93 ANTIMICROBIAL ACTIVITY AND HPLC ANALYSIS OF DAPHNE BLAGAYANA L. (THYMELAECEAE) EXTRACTS Sovrlić M. 1 , Vasiljević P. 2 , Jušković M. 2 , Mašković P. 3 , Manojlović N. 1 1 Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia 2 Faculty of Agriculture, University of Kragujevac, Čačak, Serbia 3 Faculty of Science, University of Niš, Niš, Serbia The aim of this study was to investigate the antimicrobial activity and HPLC analysis of the methanol and chloro- form extracts of leaves and twigs of endemic species Daphne blagayana collected from the area of Suva Mountain, Serbia. The antimicrobial activity, expressed as the values of MIC, were investigated on the 6 standardized bacterial strains and two strains of fungi. Amracin and ketoconazole are used as standard antibiotics. MIC values of the tested extracts were ranged from 15.62 to 125μg/ml. The strongest effect of the extracts exhibited against Klebsiella pneu- moniae, Staphylococcus aureus and Proteus mirabilis. Results of the HPLC analysis showed the presence of a variety of secondary metabolites, one of which is 7,8-dihydroxycoumarin (daphnetin), which was identified in all the extracts tested. Daphnetin, as biologically active substance, is most responsible for the antimicrobial activity of the tested ex- tracts, but its role can not exclude the influence of other secondary metabolites (phenols and flavonoids) present in the extracts, which may have a synergistic effect. Keywords: Daphne blagayana, HPLC, dafnetin, antimicrobial activity. INTRODUCTION Daphne blagayana (traditional names: sling, oputnik, jeremicak) was first described by Slovenian botanist Henrik Freyer 1837 [1]. It belongs to the family Thymeleaceae. It is an evergreen perennial prostrate shrub, yellowish fragrant flowers, up to 30 cm [2]. It occurs as Balkan endemic species with habitats in the western parts of Serbia, northern Montenegro, Bosnia and Herzegovina, and can be found in Slovenia, Romania, Bulgaria, Macedonia, Albania and Italy [3,4,5]. Plant spe- cies of the genus Daphne are widely used in folk medi- cine to treat various forms of cancer, skin diseases, rheumatism and toothache [6,7]. Various compounds isolated from these plants show antileucemic and anti- tumor activities [8,9,10]. Recent studies have also shown that isolated molecules from Daphne species exhibit anti- inflammatory and antinociceptive effect by reducing the levels of cytokines that are directly involved in the in- flammatory process, which indicates their effectiveness and use in the treatment of inflammatory diseases [11]. Previous studies have showed the presence of various secondary metabolites in Daphne species including lig- nans, lignins, flavonoids, coumarins and terpenoids [12,13,14]. Keeping in mind that secondary metabolites detected in these plant species possess pronounced bio- logical activity, they are of a great importance for fur- ther investigation of the unexplored Daphne species. These species are a good source for the isolation of pure and natural substances that can potentially be used in the treatment of various diseases. The results of our pre- vious studies have shown that the leaves and twigs of Daphne blagayana have a high content of phenols and flavonoids and potential antioxidant activity [15]. STUDY OBJECTIVE The aim of this work was to investigate the antim- icrobial activity of the chloroform and methanol extracts of twigs and leaves of the species Daphne blagayana and the identification of secondary metabolite daphnetin by HPLC method. MATHERIAL AND METHODS Plant material The twigs and leaves of the plant Daphne blagayana were collected on Suva Planina in May 2011. Determina- tion of the plant was carried out at the Department of Biology and Ecology, Faculty of Sciences, University of Nis, and the samples were deposited under number 5497. The strains of microorganisms Antimicrobial activity of the extracts of Daphne bla- gayana was investigated in 6 standard bacterial strains: Staphylococcus aureus ATCC 25923, ATCC 13883 Kleb- ORIGINAL ARTICLES AUTHORS SUMMARY KORESPONDENT MIROSLAV SOVRLIĆ Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia  sofke-ph@hotmail.com