Lipid metabolites with free-radical scavenging activity from Euphorbia helioscopia L.

Article abstract of DOI:10.1016/j.chemphyslip.2014.03.001

The methanolic extract of the plant Euphorbia helioscopia L. exhibited an interesting free-radical scavenging activity. From the aerial parts of Euphorbia helioscopia L. (Euphorbiaceae), a complex mixture of seven cerebrosides together with glucoclionasterol, a digalactosyldiacylglycerol and a diacylmonogalactosylglycerol were identified. The structures of the cerebrosides were characterized as 1-O-β-d-glucosides of phytosphingosines, which comprised (2S, 3S, 4E, 8E)-2-amino-4(E),8(E)-octadecadiene-1,3-diol, (2S, 3S, 4E, 8Z)-2-amino-4(E),8(Z)-octadecadiene-1,3-diol, (2S, 3S, 4R, 8Z)-2-amino-8(Z)-octadecene-1,3,4-triol as long chain bases with seven 2-hydroxy fatty acids of varying chain lengths (C₁₆, C_24:1, C _26:1, C₂₄, C₂₆, C_28:1) linked to the amino group. The glycosylglycerides were characterized as (2S)-2,3-O-di-(9,12, 15-octadecatrienoyl)-glyceryl-6-O-(α-d-galactopyranosyl) -β-d-galactopyranoside and (2S)-2,3-O-di-(9,12,15-octadecatrienoyl)- glyceryl-1-O-β-d-galactopyranoside. The structures were established on the basis of spectroscopic data and chemical reactions.

Full text of DOI:10.1016/j.chemphyslip.2014.03.001

Chemistry and Physics of Lipids 181 (2014) 90–98
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Lipid metabolites with free-radical scavenging activity from Euphorbia
helioscopia L.
F. Catenia,∗, J. Zilica, T. Altieria, M. Zacchignaa, G. Procidaa, R. Gaggerib,
D. Rossib, S. Collinab,c
a Department of Chemical and Pharmaceutical Sciences, University of Trieste, P.zle Europa 1, 34127 Trieste, Italy
b Department of Drug Sciences, University of Pavia, Via Taramelli 12, Pavia, Italy
c Center for Studies and Researches in Ethno Pharmacy (C.I.St.R.E.), University of Pavia, Via Taramelli 12, 27100 Pavia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The methanolic extract of the plant Euphorbia helioscopia L. exhibited an interesting free-radical scav-
enging activity.
Received 16 December 2013
Received in revised form 5 March 2014
Accepted 6 March 2014
From the aerial parts of Euphorbia helioscopia L. (Euphorbiaceae), a complex mixture of seven cerebro-
sides together with glucoclionasterol, a digalactosyldiacylglycerol and a diacylmonogalactosylglycerol
were identified. The structures of the cerebrosides were characterized as 1-O-␤-d-glucosides of phy-
tosphingosines, which comprised (2S, 3S, 4E, 8E)-2-amino-4(E),8(E)-octadecadiene-1,3-diol, (2S, 3S, 4E,
8Z)-2-amino-4(E),8(Z)-octadecadiene-1,3-diol, (2S, 3S, 4R, 8Z)-2-amino-8(Z)-octadecene-1,3,4-triol as
Available online 19 March 2014
Keywords:
Euphorbia helioscopia L.
Free-radical scavenging activity
Cerebrosides
Diacyldigalactosylglycerol
Diacylmonogalactosylglycerol
long chain bases with seven 2-hydroxy fatty acids of varying chain lengths (C16, C24:1, C26:1, C24, C26
28:1) linked to the amino group.
The glycosylglycerides were characterized as (2S)-2,3-O-di-(9,12,15-octadecatrienoyl)-glyceryl-6-O-
,
C
(˛-d-galactopyranosyl)-␤-d-galactopyranoside and (2S)-2,3-O-di-(9,12,15-octadecatrienoyl)-glyceryl-
1-O-␤-d-galactopyranoside. The structures were established on the basis of spectroscopic data and
chemical reactions.
© 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
now, more than 30 diterpenoids have been isolated and struc-
turally characterized from E. helioscopia (Lu et al., 2008). As a part
Cellular damage or oxidative injury arising from free radicals or
reactive oxygene species (ROS) are involved in various human neu-
rodegenerative disorders, diabetes, inflammation, viral infections,
autoimmune pathologies and digestive system disorders.
