Screening of Antibacterial and Antifungal Activities of Selected Macedonian Wild Mushrooms

Зборник Матице српске за природне науке / Jour. Nat. Sci, Matica Srpska Novi Sad, № 124, 333—340, 2013

UDC 582.28:577.18(497.7) DOI: 10.2298/ZMSPN1324333N

Daniela Nikolovska Nedelkoska1*, Natalija Atanasova Pančevska2, Haris Amedi2, Dafina Veleska2, Emilija Ivanova2, Mitko Karadelev2, Dzoko Kungulovski2

1 Faculty of Technology and Technical Sciences, St. Kliment Ohridski University, P. Prlickov 42, 1400 Veles, Republic of Macedonia 2 Faculty of Natural Science and Mathematics, Ss. Cyril and Methodius University, Arhimedova 5, 1000 Skopje, Republic of Macedonia

SCREENING OF ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES OF SELECTED MACEDONIAN WILD MUSHROOMS

ABSTRACT: Regarding the development of novel safe antimicrobials of natural origin, macrofungi became attractive for the researchers in the last decade. In this study, antimicrobial potential of methanolic extracts of six wild macromycetes (Boletus lupinus, Flammulina velutypes, Phellinus igniarius, Sarcodon imbricatus, Tricholoma aurantium, Xerocomus ichnusanus) was evaluated. In vitro antimicrobial activity was investigated by the microdilution method and minimum inhibitory concentration (MIC) was determined. Testing was conducted against eleven microorganisms, including six strains of bacteria and five species of fungi. Extracts showed selective antimicrobial properties while the activities depended both on the species of microorganism and on the type and concentration of extract. The evaluated extracts demonstrated antimicrobial activity, exhibiting more potent inhibitory effects on the growth of bacteria than on fungi. The highest antibacterial and antifungal activity was observed in methanolic extract of polypore fungus P. igniarius. KEY WORDS: Microdilution method, antimicrobial activity, mushrooms, Macedonia

INTRODUCTION

Antibiotic resistance has become a global concern in recent years (W e s t h et al., 2004). Although a number of natural and synthetic antimicrobial agents have been isolated or synthesized against pathogenic microorganisms, infectious diseases remain one of the major threats to human health. The increasing failure of chemotherapeutics and antibiotic resistance exhibited by pathogenic microorganisms has led to the screening of novel sources for their

* [email protected]

333 potential antibacterial and antifungal activity (C o l o m b o and B o l s i s i o, 1996; I w u et al., 1999). The fact that basidiomycetes have been insufficiently investigated, together with a variety of structural types of antibiotics which are produced by these organisms, suggests that they may be a source of new and useful bioactive compounds (A n k e, 1989). As a matter of fact, macrofungi need antibacterial and antifungal compounds to survive in their natural environment. Therefore, antimicrobial compounds could be isolated from many mushroom species and could be of benefit for humans. Bioactive mol- ecules have been isolated not only from edible, but also from inedible species (Q u a n g et al., 2006). Reported bioactivities of mushrooms include antibacterial, antifungal, antioxidant, antiviral, anti-tumor, cytostatic, immunosup- pressive, antiallergic, antiatherogenic, hypoglycemic, anti-inflammatory and hepatoprotective activities (W a s s e r and W e i s, 1999; L i n d e q u i s t et al., 2005). The responsible bioactive compounds belong to several chemical groups which are often polysaccharides or triterpenes (K i m et al., 2000; S u n and L i u, 2009). One macrofungi species can have various bioactive compounds and pharmacological effects (L i n d e q u i s t et al., 2005). Generally, although many antimicrobial compounds have been isolated from mushrooms, the antimicrobial compounds from microscopic fungi still dominate as antibiotics on the market. Thus, the aim of this study was to ex- amine in vitro antimicrobial activity of methanolic extracts from selected macromycetes: Boletus lupinus, Flammulina velutypes, Phellinus igniarius, Sarcodon imbricatus, Tricholoma aurantium, Xerocomus ichnusanus.

MATERIAL AND METHODS

Fruiting body selection

Samples of the wild macromycetes Boletus lupinus, Flammulina velutypes, Phellinus igniarius, Sarcodon imbricatus, Tricholoma aurantium, Xe- rocomus ichnusanus were collected from different locations in Macedonia, in autumn 2011. Geographical location and natural habitat of the mushroom specimens are shown in Table 1. Taxonomic identification was done in Myco- logical Laboratory at the Institute of Biology, Faculty of Natural Sciences and Mathematics in Skopje, by implementing standard methods of microscopic and chemical techniques (coloring of fruit bodies and spores), and appropriate literature (A l e s s i o, 1985; B e r n i c c h i a, 1990; B r e i t e n b a c h and K r ä n z l i n, 1986, 1991, 1995; D a n k e, 2004; F e r n á n d e z, 1997; H o r a k, 2005; J a h n, 1990; J ü l i c h, 1984; K n u d s e n and V e s t e r h o l t, 2012; K r i e g l s t e i n e r, 2000a, 2000b, 2001; R y v a r d e n, 1991; R y v a r d e n and G i l b e r t s o n, 1994). The representative voucher specimens were deposited in Macedonian Collection of Fungi (MCF) at the Institute of Biology.

