Infectious diseases represent one of the major threats worldwide due to the growing prevalence of drug resistance microorganisms (Wright, 2010). Within infectious diseases, the dermatologic infections are a public health problem, since it is estimated that between 30-70% of the world population is affected by at least one type of skin disease (Hay et al., 2014). Bacteria and fungus are considered responsible for dermatologic infections in humans, among the most frequents are Staphylococcus aureus, Streptococcus pyogenes and the species of the genus Candida (Riain, 2013). These microorganisms have been developed resistance to some commercial’s antibiotics. For this reason, it is necessary to continue the search for new compounds capable of their inhibition. Whereas the damage derived by the uncontrolled production of free radicals promotes the development of diseases like cancer, rheumatoid arthritis, cirrhosis, arteriosclerosis, and degenerative processes associated with aging (Elmastas, Isildak, Turkekul & Temur, 2007, Ren et al., 2014). Preliminary research has shown that macromycetes fungi produce a wide variety of bioactive compounds like phenolic compounds, which display an important role in the protection against oxidative damage, and the defense against bacteria, viruses, and insects (Leyva, Pérez- Carlón, González-Aguilar, Esqueda & Ayala-Zavala, 2013).

Macromycetes fungi have been recognized worldwide as sources of medicine and food (Quiñónez-Martínez et al., 2014). In Mexico, they have been incorporated into the diet of some ethnic groups and have been used for the treatment of different diseases since prehistoric times (Ruiz, Pérez-Moreno, Almaraz-Suárez &Torres-Aquino, 2013). Specifically, the Sierra Tarahumara of Chihuahua, Mexico has been known to have around 500 species of macromycete fungi (Gómez-Flores et al., 2019). Some of which have been reported to have medicinal and nutritional characteristics, like A. hygrometricus and L. perlatum, whose carpophores are used for acne problems as well as pain relievers, and burns cuts swelling (Quiñónez-Martínez et al., 2014). Likewise, L. laccata and A. rubescens show relevance due to their nutritive value, the last one is collected by the inhabitants for consumption and sale. Although there are many studies about the antimicrobial and antioxidant activities of macromycete fungi in the world, there is little information available about the traditional knowledge of mushrooms from the Sierra Tarahumara at Chihuahua, Mexico. The objective of this study was to evaluate the antimicrobial activities, the phenolics compounds and the antioxidant activity of four species of macromycete fungi from Chihuahua.

Macromycete fungi represent a source of bioactive compounds that can be beneficial to humans (González-Barranco et al., 2009), it is known that their compounds could present different pharmacological activities like antimicrobial and antioxidant activities (Ren et al., 2014). In our study, we found that the four studied mushrooms species presented both activities. The antimicrobial activity of the mushrooms was tested using two different methods, disk diffusion assay and minimum inhibitory concentration (MIC). Only three macromycete fungi (A. hygrometricus, L. laccata and L. perlatum) presented inhibitory action against S. agalactiae and Candida sp. in the disk diffusion method. Nonetheless, employing the MIC method, all the mushroom extracts shown antimicrobial activity against the three-microorganism tested, which coincides with other studies that report greater effectiveness of the broth dilution method compared to the disk diffusion method when evaluating the antimicrobial activity of fungal extracts (Ren et al., 2014, Hleba et al., 2016). The disk diffusion method, as its name implies, depends primarily on the diffusion capacity of the substance present in the extracts (Ren et al., 2014), resulting in little mobility of the compounds in the agar due to its low solubility or high molecular mass.

The solvent with the best results was ethanol. This may be due to the differences in polarity with methanol and dielectric constant, which allows increasing the solubility of compounds with antimicrobial activity, such as polyphenols (Adhikari, Pandey, Agnihotri & Pande, 2018; Do et al., 2014).

The best antimicrobial activity was presented byA. hygrometricus and L. perlatum, due to the similarity of recorded values. For the disk diffusion method, the high inhibition zones were obtained by the ethanolic extract of L. perlatum against S. agalactiae (8.25 mm). These results accord with those found by Dulger (2005) who reports the antimicrobial activity of the ethanolic extract of L. perlatum, but against S. pyogenes. There are several compounds to which antimicrobial activity in L. perlatum can be attributed, such as those found in ethanolic and methanolic extracts, mainly phenolic compounds, alkaloids, carbohydrates, tannins, saponins, glycosides, and proteins (Akpi, Odoh, Ideh & Adobu, 2017).

