International Conference on Biological and Environmental Sciences MOLECULAR PHYLOGENY AND DISTRIBUTION OF ANTRODIA JUNIPERINA (MURRILL) NIEMELA & RYVARDEN AND PYROFOMES DEMIDOFFII (LEV.) KOTL. & POUZAR IN THE REPUBLIC OF MACEDONIA AND CORRELATION TO THEIR POTENTIAL ANTI- CANCEROGENIC AND ANTI-ATHEROGENIC ACTIVITIES GREBENC Tine', DIMITROVSKA Shumkovska Jasmina?, RUSEVSKA Katerina’, KRAIGHER Hojka', KARADELEV Mitko* ‘Slovenian Forestry Institute, Vetna pot 2, 1000 Ljubljana, Slovenia “Institute of Biology, FNS and Mathematics, Arhimedova 5, 1000 Skopie, Republi of Macedonia E-mail: sine grebenc@vordis.si: mitkok@iunon jasminad@iunona, prof ukim,ed ak ABSTRACT Antrodia juniperina (Muriiit) Niemeld & Ryvarden and Pyrofames demidofft (Lév.) Koil. & Pouzar are basidiomycete fungi commonly found on specific wood substrate of Greek juniper (Juniperus excelsa M. Bieb.} occurring exclusively in warm regions of Europe (south-eastern Balkan Peninsula and Turkey). They exhibit high potential for use in human imedicine as anti-cancer and anti-atherogeni¢ drugs of natural origin. ITS regions were applied for comparison of phylogenetic relationship among species closely related to Antrodia juniperina and Pyrofomes demidofit after NI analysis, Both species were confirmed as clear and stable species. Preliminary results of tests for the biological activity of whole sporocarp extract or dried and homogenised maiure sporocarps of basidiomycete fungi Antrodia juniperina and Pyrofomes demidofi, as separate and independent applications, indicated their relative high antioxidant activity in vitro and on cancer-induced mouse. The phylogenetic distribution of close relatives based on molecular markers did not show phylogenetic closeness or common lineages of both analysed species to species or clusters with known biologically active ingredients with potential pharmaceutical value. INTRODUCTION The kingdom Fieg! is currently represented by ca 80.060 described species. The overall number of fungi is estimated t0 1.500.000 (Bridge & Spooner, 2001). About (0% out of them are mushrooms or “higher fungi” and no more then 700 species are known to possess significant pharmacological properties (Wasser, 2002). Mushroom,metabolites are increasingly being utilized to treat a wide variety of diseases, particularly due 10 theie simple application thru diet and ability of being used orally. A lot of scientific works have been done to discover possible functional properties of fungal sporocarps and subsequent disease treatments as cancer, thrombosis, various infections etc, (Lull et al., 2005) or other biclogicat characteristics with putative use in medicine were tested, as for example proteolytic activities (Sabotié et al., 2007). Commonly tested fungi were the one previously used in traditional medicine. Fungi (commonly called medica! mushrooms) were used for hundred of years in Asia (Korean, China and Japan) and some parts of Russia (Wasser, 2002), Most of traditional knowledge and practice originates from these regions so as the majority of traditionally used fungal species with keown biological activities (Lull et al., 2005). Only recently this knowiedge became broader with more species and regions analysed. Not many data is available for fungal species with centre of origin in the Balkan area, Through the geological history Balkan area served as refugia sites for numerous European plant species and associated fungi, and represents the centre of recent plant 24)University of Tirana, Faculty of Natural Sciences diversification (Bennet et al., 1991). According to some more recent data this region is still very diverse in flora and fauna (Myers & Cowling, 1999) bearing high level of endemism throughout the Dinaric areas (Trigas et al., 2007) mainly due numerous glacial refugia and diverse ecosystems and niches. One of the important characteristics of medicinal mushrooms is the abitity to fight cancer or exhibit other biological/medical activities in a Cinumber of ways thru biologically active substances as polysaccharides (Yoon et al., 2003), proteolytic substances (Sabotit et al., 2007), polysaccharopeptides, proteins, terpens, lipids, phenols etc. (Lull et al. 2005), Not much is known about the heredity and distribution among relatives of particular components, secondary compounds, and subsequently their biological activities but we expect (in concordance with morphological species