Over the past two decades, evidences derived from epidemio-
logical and laboratory studies have demonstrated that some edible
plants as a whole, or their identified compounds with antioxidant
properties have protective effects on human carcinogenesis (Dixit
and Ali, 2010).
of our ethnobotanical-directed search for novel bioactive agents,
herein we report a phytochemical study of Euphorbia helioscopia L.
methanolic leaves extract. In detail, bioassay-guided phytochemi-
cal study on E. helioscopia revealed seven known cerebrosides (1–7),
glucoclionasterol (8), a digalactosyldiacylglycerol (9) and a mono-
galactosyldiacylglycerol (10), all were isolated from this species for
the first time. The isolation and structure elucidation of different
lipid metabolites (compounds 1–10) by spectroscopic methods and
chemical reactions was described.
The genus Euphorbia is the largest genus of the plant family
Euphorbiaceae, and has been reported to be a rich source of skin-
irritating, cytotoxic and tumor-promoting diterpenoids (Singla and
Kamala, 1990). Euphorbia helioscopia L. is a biennial herb of Euphor-
biaceae, which grows wild in Europe, China and Japan. It has been
used as a traditional folk medicine for the treatment of malaria,
bacillary dysentery and osteomyelitis (Hua et al., 1999). Up to
Moreover, the free-radical scavenging activity of the crude
extract as well as of each isolated fraction was assayed and com-
pared to that one of a commercial standardized antioxidant extract
of green the.
2. Materials and methods
2.1. Chemistry
2.1.1. General experimental procedures
∗
NMR-spectroscopy: nuclear magnetic resonance spectra were
Corresponding author. Tel.: +39 040 5583722; fax: +39 040 52572.
E-mail address: cateni@units.it (F. Cateni).
recorded with a
Varian Unity 400 spectrometer. 13C NMR:
http://dx.doi.org/10.1016/j.chemphyslip.2014.03.001
0009-3084/© 2014 Elsevier Ireland Ltd. All rights reserved.

F. Cateni et al. / Chemistry and Physics of Lipids 181 (2014) 90–98
91
100.4 MHz, Unity 400 spectrometer. NMR spectra were obtained
by using C5D5N as solvent; chemical shifts are expressed as
ı units (ppm) relative to tetramethylsilane (TMS) as internal
standard. The abbreviations s, d, dd, t, q m and br s refer
to singlet, doublet, doublet of doublet, triplet, quartet, multi-
plet and broad singlet, respectively. The PI-FD spectra (CH2Cl2)
were obtained using double-focusing MAT 95 mass spectrometer.
FAB-MS: Kratos MS 80 RFA. FAB-MS: (8 Kv, Xe, methanol as sol-
vent and glycerol matrix + NaCl). Electrospray analysis: API Perkin
Elmer (voltage + 5600 with orifice 90 and/or 120). Silica gel col-
(M−H, 30%)−, 533 (M−H–C6H12O6, 25%)− (Shibuya et al., 1990a,b).
1H and 13C NMR data are reported in Table 1.
Cerebroside 3. Amorphous powder. IR (KBr) cm−1
: 3415
(hydroxyl), 1647, 1537 (amide). Positive-ion FAB MS: m/z = 842
(M+H, 28%)+, 824 (M+H–H2O, 8%)+, 680 (M+H–C6H11O5, 31%)+, 662
(M+H–C6H12O6, 18%)+, 500 (M+Na–C24H45O2)+. Negative ion FAB
MS: m/z = 840 (M−H, 62%)−, 678 (M−H–C6H11O5, 14%)− (Shibuya
et al., 1990a,b). 1H and 13C NMR data are reported in Table 1.
Cerebroside 4. Amorphous powder. IR (KBr) cm−1
: 3413
(hydroxyl), 1646, 1540 (amide). Positive-ion FAB MS: m/z = 892
(M+Na, 63%)+, 870 (M+H, 84%)+, 852 (M+H–H2O, 51%)+, 730
(M+Na–C6H11O5, 17%)+, 708 (M+H–C6H11O5, 55%)+, 690
(M+H–C6H12O6, 20%)+, 476 (M+H–C26H50O2, 16%)+. Negative
ion FAB MS: m/z = 868 (M−H, 100%)−, 706 (M−H–C6H11O5, 31%)−
(Shibuya et al., 1990a,b). 1H and 13C NMR data are reported in
Table 1.
