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Lichen: from genome to ecosystem in a changing world 3I-P 3I: New approaches to understanding biosynthesis and ecological roles of metabolites in lichens (3I - P1) Submission ID: IAL0113-00002 CHEMICAL ANALYSES AND MOLECULAR STUDIES OF SELECTED CETRARIOID LICHENS (PARMELIACEAE) AND THEIR CULTURED MYCOBIONTS Hametner C. 1 , Stocker-Wörgötter E. 1 1 Organismic Biology, University of Salzburg, Salzburg, Austria Lichens produce a great variety of primary (intracellular) and secondary metabolites (extracellular); a considerable number of them has not been structurally identified and are still recognized as “unknowns”. In this study, three cetrarioid lichens, e.g. Cetraria ericetorum, C. islandica and Flavocetraria nivalis, were chosen for molecular and chemical investigations. HPLC and TLC analyses have to be complemented by mass spectroscopic methods. In further series of experiments, the fungal symbionts of the selected lichen thalli were isolated by means of the modified Yamamoto-method and sub-cultured on different nutrient media. By using electronically adjustable culture chambers, a number of environmental conditions were tested to provide best growth reactions for the three selected lichen fungi. For identification of the cultured fungal isolates and the three voucher specimens, DNA-analyses were performed by using the primers ITS1F and ITS4 for the PCR-reactions to amplify the internal transcribed spacer 1 region, 5.8S region, and the internal transcribed spacer 2 region of genomic rDNA. The sequencing was accomplished by MWG Biotech AG (Germany) and Macrogen Inc. (Europe). The identities of the lichen and fungal sequences were examined via Blastn search of the NCBI GenBank. Consequently, the mycobiont of C. islandica was grown aposymbiotically for the first time. Finally, the mycobiont cultures were investigated by using similar chemical analyses as mentioned before for the original lichen thalli and differences in the metabolite compositions were documented. Regarding C. islandica, which was previously found to contain biologically active substances, the chemical constituents of the “crude” lichen extract, could be of high interest for future pharmaceutical applications. 132
The 7 th International Association for Lichenology Symposium 2012 (3I – P2) Submission ID: IAL0115-00001 ANTIOXIDANT AND ANTIMICROBIAL ACTIVITY OF PARMELIA SAXATILIS ETHANOL EXTRACT Stajić M. 1 , Ćilerdžić J. 1 , Vukojević J. 1 1 Institute of Botany, University of Belgrade, Faculty of Biology, Belgrade, Serbia The aim of this study was to evaluate the antioxidant and antimicrobial activity of ethanol extract of Parmelia saxatilis (L) Ach. P. saxatilis originated from Tara Mountain (Serbia) was used for the study. 4.5 g of air dried sample was extracted by stirring with 30-fold higher volume of 70% ethanol at 30 º C for 72 hours and filtering through Whatman No. 4 filter paper. The obtained ethanol extract was evaporated at 40 º C to dryness, redissolved in 96% ethanol (antioxidant test) and 5% DMSO (antimicrobial test), respectively, to a final concentration of 20 mg/ml. Antioxidant capacity was determined spectrophotometrically by methanol solution of DPPH at 517 nm and compared with commercial antioxidant, BHA. The concentrations of phenol and flavonoid compounds in the extract were measured spectrophotometrically by Folin-Ciocalteu reagent at 740 nm and ethanol/aluminium nitrate/potassium acetate mixture at 415 nm, respectively. Antimicrobial capacity of the extract was evaluated on 5 bacterial and 8 fungal species. Tests were carried out by various extract dilutions, in triplicate. Six extract concentrations ranged from 10 mg/ml to 0.312 mg/ml, obtained by double disolutions, caused decrease of DPPH free radical-scavenging in the range from 79.76% to 14.76%. Defined EC 50 value was 1.81 mg/ml. The tested extract concentrations had good antioxidant activity compared to BHA one (92.61%). The amount of total phenol compounds was ranged from 193.95 µg/ml (10 mg/ml) to 30.10 µg/ml (0.312 mg/ml). The total flavonoid content was less, ranged from 134.48 μg/ml (10 mg/ml) to 1.90 μg/ml (0.312 mg/ml). Scavenging effect directly correlates with the different phenol content (r 2 = 0.9981) and less with flavonoid content (r 2 = 0.6634). The extract showed insignificant antibacterial activity, while antifungal activity was absent. The growth of Escherichia coli, Enterococcus faecalis and Pseudomonas aeruginosa was inhibited by the highest extract concentration (10 mg/ ml), while Klebsiella pneumoniae and Staphylococcus aureus were more sensitive (1.25 mg/ml and 2.5 mg/ml, respectively). (3I – P3) Submission ID: IAL0168-00002 ACETYLCHOLINESTRASE INHIBITORY AND NEUROPROTECTIVE EFFECTS OF BIRULOQUINONE, ISOLATED FROM LICHEN-FORMING FUNGUS CLADONIA MACILENTA Luo H. 1 , Li C. 2 , Liu Y. 3 , Hur J. 3 1 Korean Lichen Research Institute, Jiangsu University of Science and Technology, Jiangsu, People’s Republic of China 2 College of Chinese Medicine Materials, Jilin Agricultural University, Changchun, People’s Republic of China 3 Korean Lichen Research Institute, Sunchon National University, Sunchon, Korea Alzheimer’s disease (AD) is the most common type of dementia in aging population. At present, acetylcholinestrase (AChE) inhibitors are the first group of drugs approved by the Food and Drug Administration to treat mild to moderate AD. In present study, one AChE inhibitor, biruloquinone, was isolated from the extract of lichen-forming fungus Cladonia macilenta, which showed the most potent AChE inhibitory activity in previous screening experiment. The IC 50 and inhibitory kinetic assay suggested biruloquinone is a mixed-II inhibitor on AChE, with the IC 50 value of 27.1 µg/ml (83.1 µM). The molecular modeling results indicated that biruloquinone is supposed to bind AChE through the stable H-bond with two active sites of the protein. The neuroprotective effects of biruloquinone on the H 2 O 2 and β-amyloid-induced cell injuries were also evaluated. The results indicated that biruloquinone remarkably improved the viability of the injured cells at 1 to 25 µg/ml. The protective effects are proposed to be related to the potent antioxidant activities of biruloquinone. These results implied that biruloquinone has the potential to be used as multi-functional anti-AD agent. 133 3I-P
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The 7 th IAL Symposium 2012 Lichens
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The 7 th International Association
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