Further studies of biology and genetics of edible and medicinal mushrooms are perspective and will assist biotechnological application of these organisms for ...
23. Badalyan S.M., C.Z. Sakeyan: In “Ecol. Fung. Comm.” BMS Annual Meeting, Manchester, UK, 2007, 68. 24. Sakeyan C.Z.: NAS RA, Elect. J., Natural Sci., 2006, 1, 21-24. 25. Badalyan S.M., Kües U., Avetisyan H.K.: In “Advan. Med. Mycology” Moscow, Russia. 2005, 176-178.
PREVENTIVE-TREATMENT OF RADIATION INJURIES WITH CU (II) CHELATES OF AMINO ACID SCHIFF S.A.Bajinyan, M.H. Malakyan, V.H. Matosyan, K.N. Babayan, D.E.Yeghiazaryan, L.A. Vardevanyan, *N.H. Karapetyan Centre of Radiation Medicine and Burns, *Yerevan State University Introduction: In spite of the existence of synthetic and natural agents in the medical armamentarium for radiation protection and radiation recovery [1], there are no existing safe and effective treatments of radiation injury, since the currently available radioprotectors are very toxic to humans and their general use is further compromised by their lack of efficacy when given after irradiation. In a search for novel less toxic radioprotective agents, studies of essential metalloelement chelates as radioprotectants and radiation recovery agents, which cause an increase in survival, seems worthwhile and offers a new approach to overcome the pathological effects of ionizing radiation suggesting their use to prevent or perhaps predominantly facilitate recovery from radiation injury [2, 3]. Aimed to develop novel anti-radiation remedies of high efficacy and low toxicity, the chelates of Cu(II) with Schiff bases derived from L-Tyrosine and L-Tryptophan were synthesized. The compounds obtained were studied for their radioprotective and DNA protecting activities. Materials and Methods: To evaluate the radioprotective properties of Cu(II)(Nicotinyl-LTyrosinate)2 and Cu(II)(Nicotinyl-L-Tryptophanate)2, white rats were treated subcutaneously (SC) or per oral (PO) with the single dose of these substances at the dose level of 10 mg/kg, 20 mg/kg or 40mg/kg 1, 3, 6 or 24 hours prior to X-ray performed on ‘RUM-17’ facility at dose level of radiation exposure that corresponded to LD100/30 (870 R). Indices of animal survival were determined in 30 days after irradiation. In order to study the potential of Cu(II)(Nicotinyl-L-Tyrosinate)2 and Cu(II)(Nicotinyl-L-Tryptophanate)2 to prevent or to repair DNA structure resulted from radiation exposure, additional experimental
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Further studies of biology and genetics of edible and medicinal mushrooms are perspective and will assist biotechnological application of these organisms for obtaining new mycelium based pharmaceuticals, nutriceuticals and cosmetic commercial products with mushroom origin. Support by DAAD (#548.104401.174), NATO (#980764), Deutsche Bundesstiftung Umwelt (DBU) and NSF (#DEB-9521526) is gratefully acknowledged.
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13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Kües U., Künzler M., Bottoli et al.: In “Fungi in Human and Animal Health” Jodhpur, India. P. 431-470. Badalyan S.M.: (Review). Probl. Med. Mycology. 2001, 3, 16-23. Badalyan S.M.: Int J Med Mushr, 2003, 5, 277-286. Wasser S.P., E. Nevo. et al. Int J Med Mushr, 2000, 2, 1-19. Chihara G.: In “Mushr. Biol. Mushr. Products”(Chang S.T., Buswell J.A., and Chiu S.W., eds.) 1993. Badalyan S.M., N.G. Gharibyan, A.E. Kocharyan: Int. J. Med. Mushr, 2007, 9, 275-276. Badalyan S.M., H.K. Avetisyan, U. Kües: Abstracts IMC-8. Cairns, Australia, 2006, P. 289. Gunde-Cimerman N, Cimerman A: Exp. Mycology, 1995, 19, 1-6 Ikekawa T.: Food Rev. Intern. 1995, 11, 203-206. Badalyan S.M., Sakeyan C.Z.: In “Adv. Med. Mycol.” Moscow, Russia, 2005, 178-181. Badalyan S.M., Kües U., Melikyan L.R., Navarro-Gonzáles M.: Int. J. Med. Mushr, 2005a, 7, 378-380. Badalyan S.M., Gharibyan N.G., Sakeyan C.Z.: Catalogue of the Fungal Culture Collection at the Yerevan State University. Yerevan, YSU. 2005b. Badalyan S.M., Hughes K.W.: In §Sci. Cult. Ed. Med. Fungi.¦ Miami Beach, Florida, USA, 2004, 149-154. Naumann A., Navarro-Gonzáles M., Sanchez-Hernandez O., Hoegger P. J., Kües U.: Curr. Trenes Biotechnol. Pharm. 2007, 1, 41-61. Badalyan S.M., H.K. Avetisyan, M. Navarro-González, U. Kües: XV Congr. Europ. Mycol., S.-Petersburg, Russia, 2007, 28-29. Badalyan S.M.: Int J Med Mushr, 2004, 6, 127-134. Badalyan S.M., Innocenti G. et al.: Phytop. Mediterr, 2004, 43, 44-48. Badalyan S.M., Sisakyan S.H.: Int J Med Mushr, 2005, 7, 382-383. Soponsathien S.:. J. Gen. Appl. Microbiol, 1998, 44, 337-345. Badalyan S.M. et al. Helmbrecht: Int. J. Med. Mushr, 2006a, 8, 263-278. Badalyan S.M., H.K. Avetisyan, M. Navarro-González, U. Kües: VI Int. Conf. Mushr. Biol. Mushr. Prod. Bonn, Germany (accepted). 2008. Badalyan S.M., Sakeyan C.Z. Int J Med Mushr, 2004, 6, 347-360.
