816
F. Cateni et al. / Il Farmaco 58 (2003) 809ꢀ817
/
bonds and were thus assigned to C-7 and C-10 (dꢃ
27.7) for the compounds 1ꢀ4 and to C-6, C-7 and C-10
(dꢃ33.1, 32.9, 33.1) for the compound 5. Thus, the
olefin groups in the LCB of 1ꢀ4 were determined to
have cis (Z) geometry (Table 1), while in the LCB of 5
trans (E) geometry has been assigned (Table 2).
The FAMs obtained from the methanolysis of the
cerebrosides 1, 2 exhibit 13C NMR signals at about
175.9, 131.3 and 130.2 expected for monounsaturated
fatty acid methyl esters. The resonance at about 27.8,
27.9 ppm confirms the Z geometry of the double bonds
in the long-chain fatty acids (Table 1). The position of
the double bonds in the monounsaturated fatty acid
methyl esters was determined by EI MS analysis of the
corresponding dimethyl disulfide (DMDS) derivatives
/
27.8,
4R, 8Z)-2-[(2?R)-2?-hydroxydocosanoilamino]-8 (Z)-oc-
tadecene-1, 3, 4-triol (3), 1-O-(b- -glucopyranosyl)-(2S,
3S, 4R, 8Z)-2-[(2?R)-2?-hydroxytetracosanoilamino]-8
(Z)-octadecene-1,3,4-triol (4) and 1-O-(b- -glucopyra-
nosyl)-(2S, 3S, 4E, 8E)-2-[(2?R)-2?-hydroxyhexadeca-
/
D
/
D
/
noylamino]-4(E),8(E)-octadecadiene-1,3
respectively.
diol
(5),
In conclusion, compounds 4 and 5, have been found
for the first time in Euphorbiaceae. Compounds 1ꢀ
have been found to be identical to the cerebrosides
/
3
isolated from Euphorbia characias L. [15].
3.1. Antiproliferative activity of the extract containing
cerebroside molecular species
[11]. The characteristic fragments at m/zꢃ317, 344 and
/
The cerebrosides 1ꢀ5 were isolated from the cerebro-
/
173, obtained from the cleavage between the sulphide
carbons, indicates the position of the double bonds in 1,
2 (Fig. 3).
side molecular species obtained from the less polar
fraction of the MeOH extract of E. nicaeensis All.
A preliminary biological assay has been carried out in
order to value the antiproliferative activity of the extract
1
1D and 2D H NMR spectroscopy, DQF-COSY and
HMQC indicated that the head group consists of a
single glucose residue in the b configuration. The
glucose configuration was determined by the character-
istic chemical shifts, the spin-spin splitting and the
multiplicity of the characteristic resonance of the H-4ƒ
proton, as well as by the splittings of the other ring
protons.
containing the complex mixture of cerebrosides 1ꢀ5 in
/
cell culture; KB cells were exposed at 1.25, 2.50, 5.00,
10.0 and 20.00 mg/ml of product for 72 h at 37 8C.
Results are reported in Table 3 and are expressed as
growth inhibition percentage to respect to the control
cultures.
It is possible to note that at the high concentration
used (20.0 mg/ml) extract appear to be effective to
The stereochemistry of the ceramide moiety was
1
determined by comparison of the H NMR data of the
blocking cell proliferation of 26.0791.91%. Significant
/
cerebrosides isolated from E. nicaeensis All. with that of
synthetic analogs as reported in literature in terms of the
signals due to 1-H to 4-H [13]. The absolute configura-
tion of the glucopyranose moiety was determined to be
cytostatic effect is observed at 2.5 mg/ml of extract (P B
/
0.05) but the entity of reduction in cell proliferation is
small.
the
Therefore the structures of 1ꢀ
1-O-(b- -glucopyranosyl)-(2S, 3S, 4R, 8Z)-2-[(2?R)-2?-
hydroxytetracosenoilamino]-8 (Z)-octadecene-1,3,4-
triol (1), 1-O-(b- -glucopyranosyl)-(2S, 3S, 4R, 8Z)-
2-[(2?R)-2?-hydroxyhexacosenoilamino]-8 (Z)-octade-
D
-form using the Hara method [14].
/5 were proposed to be
References
D
[1] H. Hemmers, P.G. Gulz, Epicuticular waxes from leaves of five
¨
D
Euphorbia species, Phytochemistry 25 (1986) 2103ꢀ2107.
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¨
[2] S. .Oksikz, H.L. Shich, J.M. Pezzato, N. Ozhatay, G.A. Cordeli,
Biologically active compounds from Euphorbiaceae; Part 1.
Triterpenoids of Euphorbia nicaeensis subsp. Glaerosa, Planta
cene-1,3,4-triol (2), 1-O-(b-
D-glucopyranosyl)-(2S, 3S,
Med. 59 (1993) 472ꢀ473.
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Table 3
Antiproliferative activity of the extract of Euphorbia nicaeensis All.
[3] G. Falsone, F. Cateni, G. Visintin, V. Lucchini, H. Wagner, O.
Seligmann, Constituents of Euphorbiaceae 12. Isolation and
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Concentration (mg/ml)
% Cell growth inhibition versus controls
biglandulosa Desf, Farmaco 49 (1994) 167ꢀ174.
/
[4] G. Falsone, F. Cateni, F. Katousian, H. Wagner, O. Seligmann,
G. Pellizer, F. Asaro, Constituents of Euphorbiaceae 10. New
cerebrosides from Euphorbia characias L, Z. Naturforsch., Teil b
1.25
2.50
0.169
3.689
4.639
13.649
26.079
/
0.04
/
0.13 *
0.21 *
1.26 **
1.91 **
5.00
/
48 (1993) 1121ꢀ1126.
/
10.00
20.00
/
[5] G. Falsone, F. Cateni, J. Jurman, H. Wagner, O. Seligmann,
Constituents of Euphorbiaceae 11. Isolation and structure eluci-
dation of seven cerebrosides from Euphorbia wulfenii Hoppe ex
/
KB cells were seeded on 96 well plastic plate. After 24 h medium of
each well was substituted with 200 ml of different extract concentra-
tions and incubated at 37 8C for 72 h. Then, medium was discarded
and cells treated as described in Section 2. Data are means9
experiments assayed in quintuplicate.
* P B0.05.
** P B0.01. Student unpaired t-test.
Koch, Farmaco 48 (1993) 1617ꢀ1629.
/
[6] Y.A. Hannun, R.M. Bell, Functions of sphingolipids and
sphingolipid breakdown products in cellular regulation, Science
/ES of two
243 (1989) 500ꢀ
[7] J. Adams, Q. Ann, Structure determination of sphingolipids by
mass spectrometry, Mass Spectrom. Rev. 12 (1993) 51ꢀ85.
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/
/
Cateni
