1. Introduction
Wood decay fungi are crucial links in the ecosystems of forests. As saprobionts, they decompose dead organic matter and release elements into circulation in nature. They are the main group of organisms—and often the only ones—capable of decomposing the high-molecular components of wood (cellulose, hemicellulose, lignin), which are the most complex and slowest natural substances to deteriorate in nature [
1]. As a result of their decomposition, humus is formed. The direct way the soil humic compounds form is their synthesis from fragments, such as polyphenols, with the involvement of organically originated nitrogen. The polyphenol source may be the lignin decomposition processes and the transformation of the carbohydrates; many polyphenols are formed as the metabolites of various microorganisms [
2].
The fungi lead to the polyphenols’ oxidation and the formation of the chinoid compound in subsequent stages. The aforementioned processes are observed in various fungi biological systems, indicating interesting studies using the saprotrophs fungi mycelium as an alternative source of polyphenols and other bioactive compounds [
3,
4,
5].
Common edible wood-decay fungi include
Kuehneromyces mutabilis (Schaeff.) Singer & A.H. Sm. and
P. ostreatus (Jacq.) P. Kumm.
Kuehneromyces mutabilis grows in large numbers in deciduous and mixed forests [
6,
7];
Pleurotus ostreatus is a fungus found in the wood of the deciduous species [
8,
9]. As edible mushrooms, they provide easily digestible protein, folic acid, vitamins (thiamin, riboflavin, and niacin) [
7,
10,
11,
12,
13], minerals (potassium, magnesium, copper, calcium, phosphorus, iron, selenium) [
14,
15], and substances that lower blood cholesterol level (lovastatin) [
16]. In addition, antioxidant, antidiabetic, antimicrobial, immunostimulating, and antitumor substances have been detected in the fruiting bodies of
P. ostreatus [
17,
18].
Poisonous fungi commonly found in the forest environment include
H. fasciculare (Huds.) P. Kumm. [
19]. A compound with an antibiotic effect against
Staphylococcus aureus was isolated from its fruiting bodies. The antagonistic activity of
H. fasciculare against other soil fungi and antibacterial activity were also found in the case of
Paraphaeosphaeria minitans (W.A. Campb.) Verkley, Göker & Stielow [
20],
Penicillium citrinum Thom [
21], and
Pleurotus tuber-regium (Fr.) Singer [
21,
22,
23,
24,
25,
26,
27]. Another common saprobiont throughout Europe is
T. versicolor (L.) Lloyd. The fruiting bodies can be found on the trunks or branches of dead shrubs and angiosperm and gymnosperm tree [
28], which cause white rot in the wood [
29]. In Europe, it is considered an inedible mushroom [
19]; however, the mushrooms list prepared for the F.A.O. listed the mushroom as edible in China, Hong Kong, Laos, and Mexico [
30]. Mycological and chemical research has proven the presence of many compound groups in
T. versicolor fruiting bodies, which are responsible for many therapeutic effects, including polysaccharides, protein-polysaccharide complexes, polyphenolic compounds, and terpenes [
31,
32].
Another mushroom with healing properties is
I. obliquus (Fr.) Pilát, which also causes white rot in wood. In Poland,
I. obliquus occurs infrequently, and it is mainly found on birch and rarely on other deciduous trees; therefore, it is under partial species protection [
6]. In Poland, the mushroom is considered inedible, while according to the F.A.O. list, it is an edible mushroom in Canada and Russia (Journal of Laws No. of 2014, item 1408) [
33].
