izolation and characterization of diterpenoids from plant species

Milica Puđa
Center for talented youth Belgrade II, milicapudja@gmail.com
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
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,
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
–isovaleryloxy–12– deoksiforbol
In this paper a method of a new diterpene ester (13-acetoxy20-isovaleryloxy-12-deoxyphorbol)
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.