As the first step in screening antiviral activity, the cytotoxicity of various concentrations of mycelial extracts was evaluated using an inhibition assay in MDCK and RK-13 cell plaques (with similar cytopathic effect). A maximum tolerated concentration of 25.0 mg/mL was determined for four species (A. aurea, F. fomentarius, F. velutipes, and T. versicolor), while the extracts of the other species, particularly two edible mushrooms (P. eryngii and L. edodes), were more toxic (Table 1).
Toxic dose of sample (mg/mL)
Toxic dose of sample (mg/mL)
50 25 12.5 6.2 3.1 1.55 0.77 50 25 12.5 6.2 3.1 1.55 0.77 Substrate* 10/10 0/10 0/10 0/10 0/10 0/10 0/10 10/10 0/10 0/10 0/10 0/10 0/10 0/10 F. fomentarius 10/10 0/10 0/10 0/10 0/10 0/10 0/10 10/10 0/10 0/10 0/10 0/10 0/10 0/10 F. velutipes 10/10 0/10 0/10 0/10 0/10 0/10 0/10 10/10 0/10 0/10 0/10 0/10 0/10 0/10 A. aurea 10/10 0/10 0/10 0/10 0/10 0/10 0/10 10/10 0/10 0/10 0/10 0/10 0/10 0/10 T. versicolor 10/10 0/10 0/10 0/10 0/10 0/10 0/10 10/10 0/10 0/10 0/10 0/10 0/10 0/10 P. ostreatus 10/10 10/10 0/10 0/10 0/10 0/10 0/10 10/10 10/10 0/10 0/10 0/10 0/10 0/10 S. commune 10/10 10/10 0/10 0/10 0/10 0/10 0/10 10/10 10/10 0/10 0/10 0/10 0/10 0/10 G. lucidum 10/10 10/10 10/10 0/10 0/10 0/10 0/10 10/10 10/10 10/10 0/10 0/10 0/10 0/10 L. shimeji 10/10 10/10 10/10 10/10 0/10 0/10 0/10 10/10 10/10 10/10 10/10 0/10 0/10 0/10 P. eryngii 10/10 10/10 10/10 10/10 10/10 0/10 0/10 10/10 10/10 10/10 10/10 10/10 0/10 0/10 L. edodes 10/10 10/10 10/10 10/10 10/10 0/10 0/10 10/10 10/10 10/10 10/10 10/10 0/10 0/10 0/10: no cytopathic effect; 10/10: cytopathic effect (complete destruction of monolayer cells). *: 60 g amaranth in 1 L distilled water (the liquid culture medium). All experiments were confirmed in three independent replicates.
Table 1. Cytotoxicity of mycelial extracts from of MDCK and RK-13 cells
The investigated mycelia had different potential antiviral activities against influenza virus strain A/FM/1/47 (H1N1), with inhibition of infectious titers ranging from 2.0 to 6.0 lg ID50 . Antiviral activity according to the inhibition of infectious titer increased in the following order: A. aurea=F. fomentarius > P. ostreatus=L. shimeji=L. edodes > P. eryngii=F. velutipes=G. lucidum > S. commune > T. versicolor. G. lucidum and T. versicolor generated the strongest antiviral effects, with T. versicolor showing the highest activity (Table 2). The anti-influenza activities of P. eryngii, L. shimeji, F. velutipes, and A. aurea have not previously been presented in the literature.
P. eryngii 1.55 5 0 L. shimeji 3.1 0.62 5.0 P. ostreatus 12.5 2.5 6.0 S. commune 12.5 0.62 20.16 L. edodes 1.55 0.077 20.12 F. velutipes 25 1.25 20.0 F. fomentarius 25 0.62 40.32 A. aurea 25 0.62 40.32 G. lucidum 0.2 0.077 80.5 T. versicolor 25 0.077 324.67 EC50: half maximal effective concentration; MTC: maximum tolerated concentration. All experiments were confirmed in three independent replicates
Table 2. Antiviral activity of samples in MDCK cells infected with influenza virus strain A/FM/1/47 (H1N1)
Only four of the 10 studied species demonstrated activity against HSV-2: mycelial extracts of P. ostreatus, F. fomentarius, A. aurea and T. versicolor significantly inhibited HSV-2 replication in RK-13 cells (Table 3). The highest therapeutic indices (selectivity indices) were identified for A. aurea and T. versicolor, at 161.29 and 324.67, respectively. This study is the first to demonstrate the activity of A. aurea mycelium against HSV-2.
