan classes. Polysaccharides that include residues of only one type of monosaccharide unit are known as homoglycans, while residues of two or more types of monosaccharide molecules are categorized as heteroglycans (Kozarski et al. 2014).
As concerns, the extraction and purification procedures, usually the polysaccharides are isolated by successive hot-water extractions followed by ethanol precipitation. Chromatographic methods such as size-exclusion (SEC) and ion-exchange chromatography (IEC) are used as purification procedures of the crude polysaccharides, while chemical reactions of hydrolysis and derivatization together with NMR experiments are useful in providing information for their structural elucidation (Sun et al. 2010a).
Polysaccharides isolated and identified from mushrooms differ in their physical–chemical properties such as in their water solubility, molecular weight, size of the molecule, and structure (Table 4). Recently, polysaccharides isolated from mushrooms have attracted increasing attention for their wide spectrum of biological properties, such as antioxidative, antitumor, immunomodulation (BRMs), and anti-inflammatory effects (Selvamani et al. 2018). The major pharmaceutical properties of mushrooms, i.e. antitumor activities and immunity potentiation, are ascribed to β-glucans. Many fungal β-glucans stimulate both innate and adaptive immunity. They activate innate immune system components such as natural killer (NK) cells, neutrophils, macrophages, and cytokines. These cytokines, in turn activate adaptive immunity with the stimulation of B-cell for antibodies production and promotion of T-cell differentiation to T-helper cells, which mediate cell and humoral immunities (Oloke and Adebayo 2015).
Pleuran, a water soluble polysaccharide [β-(1,3/1,6)-d-Glucan], is the best-known β-glucan isolated from P. ostreatus with a molecular weight of 762 KD. It is composed of a backbone (1 → 3) linked β-d-glucose with a side chain of a β-(1 → 6) or β-(1 → 4)-d-glucosyl residue of ever fourth glucose unit. The compound exhibit anti-neoplastic properties against different cells, including breast cancer MCF-7, prostate cancer cells PC-3 and colorectal HT-29 cancer cells. It possesses also antiviral and anti-oxidative properties (Golak-Siwulska et al. 2018).
The purified polysaccharides PEPE-A1 and PEPE-A2 from P. eryngii are characterized by a β-(1 → 3)-glucan as the backbone accompanied by α-(1 → 6)-d-glucosyl residues side chains. They showed a strong inhibitory effect on lipid accumulation (Fu et al. 2016). Recently, a mannogalactan with the main chain of (1 → 6)-linked-α-D-galactopyranosyl and 3-O-methyl-α-d-galactopyranosyl residues, both partially substituted at OH-2 by β-d-Manp units was isolated from P. eryngii and tested against murine melanoma cells (Biscaia et al. 2017).
Zhang et al. (2014) isolated three subfractions of intracellular zinc polysaccharides (IZPS) from P. cornucopiae. All the subfractions have shown antioxidant activities in vitro and in vivo. They were found able to act as upregulatation of the superoxide dismutase, GSH peroxidase and catalase, and significantly decreased the contents of malondialdehyde and lipid peroxidation in vivo. PCPS from P. cornucopiae mushroom extract is a β-(1 → 6)-glucan possessing a proinflammatory effect on innate immune cells (Minato et al. 2017).
From A. bisporus a new heteropolysaccharide consisting of ribose, rhamnose, arabinose, xylose, mannose, glucose, and galactose with 1 → 2 and 1 → 4 glycosidic bonds and probably 1 → 3 glycosidic bonds was isolated and identified with high in vitro immunobiological activity (Liu et al. 2020a, b, c).
Motoshima et al. (2018) identified a fucogalactan from C. aegerita (FG-Aa) characterized by (1 → 6)-linked α-D-galactopyranosyl main chain, substituted at O-2 by non-reducing end units of α-L-Fucp, on the average of one to every second residue of the backbone. The obtained fucogalactan was evaluated against arginase from Leishmania amazonensis.
A water-insoluble (1 → 3)-β-d-Glucan was firstly isolated from the fresh fruiting bodies of R. virescens, and then the sulfated derivative was synthesized with sulfur trioxide-pyridine complex. The sulfated derivative exhibited enhanced anti-tumor activities against Sarcoma 180 tumor cell (Li et al. 2020). Besides, a water-soluble poaysaccharide (RVP) with anti-oxidant properties was isolated from the fruiting bodies of R. virescens consisting of (1 → 6)-linked-α-d-galactopyranosyl and (1 → 2,6)-linked-α-d-galactopyranosyl residues that terminated in a single non-reducing terminal (1 →)-α-d-mannopyranosyl residue at the O-2 position of each (1 → 2,6)-linked-α-D-galactopyranosyl residues along the backbone (Sun et al. 2010a). Also RVP was sulfated and in vitro activity test data indicated that the SRVPs showed better antioxidant, anticoagulant, antitumor and antibacterial activities compared with RVP.
