IZOLATION AND CHARACTERIZATION OF DITERPENES FROM PLANT SPECIES EUPHORBIA PALUSTRIS Milica Puđa Center for talented youth Belgrade II, firstname.lastname@example.org Supervisors: Dr Vele Tešević, associate professor of University of Belgrade Faculty of Chemistry M. Sc Gordana Krstić, teaching assistant of University of Belgrade Faculty of Chemistry 1. Introduction Plant species pond spurge (Euphorbia palustris) is very rich source of milky juice-latex.1 It is known that the latex of these plants contains a large number of organic compounds, such as diterpenes, phenols, and flavonoids.2 An class of diterpenes that has been isolated from the plant genus Euphorbia is a class of tiglianes. It was found that that tiglianic type of diterpenoids have cytotoxic, analgoantipyretic 3 and sedative effects.4 They also acts as irritants.5 C7 (C6-133.9 ppm, C7–134.8 ppm). It also can be noticed the presence of oxygenated carbon atoms (C4–73.6 ppm, C13– 63.2 ppm, C20–69.8 ppm) (Picture 2). Based on these data, the structure of 13–acetoksi–20–izovaleriloksi–12– deoksiforbol was determined (Picture 3). 2. Objective The aim of this work is the isolation of diterpenes from plant species Euphorbia palustris, and their characterization. Picture 2: 1H NMR spectrum of fraction 25 3. Materials and methods Lyophilized milk of Euphorbia palustirs was extracted twice with hexane. The extract was applied to a dry-flash chromatography column, packed with silica gel. Elution was carried out by solvent system hexane/ethyl acetate, with the solvent polarity increase, i.e. percent of ethyl-acetate increased. Gradient elution was monitored by TLC. Total of 17 fractions from dry–flash column was eluted. Fractions 6, 7 and 8, that were equal on the basis of Rf values, were collected and subsequently separated on column chromatography. A silica gel was used as stationary phase, while solvent system hexane/ethyl-acetate was used as a mobile phase. Elution was isocratic, with a mixture of hexane/ethyl-acetate (V/V 8/2) and monitored by TLC also. Total of 27 fractions were eluted by the gravity column. Fraction 25th was further analyzed. 4. Results and discussion Based on the results of TLC fraction 25th was prepared for recording NMR spectra. NMR spectrum of 1H and 13C were recorded. Based on the appearance of 1H NMR spectra, it can be concluded that the compound contains two protons, which are located on the sp2-hybridized carbon (H–1 7.61 ppm, H–7 5.72). It can also be observed the presence of a proton which is oxygenated to carbon (H20–4:45 ppm) (Picture 1). From the 13C NMR spectrum was observed: keto group (C3–209.1 ppm), ester carbonyl groups (isovaleroxy 170.8 ppm, acetoxy 175.2 ppm), as well as two double bonds at positions C1–C2 (C1-161.4 ppm, C2–132.8 ppm), and C6- Picture 3: 13C NMR Picture 4: Structure of 13– acetoxy–20 spectrum of fraction 25 5. –isovaleryloxy–12– deoksiforbol Conclusion In this paper a method of a new diterpene ester (13-acetoxy20-isovaleryloxy-12-deoxyphorbol) isolation and characterization was described. Characterization of the isolated compounds was performed by NMR spectroscopy. In further research the biological activity of the isolated compounds will be tested. 6. Literature 1. Jančić R. (2004):Botanika farmaceutika. Službeni list SCG. 2. Eke,T., et al. (2000): Arch. Ophthalmol (AMA). 18, 13–16. 3. Quing–Wen S. et al. (2008): Chem. Rev. 108, 4295 – 4327 4. Qing–Gao M. et al. (1997): J. Phytochemistry. 44, 663–666. 5. Baloch I. B. et al. (2007): Eur. J. Med. Chem. 43, 274–281.