581 Ashok Chittaragi and Raja Naika. / International Journal of Biological & Pharmaceutical Research. 2014; 5(7): 581-589.

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ANTIFUNGAL PROPERTIES OF HYGROCYBE CANTHARELLUS (SCHWEIN.) MURRILL

Ashok Chittaragi1* and Raja Naika2

*1Dept. of P. G. Studies and Research in Applied Botany, Mycology Lab, Bio-Science Complex, 2Jnana Sahyadri, Kuvempu University, Shankaraghatta-577451, Shivammogga (District) Karnataka, India.

ABSTRACT The aim of present study is to investigate the antifungal properties of crude extracts of fruit body in different polar solvents (petroleum ether, chloroform, Methanol and aqueous) of Hygrocybe cantharellus in order to use it as a possible source for new antifungal substances. We evaluated the antifungal activity of crude extracts of fruit body of H.cantharellus against some human as well as plant pathogen viz.M. gypseum, T. equinum, T. kanei, C. albicans, C. indicum, C. krusei, C. merdarium, C. zonatum, E. floccosum and T. rubrum andplant pathogenic fungi viz., A. alternate, A. flavus, A. solani, A. tomentosa, C. capsici, C. dematium, C. lindemuthianum, F. oxysporum and F. solani. The dried and powdered fruiting bodies were successively extracted with a series of non polar to polar solvents using Soxhlet assembly. The antifungal assay was done by agar well diffusion method. Petroleum ether and chloroform extract of H. cantharellus show highest activity against C. merdarium and T. equinum (human pathogen), as well as in C. capsici and F. oxysporum (plant pathogen) to varying degrees, by most of the extracts. Clotrimazole, Fleuconazole, Mancozeb and Captan were used as the standard antibiotics against human and plant pathogenic fungi. The extracts of H. cantharellus also significantly inhibited the fungal growth. The inhibitory effect is very identical in magnitude and comparable with that of standard antibiotics used.

Key Words: Antifungal properties, Solvent extracts, Pathogens, Hygrocybe cantharellus, Fruiting bodies.

