P. badius, P. gilvus and P. rimosus were collected at the National Forest Reserve and Wildlife Refuge Ajos-Bavispe, located at Sonora, Mexico. Sampling was performed seasonally from fall 2004 to summer 2005. Phellinus specimens were collected and conserved following conventional mycological techniques. Taxonomic identification was reached according to Gilbertson and Ryvarden8 and Larsen & Cobb-Poulle.14 Three major species were identified as P. badius, P. gilvus and P. rimosus. Ten organisms with an approximate weight of 100g per organism per species were selected and grouped. Mycelia from each organism of each species were mixed, freeze dried and vacuum stored at −35°C until extraction.

Methanolic extracts of Phellinus spp. were obtained as follows: 1g of freeze-dried sample was mixed in 15mL of 80% (v/v) methanol, and homogenized with an Ultra-Turrax T 25 basic (IKA® WERKE, Germany), thereafter the homogenate was sonicated for 15min and centrifuged at 10,000rpm at 5°C for 15min. The sample was vacuum-filtered through Whatman No. 1 filter paper. This procedure was repeated twice to ensure the maximum extraction of bioactive compounds using 20mL of methanol. The extracts were collected and made up to final volume of 50mL. The final concentration of the extract was 0.02g/mL, which was used for total phenolics, total flavonoids and antioxidant activity determinations.

Concentrations of total phenolic compounds were measured by the methods described by Singleton and Rossi26 with some modifications. Extracts (50μL) were mixed with 3mL of H2O, and 250μL of Folin–Ciocalteu's phenol reagent 1N. After 8min of equilibration time, 750μL of Na2CO3 (20%) and 950μL of H2O were added to the extracts. After incubation for 30min at room temperature, the absorbance was read at 765nm with an UV–vis spectrophotometer (Cary, model 50 Bio, Varian, Italy). Concentration of total phenol compounds was calculated using a standard curve of gallic acid equivalents (GAE) and expressed as milligrams per g of dry weight.

Flavonoid content was determined based on the methods described by Zhishen et al.32 modified as required. Flavonoids were measured in 1mL of each methanolic extract and it was mixed with 5% NaNO2, 10% AlCl3 and 1mol/L NaOH and measured spectrophotometrically at 415nm using quercetin as standard. The results were expressed as mg of quercetin equivalents (QE) per g of dry weight.

This experiment was based on a completely randomized design with equal replications. Analysis of variance for the treatments was done using Number Cruncher Statistical System version 6.0 software.11 Mean comparisons of the studied parameters among treatments were done using the least significant difference test at a 5% level (P<0.05).

Eleven macrofungus Phellinus species were found in the Ajos-Bavispe natural protected area, three of them were more commonly collected and studied in the present study. P. badius (Berk. ex Cooke) G. Cunn. was found in subtropical scrub and mezquital (plants from the genus Prosopis). It has a circumglobal distribution in the tropics and subtropical regions.8P. gilvus (Schwein.) Pat. was collected in pine-oak wood, oak wood and mezquital (plants from the genus Prosopis). It is a common fungus on living and dead hardwoods in many genera, and it is more common to be found on oaks. P. rimosus (Berk.) Pilát was observed in pine-oak forest, subtropical scrub and mezquital (plants from the genus Prosopis). This macrofungus has been commonly collected in the Sonoran mycobiota.19

Table 1 shows the contents of total phenolic compounds expressed as gallic acid equivalents determined for P. badius, P. gilvus and P. rimosus, respectively. A significant difference (P<0.05) was observed among P. gilvus with the highest total phenolic content (49.31mgGAE/g), followed by P. rimosus (46.51mgGAE/g) and P. badius (44.76mgGAE/g). Similarly, the total flavonoid content of Phellinus methanolic extracts showed the same behavior already described for phenolic compounds (Table 1), where P. gilvus presented the highest flavonoids content (30.58mgQE/g), followed by P. rimosus (28mgQE/g) and P. badius (26.48mgQE/g).

Comparison with other kind of macrofungus reported in previous publications showed that Phellinus spp. from Sonora contains higher levels of phenolic compounds. The phenolic content of Leucopaxillus giganteus, Sarcodon imbricatus, and Agaricus arvensis was 6.29, 3.76, and 2.83mg/g, respectively.3 However, Phellinus baumii samples presented significantly higher levels of phenolic compounds (338μg/mL).18 In contrast, other species of fungus such as Endoptychum arizonycum and Disciseda verrucosa presented lower values of phenolic compounds (9.81 and 16.1mg/g, respectively; unpublished data). Total phenolic and flavonoid contents of different solvent extracts of Phellinus igniarius ranked from 6 to 23mg/g of extract, and 0.05 to 4.26mg/g, respectively.17 Clearly the phenolic content of P. badius, P. rimosus and P. gilvus are higher than those published values in other macrofungus species.