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fomentarius, G. applanatum and L. sulphureus. Mycelial growth was not observed in any of the strains at 5 and 100 C. They formed dense and/or not dense, smooth and/or filamentous mycelial pellets during submerged growth [22, 23]. Different levels of AFA of screened medicinal mushrooms were observed in dual culture experiment [17]. Among tested Aphyllophoromycetideae species, D. quercina and F. fomentarius were more active against filamentous fungi [24]. The highest AFA toward phytopathogens and their antagonists (G. roseum and Trichoderma spp.) possessed F. velutipes and P. ostreatus strains. CMs, particularly Copinellus disseminatus, C. micaceus, C. domesticus and Coprinopsis strossmayeri suppressed the growth of humans, animals and plant pathogens (except F. culmorum), however the Trichoderma spp. showed the highest combative activity against these mushrooms. Among CMs mycelia of Coprinellus species (C. disseminatus, C. micaceus, C. domesticus) and Coprinopsis strossmayeri are markedly suppressed the growth of test-bacteria. Strong APA against P. caudatum was present in CL of C. comatus and F. velutipes, whereas different concentrations of ME showed up to 2.8-fold MGA [18]. The 3-4-fold increase of stimulatory effects on wheat and maize seeds’ growth of CL and ME samples of G. lucidum, D. quercina and P. betulinus have also been detected [23]. The relatively higher indicators of AOA were revealed in CL samples, rather than ME samples of tested species [3, 25]. Significantly higher indicators of AOA were detected in C. comatus, Coprinellus disseminatus, C. domesticus, C. radians and F. velutipes. Different levels of enzymatic (peroxidases, proteases) activity was revealed [7]. Strong laccase activity showed species from Coprinus and Coprinellus (disseminatus, micaceus, radiata, xanthothrix, curtus) clades. Milk peptonization was detected in CL of Coprinellus species, Coprinopsis strossmayeri, F. velutipes and P. ostreatus. PLA was expressed by coagulation reaction in P. plicatilis and it was absent in C. comatus. The species possessing milk-peptonizing properties could be applicable in medicinal industry for obtaining thrombolytic and fibrinolytic biotech-products, whereas revealed milk-coagulating activity allows recommending them as source of proteases in food (dairy) industry. Almost all screened mushroom species/strains, particularly xylotrophes, which possess in vitro fruiting ability. Conclusion:
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was tested by their anti-oxidative potential to inhibit the reactions of free radical peroxide oxidation of lipids (POL) in rat brain homogenate [3]. Different tests were applied to estimate extracellular phenoloxidases (laccases) and proteases in mycelia of mushroom species/strains [7, 19]. Results and Discussion: Biological, particularly morphological and ecological characteristics of mycelia of screened mushroom collections were thoroughly described and their growth parameters were detected on various nutrient media. The MEA and PDA media were favorable for cultivation of mycelia of all tested species. Based on morphological and genetic studies two species-specific morphotypes (A, B) and 3 subtypes (A-B, A1, A2) of mycelial colonies within Armenian collections of F. velutipes were observed [20]. The discovery of two unknown Flammulina (SB71) and Pleurotus (SB27) biotypes, high levels of sequence variability and heterozygosity in Armenian collections of P. ostreatus and F. velutipes when compared to their Western European collections suggest that Armenian populations may retain significant variability that could provide genetic stocks for further medicinal usage of these species. Armenia may also have served as part of a refugium for fungal species