INTRODUCTION It has been known that macrofungi are used as a It is believed that mushrooms need antibacterial valuable food source and traditional medicines since Greek and antifungal compounds to survive in their natural and Roman antiquity (Anke, 1989). Dioscorides, first environment. Antimicrobial compounds could be isolated century Greek physician, knewthat Laricifomes from many mushroom species and some proved to be of (Fomitopsis) officinalis (Vill.) Kotl and Pouzar benefit for humans (Lindequist et al., 2005). As an (Fomitopsidaceae) can be used for treatment of antifungal and antibacterial compound, Sparassol was “consumption”, a disease now known as tuberculosis isolated in the early 1920s from Sparassis crispa. Since (Stamets, 2002). The famous 5300year-old Otzi, or Ice then, several antifungal and antibacterial compounds have Man, had Piptoporus betulinus (Bull.)P. Karst. been isolated from different macrofungi species. (Fomitopsidaceae) and Fomes fomentarius (L.) J.J.Kickx The scientific community, while searching for (Polyporaceae) with him when his body was discovered new therapeutic alternatives, has studied many kinds of (Stamets, 2002). mushrooms and has found various therapeutic activities such as anticarcinogenic, anti-inflammatory, immuno- Corresponding Author suppressor and antibiotic, among others. In recent decades, various extracts of mushrooms and plants have been of Ashok Chittaragi great interest as sources of natural products (Turkoglu et Email: [email protected] al., 2007). Some mushrooms serve as food because of their 582 Ashok Chittaragi and Raja Naika. / International Journal of Biological & Pharmaceutical Research. 2014; 5(7): 581-589. nutrient contents while some have been used extensively in morphological, anatomical and physiological traditional medicine (Stamets, 2000; Lindequist et al., characteristics with the help of standard literatures 2005). The effects of different mushroom extracts on (Purkayastha and Chandra, 1985; Singer, 1986; Roy and pathogens and microorganisms are studied by a very large De, 1996; Das and Sharma, 2005). number of researchers in different parts of the world (Jonathan and Fasidi, 2003; Rosa et al., 2003; Uzun et al., Preparation of crude extract 2004; Gbolagade and Fasidi, 2005; Gezer et al., 2006; Various extracts of the experimental fruit body Solak et al., 2006; Turkoglu et al., 2006; Barros et al., was prepared according to the methodology of Indian 2007; Demirhan et al., 2007). Pharmacopoeia (Anonymous, 1966). The fresh fruit bodies Several compounds with important were dried in shade conditions and the dried materials were pharmaceutical properties have been isolated from these pulverized in a blender to get coarse powder. The coarse organisms. Substances that act as anti-aging, in longevity, powder material was used to successively with 2000 ml modulating the immune system, having hypoglycemic petroleum ether following chloroform and methanol with a activity and to inhibit tumor growth have been isolated Soxhlet extractor for 48 h at temperature not exceeding the from mushrooms, such as polysaccharides. boiling point of the solvent (Lin et al., 1995).These Polysaccharides can inter connect several points forming a extracts were concentrated to dryness in flash evaporator wide variety of branched or linear structures, for example, under reduced pressure and controlled temperature (40- ß4 glucans (Ooi and Liu, 2000). Furthermore, other 50˚C). The yield of extracts obtained from petroleum ether bioactive substances such as triterpenes, lipids and phenols was 10.88 gm, followed by chloroform 11.21 gm and have also been identified and characterized in mushrooms methanol 51.16 gm (Table-1). The highest % of extracts with medicinal properties (Maiti et al., 2008). Mushroom was obtained from methanol of H. cantharellus (Piechart- contain vitamins A and C of ß-carotene and a great variety 1).Each extract was transferred to glass vials and kept at of secondary metabolites such as phenolics compounds, 4˚C before use.The residence was dissolved with dimethyl polyketides, terpenes, steroids and phenols, all have sulphoxide (DMSO) with different concentrations and protective effects because of their antioxidant properties checked it for antifungal activity. (Jayakumar et al., 2009; Soares et al., 2009). In disc diffusion method, the discs are very Test organisms expensive and their acquisition in developing countries is All experimental fungi were obtained from the sometimes difficult. In an attempt to combat this, in 1997 Microbial Type Culture Collection and Gene Bank Magaldi developed a modification of the disc diffusion (MTCC), Institute of Microbial Technology (IMTECH), method which she named the „well diffusion‟ method Chandigarh. American Type Culture Collection (ATCC). (WD). The procedure is similar; the discs are The viability of the organisms was maintained by regular supplemented with dilutions of the drug placed in wells transfer into freshly prepared on potato dextrose agar which have been cut out in the agar. This allows the use (PDA) at 28˚C and stored at 4˚C until used. For the present and standardization of various concentrations of any drug study pure fungal cultures were taken (Table-2). for different fungal species. It has proven to be a cheap, simple and reliable method of antifungal drug ANTIFUNGAL STUDIES susceptibility testing for Candida spp., and it produces Fungal media (PDA) results comparable with the disc diffusion test (Magaldi 200 gms of potato slices were boiled with distilled and Camero, 1997; Magaldi, 2004). water. The potato infusion was used as water source of The aim of the present work is to antifungal media preparation. 20gm of dextrose was mixed with properties of Hygrocybe cantharellus extracts with the help potato infusion. 20gm of agar was added as a solidifying of petroleum ether, chloroform and methanol solvent agent. These constituents were mixed and autoclaved. The system against to the plant and human pathogenic fungi solidified plates were bored with 6mm diameter by cork were investigated. borer.

MATERIALS AND METHODS Agar well diffusion method Collection of mushroom fruit body Antifungal activity of the mushrooms extract was The Hygrocybe cantharellus were collected from tested using agar well diffusion method (Bauer et al., Haniya, Hosanagar taluk, Shivammogga district, 1996). The prepared culture plates were inoculated with Karnataka, India, during the month of August 2013. The H. different fungus by using plate method. Wells were made cantharellus of mushroom was picked from the litter and on the agar surface with 6mm cork borer. The different decaying soil surface, with help of forceps and then they solvent extracts of Hygrocybe cantharellus were loaded to were cleaned and air dried in an oven at 40˚C for 48 h. the four wells by using 100μl micropipette in 4 different dried mushroom samples were powdered mechanically for concentrations i.e., 12.5 mg/ml, 25 mg/ml, 50 mg/ml further use. Identification was done by comparing their and100 mg/ml respectively. Clotrimazole, Fleuconazole, 583 Ashok Chittaragi and Raja Naika. / International Journal of Biological & Pharmaceutical Research. 2014; 5(7): 581-589.