I. obliquus sclerotia (black, cracked, lumpy formations) from birch trunks are used for medicinal purposes. The results of contemporary studies of the metabolites isolated from
I. obliquus sclerotia [
34] indicate their broad biological activity and potential properties, including hypoglycemic [
35], antiviral [
36,
37], antimutagenic [
38], anticancer, and cytostatic characteristics [
35,
39,
40,
41,
42,
43,
44,
45,
46], as well as strong antioxidative properties [
45,
47,
48,
49,
50,
51]. Common white rot fungi are:
B. adusta (Willd.) P. Karst. and
P. squarrosa (Vahl) P. Kumm.
The presented study aimed to determine the content of selected antioxidant active compounds (phenolic acids, flavonoids, carotenoids) of selected fungi (saprobionts) commonly found in the European forests: H. fasciculare, B. adusta., I. obliquus, K. mutabilis., T. versicolor, P. ostreatus., and P. squarrosa. The antioxidant activity measured by the ABTS radical and the free phenolic acids (FPA) content was also tested. In addition, the content of ergosterol, the main component of the fungal cell membrane, was determined.
4. Discussion
The studied saprobiont fungi occurring in the various Polish forest habitats were characterized by a diversified content of the biologically active compounds. The first group of compounds analyzed in this study was phenolic acids, which included
p-coumaric, ferulic, chlorogenic, and sinapic acids as the major components. Gallic, p-hydroxybenzoic, protocatechuic, vanillic, syringic, caffeic, ferulic,
p-coumaric, and sinapic acids are the most common acids in the plant world [
56]. In the tested isolates, the presence of 13 acids was observed, including the highest concentrations of
p-coumaric, ferulic, chlorogenic, and sinapic acid. The greatest antioxidant capacity (protection of cells against hydrogen peroxide) is shown by the following acids: vanillic and caffeic acid [
57]. The latter was present in all tested isolates, and the highest concentration was observed in
H. fasciculare (Hf),
I. obliquus (Io), and
K. mutabilis (Km). The other acids determined in the isolates tested, which were characterized by in vitro and in vivo antioxidant, antibacterial, antiviral, and antifungal properties, are p-hydroxybenzoic, gallic, and protocatechuic acid [
58].
A rich profile of phenolic acids in
I. obliquus was presented in this study (
Table 2) Numerous reports on the presence of phenolic acids in
I. obliquus (Io) confirmed the presence of the following acids: gallic, protocatechuic, and p-hydroxybenzoic acid [
34,
50,
59,
60,
61]. Ju et al. [
62] isolated vanillic, protocatechuic, and 2.5-dihydroxyterephthalic acids.
Similar results were obtained by Janjušević et al. [
63], who identified ten phenolic acids in
T. versicolor (Tv) harvested in northwest Romania, six of which were confirmed in this study, among others: quinic, malic, vanillic, and caffeic acid. Other researchers found that the methanol extract of
T. versicolor fruiting bodies showed the presence of three free phenolic acids: gallic (73 µg/g DM), protocatechuic (48 µg/g DM), and caffeic acid (154 µg/g DM) [
58].
In the conducted analyses, six phenolic acids were determined in
P. ostreatus (
Table 1). Sinapic acid was present in a high concentration (100.72 mg/kg DM) with 141.82 mg/kg DM total acids content. Muszyńska et al. [
64] found the presence of five acids in
P. ostreatus: protocatechuic, p-hydroxybenzoic,
p-coumaric, sinapic, vanillic, ferulic (1.28–21.38 mg/kg DM), and cinnamic acid (from 1.09 to 8.73 mg/kg DM). Similar results were obtained by Gąsecka et al. [
65], but they did not measure sinapic acid. However, Kim et al. [
66] noted a higher concentration of chlorogenic acids in the methanol extract of
P. ostreatus. The study also quantified two other HBA derivatives and protocatechuic acids.
Badalyan et al. [
67] determined the following acids in
P. squarrosa and
P. ostreatus: 4-hydroxybenzoic, 4-hydroxycinnamic, 4-hydroxy-3-methoxybenzoic, and 3,4-dihydroxyphenylacetic. Woldegiorgis et al. [
68] also confirmed the presence of caffeic, gallic, and p-hydroxybenzoic acids in the methanolic extract of
P. ostreatus cultivated in Ethiopia and determined that caffeic acid had the highest concentration among the phenolic acids. A different result was obtained by Palacious et al. [
69], who found a low level of caffeic acid in
P. ostreatus. Barros et al. [
70] isolated the following acids from
H. fasciculare: protocatechuic, p-hydroxybenzoic, and
p-coumaric acids. In our research, we also quantified thirteen phenolic acids, and the highest concentration was observed for
p-coumaric acid.