and their plant hosts [13]. CMs were different based on their average GR and GC indicators. The highest GCs possess Coprinellus (75) and Coprinopsis (69) species. Hyphal clamps were present in Coprinopsis, whereas they are rare or absent in Coprinellus species. Asexual spores (oidia) were described in Coprinellus species and they were rare or absent in Coprinopsis, Coprinus and Parasola species. Screened CMs, particularly Coprinellus species/strains grew well over a wide range of pH (5-12) and can therefore be classified as “ammonia fungi”. They are mesophilic species having a growth temperature range of 25-37°C. However, Coprinellus species are more tolerant, rather than Coprinopsis species. None of the tested strains grew at pH 4 and 5°C [15]. Submerged cultures of Coprinopsis species formed dense and smooth pellets whereas for Coprinellus species, dense filamentous pellets are typical [21]. Mycelial morphology, growth parameters and pellet formation in Aphyllophoromycetideae species/strains were described, as well. The range of favorable growth temperature (25-30 0C) and pH (3-10) was revealed. At 350C, poor growth was detected in D. quercina F.
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Studies of morphological, ecological, physiological characteristics and definition of mycelial growth parameters (growth rate – GR, growth coefficient-GC) of fungal cultures were evaluated on different agar (malt-extract agar-MEA, potato-dextrose agar-PDA, glucosepeptone agar-GPA) and liquid (malt-extract) media under different ecological (pH 3-12 at 25 C and pH 6 at 5, 15, 20, 25, 30, 37 C) and culture (static and submerged) conditions. Pellets formation and morphology during submerged mycelial growth (at pH 6.5, 25 0C, 200 rpm) as required characteristic for optimizing product yield were described. Genetic identification of Coprinoid mushrooms (CMs species from traditional genus Coprinus Pers. currently separated into clades Coprinellus, Coprinopsis, Coprinus and Parasola), P. ostreatus and F. velutipes collections was realized using the ribosomal DNAITS sequence analysis as a species identification tool in order to ensure accurate species names for their further biotechnological cultivation and medicinal studies. The genetic variability within P. ostreatus and F. velutipes collections in Armenia was examined [13, 14]. Taxonomically and phylogenetically valuable “morphological” and “molecular” characteristics, such as hyphal clamps, asexual spores, DNA-ITS sequences data, etc. were estimated [17]. In vitro antifungal (AFA), antibacterial (ABA), antioxidant (AOA), antiprotozoal (APA), mitogenic (MGA) and proteolytic (PLA) activities of screened mushrooms samples (cultural liquid – CL, mycelial extract - ME) obtained after submerged cultivation of mycelia were investigated by appropriate methods previously described in our publications [3, 16-18]. AFA of growth mycelia was studied towards phytopathogenic fungi (Bipolaris sorokiniana, Rhizoctonia cerealis, Rh. solani, Fusarium culmorum, F. tricinctum, Gaeumannomyces graminis var. triticii, Verticillium dahliae) and their antagonists (Gliocladium roseum, Trichoderma asperellum, T. harzianum, T. pseudokoningii, T. viride), as well as potentially pathogenic for humans and animals filamentous fungi (Chrysosporium keratinophilum, Microsporum gypseum, Stachybotris chartarum, Trichophyton ajelloi). Several test-bacteria (Bacillus subtilis, Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus epidermidis, S. aureus, Streptococcus haemoliticus) were used for ABA screening of CL and ME samples of investigated mushrooms. Their APA was tested against Paramecium caudatum. For study of MGA the Paramecia culture, wheat and maize seeds were used as test-organisms. The AOA of CL and ME samples