Three crude polysaccharides (BEPF30, BEPF60, and BEPF80) were isolated from the fruiting bodies of B. edulis and investigated for their antioxidant activities. BEPF60 showed significant reducing power and chelating activity together with the highest inhibitory effects on hydroxyl and superoxide radicals (Zhang et al. 2011). Other crude water-soluble polysaccharides (BEBPs) were extracted from B. edulis and evaluated for their antioxidant activities. BEBP-3 showed a significant anti-oxidant activity (Luo et al. 2012).
Lactarius deliciosus is an important source of polysaccharides. Su et al. (2019) reported the structural characterization and immune regulation activity of a novel polysaccharide (LDG-M) from L. deliciosus Gray. LDG-M was composed of β-d-glucose and α-d-lyxose with ratio 2:1. The proposed structure of LDG-M was a backbone of 1,6-linked-β-d-glucose and 1,4,6-linked-β-d-glucose, with branches composed of one (1 → 4)-linked-α-d-lyxose residue (Table 4). The structural elucidation of LDG-A indicated a backbone of 1,6-disubstituted-α-L-mannopyranose with branches at O-2 mainly composed of a (2 → 3)-α-D-xylopyranose residue. LDG-A exhibited marked antitumor activities in vivo. A new heteropolysaccharide (LDG-B) with a backbone of (1,6)-linked-d-galactose and (1,2,6)-linked-d-galactose with branches composed of 4-linked-d-glucose and 6-linked-d-galactose residue was identified from L. deliciosus. Cell cycle test data showed that LDG-B could promote the proliferation of B cells and macrophage cells by affecting G0/G1, S and G2/M phases (Hou et al. 2016). Besides, also the structure elucidation and anti-tumor activity of water-soluble oligosaccharides (LDGO-A) were reported by Ding et al. (2015).
A modified polysaccharide named MPCC was obtained by snailase hydrolysis from C. comatus with antioxidant and hepatoprotective properties (Zhao et al. 2019). The structural investigation of CCPP-1 from C. comatus has shown that CCPP-1 was α-D-(1 → 4)-glucan with branches at C-6 consisting of non-reducing terminal approximately every fourteen residues. While the crude polysaccharide fractions CCPF showed significant hypoglycemic activity, CCPP-1 was not useful on reducing blood sugar (Liu et al. 2013).
As concerns Tuber fruiting bodies and fermentation system, the structure, the physicochemical and biological properties of the polysaccharides have not been thoroughly investigated. Tejedor-Calvo et al. (2020) reported a preliminary screening of the main bioactive compounds for T. magnatum, T. melanosporum and T. borchii by using pressurized liquid extractions (PLE). The polysaccharide composition of the obtained extracts was investigated by NMR analysis and their immunomodulatory activity tested in vitro with cell cultures. NMR investigation revealed that the extracted polysaccharides were β-(1 → 3)-glucans and a heteropolymer consisting of galactose and mannose.
Proteins, peptides and lectins
Other macromolecular mycochemicals isolated from mushrooms with high molecular weight are proteins, peptides, and lectins.
The proteins in mushrooms, as in other plants, are high molecular weight polymers of amino acids. The amino acids are arranged in a particular linear order and each protein has a specific amino acid sequence. Proteins are usually purified according to molecular weight so they are subjected to gel filtration on a column of Sephadex. Separation of proteins by gel electrophoresis is also partly determined by their molecular size since their mobility on the gel is closely related to their charge properties (Oloke and Adebayo 2015).
The composition of mushroom proteins seems to be of higher nutritional value concerning most plant proteins. Mushrooms proteins contain all nine essential amino acids required by humans and can be used as a substitute for meat (Kakon et al. 2012). High contents of proteins 38.9 and 36.9% were observed in A. bisporus and B. edulis, respectively (Nagy et al. 2017). Mushrooms are a rich source of proteins with several properties for biotechnological and medicinal applications. Immunomodulatory proteins (FIPs) are a group of fungal proteins able to alter the cytokine response (Oloke and Adebayo 2015). Proteins isolated from selected mushrooms exhibited antiviral, antitumor, antifungal, and antibacterial properties (Table 5). Moreover, the fruiting bodies and mycelium of several mushrooms are an abundant source of ergothioneine, an unusual sulfur-containing derivative of histidine, with antioxidant properties (Chen et al. 2012).