˚
umn chromatography: Kieselgel 60 (230–400 Mesh, 60 A Merck).
FT-IR spectra: Jasco IR-700 infrared spectrophotometer. Flash chro-
matography reversed-phase: Lichoprep RP-18 (40, 63 ␮m, Merck).
All solvents were distilled before use. TLC: Kieselgel 60 F254
(20 cm × 20 cm; 0.2 mm, Merck). HPTLC: HPTLC-fertigplatten RP-18
F254 (10 cm × 10 cm, Merck).
The fatty acid composition of was released as methyl esters by
the official A.O.A.C. methylation procedure, and analyzed by gas
chromatography (GLC) (Helrich, 1990). A Shimadzu GC 14A (Kyoto,
Japan) instrument, equipped with a split/splitless injector (1:20)
and a flame ionization detector, was used. A SP 2330 fused sil-
ica capillary column, 30 m × 0.32 mm I.D., 0.20 ␮m film thickness
(Supelco Inc., Bellefonte, PA) was employed. The chromatographic
conditions were: column temperature was programmed from
150 ◦C (kept for 2 min) to 250 ◦C at 10 ◦C/min (maintained for
5 min), injector and detector temperature 280 ◦C, carrier gas
(helium) and flow rate 2.0 mL/min.
Cerebroside 5. Amorphous powder. IR (KBr) cm−1
: 3415
(hydroxyl), 1646, 1542 (amide). Positive-ion FAB MS: m/z = 866
(M+Na, 90%)+, 844 (M+H, 21%)+, 826 (M+H–H2O, 29%)+,
682 (M+H–C6H11O5, 78%)+, 664 (M+H–C6H12O6, 53%)+, 500
(M+Na–C24H47O2, 10%)+. Negative ion FAB MS: m/z = 842 (M−H,
96%)−, 680 (M−H–C6H11O5, 33%)−, 663 (M−H–C6H12O6, 19%)−
(Shibuya et al., 1990a,b). 1H and 13C NMR data are reported in
Table 1.
Cerebroside 6. Amorphous powder. IR (KBr) cm−1
: 3417
(hydroxyl), 1645, 1540 (amide). Positive-ion FAB MS: m/z = 920
(M+Na, 87%)+, 898 (M+H, 23%)+, 880 (M+H–H2O, 34%)+, 735
(M+H–C6H11O5, 65%)+, 718 (M+H–C6H12O6, 55%)+. Negative ion
FAB MS: m/z = 896 (M−H, 95%)−, 733 (M−H–C6H11O5, 39%)−, 716
(M−H–C6H12O6, 23%)− (Shibuya et al., 1990a,b). 1H and 13C NMR
data are reported in Table 1.
2.1.2. Plant material
E. helioscopia (1 kg) was collected from wild stock growing in San
Dorligo (Trieste), Italy in May 2010. A voucher specimen of the plant
material has been deposited at the Herbarium of the Department
of Biology (TSB-11202) of the University of Trieste (Italy).
Cerebroside 7. Amorphous powder. IR (KBr) cm−1
: 3413
(hydroxyl), 1647, 1545 (amide). Positive-ion FAB MS: m/z = 894
(M+Na, 95%)+, 872 (M+H, 34%)+, 854 (M+H–H2O, 42%)+,
709 (M+H–C6H11O5, 60%)+, 692 (M+H–C6H12O6, 53%)+, 499
(M+Na–C26H51O2, 15%)+. Negative ion FAB MS: m/z = 870 (M−H,
88%)−, 707 (M−H–C6H11O5, 31%)−, 690 (M−H–C6H12O6, 24%)−
(Shibuya et al., 1990a,b). 1H and 13C NMR data are reported in
Table 1.