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additives (“nutriceuticals”) [4]. Biopreparations lentinane, krestin, coriolan and schyzophyllan are successfully used for the treatment of different diseases in combination with chemotherapy [5]. Nutritive, anti-inflammatory, regenerative and antioxidant properties of medicinal and edible mushrooms make their usage perspective also in cosmetology. Mushroom’s extracts are applicable in the manufacturing of hair and skin care products [6]. Extracellular fungal proteases attack mainly casein and fibrin and could be used to obtain milk-coagulating, proteolytic, thrombolytic and fibrinolytic biotech-products [7]. Cultivation of both mycelium and fruiting bodies of mushrooms has significant industrial potential. Study of biological characteristics and elaboration of optimal growth conditions of mycelia will allow controlling biotechnological cultivation process of edible and medicinal species for obtaining high yield biomass and desired biotechproducts. However reported medicinal properties of mushrooms have not always confirmed the morphological identification, particularly their cultures/strains and in many cases, incorrect species epithets have been used. It makes genetic identification of mycelial cultures, particularly of industrial species/strains, necessary. Medicinal properties (antibacterial, antiviral, immune-modulating, hypocholesterolemic, regenerative, etc.) of Pleurotus ostreatus and Flammulina velutipes, as well as species from Coprinoid and Aphyllophoromycetideae mushrooms groups have been reported in many publications and reviews [8-11]. In this paper data related biological characteristics, genetic variability and perspective of biotechnological application of these medicinal mushrooms are presented. Materials and Methods: In screening program, 26 species and 149 strains of Basidiomycetes mushrooms from Fungal Culture Collection of Yerevan State University have been involved [12]. Armenian collections of P. ostreatus and F. velutipes include 20 and 61 strains, respectively. Among screened cultures 20 species and 49 strains belong to Coprinoid (Coprinus comatus, Coprinellus curtus, C. disseminatus, C. domesticcus, C. ellisii, C. radians, C. micaceus, C. radians, C. xanthothrix, Coprinopsis atramentaria, C. cinerea, C. cothurnata, C. episcopalis, C. gonophylla, C. lagopides, C. radiata, C. romagnesiana, C. scobicola, C. strossmayeri, Parasola plicatilis) and 6 species and 19 strains to Aphyllophoromycetideae (Daedalea quercina, Fomes fomentarius, Ganoderma applanatum, Ganoderma lucidum, Laetiporus sulphureus, Piptoporus betulinus) mushrooms.
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3. 4. 5. 6. 7. 8. 9.
Pool-Zobel BL, Leucht U: Mutation Research, 1997, 375, 105–116. Sobels FH: Archives of Toxicology, 1980, 46, 21-30. Gasparyan G, Hovhannisyan G et al.: International Journal of Toxicology, 2007, 26, 1-6. Tice RR, Agurell E, Anderson D et al.: Mol. Mutagen. 2000, 35,206-221 Ghazaryan RK, Sahakyan LA, Tovmasyan AG: Patent of the Republic of Armenia No 1715 A2. 2006. Singh NP, McCoy MT, Tice RR, Schneider EL: Exp. Cell Res., 1988, 175, 184-191. Hartmann A, Agurell E et al.: Mutagenesis, 2003, 18, 1, 45-51.
BIOLOGY, GENETIC VARIABILITY AND BIOTECHNOLOGICAL APPLICATION OF SEVERAL MEDICINAL MUSHROOMS S.M. Badalyan1, K.W. Hughes2 and U. Kües3 1 Yerevan State University, 2University of Tennessee, Knoxville, 3 Georg-August-University Göttingen. Introduction: Extensive research on biology and genetics of Basidiomycetes mushrooms has markedly increased owing to their potential use in biotechnology. Mushrooms are rich in proteins, carbohydrates, fibers, unsaturated fatty acids, vitamins, minerals and considered a very good dietary food [1]. They are also regarded as sources of a wide range of bioactive metabolites (polysaccharides, glucans, terpenoic, indolic and phenolic compounds, etc.) and different enzymes used in medicine, food and cosmetic industries. Since ancient times, anti-inflammatory, analgesic, blood-coagulating and wound-healing properties of several mushrooms (Ganoderma lucidum, Lentinula edodes, Coriolus versicolor, Cordyceps sinensis, Grifola frondosa, Auricularia auricula- judae, and Schizophyllum commune) were recognized in China, Korea, Japan, Africa, as well as in Central and North American countries. Scientific data have documented that bioactive compounds of mushroom origin possess antibacterial, antifungal, immunemodulating, antioxidant, hypocholesterolemic, hypoglycemic and other therapeutic activities [2, 3]. Nowadays, the commercial mushroom biotech-products obtained from both fruiting bodies and/or mycelia are largely consumable in the world market. They are not considered strictly pharmaceutical products (medicines), but represent a novel class of dietary supplements (DSs) or functional food
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