Sarcodon Mushrooms: Biologically Active Metabolites

Thelephorales Corn ex. Oberw. belong to the Agaricomycetes (Basidiomycota, Fungi) and comprise more than 177 species (Kirk et al., 2008) that are classified into two families: the Bankeraceae Donk (mostly with colourless basidiospores) and the Thelephoraceae Chevall. (mostly with brown to yellowish basidiospores) (Yorou & Agerer, 2007). Bankeraceae are characterized by a typical fenugreek odour, with pileate and stipitate fruit bodies, hydnoid to spinose hymenophores with brown and lobed as well as colourless and evenly ornamented basidiospores. Bankeraceae comprise 5 distinct genera: Bankera Coker & Beers ex Pouzar, Hydnellum P. Karsten, Phellodon P. Karsten, Sarcodon Quel. ex P. Karst and Boletopsis Fayod.

The fungus can be bitter, although this is less apparent in younger specimens. Submerging the mushrooms in boiling water will remove this. It can be pickled or dried and used as flavouring. In Bulgaria it is collected, dried and finely ground to be used as an aromatic mushroom flour. It is reported as edible, but of poor quality in the United States by some sources but as deliciously edible by others. In China Sarcodon aspratus, also known as "black tiger's paw", is a popular natural edible mushroom. It is used for lowering cholesterol level, muscles relaxation and blood circulation. Its special musky aroma enhances the taste in meat's dishes, especially hams (S.K. Kim, 2006), and particularly brings out the sweet flavour when making a clear soup out of it.

Phytochemistry of Sarcodon imbricatus
In view of its use as a food, the phytochemical composition of S. imbricatus has been studied.
Proximate constituents (moisture, fat, crude protein, ash and carbohydrates) of this mushroom have been reported (Barros et al., 2007a). On the basis of the proximate analysis, it can be calculated that a portion of 100 g of this mushroom provides, on average, 24 kcal (100 kJ). The analysis of fatty acid composition allowed to determine 15 fatty acids.
Other interesting metabolites isolated and identified from S. aspratus are polysaccharides. Han and coworkers isolated a water soluble polysaccharide (HCP) with molecular mass of 6.7 x 105 Da (Han et al., 2011). HCP resulted to be a linear glucan with a backbone structure of (1→6)-linked--glupyranosyl residues. Another polysaccharide (HBP) with a molecular weight of 4.3 x 105 Da was isolated by the same author in 2010. Even in this case the backbone structure was constituted by (1→6)-linked--glupyranosyl residues, which occasionally branched at O-3 position (Han et al., 2010).

Sarcodon cyrneus Maas Gest
Sarcodon cyrneus, according Myco Bank (www.mycobank.org), has a "pileus up to 65 mm across, planoconvex to somewhat depressed in centre, finely tomentose at margin, becoming felted farther back, or tomentum collapsed to form smooth and shiny pellicle. Stipe 15-30 x 6-15 mm, broader when fused, equal or somewhat enlarged below, with abruptly pointed base, straight to curved, tomentose, in places glabrescent, pale grey-brown, darkening with age and becoming more or less concolorous with, pileus, at extreme base with yellowish grey mycelium. Spines up to 3 mm long, 0.1-0.3 mm broad, long decurrent, often almost reaching base of stipe, crowded, subulate, first whitish, becoming purplish brown. Context pallid (whitish suffused with pinkish when fresh, according to the collector), not greenish in base of stipe" (Fig. 3). The taste is bitter and the mushroom is not edible.

Phytochemistry of Sarcodon cyrneus
In the literature only two papers regarding the secondary metabolites isolated from S. cyrneus have been reported (Marcotullio et al., 2006b;Marcotullio et al., 2007) and they deals with the structural identification of five cyathane diterpenes. These compounds were named cyrneine A-D (1-4) (Fig. 4) and glaucopine C (7) (Fig. 6). According MycoBank (www.mycobank.org) the mushroom has a "pileus up to 110 mm across, plano-convex to somewhat depressed; at first tomentose, then matted, forming a cuticle which breaks up into scales near margin, into areoles in centre; scales adhering to appressed, yellow-brown with vinaceous shade or pale to dark purplish brown, occasionally locally violet-grey (giving a peculiar leaden grey impression), at times very dark brown in centre, contrasting with dingy yellowish ground colour, not infrequently covered with minute yellowish dots of excreted matter when dried. Stipe 27-75 x 10-40 mm, cylindrical, tapering below or somewhat broadened below, tomentose, fibrillose, covered with adnate fibrillose squamules or partly matted, dingy whitish, soon pinkish brown to purplish brown above, grey-green below, with pointed, whitish base. Spines up to 5 mm long, 0.1-0.2 mm broad, whitish, finally purplish brown" (Fig. 5). The taste is bitter and the mushroom is not edible. www.intechopen.com