2.1.3. Extraction and Isolation
The stems and the leaves of the plant were cut and extracted
with MeOH (31) for 7 days. The extract was filtered, methanol
was concentrated in vacuo to give a MeOH extract (24.7 g),
which was chromatographed on silica gel (CH2Cl2:MeOH/10:1/v:v,
CH2Cl2:MeOH/10:2/v:v and MeOH) to give five fractions. The frac-
tion 5 eluted with CH2Cl2:MeOH (10:2/v:v) was concentrated
in vacuo and submitted to reversed phase column ‘flash chromatog-
raphy’ using MeOH as eluent to afford cerebroside 1 (25 mg), 2
(30 mg), 3 (17 mg), 4 (15 mg), 5 (28 mg), 6 (12 mg), 7 (22 mg) each
showed a single spot on reversed phase TLC (MeOH). Rf = 0.25
(1), Rf = 0.27 (2), Rf = 0.076 (3), Rf = 0.15 (4), Rf = 0.10 (5), Rf = 0.29
(6), Rf = 0.14 (7). From the fraction 3 eluted with CH2Cl2:MeOH
(10:1/v:v), the compound 8 (60 mg) was isolated and identi-
fied Rf = 0.48 (8). From the fraction 4 eluted with CH2Cl2:MeOH
(10:1/v:v), the compounds 9 and 10 have been isolated with
Rf = 0.15 and identified in mixture.
2.1.4. Methanolysis of 1–7
Compounds 1 (30 mg) was refluxed with 0.9 M HCl in 82% aq.
MeOH (10 mL) for 18 h. The mixture was extracted with n-hexane
and the combined organic phase was washed with water and dried
over Na2SO4. Removal of the solvent gave a colorless wax which
was (19.0 mg) which was chromatographed on silica gel [hex-
ane/EtOAc (5:1)] to yield fatty acid methyl ester as a colorless wax
(11.5 mg).
The compounds 2–7 were methanolyzed using the same
method described above. The esters were analyzed by GC–MS. The
results were as follows: FAMs-1,2 (methyl 2-hydroxypalmitate),
EI-MS: m/z = 286 [M]+, 254 [M−CH3OH]+, 227 [M−CH3COO]+,
FAM-3 (methyl 2-hydroxytetracosenoate), EI-MS: m/z = 396
[M]+, 364 [M−CH3OH]+, 337 [M−CH3COO]+, FAM-4 (methyl 2-
hydroxyhexacosenoate), EI-MS: m/z = 424 [M]+, 392 [M−CH3OH]+,
365 [M−CH3COO]+, FAM-5 (methyl 2-hydroxytetracosanoate),
EI-MS: m/z = 398 [M]+, 366 [M−CH3OH]+, 339 [M−CH3COO]+,
FAM-6 (methyl 2-hydroxyoctacosenoate), EI-MS: m/z = 452 [M]+,
420 [M−CH3OH]+, 393 [M−CH3COO]+, and FAM-7 (methyl 2-
hydroxyhexacosanoate), EI-MS: m/z = 426 [M]+, 394 [M−CH3OH]+,
367 [M−CH3COO]+.
Cerebroside 1. Amorphous powder. IR (KBr) cm−1
: 3415
(hydroxyl), 1645, 1543 (amide). Positive-ion FAB MS: m/z = 736
(M+Na, 46%)+, 714 (M+H, 7%)+, 696 (M+H–H2O, 52%)+, 534
(M+H–C6H12O6, 100%)+, 516 (M+H–C6H12O6–H2O, 28%)+, 482
(M+Na–C16H31O2, 15%)+, 336 (M+Na–C6H11O5–C16H29O)+, 319
(M+Na–C6H12O6–C16H29O)+. Negative ion FAB MS: m/z = 712
(M−H, 30%)−, 533 (M−H–C6H12O6, 25%)− (Shibuya et al., 1990a,b).
1H and 13C NMR data are reported in Table 1 .
Cerebroside 2. Amorphous powder. IR (KBr) cm−1
: 3415
(hydroxyl), 1645, 1543 (amide). Positive-ion FAB MS: m/z = 736
(M+Na, 46%)+, 714 (M+H, 7%)+, 696 (M+H–H2O, 52%)+, 534
(M+H–C6H12O6, 100%)+, 516 (M+H–C6H12O6–H2O, 28%)+, 482
(M+Na–C16H31O2, 15%)+, 336 (M+Na–C6H11O5–C16H29O)+, 319
(M+Na–C6H12O6–C16H29O)+. Negative ion FAB MS: m/z = 712
The aq. MeOH layer was neutralized with NH4OH and extracted
with EtOAc. The combined EtOAc extract was washed with H2O,
dried over Na2SO4 and evaporated to give the long-chain base
(LCB) as a slightly yellow wax (14.5 mg). The aq. MeOH layer was
then evaporated to dryness and chromatographed on silica gel

92
F. Cateni et al. / Chemistry and Physics of Lipids 181 (2014) 90–98
Table 1
1H and 13C NMR data for compounds 1–7.