334 Tab. 1. – Geographical Location and Natural Habitat of the Mushroom Species Studied for Antimicrobial Potential mushroom species natural habitat geographical location Boletus lupinus mycorrhizal (on ground in open oak forest) Galichica Mt. (Ohrid side) Flammulina velutypes saprotrophic (on stump of deciduous trees) Botanical garden, Skopje Vicinity of the town of Phellinus igniarius parasitic (on living willow trunks) Kumanovo Sarcodon imbricatus saprotrophic (on soil in conifer forest) Suva Gora Mt. Tricholoma aurantium mycorrhizal (on ground in pine forest) Suva Gora Mt. Xerocomus ichnusanus mycorrhizal (on ground in open oak forest) Galichica Mt. (Prespa side)

Preparation of methanolic extracts of mushrooms

The fruiting bodies were cleaned to remove any residual compost/soil and subsequently air-dried in the oven at 40oC. Dried specimens were ground to fine powder and extracted by stirring with 80% (v/v) methanol in ultra- sonic bath for 30 min at 4ºC, and then centrifuged at 12000 rpm for 15 min. Supernatants were used for the evaluation of antimicrobial potential of the samples. The organic solvent in the extracts was evaporated until dry, under vacuum. The yields of methanolic extracts of the fruiting bodies are presented in Table 2.

Tab. 2. – Yield of Mushroom Methanolic Extracts yield of extractsa sample mushroom species (g/100 g of dry mushroom) 1 Boletus lupinus 37.67 ± 4.04 2 Flammulina velutypes 30.00 ± 0.00 3 Phellinus igniarius 2.20 ± 0.17 4 Sarcodon imbricatus 39.00 ± 1.15 5 Tricholoma aurantium 36.67 ± 1.15 6 Xerocomus ichnusanus 38.50 ± 2.12 a Each value is the mean of three replicate determinations ± standard deviation.

In vitro antimicrobial assay

Test microorganisms Antimicrobial activities of methanol extracts were tested against 11 microorganisms, including 6 strains of bacteria: Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Bacillus pumilus NCTC 8241, Sarcina lutea ATCC 9341 and Bacillus subtilis ATCC 6633, and 5 species of fungi: Saccharomyces cerevisiae ATCC 16404, Candida albicans ATCC 10231, Aspergillus niger I.N. 1110, Aspergillus sojae CCF and Penicillium spp. FNS FCC 266.

335 The microorganisms were provided from the collection held in the Micro- biology Laboratory, Faculty of Natural Sciences and Mathematics in Skopje.

Suspension preparation Microbial suspensions were prepared in accordance with the direct colony method. The turbidity of initial suspension was adjusted by comparison with 0.5 McFarland’s standard (A n d r e w s, 2005). The initial suspension contained about 108 colony forming units (CFU)/mL. Additionally, 1:100 dilutions of initial suspension were prepared in sterile 0.9% saline.

Microdilution method Antimicrobial activity was tested by determining the minimum inhibitory concentration (MIC) using the microdilution method with resazurin, an indicator of microbial growth (Sarker et al., 2007). The 96-well plates were prepared by dispensing 50 μL of nutrient broth, Mueller-Hinton broth for bacteria and Sabouraud dextrose broth for fungi in each well. A volume of 50 μL from the stock solution of tested extracts (concentration: 100 mg/mL for mushroom specimens Boletus lupinus, Flammulina velutypes, Sarcodon imbricatus, Tricholoma aurantium, Xerocomus ichnusanus and 20 mg/ml for polypore fungus Phellinus igniarius) was added into the first row of the plate and then two-fold serial dilutions of extracts were performed. The obtained concentration range was from 50 mg/mL to 0.025 mg/mL for extracts of Boletus lupinus, Flammulina velutypes, Sarcodon imbricatus, Tricholoma aurantium, Xeroco- mus ichnusanus, and from 10 to 0.0049 mg/mL for Phellinus igniarius. MIC was defined as the lowest concentration of the tested extracts that prevented a resazurin color change from blue to pink. The tested extracts were dissolved in 10% (v/v) DMSO in sterile water. A solvent control test was performed to study the effect of DMSO on the growth of microorganisms. The test proved that DMSO had no inhibitory effect on the tested organisms. Each test plate included growth control and sterility control. All tests were performed in duplicate and MIC values were constant.

RESULTS AND DISCUSSION

In vitro antimicrobial activity was investigated by the microdilution method and MIC was determined. The data relating to the antimicrobial activities of fruit body samples is summarized in Table 3. Antimicrobial activity was observed in all species included in the study. It was detected that all of them had antimicrobial activity against at least four of the test microorganisms employed. Most of these activities were antibacterial.

