İrez et al. 2021, Biologica Nyssana 12(1) 12 (1) September 2021: 55-62 DOI: 10.5281/zenodo.5523017 Fomes fomentarius (L.) Fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents Original Article Eylül İrem İrez Department of Biology, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University, Çanakkale, Turkey eyluliremirez@gmail.com Nurcihan Hacioğlu Doğru Department of Biology, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University, Çanakkale, Turkey nurcihan.n@gmail.com (corresponding author) Neslihan Demir Department of Biology, Faculty of Arts and Sciences, Çanakkale Onsekiz Mart University, Çanakkale, Turkey neslihandemir@comu.edu.tr Received: Mart 03, 2020 Revised: December 22, 2020 Accepted: February 08, 2021 Abstract: This paper evaluated antioxidant, antimicrobial and antibiofilm activities of ethanol, methanol, acetone and chloroform extracts of lignicolous fungal species Fomes fomentarius (L.) Fr. (Polypodiaceae). Antiradical activity was evaluated by using DPPH (2,2-diphenyl-1-picrylhydrazyl) assay. The antimicrobial screening was carried out via disc diffusion and microdilution methods in order to estimate minimum inhibitory (MIC) and minimum bactericidal concentration (MBC) of analyzed extracts against seven standard bacteria and one yeast: Escherichia coli NRRL B-3704, Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris ATCC 13315, Acinetobacter baumanii ATCC 19606, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, S. haemolyticus ATCC 43252 and Candida albicans ATCC 10231. In vitro antibiofilm activities were investigated based on crystal violet binding assay. All the extracts showed higher antioxidant activity compared to BHT (butylated hydroxytoluene) which was used as a standard. Ethanol, methanol and acetone extracts of the tested macrofungus also showed higher antimicrobial effect against P. aeruginosa ATCC 27853 in comparison to the antibiotic penicillin (P10). The lowest MIC was recorded by ethanol extract against S. haemolyticus ATCC 43252 (0.625 µg/mL). The highest antibiofilm activity was also noticed against biofilm formed by S. haemolyticus ATCC 43252. The results indicated that the F. fomentarius tested represent potential source of natural bioactive compounds with respect to antimicrobial, antibiofilm and antioxidant activities. Key words: antimicrobial, antibiofilm, antioxidant activity, Fomes fomentarius Apstract: Fomes fomentarius (L.) Fr. ekstrakti kao izvori antioksidanasa, antimikrobnih i antibiotskih sredstava Ovaj rad se bavi procenom antioksidativne, antimikrobne i antibiofilm aktivnosti etanolnog, metanolnog, acetonskog i hloroformnog ekstrakta lignikolne gljive Fomes fomentarius (L.) Fr. (Polyporaceae). Antiradikalna aktivnost je utvrđena korišćenjem DPPH (2,2-difenil-1-pikrilhidrazil) eseja. Antimikrobni skrining je izvršen putem disk difuzione i mikrodilucione metode kako bi se utvrdile minimalna inhibitorna (MIK) i minimalna baktericidna koncentracija (MBK) analiziranih ekstrakata u odnosu na sedam standardnih bakterija i jednu gljivu: Escherichia coli NRRL B-3704, Pseudomonas aeruginosa ATCC 27853, Proteus vulgarisATCC 13315, Acinetobacter baumaniiATCC 19606, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, S. haemolyticus ATCC 43252 and Candida albicans ATCC 10231. In vitro antibiofilm aktivnost je ispitana korišćenjem kristal violet eseja. Svi ekstraktu pokazali su veću antioksidativnu aktivnost u poređenju sa BHT (butilovanim hidroksitoluenom) koji je korišćen kao standard. Takođe, etanolni, metanolni i acetonski ekstrakti testirane makrogljive su u poređenju sa antibiotikom penicilinom (P10) pokazali veći antimikrobni efekat na P. aeruginosa ATCC 27853. Najniža MIK vrednost je zabeležena zza etano0lni ekstrakt i to protiv S. haemolyticus ATCC 43252 (0.625 µg/mL). Najviša antibiofilm aktivnost uočena je kod biofilma formiranog od strane S. haemolyticus ATCC 43252. Rezultati su ukazalu da testirana vrsta F. fomentarius predstavlja potencijalni izvor prirodnih bioaktivnih jedinjenja u smislu antimikrobne, antibiofilm i antioksidativne aktivnosti. Ključne reči: antimikrobna, antibiofilm, antioksidativna aktivnost, Fomes fomentarius © 2021 İrez et al. This is an open-access article distributed under the terms of the Creative Commons Attribu- tion License, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 55 Introduction In recent years, the problem of antibiotic resistance, where bacterial and fungal pathogens developed numerous defense mechanisms against antimicrobial agents is increased, and scientific studies presented new and more powerful agents as an