Mushrooms as Possible Antioxidant and Antimicrobial Agents

Introduction

   Reactive oxygen species (ROS) are an entire class of highly reactive molecules derived from the metabolism of oxygen. At normal physiological concentrations ROS are required for cellular activities, however, at higher concentrations, ROS can cause extensive damage to cells and tissues, during infections and various degenerative disorders, such as cardiovascular disease, aging, and neurodegenerative diseases like Alzheimer’s disease,  mutations and cancer (1, 2).  

   Antioxidants, both synthetic or natural, can be effective to help the human body in reducing oxidative damage by ROS (3). However, at the present time, suspected that synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ) and propyl gallate (PG) have toxic and carcinogenic effects (4). Therefore, the development and utilization of more effective antioxidants of natural origins are desired. In recent years, the antioxidant properties of numerous plants, lichens and mushrooms have been widely reported. In order to find new natural sources of antioxidants, our attention was focused on mushrooms.

   Mushrooms possess high contents of qualitative protein, crude fibre, minerals and vitamins. Apart from their nutritional potentials, mushrooms are also sources of physiologically beneficial bioactive substances that promote good health. They produce a wide range of secondary metabolites with high therapeutic value. Health promoting properties, e.g. antioxidant, antimicrobial, anticancer, cholesterol lowering and immunostimulatory effects, have been reported for some species of mushrooms.  Both fruiting bodies and the mycelium contain compounds with wide ranging antioxidant and antimicrobial activities (5-8).

   Thus, the aim of this study was to examine in-vitro antioxidant and antimicrobial activity of the acetonic and metanolic extract of the mushrooms Boletus aestivalis, Boletus edulis and Leccinum carpini.

Experimental

Fungal materials

   Fungal samples of Boletus aestivalis (Paul.) Fr., Boletus edulisBull. Fr.,and Leccinum carpini (Schulzer)  Moser ex Reid were collected from Kopaonik, Serbia, in June of 2010. The demonstration samples have been preserved in facilities of the Department of Biology and Ecology of Kragujevac, Faculty of Science. Identification of mushrooms was done  using standard keys (9-11).

Extraction

   Fresh fungal material was milled by an electrical mill. Finely ground mushrooms (50 g) were extracted using acetone and methanol for 24 h. The extracts were filtered and then concentrated under reduced pressure in a rotary evaporator. The dry extracts were stored at -18°C until used in the tests. The extracts were dissolved in 5% dimethyl sulphoxide (DMSO) prior to performing the tests.

Antioxidant activity

Scavenging DPPH radicals

     The free radical scavenging activity of mushrooms extracts was measured by using 1,1-diphenyl-picryl-hydrazil (DPPH). A previously reported method was used (12, 13), with some minor modifications. Two mL of a methanolic solution of DPPH radical in the concentration of 0.05 mg/mL and 1 mL of extract were placed in cuvettes. The mixture was shaken vigorously and left to stay at room temperature for 30 min. Then, the absorbance of the solution was measured at 517 nm using a Janway spectrophotometer (Bibby Scientific Limited, Stone, UK). Ascorbic acid, butylated hydroxyanisole (BHA) and α-tocopherol were used as positive control. Inhibition of free radical DPPH expressed as percentage [I (%)] was calculated as follows:

I (%) = ((A₀ - A₁)/ A₀) x 100

where A₀  is the absorbance of the negative control and A₁ is the absorbance of reaction mixture or standards.

   The inhibition concentration at 50 % inhibition (IC₅₀) was the parameter used to compare the radical scavenging activity. A lower IC₅₀ means better radical scavenging activity.

Reducing power

    The reducing power of extracts was determined by the method of Oyaizu (14). One mL of the extracts were mixed with 2.5 mL of phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide [K₃Fe(CN)₆] (2.5 mL, 1%). The mixtures were incubated at 50^oC for 20 min. Then,  trichloroacetic acid (10%, 2.5 mL) was added to the mixture and centrifuged. Finally, the upper layer were mixed with distilled water (2.5 mL) and 0.5 mL of 0.1% ferric chloride (FeCl₃). The absorbance of the solution was measured at 700 nm by the spectrophotometer. Higher absorbance values of the reaction mixture indicates that the reducing power is increased. Ascorbic acid, BHA and α-tocopherol were used as positive control.