C, H
1
2
3
4
1H NMR ı (m, J
13C NMR ı
1H NMR ı (m, J
13C NMR ı
1H NMR ı (m, J
13C NMR ı
1H NMR ı (m, J
13C NMR ı
in Hz)
in Hz)
in Hz)
in Hz)
NH
1a
1b
8.42 (d, 8)
4.76 (dd, 12, 6)
4.59 (m)
–
8.41 (d, 8)
4.76 (dd, 12, 6)
4.55 (m)
–
8.53 (d, 8)
4.70 (dd, 12, 7)
4.52 (m)
–
8.55 (d, 8)
4.73 (dd, 11, 5.5)
4.50 (m)
–
70.23
70.21
70.61
70.53
2
3
4
5
4.80 (m)
4.80 (m)
6.00 (dd, 15, 6)
5.92 (dt, 15, 6)
54.60
72.43
132.10
132.34
4.80 (m)
4.80 (m)
6.00 (dd, 15, 6)
5.92 (dt, 15, 6)
54.63
72.32
132.06
132.19
5.22 (m)
4.28 (dd, 4.5, 3)
4.22 (obs)
51.49
76.13
72.63
5.22 (m)
4.26 (dd, 4, 3.5)
4.23 (obs)
51.19
76.25
72.60
5a
5b
6
6a
6b
7
8
9
10
11
11–15
12–15
16
2.40 (m)
1.88 (m)
34.15
26.17
2.41 (m)
1.91 (m)
34.10
26.05
2.08 (m)
33.17
2.07 (m)
32.93
1.70 (m)
1.65 (m)
2.20 (m)
5.40 (dt, 10.8, 7.1)
5.45 (dt, 10.8, 6.8)
2.20 (m)
1.40 (m)
1.30 (br s)
–
1.25 (br s)
1.25 (br s)
0.85 (t, 7)
–
4.57 (dd, 8, 3.7)
2.20 (m)
2.00 (m)
1.94 (m)
1.70 (m)
1.30 (m)
1.75 (m)
1.65 (m)
2.21 (m)
5.45 (m)
5.45 (m)
2.21 (m)
2.10 (m)
5.45 (t, 4.9)
5.45 (t, 4.9)
1.80 (m)
1.40 (m)
–
1.30 (m)
1.25 (br s)
1.25 (br s)
0.90 (t, 6)
–
4.55 (dd, 8, 4.2)
2.12 (m)
1.94 (m)
1.95 (m)
1.70 (m)
1.30 (m)
32.83
130.06
131.28
33.20
29.65–30.40
–
29.65–30.40
32.40
23.16
2.09 (m)
5.39 (t, 4.9)
5.39 (t, 4.9)
1.90 (m)
1.40 (m)
1.30 (m)
–
1.26 (br s)
1.26 (br s)
0.95 (t, 6)
–
4.55 (dd, 8, 4.2)
2.10 (m)
1.90 (m)
1.96 (m)
1.70 (m)
1.30 (m)
27.63
130.64
129.42
27.36
29.65–30.40
29.61–30.05
–
32.15
22.97
14.31
175.69
72.50
27.81
130.67
130.25
27.75
29.65–30.40
29.43–30.42
–
32.45
23.47
14.64
175.78
72.57
27.81
130.47
130.22
27.70
1.32 (br s)
–
1.25 (br s)
1.25 (br s)
0.90 (t, 7.1)
–
4.60 (dd, 8, 4.3)
2.22 (m)
2.00 (m)
1.94 (m)
1.73 (m)
29.31–30.75
–
32.35
23.42
14.21
175.29
72.43
35.92
17
18
14.53
175.85
72.58
1ꢀ
2ꢀ
3aꢀ
35.88
35.68
35.69
3bꢀ
4aꢀ
26.10
25.95
26.75
26.48
4bꢀ
5ꢀ–13ꢀ
5ꢀ–15ꢀ
16ꢀ–19ꢀ
14ꢀ
29.65–30.40
29.61–30.0
29.43–30.42
1.32 (m)
2.21 (m)
29.31–30.75
27.93
1.25 (m)
1.25 (m)
0.90 (t, 6)
32.40
23.16