Mancozeb and Captan are used as a positive by 10 mm against C. albicans and minimum (7.3 mm) by control and DMSO is used as a negative control. All the 50mg/ml of extract against C. indicum. In the same time, plates were incubated at 23±2˚C fungal growth was does not show any zone of inhibition against E. floccosum determined by measuring the diameter of zone of inhibition followed by C. krusei and C. zonatum in three solvents after 5 days of incubation. The test was done in triplicates extracts (Fig -3, 4 and 5). to arrive concordant result. The agar plates were incubated The zone of inhibition against plant pathogens at 37˚C for 24hrs. (Table-4) ranged between 3.6-8.6 mm in petroleum ether, respectively 3.6-9 mm in chloroform and 4-9 mm in RESULTS methanolic extract. The maximum activity (10,9 and 8.6 The petroleum ether, chloroform and methanol mm) was recorded from 100mg of petroleum ether and extracts of the Hygrocybe cantharellus fruit body were methanolic extract against C. capsici followed by F. screened against ten human and nine plant pathogenic oxysporum, and A. alternate, respectively and minimum fungi to check antifungal activities by agar well diffusion (3.6 mm) by C. lindemuthianum at 12.5 mg/ml level method which showed valuable zone of inhibition. The whereas, the chloroform extract showed the moderate specific zone of inhibition against various types of activity (7 mm) was recorded from 100mg against F. pathogenic fungi was shown in Table-3 and 4.All the solani and minimum (3.6 mm) by 12.5 mg/ml against A. solvents extract were effective against both human and solani. The concentration at 25 and 50 mg/ml in all three plant pathogens and chloroform and petroleum ether extracts shows minimum (4.3-7 mm) against A. solani and extract was better than methanol extract against human C. lindemuthianum. C. dematium and A. flavus was not pathogens but in the case of plant pathogens in vice versa. responding for all the solvent extracts. In the same time F. The zone of inhibition against human pathogens oxysporum and A. tomentosa are also not responding for (Table-3) ranged between 5.3-14 mm both in petroleum both petroleum ether and chloroform extracts (Fig-3, 4 and ether extract, chloroform and 6-13 mm in methanolic 5). extract. The maximum activity (14-13.2 mm) was recorded The antifungal activity of different solvent from 100mg/ml of petroleum ether, chloroform and extracts of mushroom is changeable and has a lower methanolic extract of H. cantharellus against M. gypseum, antifungal activity as to comparison of antibiotics viz., respectively C. merdarium, T. equinum followed by 13 Clotrimazole, Fleuconazole, Mancozeb and Captan (Table- mm against T. kanei, C. albicans and minimum (5.3 and 8 5) and also various solvents extracts of mushrooms mm) against T. rubrum, and C. albicansat 12.5 mg/ml inhibited the growth of some microorganisms at different level whereas, the extract showed the moderate activity (11 concentrations which account for the differential and 12.3 mm) was recorded from 25 and 50 mg/ml of fruit antimicrobial effect, as suggested (Fig-1 and 2). body extract against C. merdarium, M. gypseum followed

Piechart 1. Showing total yield in % from different Fig. 1. Showing antifungal activity of standard drugs solvents extracts of H. cantharellus and control against human pathogens

Fig. 2. Showing antifungal activity of standard drugs and Fig. 3. Comparative studies of petroleum ether extract of control against plant pathogens H. cantharellus

584 Ashok Chittaragi and Raja Naika. / International Journal of Biological & Pharmaceutical Research. 2014; 5(7): 581-589.

Fig. 4. Comparative studies of chloroform extract of H. Fig. 5. Comparative studies of methanol extract of H. cantharellus cantharellus

Table 1. Total yield of mushrooms extracts obtained various organic solvents (200 gm in 2000 ml) Mushroom species Organic solvents Yield of extract in gm Petroleum ether 10.88 Hygrocybe cantharellus Chloroform 11.21 Methanol 51.16

Table 2. Microorganisms Numbers Name of the Fungal MTCC ATCC Human pathogens Chrysosporium indicum 4266 Epidermophyton floccosum 613 Trichophyton rubrum 1538 Microsporum gypseum 2157 Trichophyton kanei 2091 Candida albicans 10231 Candida krusei 6258 Chrysosporium merdarium 900628 Chrysosporium zonatum 845981 Trichophyton equinum 6275 Plant pathogens Colletotrichum lindemuthianum 90028 Colletotrichum dematium 60192 Alternaria tomentosa 16404 Alternaria solani 26934 Aspergillus flavus 9170 Fusarium solani 2935 Fusarium oxysporum 2485 Colletotrichum capsici 2071 Alternaria alternate 7202