S. commune 12.5 0 0 F. velutipes 25 0 0 P. eryngii 1.55 0 0 L. shimeji 3.1 0 0 L. edodes 1.55 0 0 G. lucidum 6.2 0 0 F. fomentarius 25 0.62 40.32 P. ostreatus 12.5 0.155 80.64 А. aureа 25 0.155 161.29 T. versicolor 25 0.077 324.67 EC50: half maximal effective concentration; MTC: maximum tolerated concentration. All experiments were confirmed in three independent replicates.
Table 3. Antiviral activity of samples in RK-13 cells infected with herpes simplex virus type 2, strain BH
Recently, the search for natural substances as raw materials for the pharmaceutical industry has revived interest in medicinal and edible mushrooms. While most studies have isolated therapeutically active substances from the fruiting bodies, the use of mycelium makes it possible to obtain products of consistent quality more quickly and at lower cost. Data regarding the absence of toxicity of various products have been obtained from cultivation, mainly on synthetic or semisynthetic substrates. The mycelium toxicity (non-critical) obtained in our studies (Table 1) can be related to the natural substrate (its toxicity), has been used by us.
The influenza and herpes viruses have been a particular focus of research. The antiviral activity of mushroom preparations has been evaluated by researchers using different indicators, including the index of neutralization (Amoros M, et al., 1997; Ibragimova Zh D, et al., 2012; Teplyakova T V, et al., 2012; Filippova E I, et al., 2012; Kostina N E, et al., 2013) and the therapeutic index (Eo S-K, et al,. 1999; Oh K-W, et al,. 2000; Liu J, et al., 2004; GuC-Q, et al., 2007; Cardoso FTGS, et al., 2011). In the current study, we have shown that the magnitude of the neutralization index is not directly proportional to the magnitude of the therapeutic index (Table 2 to 4). This is to be expected, since the therapeutic index is the ratio of the minimal effective dose of a chemotherapeutic agent to the maximal tolerated dose, and this ratio can be very small (even if the neutralization index is large). So, water- and methanol-soluble substances isolated from the carpophores of G. lucidum have shown antiviral activity against influenza A virus A/Equine/2/Miami/1/63 strain, but the therapeutic index was zero (Eo S-K, et al,. 1999). In contrast, the current study found a high therapeutic index value (80.5) for G. lucidum mycelial activity against influenza virus strain A/FM/1/47 (H1N1). According to our data, F. fomentarius mycelium shows similar activity (Table 4) to preparations from F. fomentarius 11-72, which has been reported to inhibit influenza virus A/Aichi/2/68 in MDCK cell culture at 2.4 lg (Ibragimova Zh D, et al., 2012). The fruiting bodies of T. versicolor have been reported to slightly inhibit influenza virus A/Chicken/Kurgan/05/2005 (H5N1) in vivo (Ibragimova Zh B, et al., 2012). Our results (Table 4) for T. versicolor 353 mycelium were significantly higher (6.0 lg) than those reported in similar studies: water extracts of T. versicolor 2263 mycelium have been reported to repress viruses A/Chicken/Kurgan/05/2005 (H5N1)(2.5 lg) and A/Aichi/2/68 (H3N2)(0.5 lg) on MDCK cells (Teplyakova TV, et al., 2012). In the current study, L. shimeji, L. edodes and P. ostreatus mycelia showed neutralization indices for influenza virus strain A/FM/1/47 (H1N1) that were similar to the values obtained by Filippova EI, et al.(2012) for fruiting bodies of Ganoderma applanatum, Inonotus obliquus and Laetiporus sulphureus against pandemic influenza virus A/Moscow/226/2009 (H1N1).
Sample virus H1N1, strain A/FM/1/47 virus HSV-2, strain BH Infectivity of the virus (VAF titer) in MDCK cells
(ID50 in lgTCID50/mL)
(ID50 control – ID50 exp.), lg
Infectivity of the virus (VAF titer) in RK-13 cells
(ID50 in lgTCID50/mL)
(ID50 control – ID50 exp.), lg
P. eryngii 2.0 4.0 6.0 0 L. shimeji 3.0 3.0 6.0 0 P. ostreatus 3.0 3.0 3.5 2.5 S. commune 1.0 5.0 6.0 0 L. edodes 3.0 3.0 6.0 0 F. velutipes 2.0 4.0 6.0 0 F. fomentarius 4.0 2.0 3.0 3.0 A. aurea 4,0 2.0 4.0 2.0 G. lucidum 2.0 4.0 6.0 0 T. versicolor 0 6.0 0 6.0 Substratea 6.0 0 6.0 0 Control (virus) 6.0b – 6.0c – Note: HSV-2, herpes simplex virus type 2; TCID50, median tissue culture infective dose. The standard deviation for the reduction of virus titer was approximately 0.5 log10. a: 60 g amaranth in 1 L distilled water (the liquid culture medium). b: virus H1N1, strain A/FM/1/47; c: virus HSV-2, strain BH. All experiments were confirmed in three independent replicates.