Determination of total phenolic compounds

   Total soluble phenolic compounds in the mushrooms extracts were determined with Folin-Ciocalteu reagent according to the method described by Slinkard and Slingleton (15), using pyrocatechol as a standard phenolic compound. Briefly, 1 mL of the extract (1 mg/mL) in a volumetric flasc was diluted with distilled water (46 mL). One milliliter of Folin-Ciocalteu reagent was added and the content of the flask was mixed thoroughly. After 3 min 3 mL of 2% sodium carbonate (Na₂CO₃) was added and the solution was left to stay for 2h with intermittent shaking. The absorbance of the solution was measured at 760 nm  by the spectrophotometer. The total concentration of phenolic compounds in the extract was determined as microgram of pyrocatechol equivalent per milligram of dry extract, using the equation which derived from standard pyrocatechol graph as follows:

Absorbance = 0.0021 x total phenols (μg pyrocatechol equivalent/mg of extract) - 0.0092

(R² = 0.9934)

Total flavonoid content

    The total flavonoid content was determined using the Dowd method (16). Two mL of 2 % aluminium trichloride (AlCl₃) in methanol was mixed with the same volume of the extract solution (1 mg/mL). The mixture was left at room temperature for 10 min, and the absorbance was measured at 415 nm against blank samples. The total flavonoid content was detemined as microgram of rutin equivalent per milligram of dry extract, using an equation that was obtained from standard rutin graph as follows:

Absorbance = 0.0144 x total flavonoid (μg rutin equivalent/mg of extract) + 0.0556

(R² = 0.9992)

Antimicrobial activity

Microorganisms and media

     The following bacteria were used as test organisms in this study: Staphilococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 70063), Pseudomonas aeruginosa (ATCC 27853) and Enterococcus faecalis (ATCC 29212). All the bacteria used were obtained from the American Type Culture Collection (ATCC). Their identification was confirmed at the Microbiological Laboratory of Kragujevac, University of Kragujevac, Department of Biology. The fungi used as test organisms were: Aspergillus flavus (ATCC 9170), Aspergillus fumigatus (DBFS 310), Candida albicans (IPH 1316), Paecilomyces variotii (ATCC 22319), Penicillium purpurescens (DBFS 418). They were obtained from the American Type Culture Collection and the mycological collection maintained by the Mycological Laboratory within the Department of Biology of Kragujevac, University of Kragujevac, Faculty of Science (DBFS). Bacterial cultures were maintained on Müller-Hinton agar substrates (Torlak, Belgrade). Fungal cultures were maintained on potato dextrose (PD) agar and Sabourad dextrose (SD) agar (Torlak, Belgrade). All cultures were stored at 4°C and subcultured every 15 days.

     The sensitivity of microorganisms to acetone and methanol contained in the extracts of the examined species of mushrooms was tested by determining the minimum inhibitory concentration (MIC).

     Bacterial inoculi were obtained from bacterial cultures incubated for 24 h at 37°C on Müller-Hinton agar substrate and brought up by dilution according to the 0.5 McFarland standard to approximately 10⁸ CFU/mL. Suspensions of fungal spores were prepared from fresh mature (3- to 7-day-old) cultures that grew at 30°C on a PD agar substrate. Spores were rinsed with sterile distilled water, used to determine turbidity spectrophotometriacally at 530 nm, and then further diluted to approximately 10⁶ CFU/mL according to the procedure recommended byNCCLS (17).

Minimum inhibitory concentration

    The MIC was determined by the broth microdilution method using 96-well micro-titer plates (18).A series of dilutions with concentrations ranging from 40 to 0.156 mg/mL for extracts were used in experiment against every microorganism tested. The starting solutions of the extracts were obtained by measuring off a certain quantity of each extract and dissolving it in DMSO. Two-fold dilutions of the extracts were prepared in Müller-Hinton broth for bacterial cultures and SD broth for fungal cultures. The MIC was determined by establishing visible growth of microorganisms. The boundary dilution without any visible growth was defined as the MIC for the tested microorganism at the given concentration. As a positive control of growth inhibition in our experiments, streptomycin in the study on bacteria and ketoconazole in the study on fungi were used, respectively. A DMSO solution was used as a negative control. All experiments were performed in triplicate.