delimitation) closely related species to exhibit similar characteristics. In genus Antrodia close relatives to A. juniperina are known for their medicinal uses including curative effect on cancer (Wu et al,, 1997). Based on their phylogenetic position in the genus, analysed for their anti-cancer and anti-atherogenic, we propose further tests within the close relatives. We envision that integration of approaches applied in the project will enable us to select potentially applicable fungat species, to idemtify end purify natural compounds from novel endemic and rare fungi, test their antioxidant properties. and to learn the mechanism of cancer preventive effects of flngal extracts in order to achieve hew insight in the scavenger molecular processes related to carcinogenesis. Reactive oxygen species are considered to play a major role in intracellular damage either as mutagens leading to cancer or being part of processes related t¢ injury or inflammation. Various studies have shown the action of free radicals at many stages of pathogenesis of age-dependent degenerative disorders, including atherosclerosis, (Halliwell & Gutteridge, 1985; Halliwell & Gutteridge, 1999; Janero, 1990). Although the antioxidant defence systems includes both endogenously and exogenously derived compounds, dietary plants based antioxidant properties have received a great attention (Niki et al., 1994). Where effective, dietary management may help to reduce or eliminate the need for conventional drugs, some or which are associated with adverse secondary effects. Hence many studies have been performed to identify antioxidant compounds with pharmacological activity and a limited toxicity from medicinal mushrooms. In this context, ethno pharmacology plays a significant part in the search for interesting and therapeutically useful fungi and plants. In order to contribute to the knowledge of rare or endemie fungi from Macedonia, in the present study, we primarily screened the free radical scavenging and antioxidant activity from Pyrofomes demidoffi. The standard approach in study of biofogical activities of fungi has been to isolate, and administer the active constituents, Our next step are animal models with induced oxidative stress and practical application Of several fungal sporocarps as diet supplementation to regular feed on ApoE knockout mice (B6.129P2- apoE"'NI1) and their wild type (WT) background strain CS7BL/6 mice. The animals are already subjected to the chemical carcinogen and endothelial inflammatory drug 7, 12-dimethylbenz{aJantracene (DMBA). Since different components in mushrooms may have synergistic activities (Vickers, 2002) or can be easily lost during the extraction process, we propose the use of crude fungal material or whole sporocarp extracts to give better chances to record the desired effect(s). MATERIALS AND METHODS Sporocarp collection, handling and preparation of mouse food pellets Mature sporocarps of Antrodia juniperina (Morrill) Niemeia & Ryvarden and Pyrofomes demidoffii (Lév.) Kotl, & Pouzar exhibiting all necessary morphological characters for correct identification were collected during the growing season from the host tree Juniperus excelsa M. Bieb. Due their relative rare occurrence and connection to specific wood substrate, sporocarps were coilected only from several sites in central Macedonia. Sporocarps were air-dried without use of high temperature (max. 40°C), immediately after the transportation to laboratory. Dried sporocarps were kept in cold and dry place until further use. Preparation of fingal applicable forms 50 g of dried and powdered mature sporocarp from both fungi was extracted at room temperature with 200 mL of methanol under constant shaking for 24 hours. After filtration and centrifugation, the upper layer was evaporated to dryness to obtain the methanol extract. The later was used for further biological assays. All the 242International Conference on Biological and Environmental Sciences chemical and reagents used for the analysis were of analytical grade. Additionally, powdered sporocarps were mixed with common laboratory mouse dry food. The components of the diet as listed by manufacturer were: crude fat min, 5.7%, crude proteins min.18 %, and carbohydrates min, 60 %, fibre max. 4.5%, ash max. 8% (NIH#31M, prepared by Filpaso, Skopje). Mixing ratio of dried fungus and common laboratory mouse food was 2:8. The mixture