alternative to antibiotic therapy (Heleno et al., 2013). The interest in natural compounds has been a common point for most biotechnology companies to be used in the production of new antimicrobial drugs (Butler, 2004). Microbial biofilms are communities of bacteria, embedded in a self-producing matrix, forming on living and nonliving solid surfaces (Vasudevan, 2014). They are considered as an important virulence factor that causes persistent chronic and recurrent infections; they are highly resistant to antibiotics and host immune defenses. Biofilm resistance is due to several reasons, like restricted diffusion of antibiotics into biofilm matrix, expression of multidrug efflux pumps, type IV secretion systems, decreased permeability, and the action of antibiotic- modifying enzymes (Alekshun and Levy, 2007). The increased biofilm resistance to conventional treatments enhances the need to develop new control strategies (Simões et al., 2007; Sánchez et al., 2016). It is common to believe that it is difficult to prevent and treat biofilm infections. Current findings regarding the fact that biofilms have a highly heterogeneous structure necessitates the development of new treatment strategies unlike the treatment strategies used in the destruction of microorganisms in the planktonic phase (Clatworthy et al., 2007). Besides its antimicrobial effects, some natural product extracts are known to prevent biofilm formation. Due to the inadequate antimicrobial therapies in combating biofilms, researchers are directed towards the discovery and identification of new antimicrobial agents, especially natural (Karaca et al., 2017). Antioxidants have the ability to protect the body against damage caused by oxidative stress. The interest in natural antioxidants is increasing day by day. For example, polyphenols, found in medicinal plants and foods, can help prevent oxidative damage (Silva et al., 2005). Mushrooms are rich sources of antioxidant compounds such as phenolic compounds (phenolic acids and flavonoids) and tocopherols (Cheung et al., 2003; Ferreira et al., 2009; Heleno et al., 2010; Sun et al., 2011; Dündar et al., 2016). Fomes fomentarius (L.) Fr., Polyporaceae, tinder fungus is a woody, perennial fungus, large in size which develops as a parasite or saprophyte on the beech (Fagus sylvatica L.) and other deciduous species. It is a white root fungus causing heart rot of the wood. In recent years, the active compounds of F. fomentarius have been studied extensively and most of its biological activities have been put forward. It is found that F. fomentarius have significant effects such as antioxidant (Lee, 2005; Vazirian et al., 2014; Dündar et al., 2016; Bal et al., 2017), antimicrobial (Kolundžić et al., 2016), antifungal (Dresch et al., 2015), anti-inflammatory (Vazirian et al., 2014), cytotoxic (Kolundžić et al., 2016), antitumor (Chen et al., 2008), antiviral (Aoki et al., 1993), DNA protective (Bal et al., 2017), fibrinolytic activity (Sánchez-Santillán et al., 2015). This extended study was to evaluate the antimicrobial, antibiofilm and antioxidant activities of ethanol, methanol, acetone and chlorofom extracts of F. fomentarius. Although there have been extensive researches to reveal antioxidant and antimicrobial activity of F. fomentarius, studies on antibiofilm activity have not been found in the literature. We also aimed to determine antibiofilm potential of the four extracts obtained from F. fomentarius against important pathogen microorganisms for the first time. Materials and Methods The macrofungal material Fomes fomentarius was collected from Çanakkale, Turkey on decaying stump in January, 2018 and identified by Dr. Ersin KARABACAK. Preparation of extracts The sample of the dried macrofungus (15 g) was extracted with four solvents- ethanol, methanol, acetone and chloroform (150 ml) using the Soxhlet apparatus (ISOLAB). The extraction was continued until the final extract was colorless according to Khan et al. (1988). The solvents were removed under reduced pressure and dried using a rotary evaporator (Heidolp, Laborota 4001, Germany) at 55 °C. Dry extract was taken into glass bottles with dark lid and stored at +4 °C. For the experiments, dissolution in dimethyl sulfoxide (DMSO) (10%) and different concentrations of the prepared extract were sterilized by a membrane filter (0.2 µm) and prepared for screening. Evaluation of antiradical activity DPPH assay The antioxidant activity of the extracts was measured by using commercial free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) following the procedure described by Brand-Williams