Astraeus hygrometricus obtained the best inhibition values against Candida sp. since it was the only one capable of developing inhibition zones (4.5 mm) and the one that reported lower concentrations of the yeast by the MIC method. The antimicrobial activity of A. hygrometricus has been demonstrated in previous studies, Giri et al. (2012) reported positive zones of inhibition when extracting carpophores with methanol at a concentration of 10 mg/mL. Also, according to Lai et al. (2012), the constituents responsible for the inhibition against C. albicans in A. hygrometricus are two triterpenes known as astrakurkurol and astrakurkurone.

In the case of L. laccata, its extracts exhibited similar inhibition actions, this could be attributed to the phenolic compounds found in the carpophores (8.75 mg GAE/g DW), and in addition to the fatty acids found in the petroleum ether and ethyl acetate fractions from the ethanolic extract of L. laccata carpophores, such palmitic acid, linoleic acid, and oleic acid (Nieto & Cucaita, 2007), which have been previously reported as compounds with antimicrobial activity.

The extracts of A. rubescens did not show inhibition of the microorganisms through the disk diffusion method since this method is not appropriate to test partially or completely hydrophobic compounds (Ren et al., 2014), this specie is rich in compounds with such nature (Kalač, 2013). Among the majority compounds in the species of the Amanita genus are some peptides (Li & Oberlies, 2005), which have low solubility in a solid medium such as agar, but they manage to diffuse properly into a liquid (dilution in broth). Although A. rubescens was the specie with the highest content of total phenols (20.93 mg GAE/g DW), the low antimicrobial activity may be due to the existence of compounds linked to phenols that make them more hydrophilic, such as sugars, polysaccharides, lignins, amines, long-chainalcohols, and glycerol, as well as long-chain omega fatty acids (Naczk & Shahidi, 2006), making it difficult to diffuse during tests of antimicrobial activity. A. rubescens is the species with the least inhibitory effect using the MIC assay, along with other species of the same genus such as A. muscaria and A. phalloides which also exhibit a moderate MIC of 6.25 mg/mL against S. pyogenes (Chelela, Chacha & Matemu, 2014). S. agalactiae was the microorganism most susceptible to the inhibitory action of macromycetes, these results agree with several reports (Canli, Akata & Altuner, 2016; Alves et al., 2013). In general, some studies indicate that fungal extracts have a greater growth inhibitory effect on species belonging to the genus Streptococcus than other microorganisms, mainly in the species of S. pyogenes, S. mutans and S. sobrinus with MIC values ranging from 2.4 to 87.4 µg/mL(Doǧan, Duman, Özkalp & Aydin, 2013; Lund et al., 2009). Therefore, it is considered the microorganism more susceptible to most antibiotics, since there are few strains with characteristics of resistance to penicillin, ampicillin, and cefotaxime (Torres & Cercenado, 2010), making possible the action of the evaluated extracts against the microorganism in the present study.

Staphylococcus aureus did not show susceptibility since it is considered a microorganism with phenotypes resistant to different antibiotics such as beta-lactams and methicillin (Torres & Cercenado, 2010). However, the phenotype used in these tests of antimicrobial activity is unknown. There are studies about the null activity of fungal extracts against S. aureus such as that of Canli et al. (2016), who found no inhibition by the ethanolic extracts of Lycoperdon lividum; Barros, Venturini, Baptista, Estevinho & Ferreira (2008), whose extracts did not show antimicrobial activity using L. perlatum, and Giri et al. (2012), reporting no inhibition of the bacteria with methanolic extracts of A. hygrometricus.

Phenols compounds are widely distributed in plants and fungal species. Some studies mention that the total phenol content in fungi ranges between 6.08 and 24.85 mg GAE/g (Prasad, Varshney, Harsh & Kumar, 2015), although in the present investigation results are found in a range of 1.54 - 20.93 mg GAE/g DW. According to Mujić, Zeković, Lepojević,Vidović & Živković (2010), the range obtained of total phenolics compounds for mushrooms are in a range of 7.8 - 23.07 mg GAE/g, and for edible mushrooms it goes from 4.94 to 35.56 mg GAE/g (Alispahić et al., 2015). Even though our results are similar and are within the range reported, it is difficult to compare the results due to differences in the mode of expression of the results, extraction methods of the phenolic compounds in the fungi, and environmental conditions from the collection site.

Amanita rubescens (20.93 mg GAE/g DW) presented a high content of total phenols, which differs from that found by Keleş, Koca & Gençcelep (2011), who reported a content of 5.7 mg GAE/g DW. However, there are reports for other species of the genus Amanita with similar values than those found in the study, like A. patherina (5.27 mg GAE/g), A. muscaria (7.59 mg GAE/g), A. porphyria (14.53 mg GAE/g) and A. citrina (38.44 GAE/g) (Nowacka et al., 2015).