was pelleted without and further treatment. Food was packed and kept in cool and dry place until application. Prior to the experimental procedures, the rats were fed a standard commercial pellet feed (Flips, 52.11, Skopje, Republic of Macedonia). Experimental design and statistics Hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and test compounds for hydroxyl radical generated by Fe™- Ascorbate-EDTA-H,O, system (Fenton reaction) according to the method of Kunchandy and Rao (1990). The hydroxyl radicals attack deoxyribose that eventually results in TBARS formation. The free radical damage imposed on the substrate, deoxyribose was measured as TBARS by the method of Ohkawa et al (1979). 1.0 mL of thiobarbituric acid (1%) and 1.0 mL of trichloroacetic acid (2.8%) were added to the test tubes and were incubated at 95° C for 20 minutes, After cooling, absorbance was measured at 532.nm against control containing deoxyribose and buffer. Catechin was used as a positive control, The percentage inhibition was determined by comparing the results of the test compounds and control. Nitric oxide radical scavenging effect was analysed using nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH interacts with oxygen to produce nitrite ions which were measured by the Griess reaction. The absorbance of the chromophore formed was measured at 546 nm. The percentage inhibition of nitric oxide generated was measured by comparing the absorbance values of control and test. Curcumin was used as a reference compound. Administration of DMBA and custom tailored diet The required solutions of the carcinogenic, DMBA (7, 12 dimethylbenz (a) antracene, Sigma Chemicals) were prepared freshly by dissolving DMBA in sesame oil. For pellet sporocarp powder application, mice of the following strains are used: C57BL/6 Tac Control mice (40 of female gender) and apoE (-/-) deficient mice (B6.129P2-Apoe’ N11, of female gender), 29 weeks old, were either purchased from Taconic, Europe (Tombjerg, Denmark), or produced in our breeding colony. Animals were housed in humidity and temperature controlled room in a 12-h dark-light eyele, at the Animal Laboratory at the University of Ss Cyril and Methodius, Faculty of Biology, Skopje, Republic of Macedonia. Before fungus administration, the rats were randomized into 3 general groups: 1a +1b) control rats. WT and Apo E receiving mentioned commercial pellet ‘eed throughout the experiment, exposed to vehicle- 200,L sesame oil (each group, n=15); 2a) WT (n=10) and 2b) Apo E experimental rats receiving custom tailored fungus diet (10-15 grams per day / per animal) for a period of 14 weeks (n=12, ftom each group with each different supplement: Antrodia pellet sporocarp and Phellinus pelleted sporocarp), and 3a) WT (n =10) +3b) ApoE deficient mice which received DMBA, two times at weekly intervals (Smg/kg b.w.dissolved in 200 iL sesame oil, subcutaneous application), followed with custom tailored fungus diets (n=15, each group of fungi). All animals are allowed to continuous access to astandard laboratory chow diet in pellet form of mixed food & fungi diet. Following the exposure, animals will be sacrificed after 14 weeks of supplementation, with cardiac puncture for blood collection under ketamine: \ylazine anaesthesia (100 mg/kg i.p. and 20 mg/kg i.p., respectively) in the fasting state. Tissue samples will be removed quickly, flash frozen, measure and store at -80°C until further analysis, All procedures are in Accordance with National Institutes of Health guidelines (Skopje, Republic of Macedonia) for the care and use of experimental animals. Molecular phylogeny of fungi Selected sporocarps of Antrodia juniperina and Pyrofomes demidoffi, and their closely related species were Selected and sampled for rDNA ITS sequence analysis. About SOng of fungal material was used for DNA a na 243University of Tirana, Faculty of Natural Sciences extraction after careful removal of clean and recent part of the hymenium. The DNA was extracted by Plant DNeasy Mini Kit (Promega). Afier procedure DNA was re-suspended in pre-warmed, sterile Milli-Q water to the approximate final concentration 100ng/1. Fungal specific primers ITSIF (Gardes & Bruns, 1993) and ITS4 (White et al., 1990) were used for PCR amplification of the IT'S regions, including 5.88 rDNA. Amplification reactions were done using standard procedure described in White et al. (1990) in a total reaction