The carotenoids were another compound detected from the tested fungi. They belong to the group of chemicals that are synthesized in the organisms of bacteria, fungi, algae, and plants. Carotenoids containing 4-keto groups, monocyclic, and 13 double bonds in the structure, are characteristic of fungi [
71,
72]. The conjugated bonds are responsible for the distinctive color of these compounds (yellow, orange, or purple). These compounds protect against oxidative stress and exposure to visible light or UV radiation [
73]. Moreover, carotenoids are intermediates in the biosynthesis of physiologically active apocarotenoids and their derivatives [
74]. A large amount of reactive, conjugated double bonds present in the carotenoids causes the high activity of these compounds as antioxidants active against free radicals. These compounds stabilize cell membranes and act as photoreceptors.
All isolates were tested for β-carotene, lutein, zeaxanthin, and astaxanthin. The latter metabolite is characterized by 10-fold higher antioxidant activity than the other carotenoids: β-carotene, zeaxanthin, and lutein [
75,
76]. In our research, the highest concentration of this metabolite was observed in the
T. versicolor (Tv) isolate. Other researchers confirmed the presence of β-carotene in
P. ostreatus [
77]. Jayakumar et al. [
78] determined β-carotene in a 5-fold higher concentration than the present study. Robaszkiewicz et al. [
79], Jaworska et al. [
80], and Turfan et al. [
81] found lycopene presence in addition to β-carotene. The latter was present at a much lower concentration in
P. ostreatus. Mushroom species that contain β-carotene are
Cantharellus cibarius Fr.,
Agaricus bisporus (J.E. Lange) Imbach,
Boletus edulis Bull.,
Suillus bovinus (L.) Roussel, and
Tricholoma equestre (L.) P. Kumm. Lycopene was found in the fruiting bodies of
C. cibarius,
A. bisporus,
B. edulis,
S. bovinus, and
T. equestre. Lutein, α-carotene, and xanthotoxin were found in the fruiting bodies of
C. cibarius, while γ-carotene, auroxanthin, and neurosporin was found in
B. edulis. Barros et al. [
82,
83] spectrophotometrically proved that among the six
Basidiomycota (Whittaker ex R.T. Moore) species collected in northeast Portugal,
C. cibarius contains the highest amount of β-carotene, which was equal to 13.56 mg/g DM. For the other species, the content of this metabolite ranged from 1.95–12.77 mg/g DM [
82,
83].
Another group of active compounds isolated from the analyzed fungi is flavonoids. Today, more than 9000 different flavonoids are known, and the number continues to grow [
84]. Flavonoids are polyphenolic chemical compounds of plant-based origins on the flavone skeleton. Their name comes from the Latin word flavus, meaning yellow. They occur mainly in the form of glycosides in the higher plants’ tissues and fungi. The antioxidant activity of the flavonoids depends on the conjugated double bonds in the C-2 and C-3 position, hydroxyl groups, and the carboxyl group in the C-4 position [
85,
86]. In the conducted analyses, the highest content of these metabolites was observed in
I. obliquus (Io)—114.5 mg/kg DM. High concentrations were also observed in
H. fasciculare (Hf) and
P. squarrosa (Ps). In addition, a high concentration of naringenin, quercetin, and kaempferol above 21 mg/kg DM was found. Similar results were obtained by Zheng et al. [
34], who also isolated quercetins, naringenins, and kaempferol from
I. obliquus (Io). Conversely,
B. adusta (Ba) (0.07 mg/kg DM) was characterized by containing the lowest concentration of flavonoids.
P. squarrosa (Ps) and
T. versicolor (Tv) were poor in flavonoids in our research. Janjušević et al. [
63] obtained different results with
T. versicolor (Tv) flavonoids content harvested from northwest Romania. The obtained profile was rich and included: flavones (6 compounds, e.g., apiin, vitexin, coumarins (2 compounds), flavanols (6 compounds, e.g., quercetin and rutin), isoflavonoids, and biflavonoids (amentoflavone). The flavonoid content was 67 mg/100 g DM. Other species with significant flavonoid amounts influencing their antioxidant value were:
Lactarius deterrimus Gröger,
Boletus edulis Bull. and
Xerocomellus chrysenteron (Bull.) Šutara.