L. laccata and L. perlatum presented phenolic content in the range of those reported for this specie (8.75 and 6.05 mg GAE/g DW, respectively). Nowacka et al. (2014), found records like those of the study for L. laccata (9.38 mg GAE/g dry extract) and different for L. perlatum (13.59 mg GAE/g dry extract).According to Heleno, Barros, Sousa, Martins & Ferreira (2010), the variations found are mainly due to the substrate where the macromycetes grow, which causes modifications in the secondary metabolism of the species (shikimic acid and acetate pathway) affecting the production of phenolic compounds. In addition, the area and collection time also causes changes in the composition of total phenols (Smolskaite, Venskutonis & Talou, 2015). Astraeus hygrometricus was the species with the lowest content of total phenols (1.54 mg GAE/g DW) compared to the other species analyzed. These differences are mainly attributed to the extraction methods, due to the treatment of the carpophores with boiled water before quantification, maximizing the extraction of the compounds, since cooking results in an increase in total phenols (Pavithra, Sridhar, Greeshma & Tomita-Yokotani, 2016).

In general, fungi have great antioxidant activity (Álvarez- Parrilla et al., 2007; Elmastas et al., 2007), which is in a range that goes from 0.0741 to 7.61 mmol TE /100 g (Srikram & Supapvanich, 2016; Özyürek, Bener, Güçlü & Apak, 2014). However, in the present study lower values were found. The differences in the results of the antioxidant capacity are due to factors such as the analysis of different species, the area in which they are collected or the substrate where they grow (Heleno et al., 2010). As well as differences in extraction methods (since that there is still no standardization or consensus on the treatment of the sample before the analysis), among which are the extraction temperature and the relation of the solute with the solvent (Dai & Mumper, 2010). In the present investigation, the extraction temperature to assess antioxidant capacity was 24 °C and according to Özyürek et al. (2014), the optimal extraction temperature that improves the performance of antioxidant capacity in mushroom extracts is 80 °C, since an increase in temperature can accelerate cell rupture, generating a raise in internal pressure within the cells of fungi and promoting a higher solubility of the analyte (Dai & Mumper, 2010). Likewise, the use of solvents other than methanol may also show better results, since depending on the polarity of the solvents they will be able to extract various compounds that may have a high antioxidant capacity (Kosanić, Ranković, Rančić & Stanojković, 2016). Kaewnarin, Suwannarach, Kumla & Lumyong (2016), extracting the antioxidant compounds using ethanol, methanol, and water, with water shows highest antioxidant activity efficiency for the same methods evaluated (ABTS, DPPH, and FRAP).

ABTS assay measures the ability of the radical to donate a proton to an unstable cationic radical (ABTS•+), it is used to evaluate antioxidant capacity, making possible theanalysis ofhydrophilic and lipophilic compounds (Kaewnarin et al., 2016). The results obtained by this test showed that A. rubescens presents the highest averages of antioxidant capacity (0.0854 mmol TE/g DW), followed by L. laccata (0.0293 mmol TE/g DW), L. perlatum (0.0240 mmol TE/g DW) and A. hygrometricus (0.0074 mmol TE/g DW). This differs from that reported by Iwalokun, Usen, Otunba & Olukoya (2007), whose antioxidant activity (ABTS•+ radical) of Pleurotus ostreatus extracts was 4.4 millimol. However, they used acetone as a solvent, which can extract another class of antioxidant compound such as carotenoids and tocopherols (Liu, Jia, Kan & Jin, 2013), also the species was different.

DPPH test is considered one of the most widely used methods to evaluate antioxidant capacity because radicals are very stable and easy to use (Acharya, Khatua & Ray, 2017). The antioxidant activity of the extracts showed very similar averages among the species, with A. rubescens (0.043 mmol TE/g DW) being the species with the highest radical uptake, followed by L. laccata (0.0368 mmol TE/g DW), L. perlatum (0.0244 mmol TE/g DW) and A. hygrometricus (0.0135 mmol TE/g DW). The same behavior was observed with total phenols content, which coincides with that reported by Alothman, Bhat & Karim (2009), who mentioned that the higher the phenolic content, the higher the DPPH values. Recently Smolskaite et al. (2015), evaluated the antioxidant properties of eight macromycete species through different tests (ABTS, DPPH, and FRAP) using sequential extractions with solvents of different polarities (among which was methanol), whose valuesrangedfrom 0.12 to 9.62 µM TE/g DW for the DPPH assay is similar to the results obtained by the same method.