Table 3. Antifungal activity of Hygrocybe cantharellus against human pathogens Indicator test Concentration (mg/ml) Extracts fungi 12.5 % 25 % 50 % 100 % C. indicum - - 7.3 8.6 E. floccosum - - - - Petroleum ether T.rubrum 5.3 7.6 8 9 M.gypseum 9 11 12 14 T.kanei - - - - 585 Ashok Chittaragi and Raja Naika. / International Journal of Biological & Pharmaceutical Research. 2014; 5(7): 581-589.

C.albicans 8 10 11 12 C.krusei - - - - C.merdarium 7 9 10 12 C.zonatum - - - - T.equinum 7.3 7.6 8.3 9 C. indicum - - 9 11 E. floccosum - - - - T.rubrum 7.3 8.6 10 12 M.gypseum 8 10 12 13 T.kanei 8 9 11 12 Chloroform C.albicans 7.6 8.3 9 12 C.krusei - - - - C.merdarium 9.3 11 12.3 14 C.zonatum - - - - T.equinum 5.3 6.3 10 13.2 C. indicum - - - - E. floccosum - - - - T.rubrum 6 8 9.3 12 M.gypseum 6.3 7.3 8 10 T.kanei - - 10 13 Methanol C.albicans 7.3 9 11 13 C.krusei - - - - C.merdarium - - - - C.zonatum - - - - T.equinum 8 9 11 12 Note: „-„-No activity

Table 4. Antifungal activity of Hygrocybe cantharellus against plant pathogens Indicator test fungi Concentration (mg/ml) Extracts 12.5 25 % 50 % 100 % % C. lindemuthianum 3.6 5.6 7 8 C.dematium - - - - A.tomentosa - 3 5.3 6 A.solani 4 4.6 6 7 Petroleum A.flavus - - - - ether F.solani 6.3 7.3 7.6 8 F.oxysporum - - - - C.capsici 7 8.6 9 10 A. alternate 5.3 6.3 7.3 8.6 C. lindemuthianum - - 7 9 C.dematium - - - - A.tomentosa - - - - A.solani 3.6 4.3 6 6.3 Chloroform A.flavus - - - - F.solani 4.3 5.3 6 7 F.oxysporum - - - - C.capsici - - - - A. alternate - - - - C. lindemuthianum 4 5 6.3 7.3 C.dematium - - - - Methanol A.tomentosa - - 4 6 A.solani - - 6 7.3 A.flavus - - - - 586 Ashok Chittaragi and Raja Naika. / International Journal of Biological & Pharmaceutical Research. 2014; 5(7): 581-589.

F.solani 4 6 7 8 F.oxysporum 5 6 8.6 9 C.capsici 5.6 6.3 7 9 A. alternate 3.6 4.6 5.6 6.6 Note: „-„-No activity

Table 5. Antifungal activity of standard drug and control against human and plant pathogenic fungi Standard Control Test organism Clotrimazole Fleuconazole Mancozeb Captan DMSO Human pathogens C. indicum 32 30 x x - E. floccosum 30 31 x x - T.rubrum 32 34 x x - M.gypseum 28 32 x x - T.kanei 24.3 28 x x - C.albicans 27 26 x x - C.krusei 26 30 x x - C.merdarium 24 20 x x - C.zonatum 22.3 24 x x - T.equinum 24.6 25.3 x x - Plant pathogens C. lindemuthianum x x 25 20 - C.dematium x x 23 25 - A.tomentosa x x 26.3 26 - A.solani x x 25 22 - A.flavus x x 30 28 - F.solani x x 28 27 - F.oxysporum x x 30 29 - C.capsici x x 23 23 - A. alternate x x 25 24 - Note: „x‟-Not applicable, „–„- No activity.