14.53
1.26 (m)
1.26 (m)
0.95 (t, 6)
32.15
22.97
14.31
1.26 (m)
2.20 (m)
32.15
27.81
14ꢀ, 17ꢀ
15ꢀ
15ꢀ, 16ꢀ
16ꢀ
5.45 (m)
130.14
17ꢀ, 18ꢀ
20ꢀ, 23ꢀ
18ꢀ, 21ꢀ
22ꢀ
5.45 (m)
1.32 (m)
131.34
29.31–30.75
1.30 (m)
1.25 (m)
1.25 (m)
0.85 (t, 7)
29.43–30.42
32.45
23.47
23ꢀ
24ꢀ
14.64
1.25 (m)
1.25 (m)
32.41
23.22
14.17
–
–
–
25ꢀ
26ꢀ
0.90 (t, 7.1)
7.62 (br s)
6.81 (br s)
6.00 (br s)
6.30 (br s)
4.95 (d, 7.9)
4.02 (t, 8)
4.22 (obs)
4.19 (obs)
3.88 (m)
OH-2ꢀ
OH-3
OH-4
OH-6ꢀꢀ
1ꢀꢀ
7.72 (br s)
6.98 (br d)
–
–
7.71 (br s)
6.98 (br d)
–
–
7.60 (br s)
6.82 (br s)
6.00 (br s)
6.40 (br s)
4.95 (d, 8)
4.00 (t, 8)
4.21 (obs)
4.19 (obs)
3.87 (m)
–
–
–
–
6.57 (m)
4.95 (d, 8)
4.07 (t, 8)
4.21 (obs)
4.20 (obs)
3.90 (m)
4.52 (dd, 12, 2)
4.36 (dd, 12, 5)
–
6.57 (m)
4.95 (d, 8)
4.07 (t, 8)
4.21 (obs)
4.20 (obs)
3.90 (m)
4.52 (dd, 12, 2)
4.36 (dd, 12, 5)
–
–
105.73 (␤)
75.26
78.57
71.64
78.70
62.45
105.69 (␤)
75.14
78.47
71.50
78.62
62.64
105.31 (␤)
75.18
78.34
71.25
78.63
62.21
105.55 (␤)
75.23
78.26
71.72
78.31
62.54
2ꢀꢀ
3ꢀꢀ
4ꢀꢀ
5ꢀꢀ
6aꢀꢀ
6bꢀꢀ
4.48 (dd, 12, 2)
4.34 (dd, 12, 5)
4.48 (dd, 11, 2)
4.36 (dd, 11, 4)
C, H
5
6
7
1H NMR ı (m, J in Hz)
13C NMR ı
1H NMR ı (m, J in Hz)
13C NMR ı
1H NMR ı (m, J in Hz)
13C NMR ı
NH
1a
1b
2
3
4
5a
5b
6a
6b
7
8.54 (d, 9)
4.69 (dd, 13.3, 5)
4.56 (m)
–
8.53 (d, 8)
4.70 (dd, 12, 7)
4.52 (m)
–
8.54 (d, 9)
4.69 (dd, 13.3, 5)
4.56 (m)
–
70.57
70.61
70.57
5.20 (m)
51.90
76.09
72.52
33.91
5.22 (m)
51.49
76.13
72.63
34.15
5.20 (m)
51.90
76.09
72.52
33.91
4.29 (dd, 3, 4.7)
4.27 (obs)
2.41 (m)
1.91 (m)
1.75 (m)
1.63 (m)
2.23 (m)
5.44 (m)
5.44 (m)
4.28 (dd, 4.5, 3)
4.22 (obs)
2.40 (m)
1.88 (m)
1.70 (m)
1.65 (m)
2.20 (m)
5.45 (m)
5.45 (m)
4.29 (dd, 3, 4.7)
4.27 (obs)
2.41 (m)
1.91 (m)
1.75 (m)
1.63 (m)
2.23 (m)
5.44 (m)
5.44 (m)
26.27
26.17
26.27
27.65
130.23
129.51
27.80
27.81
130.67
130.25
27.75
27.65
130.23
129.51
27.80
8
9
10
2.23 (m)
2.20 (m)
2.23 (m)

F. Cateni et al. / Chemistry and Physics of Lipids 181 (2014) 90–98
93
Table 1 (Continued )
C, H
5