Phenolic compounds are one of the most widely occurring groups of phytochemicals which are of considerable physiological and morphological importance in fungi and plants.12,20 These compounds play an important role providing protection against pathogens and predators.20 In humans, phenolic compounds can exhibit a wide range of physiological properties, such as anti-allergenic, anti-artherogenic, anti-inflammatory, antimicrobial, antioxidant, anti-thrombotic, cardioprotective and vasodilatory effects.12,13 The beneficial effects derived from phenolic compounds have been attributed to their antioxidant activity.12

All the macrofungus methanol extracts showed high DPPH radical scavenging activity (Table 1) as expected considering the total phenolic content. The highest antioxidant scavenging effect (90.8%) was showed by the P. gilvus extract, followed by P. rimosus extract (84%) and P. badius extract (77.0%) at a concentration of 0.02g/mL. The antiradical activity evaluated as the efficient concentration of each extract to inactivate the 50% of the DPPH radical (EC50) was 0.009g/mL for P. gilvus, 0.01g/mL for P. rimosus and 0.013g/mL for P. badius. The EC50 values also indicated that the antioxidant activity of the P. gilvus extract was higher than that of P. rimosus. The antiradical scavenging activity might be attributed to the replacement of hydroxyl groups in the aromatic ring systems of the phenolic compounds as a result of their hydrogen donating ability.4

Analysis of ABTS radical scavenging activity of the methanolic extracts of Phellinus spp. expressed as trolox equivalent antioxidant capacity (TEAC) (Table 1) showed the same pattern than that of DPPH assay. P. gilvus extract presented significantly (P<0.05) higher antioxidant activity (266μmolTE/g), followed by P. rimosus (212μmolTE/g), and P. badius (122μmolTE/g). The oxygen radical absorbance capacity (ORAC) values confirmed these results (Table 1), being P. gilvus (7470μmolTE/g) the more potent antioxidant extract, followed by P. rimosus (1848μmolTE/g) and P. badius (1704.35μmolTE/g). All the tested extracts were significantly different (P<0.05) among them.

The methanolic extract of Agrocybe cylindracea strain B exhibited a DPPH scavenging ability of 93.8% at 0.005g/mL.28 Tsai et al.29 found that scavenging abilities of three ethanolic extracts were 94.9%, 89.2%, and 88.8% at 0.005g/mL for Agaricus blazei, A. cylindracea, and Boletus edulis, respectively. Furthermore, ethanolic extracts of Pleurotus citrinopileatus15 and Hypsizgus marmoreus16 showed scavenging abilities of 94.9% and 59.7% at 0.005g/mL, respectively. Our results suggest that the scavenging ability of P. gilvus was higher than that of P. rimosus and P. badius; however, an effect of the solvent extraction method could explain the differences when compared with other studies.

The antioxidant activity of phenolic compounds is due to their ability to scavenge free radicals, donate hydrogen atoms or electron, or chelate metal cations. The structure of phenolic compounds is a key determinant of their radical scavenging and metal chelating activity. The position and number of hydroxyl groups in the phenolics and flavonoids determine the capacity of the molecules to donate an electron and stabilize free radicals. Therefore, antioxidants have the ability to scavenge free radicals and prevent diseases caused by these reactive species. Antioxidants from Phellinus sensu lato are composed of hispidin and its derivatives.13 Also, the catechol moiety is responsible for inhibiting radical generation by chelating metal ions.13 Therefore, these phenolic compounds could have synergistic effects and be responsible of the high antioxidant capacity of the studied Phellinus extracts.

In this study, total antioxidant component contents (phenolic and flavonoids) were positively correlated with the values of antioxidant activity. The correlation coefficients (R2) of total phenolic compounds versus DPPH, TEAC, and ORAC were 0.9576, 0.9014, and 0.8606, respectively (Fig. 1). However, the correlation coefficients of total flavonoids versus DPPH, TEAC, and ORAC were slightly lower at 0.9214, 0.8840 and 0.8155, respectively (Fig. 2). In addition, an excellent correlation between contents of total phenolics and antioxidant activity was also obtained and pointed out that the antioxidant activity of phenolics was mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donators and single oxygen quenchers.23 Consequently, total phenols in the three methanolic extracts from Phellinus spp. were possibly the major components that contribute in higher extent to their antioxidant activities.

Fig. 1.

Correlations of total phenolic compounds and antioxidant capacity values, measured by DPPH, TEAC, and ORAC methods.

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Fig. 2.

Correlations of total flavonoid compounds and antioxidant capacity values, measured by DPPH, TEAC, and ORAC methods.

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The antifungal potential of methanolic extracts of P. badius, P. gilvus and P. rimosus was determined (Fig. 3). Their antifungal potential was tested against A. alternata, a mold highly related to plant food spoilage. The three tested macrofungus extracts were effective to inhibit the growth of A. alternata, however, no significant differences among them were observed. The antifungal activity of all the extracts did not show a concentration-dependent behavior and no significant (P>0.05) effect of concentration was observed.

Fig. 3.

Antifungal activity of methanolic extracts from Phellinus spp. against Alternaria alternata. Different letters among bars indicate significant differences among means (P<0.05).

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The antimicrobial activities of a variety of naturally occurring phenolic compounds from fungi have been studied.2 These compounds can play an important role in tissue's protection against pathogenic agents, penetrating the cell membrane of microorganisms, causing lysis.12,13 Phenolic compounds from spices such as gingeron, zingerone, and capsaicin have been found to inhibit the germination of bacterial spores.5 Polyphenols contained in green tea (Camellia sinensis) are effective against Vibrio cholerae O1, Streptococcus mutans, and Shigella.25 The antimicrobial activity of other Phellinus extracts has not been widely evaluated, only two reports from antibiotic activity of Phellinus fastuosus and Phellinus ribis were found in the literature.7 In this context, the mixture of phytochemical constituents in methanolic extracts of Phellinus spp. can be responsible for their antioxidant and antifungal activity.1