Phytochemistry of Sarcodon galucopus
No phytochemical studies regarding proximate composition have been reported for S. glaucopus. In the literature there are only two studies on the secondary metabolites of S. glaucopus, and they deal with the isolation and structure elucidation of three cyathane diterpenes named glaucopine A (5), glaucopine B (6) (Curini et al., 2005) and glaucopine C (7) (Marcotullio et al., 2006a) (Fig. 6). Index Fungorum (www.indexfungorum.org) reports for S. laevigatus (syn. S. leucopus, often the term S. laevigatum is used instead of S. laevigatus). A description of Sarcodon leucopus (Pers.) Maas Geest. & Nannf. has been reported in MycoBank: "Pileus up to ca. 200 mm across, planoconvex or slightly depressed, without concentric or radiate markings, at first finely felted; felt collapsed to form smooth, more or less shiny, innate-scaly cuticle, the latter subsequently radiately rimose near margin, breaking up into areoles in centre, here with scales somewhat more pronounced and tips sometimes slightly raised; pale purplish brown www.intechopen.com on yellowish drab ground colour or a rich purplish brown to dark brown. Stipe 40-80 x 20-60 mm, cylindrical to ventricose, finely tomentose, later with smooth or innate-scaly cuticle, concolorous with pileus or paler, whitish below, after some time with green spots (always?). Spines up to c. 15 x 1 mm, whitish, finally purplish brown. Context up to 40 mm thick near centre of pileus, whitish, suffused with purplish brown to violet tints, after some time pale greenish. Odour commonly experienced as disagreeable. Taste bitter after some time".

Phytochemistry of Sarcodon laevigatus or/and S. leucopus
In the literature no references to S. laevigatus have been reported, while it is possible to find two papers referring to S. laevigatum and to S. laevigetum. The first one reports the isolation and the structure identification of three p-terphenyls (8-10), of which 8 (named sarcodan) was a new compound (Ma et al., 2006). The second report is a Patent and deals with the preparation of compound 9 (named B1-V) by extraction from S. laevigetum (Ma, 2009).
On the other hand, Tringali and coworkers (Tringali et al., 1987) reported the isolation and structure identification of several p-terphenyls from Sarcodon leucopus.
The first two isolated compounds were 10 and 11. It is evident from these reports how the correct identification of the species is important for preventing mistakes in the identification of new secondary metabolites.
Later, the same research group isolated other nitrogen-containing p-terphenyl derivatives that they named sarcodonins. The first to be isolated was 12 in 2000 (Geraci et al., 2000) and other six related compounds (13-18) were isolated in (Cali et al., 2004 (Fig. 9).  Compounds 13-15 were named sarcodonin , , and , respectively, while compounds 16-18 epi-sarcodonin, epi-sarcodonin  and , respectively. Cali et al., (2004) from the same mushroom isolated oxidated sarcodonins and named them sarcoviolin (19) and epi-sarcoviolin (20) (Fig. 10). Recently the structures of sarcodonins and sarcoviolins have been revised by synthesis, and the new structure for sarcodonin 12 is reported in Fig. 11 (Lin et al., 2011). It is very interesting to notice that screening the literature with the term "sarcodonin" different structures with the same name have been reported, as we will see checking the compounds isolated from S. scabrosus.

Sarcodon scabrosus (Fr.) P. Karst.
Myco Bank describes S. scabrosus "with pileus up to 120 mm across (fresh), plano-convex, more or less deeply depressed in the centre, coarsely scaly, the scales erect in the centre, decumbent farther outwards, adnate and woolly near the margin, brown in various shades (brick-colour, fulvous, ferruginous, bay) on a fairly pale yellow-brown ground, in some specimens passing into a delicate lilac at the margin, with age becoming very dark purplish brown, and the scale-tips even blackish, somewhat shiny when dried. Stipe 20-120 x 10-60 mm (fresh), tapering downwards, usually with pointed base, felted to subfibrillose, pinkish brown to brick-colour, becoming concolorous with the pileus, the lower part or the base grey-green, when young covered with white mycelium. Spines up to 5 mm long (dry), slender (up to 0.3 mm), decurrent, crowded, subulate, long remaining yellowish brown, finally purplish brown. Context dingy whitish in the pileus and the top of the stipe, vinescent, brownish-marbled with age, grey-green in the base of the stipe. Greenish mycelium from the base of the stipe staining dingy pinkish brown to reddish brown in KOH solution. Odour of water-melon (Citrullus vulgaris) when cut fresh" (Fig.  12).
Due to its bitter taste it is not edible.

Phytochemistry of Sarcodon scabrosus
An analysis of the literature revealed that Sarcodon scabrosus (syn Hydnum scabrosus), together with S. imbricatus, is one of the most studied species among Sarcodon. Two classes of secondary metabolites have been isolated from this species, and both are represented by cythane diterpenes: scabronines and sarcodonins. The use of the same name for different classes of secondary metabolites clearly shows how the use of common names can give rise to mistakes.
The first report about the isolation of cyathane diterpenes from S. scabrosus was by Shibata et al. (1989) and it is about sarcodonin A (21) and G (22) (Fig. 13).