et al. (1995), with slight modifications. The extracts were evaporated to dryness and re-suspended in DMSO. Butylated hydroxytoluene (BHT) solution was used as a positive control. The absorbance 56 BIOLOGICA NYSSANA ● 12 (1) September 2021: 55-62 İrez et al. ● Fomes fomentarius (L.) Fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents 57 values of the samples were measured on a UV-VIS spectrophotometer (PG Instruments T+80) at 517 nm against a blank. The experiment was repeated three times and the arithmetic mean of the readings was taken. The radical scavenging activity of each sample was calculated using the following equation and the results were expressed as % inhibition. Inhibition (%) = [(A0 - A1) / A0] x 100 A0: Extract or non-standard control absorbance, A1: Extract or standard absorbance Evaluation of antimicrobial activity Sterilized antibiotic discs 6 mm in diameter (Schleicher and Schull No. 2668, Dassel, Germany) were impregnated with 50 μL of each extract (10 mg/disc) at concentration of 200 mg/mL. Gram negative bacteria - Escherichia coli NRRL B-3704, Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris ATCC 13315, Acinetobacter baumanii ATCC 19606, Gram positive bacteria - Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, S. haemolyticus ATCC 43252 and yeast culture - Candida albicans ATCC 10231 were used as the test microorganisms. All the bacteria were incubated at 35 ± 0.1 °C for 24 h by inoculation into Nutrient Broth (Difco Laboratories, MI, USA) and the yeast culture studied was incubated in Malt Extract Broth (Difco Laboratories, MI, USA) at 25 ± 0.1 °C for 48 h. An inoculum containing 106 bacterial cells or 108 yeast cells/mL was spread on Mueller Hinton Agar (MHA) (Oxoid Ltd., Hampshire, UK) plates (1 ml inoculum/plate). The discs injected with extracts were placed on the inoculated agar by pressing slightly. Petri dishes were placed at ±4 °C for 2 h and incubated for 24 h and 48 h for bacteria and yeast, respectively (Collins et al., 1989). At the end of the period, inhibition zones formed on the medium were evaluated in millimeters. Studies were performed in triplicate. Treatments with penicillin (P10), and nystatin (NYS30) served as positive controls and treatments with ethanol, methanol, acetone and chloroform without fungal materials served as negative controls. For quantitative antimicrobial analyses, mini- mum inhibitory concentration (MIC) values of all samples were determined. MIC and MBC were in- vestigated as recommended instruction of the Clini- cal and Laboratory Standards Institute (CLSI, 2006). Briefly, stock solution of each extract was diluted in the Muller Hinton Broth (MHB) in two-fold serial dilutions to obtain final concentrations range of 20- 0.156 mg/ml at a total volume of 100 μl per well in 96-well microtiter plates. The lowest concentration of extracts inhibiting visible growth of each test mi- croorganisms was taken as the MIC. The medium, 0.1% (w/v) streptomycin (ST), nystatin (NYS30) and 10% DMSO were used as the non-treated, posi- tive and negative controls, respectively (Teanpasian et al., 2017). Confirmation of MIC and establishment of the minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC), 10 µL of the following dilutions (40 to 2.5 µg/ml) were in- oculated into plates with MHA to evaluate microbial growth. After 24 h of incubations at 35 ± 0.1 °C for bacteria and 48 h at 25 ± 0.1 °C for the yeast culture, the plates with no apparent CFU of surviving micro- organisms were determined. Each experiment was repeated three times (Pacheco-Ordaz et al., 2018). Biofilm inhibition assay Microplate biofilm method (Merrit et al., 2005) was used to evaluate the inhibition of biofilm formation by F. fomentarius extracts against tested microorganisms. Cultures were incubated in 5 ml Tryptic Soy Broth (TSB) medium containing 5% glucose. Cultures were diluted 1:100 in TSB and loaded into each well in 4 sterile microplate. Different concentrations of fungal extracts (MIC and sub- MIC concentrations: 50, 25, 12.5% of MIC) were prepared and transferred to each microplate well. After incubation at 37 ± 0.1 °C for 48 h planktonic bacteria were removed from the wells and wells were washed twice with distilled water. Crystal violet solution (0.1%, 200 μl) was added to each well and incubated for 20 minutes. The crystal violet bounded extracts were poured and washed until the crystal violet was finally removed. The microtiter plates were inverted and the remaining liquid was drained and dried in room heat. Finally, the adhered biofilm bounded crystal violet was eluted in ethanol (95%) and the absorbance was measured at 550 nm by using an automated Elisa reader (BioTek, UK). All experiments were repeated in triplicate. The calculation of the antibiofilm effect of the extract was made by the reduction formulation percentage. % Inhibition = (Acontrol – Asample / Acontrol) x 100 Acontrol: Absorbance of the control (containing 100 µL TBS instead of fungal extract) reaction, Asample: Absorbance of the tested compound Results and discussion Two polar (ethanol and methanol), one intermediate polar (acetone) and one nonpolar (chloroform) solvent were selected for comparison of biological activity of F. fomentarius. The choice of solvents depends on the type of macrofungi, part of the materials to be extracted, nature of the bioactive BIOLOGICA NYSSANA ● 12 (1) September 2021: 55-62 İrez et al. ● Fomes fomentarius (L.) Fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents 58 compounds, the availability of solvent and the literature. Polar solvents such as ethanol, methanol and acetone were used in extraction of polar compounds, whereas nonpolar solvents such as chloroform were used in extraction of nonpolar compounds such as terpenoids, flavonoids, fats and oils (Abubakar and Hague, 2020). The findings showed that polar solvents, especially ethanol, are more successful in extraction of secondary metabolites with antioxidant, antimicrobial and antibiofilm activity. DPPH assay is widely used in determination of the antioxidant activity of compounds and different fungal extracts. In this work, it was found that the inhibition percentage of ethanol extracts from F. fomentarius was slightly higher (89.95±0.21) than when acetone, methanol and chloroform solvents were applied (88.88±0.18, 87.34±0.11 and 80.93±0.07, respectively) (Fig. 1). BHT which was used as a standard showed lower antioxidant activity (75.81±0.03) compared with all other extracts, obtained by different solvents. Studies have shown that the biological activities of the extracts can vary depending on the season, type of extract, dose and duration of application. Depending on the extraction method, the level of bioactive substances can be observed (Anlas et al., 2017). Mircea et al. (2015) and Kolundžić et al. (2016) reported that DPPH radical scavenging activity of F. fomentarius methanol extract results correlated with the polyphenol content. In addition, F. fomentarius methanol and ethanol extracts have been reported to have the strongest DPPH activity comparing to other solvents (Bojin et al., 2020). The present study indicated that ethanol extract showed slightly higher antioxidant activities than the other solvents. The antimicrobial activities of the macrofungal extracts against different test strains were assessed according to inhibition zones diameter and MIC and MBC or MFC values (Tab. 1). All extracts showed higher antimicrobial effect against P. aeruginosa, P. vulgaris, S. haemolyticus, C. albicans except chloroform extract with inhibition zone of 10-13 mm. Ethanol, methanol and acetone extracts of macrofungi also showed higher antimicrobial effect against P. aeruginosa in comparison to antibiotic P10. The lowest MIC value was recorded by F. fomentarius ethanol extract against S. haemolyticus (0.625 μg/ml). Also, ethanol extract showed higher MIC against E.coli, B. subtilis, S. aureus and S. haemolyticus bacteria when compared to antibiotic streptomycin probably due to the polar component that the ethanol extract contain. There are many scientific reports about F. fomentarius antimicrobial activity (Zhao et al., 2013; Dündar et al., 2016; Kolundžić et al., 2016; Gedik et Table 1. Antimicrobial activity of F. fomentarius extracts Fig. 1. DPPH free scavenging activity of F. fomentarius extracts Test microorganisms *Disc Diffusiona Control antibiotics MIC (µg/mL) Control antibiotics MBC (MFC) MBC (MFC)/ MIC E1 E2 E3 E4 P10 NY 100 E1 E2 E3 E4 ST NY 100 E1 E2 E3 E4 E1 E2 E3 E4 E.coli 7.0 7.0 10.0 8.0 16.0 Nt 2.5 2.5 10 20 4.0 Nt 40 2.5 40 40 16 1 4 2 P. aeruginosa 10.0 11.0 10.0 7.0 8.0 Nt 10 10 10 20 1.0 Nt 40 40 40 40 4 4 4 2 P. vulgaris 13.0 10.0 10.0 7.0 13.0 Nt 20 5 10 20 4.0 Nt 40 40 40 40 2 8 4 2 A. baumanii 7.0 11.3 8.6 9.0 12.0 Nt 5 5 10 20 2.0 Nt 40 5 40 40 4 1 4 2 B. subtilis 13.0 9.0 8.3 8.0 14.0 Nt 2.5 2.5 10 20 4.0 Nt 40 2.5 40 40 16 1 4 2 S. aureus 13.0 10.6 8.3 9.0 15.0 Nt 2.5 5 10 20 4.0 Nt 40 5 40 40 16 1 4 2 S. haemolyticus 11.6 11.6 10.3 8.0 14.0 Nt 0.6 5 10 20 5.0 Nt 40 10 40 40 64 2 4 2 C. albicans 10.0 11.0 10.0 7.0 Nt 16.0 5 5 5 20 NT 5.0 40 40 40 40 4 8 8 2 BIOLOGICA NYSSANA ● 12 (1) September 2021: 55-62 İrez et al. ● Fomes