Acknowledgements Abstract Aim: The essential oils of Seseli L. genus are known for their beneficial biological activities and present novel targets in development of safe and effective formulations from plant products. However, the data on mechanism of action and genotoxicological safety are still lacking. The objective of this work was to test essential oils of different parts of Seseli rigidum for potential genotoxicity in human leucocytes by using Comet assay. Also, we evaluated a protective quality of different concentrations of the oils in attenuation of hydrogen peroxide induced DNA damage, as well as observing the existence of dose dependency. Results: The results showed that all administered concentrations of essential oils from root, fruit and aerial parts of plant did not produce genotoxic effect. When antigenotoxic potential was tested, the essential oils of all parts of the plant significantly (P<0.05) attenuated DNA damage induced by H 2 O 2. The oils from aerial parts and fruit of the plant were effective in all concentrations, while root essential oil significantly reduced DNA fragmentation only at the lowest concentration (12.5 µg/mL). The protective effect was dose dependent for all tested oils. Conclusion: In conclusion, the results demonstrated antigenotoxic properties of Seseli rigidum essential oils and revealed appropriate and safe doses with beneficial effects under described conditions. Key words: essential oils, oxidative stress, DNA damage, comet assay Introduction Seseli rigidum Waldst. & Kit. (Apiaceae) is an important aromatic species, recognized for its therapeutic properties of its essential oils and physiologically active biochemicals which seem to have good potential against human disease. Although essential oils of genus Seseli L. proved to be effective against human pathogenic microorganisms and fungal diseases as well [1-3], their biological activities against oxidative DNA damage have not been studied yet. It is known that DNA is highly sensible to oxidative insults and numerous studies showed implications of oxidative DNA damage in the development of chronic disease and cancer [4]. The objective of this work was to evaluate essential oils of different parts of Seseli rigidum as protective agents against oxidative stress induced DNA damage. Hydrogen peroxide was used as known agent that produces DNA damage. The effect of different concentrations of plant essential oils in attenuation of DNA damage induced by hydrogen peroxide in human leucocytes was explored, as well as observing the existence of dose dependency. Since the use of essential oils in therapeutic approaches may require high doses, screening for potentially genotoxic properties of different concentrations is of great significance, in order to determine the optimal concentration for safe use. The level of DNA damage in treated cells was determined by the Single cell gel electrophoresis, known as the Comet assay [5], which is widely used for genotoxicity research. Methods & Materials Subjects Heparinised whole blood samples were obtained by venipuncture from healthy donors aged under 25 who participated in the study. Subjects were non-smokers and non-alcoholics, not receiving any therapy or medications, nor taking dietary supplements. Fig 1. Effect of different concentrations of essential oils on the level of DNA damage -genotoxic potential- Results ConclusionReferences After screening for potential genotoxicity, the results have shown that the essential oils of Seseli rigidum did not produce DNA damage under described conditions (Fig. 1). All administered concentrations of essential oils from root, fruit and aerial parts of plant did not significantly increase the total number of DNA damaged cells in regard to the positive control, which indicates that the tested essential oils did not display genotoxic effect. Fig 2. Effect of different concentrations of essential oils decreased the level of DNA damages by H2O2 -antigenotoxic potential- Antigenotoxic activities of essential oils of Seseli rigidum Waldst. & Kit. (Apiaceae) in peripheral human leucocytes against H 2 O 2 induced DNA damage Andrea Čabarkapa, Mirjana Marčetić, Jovana Vasiljević, Lada Živković, Dijana Žukovec-Topalović, Biljana Spremo-Potparević Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, Belgrade, Serbia Essential oil preparation The essential oils of Seseli rigidum were obtained from root, fruit and aerial parts of plant, and tested for antigenotoxic and genotoxic properties. Root and aerial parts during flowering of Seseli rigidum were collected in June 2010 and fruits in September 2010, in Brdjanska gorge in western Serbia. Herbarium specimens were deposited at the Herbarium of the Faculty of Pharmacy in Belgrade (3240HFF). Essential oils were obtained from dried and grounded plant material by hydrodistillation in a Clevenger-type apparatus according to procedure given in European Pharmacopoeia 7.0. Experimental design In order to assess possible genotoxic properties, the essential oils were first tested for their ability to cause DNA damage per se. Peripheral blood leukocytes from three young and healthy volunteers were treated in vitro with different concentrations of the oils from root, aerial parts and fruit of Seseli rigidum ( , 50 and 100 µg/mL) for 30 minutes at 37 ⁰ C before measuring the level of DNA damage. Subsequently, antigenotoxic effects of the essential oils were explored on the duplicates of the same cell preparations. Samples were treated in vitro with hydrogen peroxide for 20 minutes on ice, and followed by the incubation with the essential oils ( , 50 and 100 µg/mL) for 30 min at 37 ⁰ C. The positive controls were exposed only to hydrogen peroxide, while negative controls were treated with PBS. When antigenotoxic potential was tested, the essential oils of all parts of the plant significantly (P<0.05) attenuated H 2 O 2 induced DNA damage, demonstrating the genoprotective effect (Fig 2). The number of cells with DNA damage was decreased significantly (P < 0.05), comparing to the positive control (H 2 O 2 ) in all tested concentrations of the essential oils from aerial parts and fruit, while root oil significantly reduced DNA fragmentation only at the lowest concentration (12.5 µg/mL). The results showed dose dependent trend. Reduction of the concentrations of the root and fruit essential oils led to decrease in the level of H 2 O 2 - induced DNA damage. Meanwhile the essential oil of aerial parts displayed opposite trend of activity. Leucocytes treated with the aerial parts oil showed a significant dose dependent increase in DNA protection (P < 0.05) with rise of the concentration. The obtained results showed that all tested essential oils of Seseli rigidum decrease the level of DNA damage and demonstrate antigenotoxic properties, which may offer great potential for producing new agents of benefit in human use and suggest further research on the mechanism of their action. All concentrations of the essential oils used in this study were found to be non genotoxic. These results contribute to the development of safe and effective new formulations from natural plant products with potential for medical use, cosmetic and pharmaceutical industry. 1.Stojkovic, S et al. (2009) Chemical composition and antimicrobial and antioxidant activity of Seseli rigidum flower essential oil, Chemistry of Natural Compounds; 45 (2), Goncalves, M. J. et al. (2012) Composition, antifungal activity and cytotoxicity of the essential oils ofSeseli tortuosum L. and Seseli montanum subsp. peixotoanum (Samp.) M. Lainz from Portugal. Industrial Crops and Products Vol. 39, 204– Siljegović J. et al (2011) Composition and antimicrobial activity of Seseli montanum subsp. tommasinii essential oil. Natural product communications Vol. 6, Khansari N, et al.,Recent Pat Inflamm Allergy Drug Discov. 2009;3(1): Singh NP, et al.,Exp Cell Res. 1988;175(1): The research has been funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia, project no. OI Corresponding author: Andrea Čabarkapa, MSc Department of Physiology Faculty of Pharmacy, University of Belgrade Vojvode Stepe 450, Belgrade, Serbia Clevenger-type apparatus for hydrodistillation of essential oils The alkaline comet test was performed to detect DNA strand breaks and alkali-labile sites as described by Singh et al. (1988). Comets were graded visually into five categories corresponding to the following amount of DNA in the tail: (A) no damage, 95%. DNA damage was characterized as DNA migration over 5% (total number of damaged cells from comet classes B+C+D+E), and mean value was calculated for all subjects. Seseli rigidum Waldst.& Kit. (Apiaceae)