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International Journal of Phytopharmacology 581 Ashok Chittaragi and Raja Naika. / International Journal of Biological & Pharmaceutical Research. 2014; 5(7): 581-589. e- ISSN 0976 - 3651 Print ISSN 2229 - 7480 International Journal of Biological & Pharmaceutical Research Journal homepage: www.ijbpr.com IJBPR ANTIFUNGAL PROPERTIES OF HYGROCYBE CANTHARELLUS (SCHWEIN.) MURRILL 1* 2 Ashok Chittaragi and Raja Naika *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
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