fomentarius (L.) Fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents al., 2019). Similar to the present study, Kolundžić et al. (2016) tested the antimicrobial activity of F. fomentarius extracts of different polarity, especially against Gram-negative and Gram-positive bacteria. They indicated that their F. fomentarius extracts (cyclohexane, dichloromethane, methanol and aqueous) displayed strong antimicrobial activity. Gedik et al. (2019) also showed that ethanol and aqueous extracts of F. fomentarius have higher antibacterial activity against K. pneumoniae, A. baumanii, S. aureus, vancomycin-resistant enterococci (VRE+), E. coli against standard antibiotics. In our study, a similar situation was obtained against P. vulgaris, B. subtilis, S. aureus bacteria. The results of Zhao et al. (2013) demonstrated weak antimicrobial activity in isolated phenyl-eth- anediols from the fruiting bodies of F. fomentarius. Moreover, methanolic extracts of F. fomentarius have been found to be effective against M. luteus, but no activity was detected against S. aureus, E. coli, P. aeruginosa, B. subtilis, Enterococcus faeca- lis (Kolundžić et al., 2016). In the present study we obtained different results for antimicrobial activity of F. fomentarius against the tested microorganisms; that is possibly due to differences of fungal species habitat factors, ecological status, seasonal differences and variety of extraction methods. The results of potential inhibition of test microorganism’s biofilm formation by four extracts of F. fomentarius were shown in Fig. 2. The MIC values of F. fomentarius extracts were used to determine antibiofilm rates. It can be seen that antibiofilm rates of four extracts ranged from 5.03 to 95.2%. The results indicated that ethanol and methanol extracts of F. fomentarius could inhibit the biofilm formation of all the tested strains, significantly. The highest antibiofilm activity was noticed against biofilm formed by S. haemolyticus. Both acetone and chloroform extracts showed very low antibiofilm effects compared to ethanol and methanol, except for C. albicans (Fig. 2). Although recent studies on the evaluation of medically important Fomes species in this direction are few in the literature (Bin et al., 2012; Solmaz et al., 2013; Alves et al., 2014; Petrović et al., 2014; Signoretto et al., 2014; Carvalho et al., 2016; Karaca et al., 2017), there is no study on the antibiofilm effects of the F. fomentarius on the related pathogen strains. Alves et al. (2014) tested the antibiofilm capacity of different macrofungal species against E. coli, P. aeruginosa, A. baumannii and P. mirabilis strains. They found that all macrofungi methanol extracts showed strong antibiofilm activity against pathogens. Petrović et al. (2014) also reported that hot water extract from Agaricus blazei could influence on biofilm formation, twitching and swimming activity, pyocyanin production which are part of anti-quorum sensing activity. Karaca et al. (2017) investigated antibiofilm potentials of methanolic and ethanolic extracts of the three medicinal macrofungi species and found that the highest antibiofilm activity was observed with Ganoderma lucidum methanolic extract. Therefore, the findings obtained from the present study are presenting the important contributions to the literature data on this subject. Conclusions In the present study, it was determined that F. fomentarius demonstrated high antioxidant, 59 Fig. 2. Inhibition (%) of biofilms formation of F. fomentarius extracts (E1: Ethanol, E2: Methanol, E3: Acetone, E4: Chloroform) BIOLOGICA NYSSANA ● 12 (1) September 2021: 55-62 İrez et al. ● Fomes fomentarius (L.) Fr. extracts as sources of an antioxidant, antimicrobial and antibiofilm agents antimicrobial and antibiofilm activities. In conclusion, it was determined that analyzed F. fomentarius extracts could be used after chemical characterization and toxicity testing as natural resources in the fields of medicine, pharmacology, nutrition and cosmetics due to their strong antioxidant and antimicrobial activities. Acknowledgment. Authors would like to thank Assoc. Prof. Dr. Ersin Karaback for defining macrofungus species. This research is a part of Mrs. Eylül İrem İrez’s TÜBİTAK-2209 A Project which is supported with the frame of Undergraduate Students Grant in Biological Science. References Abubakar, A.R., Hague, M. 2020: Preparation of medicinal plants: basic extraction and fractionation procedures for experimental purposes, Journal of Pharmacy & BioAllied Sciences, 12(1): 1-10. 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