6
7
1H NMR ı (m, J in Hz)
13C NMR ı
1H NMR ı (m, J in Hz)
13C NMR ı
1H NMR ı (m, J in Hz)
13C NMR ı
11–15
16
17
1.31 (br s)
1.24 (br s)
1.24 (m)
0.84 (t, 7)
–
4.55 (dd, 8, 5.2)
2.19 (m)
1.98 (m)
1.93 (m)
1.69 (m)
29.68–30.42
32.26
23.13
14.68
175.65
72.52
1.31 (br s)
1.25 (br s)
1.25 (br s)
0.85 (t, 7)
–
4.57 (dd, 8, 3.7)
2.20 (m)
2.00 (m)
1.94 (m)
1.70 (m)
1.30 (m)
29.43–30.42
32.45
23.47
14.64
175.78
72.57
1.31 (br s)
1.24 (br s)
1.24 (br s)
0.84 (t, 7)
–
4.55 (dd, 8, 5.2)
2.19 (m)
1.98 (m)
1.93 (m)
1.69 (m)
29.68–30.42
32.26
23.13
14.68
175.65
72.52
18
1ꢀ
2ꢀ
3aꢀ
35.82
35.69
35.82
3bꢀ
4aꢀ
26.73
26.75
26.73
4bꢀ
5ꢀ–16ꢀ
5ꢀ–21ꢀ
5ꢀ–23ꢀ
17ꢀ, 21ꢀ
15ꢀ, 16ꢀ
19ꢀ, 20ꢀ
22ꢀ
29.43–30.42
1.31 (m)
29.68–30.42
1.31 (m)
29.68–30.42
2.20 (m)
5.45 (m)
1.30 (m)
1.25 (m)
27.81
130.14
29.43–30.42
32.45
1.24 (m)
1.24 (m)
32.26
23.19
23ꢀ
23ꢀ–27ꢀ
24ꢀ
1.25 (m)
23.47
0.84 (t, 7)
14.45
1.24 (m)
1.24 (m)
0.84 (t, 7)
32.26
23.19
14.45
25ꢀ
26ꢀ
28ꢀ
0.85 (t, 7)
14.64
–
–
–
–
105.31 (␤)
75.18
78.34
71.25
78.63
62.21
OH-2ꢀ
OH-3
OH-4
OH-6ꢀꢀ
1ꢀꢀ
7.61 (br s)
6.83 (br s)
6.00 (br s)
6.40 (br s)
4.90 (d, 8)
4.06 (t, 8)
4.23 (obs)
4.18 (obs)
3.85 (m)
–
–
–
–
7.60 (br s)
6.82 (br s)
6.00 (br s)
6.40 (br s)
4.95 (d, 8)
4.00 (t, 8)
4.21 (obs)
4.19 (obs)
3.87 (m)
7.61 (br s)
6.83 (br s)
6.00 (br s)
6.40 (br s)
4.90 (d, 8)
4.06 (t, 8)
4.23 (obs)
4.18 (obs)
3.85 (m)
–
–
–
–
105.72 (␤)
75.38
78.46
71.61
78.80
62.51
105.72 (␤)
75.38
78.46
71.61
78.80
62.51
2ꢀꢀ
3ꢀꢀ
4ꢀꢀ
5ꢀꢀ
6aꢀꢀ
4.52 (dd, 11.7, 2)
4.40 (dd, 10.6, 5)
4.48 (dd, 12, 2)
4.34 (dd, 12, 5)
4.52 (dd, 11.7, 2)
4.40 (dd, 10.6, 5)
6bꢀꢀ
[CH2Cl2/MeOH/H2O (lower layer) (20:3:1, 10:3:1, 7:3:1)] to give
methyl glucopyranoside (␤-anomer) as a colorless solid (5. 1 mg).
TLC [silica gel, CH2Cl2/MeOH/H2O (lower layer) (10:3:1)] of the
resulting methyl glucopyranoside (␣- and ␤-anomer) was identi-
cal to that of the standard methyl ␣-d-glucopyranoside and methyl
␤-d-glucopyranoside.
sealed vial. The reaction was subsequently quenched with 5% aq.