Table 4. Antiviral activity of samples
Our data demonstrating sufficiently high antiherpetic activity of P. ostreatus mycelium (neutralization index of 2.5 lg and therapeutic index of 80.64; Table 3 and 4) are in contrast to those indicating an absence of antiherpetic activity for P. ostreatus fruiting bodies in cell culture (Amoros M, et al., 1997). Our data also show significant antiherpetic activity for F. fomentarius mycelium (neutralization index 3.0 lg and therapeutic index 40.32; Table 3 and 4), in contrast to data showing the absence of such activity in fruiting bodies of this fungus in cell culture (Kostina N E, et al., 2013). Conversely, G. lucidum mycelium did not show antiherpetic action in our studies, unlike the results of other researchers who used polysaccharide–protein complexes isolated from G. lucidum fruiting bodies and mycelia, including APBP (activity against HSV-2 strain 233)(Oh KW, et al., 2000) and proteoglycan (activity against HSV-2 G strain ATCC VR-734) on Vero cells (Liu J. et al, 2004.). The selectivity index values for P. ostreatus, A. aurea, and T. versicolor mycelia were significantly higher in our studies than in similar experiments with G. lucidum mycelium against HSV-2 (Oh KW, et al., 2000; Liu J, et al., 2004). Our results indicate antiherpetic activity of T. versicolor mycelium, in contrast to other investigations with fruit bodies of this species (Amoros M, et al., 1997; Kostina NE, et al., 2013). Moreover, in the current study, T. versicolor showed the highest therapeutic index (324.67) among the studied fungi. A clinical trial in which a food additive (biomass) of T. versicolor reduced the frequency and even stopped outbreaks of HSV-2 in pregnant patients (French A, 2007) confirms the high antiherpetic activity of this fungus.
It should be noted that the effective doses of P. ostreatus and A. aurea mycelium for HSV-2 neutralization were significantly lower than those for influenza virus A, with 13-fold higher antiherpetic activity than anti-influenza activity for P. ostreatus and fourfold higher antiherpetic activity for A. aurea. In contrast, the therapeutic indices of F. fomentarius and T. versicolor were identical for both viruses. Such differences may be caused by mushroom species specificity, variability in the biologically active substances, and different mechanisms of antiviral activity at the interaction between the mycelia and the virus. The high efficacy of viral replication inhibition might hint that the inhibitory activity of the tested substances occurs late in viral replication via impairment of viral protein synthesis.
This study of the antiviral activity of the mycelia of 10 mushroom species suggests that A. aurea, F. velutipes, F. fomentarius, G. lucidum, L. edodes, L. shimeji, P. eryngii, P. ostreatus, S. commune, and T. versicolor have antiviral activity against influenza virus A/FM/1/47 (H1N1), while A. aurea, F. fomentarius, G. lucidum, and T. versicolor have antiviral activity against HSV-2, strain BH. For some of the investigated species, this is the first report of anti-influenza (P. eryngii, L. shimeji, F. velutipes, A. aurea) and antiherpetic (A. aurea) effects. To the best of our knowledge, there have been no previous reports on the potential medicinal properties of A. aurea.
The wood-decaying medicinal Basidiomycete T. versicolor showed the highest therapeutic index (324.67 for both viruses). Therefore, T. versicolor 353 and its biologically active substances may be promising source materials for the pharmaceutical industry as antiinfluenza and antiherpetic agents with low toxicity. The use of products obtained in the biotechnological processing of agricultural waste (in this case, amaranth flour after CO2 extraction) and its conversion by fungi is one of the first steps in this direction of investigations, which deserves to be further explored. Further investigations will be needed to determine the most effective solvent for extracting biologically active mycelial substances; to study the qualitative and quantitative composition, antiviral activity, and mechanisms of antiviral activity of the extracted mycelial substances; and to confirm the effectiveness of T. versicolor mycelium in vivo.