336 Tab. 3. – Minimum inhibitory concentration (MIC)a of methanolic extracts from mushroom samples.

Samples Test organisms B. lupinus F. velutypes P. igniarius S. imbricatus T. aurantium X. ichnusanus B. subtilis 6.25 12.5 2.5 6.25 3.125 6.25 B. pumilus 25 3.125 5 25 12.5 12.5 S. aureus 50 50 5 25 25 12.5 S. lutea 6.25 – 2.5 12.5 12.5 6.25 P. aeruginosa 25 50 5 25 12.5 25 E. coli – – 10 50 50 50 A. niger – – – – – – A. sojae – – – 50 – – Penicilium spp. – – 10 – 50 50 S. cerevisiae – – – – – – C. albicans – – – – – – a Minimum inhibitory concentration (MIC); values given in mg/mL. (–) No inhibition

Results from this study showed that for all examined extracts no antimicrobial activity was recorded against the tested yeasts, S. cerevisiae and C. albicans, and the mold A. niger. The highest antibacterial activity was obtained in the extract from polypore fungus P. igniarius against B. subtilis and S. lutea (MIC=2.5 mg/mL). According to our results P. igniarius exhibited inhibitory effect against all tested bacterial strains with MIC values ranging from 2.5 to 10.0 mg/mL. Also, the highest antifungal activity observed in this study was in methanolic extract of P. igniarius (MIC=10.0 mg/mL) against Penicillium spp. Those results are in accordance with earlier reported data which confirmed the antimicrobial activity of this Phellinus species (S i t t i - w e t and P u a n g p r o n p i t a g, 2008; B a l a k u m a r et al., 2011). It is also evident from these results that the Gram-positive bacteria (Ba- cillus subtilis, Bacillus pumilus, Sarcina lutea and Staphylococcus aureus) were more sensitive to the examined macrofungi extracts. The results from our study are in agreement with literature data which showed that the bacteria were more sensitive to the antimicrobial agents compared to fungi (H u g o and R a s s e l l, 1983). This observation may be explained by the differences in the cell wall structure that can produce differences in antibiotic susceptibility of the cells (W a l s h and A m y e s, 2004). It is well known that Gram- negative bacteria possess an outer membrane and a periplasmic space, both of which are absent from Gram-positive bacteria (B a s i l e et al., 1998). The fungal cell wall structure might also be the reason for relatively high resistance towards antimicrobial agents, which was observed in microfungal species tested in this study. The results from this study show that, apart from P. igniarius extract, mushroom extracts inhibited the tested fungi in relatively high concentration of 50 mg/ml. The explanation might be found in protein–polysaccharides and amorphous homo- and heteropolysaccharides

337 which are identified as constituents of the fungal wall matrix that, among other functions, cement the skeletal portion of the wall, render the elasticity, hydrophilic properties, and determine the antigenicity of the cells (F a r k a š, 2003).

CONCLUSION

The current study was undertaken to measure the antimicrobial potential of methanolic extracts from fruiting bodies of six wild macromycetes (Boletus lupinus, Flammulina velutypes, Phellinus igniarius, Sarcodon imbricatus, Tricholoma aurantium, Xerocomus ichnusanus). Results from this study showed that all analyzed mushroom extracts exhibit antimicrobial activity. With an increasing number of bacteria that have developed resistance to com- mercial antibiotics, extracts and derivatives from mushrooms hold great promise for novel medicines. Regarding the development of natural antimicrobials from macrofungi, further research could be focused on the identification and quantification of individual antimicrobial compounds in the selected mushroom extracts.

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339 СКРИНИНГ АНТИБАКТЕРИЈСКЕ И АНТИФУНГАЛНЕ АКТИВНОСТИ СЕЛЕКТИРАНИХ ГЉИВА ИЗ МАКЕДОНИЈЕ Даниела Николовска Неделкоска1, Наталија Атанасова-Панчевска2, Харис Амеди2, Дафина Велеска2, Емилија Иванова2, Митко Караделев2, Џоко Кунгуловски2 1 Технолошко-технички факултет, Универзитет „Св. Климент Охридски“, П. Прличков 42, 1400 Велес, Република Македонија [email protected] 2 Природно-математички факултет, Универзитет „ Св. Кирил и Методиј“, Архимедова 5, 1000 Скопје, Република Македонија Резиме Тренд развоја нових безбедних антибиотика природног порекла у последњој деценији је разлог да макромицете постану атрактивнe за истраживаче. У овој студији спроведена је евалуација антимикробног потенцијала метанолних екстра- ката добијених из шест врста макромицета (Boletus lupinus, Flammulina velutypes, Phellinus igniarius, Sarcodon imbricatus, Tricholoma aurantium, Xerocomus ichnusanus). In vitro антимикробна активност испитана је микродилуционом методом и одређена је минимална инхибиторна концентрација (MIC) примерка. Тестирање је спрове- дено на једанаест микроорганизама, укључујући шест сојева бактерија и пет врста гљива. Екстракти су показали селективне антимикробне карактеристике. Антими кробна активност је зависила од врсте микроорганизма и од типа и концентрације екстракта. Анализовани екстракти показали су моћније инхибиторне ефекте на раст бактерија него на гљиве. Најизраженија антибактеријска и антифунгална активност је утврђена у метанолним екстрактима врсте P. igniarius. КЉУЧНЕ РЕЧИ: микродилуциона метода, антимикробна активност, гљиве, Македонија

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