FRAP assay is based on measuring the ability of antioxidants to reduce ferric iron to its ferrous form (Kaewnarin et al., 2016). In this case, the highest value was presenting in L. perlatum (0.0298 mmol TE/g DW), followed by A. rubescens (0.0254 mmol TE/g DW), L. laccata (0.0078 mmol TE/g DW) and A. hygrometicus (0.0034 mmol TE/g DW). This differs from that reported by Srikram & Supapvanich, (2016), who obtained higher results in the species A. calyptroderma (1.98 mmol TE/100 g), A. princeps (1.14 mmol TE/100 g) and A. odoratus (07.61 mmol TE/100 g). In the same way, Yahia, Gutiérrez-Orozco & Moreno-Pérez (2017), reports higher reducing power averages for L. perlatum (17 mmol TE/100 g wet weight), A. flavoconia (7 mmol TE/100 g), A. pantherina (13 mmol TE/100 g) and A. virosa (6 mmol TE/100 g), which can be attributed to the same factors previously mentioned (different species, collection site, and extraction methods) (Kosanić et al., 2016; Nowacka et al., 2014). In our results, L. perlatum and A. rubescens were the species with the greatest reducing power, so it is believed that the phenolic compounds present in the species could have more hydroxyl-functional residues in the phenol rings than the other species, which act as electron donors in the reduction reaction (Liu et al., 2013).

Antioxidant capacity varied in a pattern like the phenolic compounds, thus the species with the highest antioxidant activity was A. rubescens, while A. hygrometricus obtained the lowest activity. According to Ferreira, Barros & Abreu (2009), the antioxidant activity of different species of macromycetes is mainly related to their phenolic content, since these compounds are known to be capable of donating hydrogen to free radicals to stop the lipid chain reaction in the initial stage (due to the presence of their hydroxyl groups) (Kosanić et al., 2016).

Some studies demonstrate a significant correlation between total phenols and antioxidant capacity using the radicals ABTS•+ and DPPH•, as well as those reported by different authors who mentioned correlation coefficients of 0.8 and 0.95 in several species of fungi (Kaewnarin et al., 2016; Smolskaite et al., 2015); confirming that phenolic compounds are important contributors to the antioxidant characteristics in fungal extracts. In the case of the ferric ion reducing ability (FRAP), there is no significant correlation, which indicates that not only are phenolic compoundsresponsible for theantioxidantactivityofthe species but also that other types of compounds with reducing power could be acting as antioxidants such as tocopherols, ascorbic acid, and carotenoids, which after phenolic compounds, are the main antioxidant compounds in macromycetes (Liu et al., 2013; Özyürek et al., 2014).

Finally, there is nodirectrelationbetween antimicrobialactivity with phenolic compounds and antioxidant activity. Since A. hygrometricus and L. perlatum showed to be the species with the best antimicrobial capacity, while A. rubescens turned out to be the species with the highest total phenol content and antioxidant capacity. These results are similar with some authors reporting that species with high phenol content and antioxidant capacity exhibit better results in tests of antimicrobial activity (Liu et al., 2013; Smolskaite et al., 2015). However, these differences may be due to the extraction methods used for each test, since in most of the studies they used the same extracts for the evaluation of the antimicrobial and antioxidant activity.

The evaluated macromycete fungal species presented antimicrobial activity, which varied depending on the solvent, the method used, and the microorganism tested, highlighting A. hygrometricus and L. perlatum. This finding promotes these species as an important source for obtaining compounds with pharmacological activities.

The optimal method to evaluate antimicrobial activity was through the minimum inhibitory concentration (MIC) technique since it allowed the correct diffusion of the extracted compounds.

The minimum concentration reached with the ethanolic extracts was of 8.75 mg/mL in A. hygrometricus and L. perlatum for S. aureus; 3.75 mg/mL for S. agalactiae with the same fungi previously mentioned, and 6.25 mg/mL for C. albicans using A. hygrometricus and L. laccata. On the other hand, for methanolic extracts the growth of S. aureus was inhibited at 8.75 mg/mL using A. hygrometricus; for S. agalactiae the MIC was 5mg/mL using A. hygrometricus, L. laccata and L. perlatum, finally, A. hygrometricus had the best response inhibiting C.albicans at a concentration of 6.25 mg/mL. Generally, the ethanol extracts exerted stronger antimicrobial activity than methanol extracts, making it possible to identify the inhibitory capacity of the mushroom species.

On the other hand, it was shown that the four fungi have phenolic compounds and antioxidant properties as measured by several tests, emphasizing A. rubescens, which, being an edible species, has the potential to be a source of natural antioxidants and other bioactive compounds for nutrition.Yet, it is important to identify the bioactive compounds responsible for the antimicrobial and antioxidant activities in these species, since they can participate in the production of new antibiotics and cope with diseases caused by oxidative stress.