DISCUSSION The methanol extract shown the normal and Many pharmaceutical substances with potent and minimum zone of inhibition against human pathogenic unique health-enhancing properties have been isolated fungi T. rubrum, M. gypseum and maximum amount of from medicinal mushrooms and distributed worldwide inhibition was recorded against T. kanei and C. albicans. In (Cairney et al., 1999). Mushroom based products either other hand, against the fungal strain maximum zone was from the mycelia or fruiting bodies are consumed in the recorded against T. equinum and minimum zone against M. form of capsules, tablets or extracts (Nitha et al., 2006). gypseum. The antifungal activity depends upon the host In present study, the selected macrofungus defense system and drug reaction were factors looked into showed antifungal activity in high level which was anti-microbial chemotheraphy but depression in host screened against the selected fungal species against plant immune activity were ignored during antifungal drug pathogenic fungi viz., A. alternate, A. flavus, A. solani, A. screening (Gunji et al., 1983) .This study indicated that tomentosa, C. capsici, C. dematium, C. lindemuthianum, F. there are differences in the antimicrobial effects of oxysporum and F. solani (Table-4).Human pathogenic mushroom groups, due to phytochemical differences fungi viz., M. gypseum, T. equinum, T. kanei, C. albicans, among species. They claimed that the sensitivity of C. indicum, C. krusei, C. merdarium, C. zonatum, E. microorganism to chemotherapeutic compounds change floccosum and T. rubrum (Table-3). All the solvents even against different strains (Chittaragi et al., 2013). The extract were effective against both human and plant results revealed that antifungal and phytochemical pathogens, petroleum ether and chloroform extract was constituents of petroleum ether, chloroform and methanol better than methanol extract against human pathogens but extracts varied in usefulness which may be attributed, the in the case of plant pathogens, petroleum ether and author also notable the antibacterial activity (Divya et al., methanol maximum activity when compare to the 2011). chloroform extracts. 587 Ashok Chittaragi and Raja Naika. / International Journal of Biological & Pharmaceutical Research. 2014; 5(7): 581-589.

This difference in response of mushroom extracts CONCLUSION to test organisms might be due to a number of factors, as Results obtained from the antifungal study by agar studies suggest that the antimicrobial activities of all well diffusion method in the present study revealed that mushroom extracts are changeable (Iwalokun et al., 2007), different solvent extracts of Hygrocybe cantharellus depending upon the nature of environment and media in showed inhibitory effect against all the tested microbial which it was grown. It also depends upon the genetic strains but had more antifungal activity against human structure of mushroom species, physical and biochemical pathogenic fungi as compared to plant pathogenic fungal constituents, extraction solvents and test organisms. The strains. The zone of inhibition study also suggests that the sensitivity pattern of microorganisms also changes to extracts had shown antifungal activity in a concentration chemotherapeutic agents depending on their strains, and dependent manner against the test microorganisms and was susceptibility or resistance to antibiotic (Zhou et al., 2005). comparable with the standard drugs. Further, from the The results of the present study strengthened the antifungal study it was observed that the order of activity outcomes of earlier works done by others that showed was in the sequence of petroleum ether extract>chloroform mushrooms produced a great variety of antimicrobial extract>methanol extract. The results presented in this agents. For instance, it is known that the extract from fruit project are only based on different extract and did not bodies of several Lactarius sp. (Bergendorf and Sterner, specify any defined antifungal substances. In present study 1988; Anke et al., 1989); Fomitopsissp. (Keller et al., the use of different solvent extract are capable to extract 1996); Boletus sp. (Lee et al., 1999); Cortinarius sp. the various biochemical compound from H. cantharellus (Nicholas et al., 2001); Ganoderma lucidum, Navesporus show various beneficial effects which was supported by a floccosa and Phellinus rimosus (Sheena et al., 2003); variety of literature. From previous studies in addition to Pleurotus tuber-regium (Ezeronye et al., 2005); Amanita the current it could be concluded that Hygrocybe caesarae, Armillaria mellea, Chroogomphus rutilus, cantharellus contain a potential metabolite and can be Clavariadelphus truncates, Clitocybege otropa, further utilized for its antimicrobial applications. Ganoderma sp., Ganoderm acarnosum, Hydnum repandum, Hygrophorus agathosmus and Stereum ostrea ACKNOWLEDGEMENT (Praveen et al., 2011) contain a wide range of Authors are thankful to The Chairman, antimicrobial activity. The result of the former and current Department of Applied Botany, Jnana Sahyadri, Kuvempu study may suggest that the fruiting body of Hygrocybe University, Shankaraghatta, Shimoga (D), Karnataka, cantharellus is a source of pharmacologically active India, for providing laboratory facilities and the University substances having diverse therapeutic applications. Grant Commission (UGC), Government of India, for Bioassay guided isolation of active principle is currently giving a research grant to carry out this study. underway to characterize the antimicrobial compound of this interesting taxon.

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