Statistical analyses

   Statistical analyses were performed with the EXCEL (version 11) and SPSS (version 13) software packages. To determine the statistical significance of antioxidant activity, student’s t-test were used. Pearson’s bivariate correlation test was carried out to calculate correlation coefficients (r) between the content of total phenolic and flavonoid and the DPPH radical scavenging activity of each extract. All values are expressed as mean + SD of three parallel measurements.

 Results and Discussion

Antioxidant activity

   DPPH radical scavenging, reducing power, determination of total phenolic compounds and  determination of total flavonoid content of the acetonic and metanolic extracts of the mushrooms Boletus aestivalis, Boletus edulis and Leccinum carpini were examined in this study.

   The scavenging DPPH radicals of the studied extracts are shown in Table 1. The inhibition concentration at 50 % inhibition (IC₅₀) was the parameter used to compare the radical scavenging activity. A lower IC₅₀ meant better radical scavenging activity. Acetonic and metanolic extracts of the tested mushrooms showed a good scavenging activity on DPPH radical. There were statistically significant differences between extracts and control (p < 0.05). The IC₅₀ values of all extracts ranged from 4.72 – 212.47 µg/mL. Acetonic extract from Boletus edulis showed the highest DPPH radical scavenging activities (IC₅₀ = 4.72 µg/mL) compared to the results obtained from other samples and its acitvity was even greater than BHA and α-tocopherol. The scavengig activity were also noticeable for the acetonic extracts from Boletus aestivalis (IC₅₀ = 8.63 µg/mL) and Leccinum carpini (IC₅₀ = 67.89 µg/mL). Methanolic extracts from tested mushrooms showed lower DPPH radical scavenging activities than acetonic extracts. IC₅₀ for the metanolic extracts were 187.73 µg/mL  for Boletus aestivalis, 212.47 µg/mL for Boletus edulis and 202.471 µg/mL for Leccinum carpini.

Table 1. IC₅₀ values of acetonic and metanolic extracts of Boletus aestivalis, Boletus edulis and Leccinum carpini by free radical scavenging method.

   The results of the reducing power assay of tested extracts are summarized in Table 2. Higher absorbance indicates higher reducing power. Measured values of absorbance varied from 0.0014 to 0.0280. The reducing power of the extracts increased as contcentration increased. Among the tested extracts, acetonic and metanolic extracts of Boletus edulis showed the highest reducing power, followed by acetonic extracts from Leccinum carpini. Other extracts showed lower reducing power.

Table 2. Reducing power of acetonic and metanolic extracts of Boletus aestivalis, Boletus edulis and Leccinum carpini. 

   Total phenolic and flavonoid constituents of the tested extracts are presented in Table 3. The amount of total phenolic compounds was determined as the pyrocatechol equivalent using an equation obtained from a standard pyrocatechol graph. Results of the study showed that the phenolic compound of the tested extracts varied from 4.64 to 8.14 μg of pyrocatechol equivalent. Highest phenolic compounds was identified in acetonic extract of Boletus edulis at a 8.14 μg of pyrocatechol equivalent, followed by acetonic extract of Boletus aestivalis with 6.73 μg of pyrocatechol equivalent.

Table 3. Total phenolics and flavonoid content of acetonic and metanolic extracts of Boletus aestivalis, Boletus edulis and Leccinum carpini.

   The amount of total flavonoid compounds was determined as the rutin equivalent using an equation obtained from a standard rutin graph. As shown in Table 3, perceptible flavonoid content was found in the acetonic extract of Boletus edulis (4.93 μg of rutin equivalent) and acetonic extract of Boletus aestivalis (3.20 μg of rutin equivalent). Other lichen extracts showed lower  flavonoid content. 

   The IC₅₀ values obtained for the tested mushroom extracts  were correlated to the total phenolic and flavonoid contents (Figure 1). Notably negative correlation was established between the phenols and IC₅₀ values of antioxidant activities (r = -0.93). Also, there is a good negative correlation between flavonoid compounds of the tested extracts and IC₅₀ values of antioxidant activities (r = -0.83). These negative linear correlations proved that the samples with higher  antioxidant contents showed higher antioxidant activity with lowest IC₅₀ values.

Figure 1. Correlations between the total phenolic (a)  and flavonoid (b) content of the extracts and the free radical scavenging activity.