Many proteins are also enzymes, catalyzing particular steps in either primary or secondary metabolism, and possess health-promoting effects. Laccases were isolated from P. ostreatus and P. cornucopiae with antiviral effect against the hepatitis C virus and HIV-1 reverse transcriptase, respectively (Table 5). Lectins are another group of mycochemicals that include polysaccharide-protein and polysaccharide-peptide complexes. Lectins derived from mushrooms exhibit antiproliferative, immunomodulatory, antitumor, HIV-1 reverse transcriptase inhibiting, cell growth-regulating, and many more properties (Oloke and Adebayo 2015). Some proteins, peptides, and lectins isolated from various selected mushrooms are reported in Table 5.
From P. ostreatus a Cibacron blue affinity-purified protein (CBAEP) was isolated with potent antitumor, anticancer and immunomodulatory activity against Sarcoma-180, Dalton lymphoma (DL)-bearing mice, and B16FO melanoma tumor-bearing mice (Sarma et al. 2018).
Besides, pleurostrin and eryngin are two proteins isolated from P. ostreatus and P. eryngii mushrooms with antibacterial and antifungal properties (Erjavec et al. 2012). The laccase isolated from P. ostreatus exhibited an antiviral effect against the hepatitis C virus (Golak-Siwulska et al. 2018). A dimeric lectin, composed of subunits with a molecular weight of 40 and 41 KDa, isolated from fresh fruiting bodies of P. ostreatus exerted antitumor activity in mice bearing sarcoma S-180 and hepatoma H-22 (Table 5).
Fu et al. (2016) reported the isolation of a laccase from P. eryngii with antiviral activity against HIV. The laccase was active against HIV-1 growth with an IC50 of 2.2 µM by inhibiting HIV-1 reverse transcriptase. Also a protease named pleureryn, extracted from fresh fruiting bodies of P. eryngii, showed (23.1 ± 0.6)% and (91.4 ± 3.2)% inhibition of HIV-1 reverse transcriptase at 3 and 30 mM, respectively (Table 5).
Hu et al. (2018) reported the functional characterization of a P. eryngii protein (PEP 1b). PEP 1b is an immunomodulatory protein with 21.9 KDa able to induce the M1-polarization of the macrophage cell line RAW 264.7 cells through the activation of the TLR4-NF-κB and MAPK signal pathways.
Two types of angiotensin I-converting enzyme (ACE) inhibitory oligopeptides were obtained from the basidioma of P. cornucopiae. The amino acid sequences of the two purified oligopeptides were found to be RLPSEFDLSAFLRA and RLSGQTIEVTSEYLFRH. Besides, from the fermentation broth of P. cornucopiae was isolated a new laccase with a molecular mass of 67 KDa. It inhibited proliferation of the hepatoma cells HepG2, the breast cancer cells MCF-7, and the activity of HIV-I reverse transcriptase with IC50 values of 3.9, 7.6 and, 3.7 µM, respectively (Wu et al. 2014). Besides, a divalent cation-dependent GalNAc-specific lectin (PCL-M) was purified from the mycelia of P. cornucopiae. It is a multimeric glycoprotein composed of 40 KDa subunits linked by disulfide bonds (Oguri 2020).
A lectin, isolated from A. bisporus (ABL) showed antiproliferative effects on different cell types and might be useful for glaucoma. Besides, the fruiting bodies of A. bisporus are associated with a protein, named FIIb-1, characterized as tyrosinase (Verma et al. 2019).
Recently, a ribotoxin-like protein, named Ageritin was isolated from the basidiomycetes C. aegerita. Several biological activities are ascribed to Ageritin such as antibacterial, antiviral, endonuclease, nuclease, antifungal, and cytotoxicity to COLO 320, HeLa and, Raji cells by promoting apoptosis (Citores et al. 2019). The lectin (AAL), isolated from C. aegerita exhibited antitumor activity by inducing apoptosis (Liu et al. 2017), while lectin-2 (AAL-2) and its complexes with GlcNAc and GlcNAcβ1-3Gal β1-4GlcNAc revealed the structural features of specific recognition of non-reducing terminal N-acetylglucosamine (Ren et al. 2015).
A novel laccase was purified and characterized by R. virescens. Its N-terminal amino acid sequence was AIGPTAELVV and it was able to degrade various phenolic compounds and to decolorize several dyes (Zhu et al. 2013).
Žurga et al. (2017) isolated novel ricin B-like lectin with a β-trefoil fold from M. procera, designated as MpL with nematocidal activity indicating a function in protecting fruiting bodies against parasites. MpL was studied for potential delivery of peptidase protein inhibitors to lysosomes showing that it is a promising carrier of protein drugs to intracellular targets.
An antiviral protein Y3 isolated from C. comatus showed an inhibitory effect on the tobacco mosaic virus. Y3 has shown anticancer potential inducing caspase-dependent apoptosis in Jurkat cells of human T-cell leukemia. Besides, also laccases from mycelia of C. comatus have shown antiproliferative and antiviral properties (Nowakowski et al. 2020) (Table 5).