Biological activities of p-terphenyl derivatives
Terphenyls are aromatic hydrocarbons consisting of a chain of three benzene rings. From a structural point of view three different isomers are possible, in which the terminal rings are ortho-, meta-, or para-substituents of the central ring. Most of the natural terphenyls are pterphenyl derivatives. In recent years, some terphenyls have been reported to exhibit significant biological activity, e.g., potent immunosuppressant, neuroprotective, antithrombotic, anticoagulant, specific 5-lipoxygenase inhibitory, and cytotoxic activities (Liu, 2007).

Antibiotic properties
Compounds 10 and 11 (Tringali et al., 1987) were tested against several Gram-positive and Gram-negative bacteria in an agar dilution test and the results showed that among Grampositive the most susceptible microorganism was Proteus mirabilis and among Gramnegative Staphylococcus aureus (MIC 75 and 50 g/ml respectively).

Antitumoral activity
Sarcodonin (12) was tested against two different tumor cell lines and it resulted moderately active toward KB (ED50 10.0 g/ml) and P-388 (ED 50 27.0 g/ml) (Geraci et al., 2000). The higher cytotoxic activity of 10 with respect to 12, coupled with mild antibacterial activity already described (Tringali et al., 1987) suggest that 10 could play a role in the chemical defense of the mushroom.

Biological activities of cyathane diterpenoids
In 1965 Brodie discovered a new bird's nest fungus of the genus Cyathus that was named C. helenae (Brodie, 1966) and he showed that the metabolites of the mushroom (cyathine complex) possessed antibiotic activity (Allbutt et al., 1971). From this first report, in these 40 years, a great number of cyathane diterpenoids have been isolated, structurally identified and tested for their biological activities.

Antibiotic activity
In 1998 Shibata (Shibata et al., 1998) and coworkers isolated from S. scabrosus sarcodonins L and M and sarcodonin A (21) and G (22). Sarcodonin L and M resulted to be identical to scabronines C (25) and B (24), respectively (Kita etal., 1998). All these compounds were tested against B. subtilis and S. aureus and the results are reported in Table 1. www.intechopen.com

Antiproliferative and antitumoral activities
The first report about the antitumor activity of a cyathane diterpene was about sarcodonin G isolated from the dichloromethane extract of Sarcolon scabrosus Karst, on HeLa cells in vitro. Sarcodonin G (22), isolated from the mushroom Sarcodon scabrosus and already reported to have anti-inflammatory activity, inhibited proliferation of HeLa cells (Dong et al., 2009). www.intechopen.com

Stimulation of NGF (Nerve Growth Factor)
Sarcodon scabrosus and S. cyrneus metabolites have been studied for their unique activity to act as neuroprotective agents being able to stimulate the production of neurotrophic factors in vitro.  have been tested for their activity to induce NGF secretion from 1321N1 human astrocytoma cells and induce neuritogenesis in PC12 cells (rat pheochromocytoma cells) (Ohta et al., 1998a(Ohta et al., , 1998bKita et al., 1998;Oshima, Y. et al., 1999). Scabronines increased the expression of mRNA for NGF, and the secretion of NGF from 1321N1 cells in a concentration-dependent mechanism . Among different scabronines, scabronine G methyl ester (SG-ME) (31) resulted to be the most active and the mechanism of action was deeply studied. SG-ME activates PKC-ζ, induces the translocation NF-κB to nucleus and enhances its transcriptional activity (Fig. 17) (Obara et al., 2001).
Cyrneine A-D (1-4) and glaucopine C (7) were tested to evaluate their activity to induce NGF production from 1321N1 cells, but they resulted much less active than scabronine G (Fig. 18) . Neverthless, cyrneine A and B showed an interesting activity, being able to induce directly differentiation on PC-12 cells (Marcotullio et al., 2006b).  Fig. 18. Evaluation of NGF synthesis from 1321N1 induced by cyrneines and glaucopine C. SG-ME used as control.

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As a transcriptional regulation is required for neurite extension, and the activity of three major transcription factors (activator protein-1 (AP-1), nuclear factor-κB, and CREB) was determined. Cyrneines A and B enhanced activation of AP-1 and NF-κB. Moreover, treatment with cyrneine A led to actin translocation and subsequently, to accumulation of Factin at the tip of neurites. Rac1 activity was increased by cyrneine A and expression of a dominant-negative Rac1 mutant significantly inhibited the cyrneine A-induced extension of neurites. These results suggest that cyrneine A induces neurite outgrowth in a Rac1dependent mechanism .

Conclusions
The analysis of the literature about Sarcodon genus showed that these mushrooms produces interesting biologically active secondary metabolites such as cyathane e triphenyl derivatives. It is interesting to note that the use of trivial names instead of systematic ones generated some confusion (e.g. the term sarcodonins has been used to name both terphenyls and cyathanes). Furthermore, it was evidenced that sometimes there are problems in the use of different synonyms for the same species. Neverthless, the interesting biological properties of Sarcodon metabolites and the nutritional value of some spp make this genus worthy of further investigations.