Antimicrobial activity

   The antimicrobal activity of the tested mushrooms extracts against the tested microorganisms was shown in Table 4.

   The acetonic and metanolic extracts of the tested mushrooms showed relatively strong antimicrobial activity. The MIC for both extracts related to the tested bacteria and fungi were 1.25 - 10 mg/mL. Generally, the acetonic extracts exerted stronger antimicrobial activity than metanolic extracts.

   The maximum antimicrobial activity was found in the acetonic extract of the mushrooms Leccinum carpini against Enterococcus foecalis (MIC = 1.25 mg/mL). The measured MIC values for Leccinum carpini against bacteria were 1.25-5 mg/mLfor the acetonic and 2.5-10 mg/mLfor the metanolic extract. Both extracts of this mushroom inhibited the tested fungi in concentrations 5 mg/mLand 10 mg/mL.

   The acetonic and metanolic extract of Boletus aestivalis and Boletus edulis had approximately equal antimicrobial activity. They inhibited bacterial and fungal growth at concentrations 2.5 mg/mL, 5 mg/mLand 10 mg/mL.

   The antimicrobial activities of the extracts were compared to streptomycin (standard antibiotic) and ketoconazole (standard antimicotic). The results showed that streptomycin and ketoconazole had stronger activity than tested extracts as shown in Table 4. In a negative control, DMSO had no inhibitory effect on the tested organisms.

Table 4. Minimum inhibitory concentration (MIC ) of acetonic and metanolic extracts of Boletus aestivaliss, Boletus edulis and Leccinum carpini.

   The extracts have a strong in-vitro antioxidant activity against various oxidative systems. The intensity of antioxidant activity depends on the tested mushroom species and the extracting solvent. The differences in the antioxidant activity of various extracts may result from different capabilities of the used solvents  to extract bioactive substances (19). When antioxidative capacities of the extracts are compared to their phenolic constituents, it could be concluded that antioxidative nature of the extracts might depend on their phenolic contents. We found that the tested extracts with the highest phenolic content exhibited the highest radical scavenging activity. Numerous researches found a positive correlations between antioxidative activities and phenolic content of the tested samples (8, 20, 21).

   Antioxidant activity of some mushroom extracts were also studied by other researchers. In the literature there are numerous data for the antioxidant activity of Boletus edulis (22-24). Similar results were reported for other mushroom. For example, Ramesh et al. (25) found strong antioxidant activity for metanolic extracts from Lycoperdon perlatum, Cantharellus cibarius, Clavaria vermiculris, Ramaria formosa, Marasmius oreades, Pleurotus pulmonarius. Murcia et al. (26) find an antioxidant activity for the Lepista nuda, Lentinus edodes, Agrocybe cylindracea, Cantharellus lutescens, and Hydnum repandum.

   Numerous mushrooms were screened for antimicrobial activity in search of the new antimicrobial agents (25, 27-29). It was found that different species of mushrooms exhibit different antimicrobial activity. The differences in antimicrobial activity are probably a consequence of the content of the components with antimicrobial activity.

   In our experiments, the tested mushroom extracts showed a relatively strong antimicrobial activity. The intensity of the antimicrobial effect depended on the species of mushroom, concentration of the extracts and the tested organisms. The examined mushroom at the same concentrations showed a stronger antibacterial than antifungal activity. These results are expected due to the fact that numerous tests proved that bacteria are more sensitive to the antibiotic compared to fungi (30). The reason for different sensitivity between the fungi and bacteria can be found in different transparency of the cell wall (31).The cell wall of the gram-positive bacteria consists of peptidoglucans (mureins) and teichoic acids, while the cell wall of the gram-negative bacteria consists of lipo polysaccharides and lipopoliproteins,whereas, the cell wall of fungi consists of polysaccharides such as hitchin and glucan (32, 33).

Conclusions

   It conclusion, it can be stated that the tested mushroom extracts have a strong in-vitro antioxidant and antimicrobial activity. Based on the results obtained, mushrooms may be considered as natural sources of antioxidants and could be valuable as food and supplement for human and animals and plant diseases. Further studies should be done on the isolation and characterization of new compounds from mushrooms, which are responsible for antioxidant and antimicrobial activity.

Acknowledgements

    This work was financed in part by the Ministry of Science, Technology, and Development of the Republic of Serbia and was carried out within the framework of project no. 173032 .