volume of 40ul with AmplyTaq Gold polymerase. The PCRs were performed after Grebene & Kraigher (2007). Controls lacking fungal DNA were run for each experiment to check the contamination of reagents. PCRs were checked for successful amplification as described in Grebenc et al. (2000). Prior to sequencing the amplification products were separated on 2% agarose gel, excised and purified using the Promega Wizard SV Gel and PCR Clean-Up System. Sequences were done at commercial available sequencing service (MacroGene). Sequencher 4.8 software (Gene Codes) was used to obtain consensus sequence from the two strands of each isolate. All consensus sequences were searched for similarities to @ GenBank nucleotide database using a nucleotide query (htip:/Awww.ncbi.nim.nih.gov/blast’Blast.cgi) to check for correct amplification. Sequences have been lodged in the EMBL database with the accession numbers indicated in results. Obtained sequences and additional sequences from the GenBank were aligned and simple NJ tree (NI tree method, Jukes-Cantor substitution model on 236 nucleotides (all ungapped sites) with 1000 bootstrap replicates was constructed using MAFFT software (http://align.bmr kyushuu.ac.jp/mafiv online/ server’) (Katoh & Toh, 2008), displayed by TreeView (hitp://taxonomy.zoology. gla.ac.uk/rod/ treeview-himl) and edited with CorelDRAW 11, Ver.11,633 (Corel Corporation 2002). RESULTS AND DISCUSSION The preliminary results of the in vitro evaluation of antioxidant activities of Pyrofomes demidoffii showed significant inhibition of lipid peroxidation, and potent hydroxyl radical scavenging activity when compared with standard drug catechin. ICSO value of methanol extracts of Pyrofomes demidofii yield 375 ug/ml in case of hydroxy! radical scavenging activity, and 370 jig/ml respectively in case of lipid peroxidation, Furthermore, methanol extracts also increase significantly nitric oxide production (774 pmole/mg dry wWhr respectively) over the control. The present results revealed that P. demidoffii might have potential therapeutic use. rDNA ITS regions were applied for comparison of phylogenetic relationship among species closely related to “Antrodia juniperina and Pyrofomes demidoffi. Phylogenetic tree for genus Antrodia, Pyrofomes and selected other close relatives constructed with simple NJ method is shown in Figure 1. Based on the phylogenetic tree both species in focus were confirmed as clear and stable within their genera. Neither Pyrofomes demidoffit nor Antrodia juniperina were closely related to cluster of A. cinnamomea / camphorata, a complex of species with know potential pharmaceutical value of their biologically active ingredients (Chiu, 2007). As previously shown on mtSSR rDNA (Kim et al. 2003), also based on nuclear rDNA ITS sequences the genus Aniro- dia is not monophyletic but several morphological genera (Perenniporia, Pyrofomes, Piptoporus, Fomitopsis, ‘Melanoporia) enter the phylogenetic tree in between different Antrodia species or groups. Phylogenetic position of analyzed endemic and potential medical mushroom species Pyrofomes demidoffi’ is close to Perenniporia, as also predicted based on morphological studies (Dogan & Karadelev, 2006) but as for A. juniperina with no direct phylogenetic correlation to known medical mushrooms. ‘There is a strong need for effective antioxidants from natural sources as alternatives to synthetic antioxidant in order to prevent the free radicals implicated diseases like cancer, emphysema, cirrhosis, atherosclerosis and arthritis (Ames et al., 1993; Lachance etal., 2001). The extracts of many plants and fungi have been investigated for their antioxidant activity (Aruoma, 1998; Amakura ct al., 2002; Da Silva et al., 1991). Dietary factors can potentially modify some of the underlying processes involved in cancerogenesis, including modulation of the inflammatory response, and protection against oxidative damage. Secondary metabolites such as polyphenols, terpens, polysaccharopeptides, etc., are not directly required for development and growth, but are involved in communication and defense (Iriti & Faoro, 2004; Paiva, 2000). These substances interact with pathogens, herbivores or fungivores, and other organisms; they protect from various oxidants, repel or poison predators and attract beneficial animals (spore dispersal!) or microbes (Bravo, 1998: Chalcker-Scot, 1999; Winkel- Shirley, 2002). 244