The compounds commonly found in mushrooms are sterols. The first reports of the presence of sterol in mushroom fruiting bodies date back to 1887. The typical mushroom sterols are distinguished by a high degree of unsaturation. Ergosterol is the main component of the fungal cell membrane and is strongly associated with cytoplasm; it is also a precursor to vitamin D2. Ergosterol can be converted to vitamin D2 through UV radiation. Finnish scientists determined the total sterol content in species of large-fruited mushrooms in the range of 625–774 mg/100 g DM [
87]; a different ERG content characterized the analyzed fungi species. The highest ERG content was observed in
I. obliquus (Io) (863.33 mg/kg). Kim et al. [
66] and Shin et al. [
88] determined ERG in
I. obliquus, but not quantitatively. Alternatively, our research found that
H. fasciculare (Hf) was characterized by a four-times lower ergosterol concentration compared with
I. obliquus. Chemical composition studies of the edible mushroom species have shown that they are a rich source of ergosterol. After several years of storing the dried fruiting bodies, vitamin D2 content was high, averaging 1.43 μg/g DM in
C. cibarius Fr. The differences in the ERG content were the results of the different sites of insolation from which the fruiting bodies came from [
89].
Agaricus bisporus (J.E. Lange) Imbach contains an average of 61.5 mg/100 g of ergosterol. Research on the influence of UV-C radiation on the formation of vitamin D2, carried out by the Center for Plant and Food Science at the University of Western Australia, showed a high conversion rate of ergosterol to vitamin D2 after short-term exposure to the UV-C radiation of these species’ fruiting bodies during development. After UV-C radiation irradiating for 2.5, 5, and 10 min, the ergosterol concentrations were 6.6, 15.6, and 23.1 μg/g DM, respectively. Based on the
Boletus edulis Bull. chemical analysis, it has been shown that the entire fruiting body contains approx. 200 mg of vitamin D2 per 100 g of DM 89].
In the conducted analyses, it was found that the antioxidant activity measured with the ABTS radical was 2.5–3.5 times higher in the isolates of
I. obliquus (Io),
P. ostreatus (Po), and
H. fasciculare (HF) compared to
P. squarrosa (Ps) and
B. adusta (Ba). The highest antioxidant activity and the highest total phenolic acid content was found in
T. versicolor (Tv) when compared to the other isolates. This relationship corroborates that the phenolic compounds’ content may affect the level of antioxidant activity. Matijašević et al. [
90] investigated the total polyphenol content of the
T. versicolor (Tv) isolate collected near Belgrade. The concentration of the FPA in the mushroom ethanol extract was 25.8 mg GAE/g. Those results were almost five times lower than the results determined in this study (
Table 4). However, these results align with Vamanu and Voica’s [
91] research, who investigated the total phenolics and antioxidant activity of the several mushrooms harvested from the Moldova region of Romania. In the different isolates of
T. versicolor (Tv) variegated growths, significant differences were noted in FPA content, flavonoids, and antioxidant capacity. These significant differences can be explained by the genetic factors (different fungal isolates), harvest site and time, type of solvent, and extraction conditions. These compounds’ direct antioxidant mechanism captures the free oxygen radicals and reactive oxygen forms and limits their cell production by inhibiting the activity of the oxidizing enzymes (e.g., lipoxygenase) due to the easy hydrogen donation from the carboxyl group, which reduces peroxides and hydroxides.