Na2S2O3 and the mixture was extracted with n-hexane. The n-
hexane layer was dried over Na2SO4, filtered and concentrated to
give the dimethyl disulphide derivative of LCB-1 as a light yellow
oil (9.4 mg). The LCBs DMDS derivatives of cerebrosides 1–7 were
subjected to MS analysis. The FAMs 3, 4 and 6 were derivatized
using the same method described above. The results are reported
in Fig. 3.
2.1.5. GC–MS analysis of TMS ethers of methyl glycosides from
1–7
The mixture of methyl glycosides obtained by column chro-
matography of the aq. MeOH layer derived from methanolysis
of 1–7 was converted to their trimethyl derivatives using BSTFA
containing 1% TMCS for 3 h at 70 ◦C. 0.1 ␮l of silylated mixture
was analyzed by gas chromatograph mass spectrometer (GC–MS)
system. The chromatographic conditions were: column temper-
ature was programmed from 100 ◦C to 260 ◦C at 8 ◦C/min, with
15 min of final isotherm, injector temperature 280 ◦C, carrier
gas (helium), flow rate 1.5 mL/min. Transfer line temperature
was kept at 270 ◦C. The mass spectrometer scanned from m/z
100 to m/z 600 at 1.0 s cycle time. The ion source was set at
180 ◦C and spectra were obtained by electron impact (70 eV).
Methyl glycosides (GC–MS): methyl ␣- and ␤-glucopyranosides
were detected. Methyl ␣-d-glucopyranoside: C7H14O6, m/z = 194
[M]+, TMS derivative, m/z = 482 [M]+, 204, 191, 217, tR (min):
12.770. Methyl ␤-d-glucopyranoside: C7H14O6, m/z = 194 [M]+,
TMS derivative, m/z = 482 [M]+, 204, 191, 217, tR (min): 14.008.
3. Pharmacology
3.1. Free radical scavenging activity
The free radical scavenging activity (FRS) of MeOH extract
as well as each isolated fraction was determined by using 2,2-
diphenyl-1-picrylhydrazyl (DPPH) assay (Gaggeri et al., 2012;
Ancerewicz et al., 1998). A commercially available standardized
green tea extract (Green Select®) was used as standard. Briefly,
both samples and standard were dissolved in ethanol containing
up to 2% DMSO at concentration of 6 mg/mL. Reaction mixture was
prepared by adding 100 ␮l of extract solution (or standard solu-
tion) to 3.9 mL of DPPH solution, freshly prepared dissolving DPPH
in methanol/KH2PO4 and NaOH buffer (50/50, v/v) at a concentra-
tion of 6 × 10−5 M, giving test solutions at final concentration of 75,
37.5, 15, and 7.5 ␮g/mL. After 30 min of incubation at room tem-
perature, the absorbance was measured at 515 nm by a UV-Visible
spectrophotometer (Lambda 25 UV/VIS spectrometer, Perkin Elmer
instruments, MA, USA).
2.1.6. Dimethyl disulphide derivatives of FAMs 3, 4, 6 and LCBs
from cerebrosides 1–7
LCB-1 (10 mg) was dissolved in carbon disulphide (1 mL) and
dimethyl disulphide (1 mL) and iodine (20 mg) were added. The
reaction mixture was then kept at −60 ◦C for 48 h in a small
FRS was expressed as a percentage compared with the control,
consisting of 3.9 mL of DPPH solution and 100 ␮l of methanol. The

94
F. Cateni et al. / Chemistry and Physics of Lipids 181 (2014) 90–98
percent inhibition of the DPPH radical by the test solution was
calculated using the following formula:
2-amino-1,3-dihydroxy-4,8-octadecadiene (LCB-1,2) and 2-
amino-1,3,4-trihydroxy-8-octadecene (LCB-3–7), respectively.
The ESI-MS mass spectra of the DMDS derivatives of 1–7 showed
remarkable fragments ion peaks at m/z 149, 187 (1, 2) and 221
(3–7), respectively, due to cleavage of the bond between the car-
bons bearing a methylthio group (Fig. 3) (Scribe et al., 1988). The
above fragment ions arising from selective fragmentation at the C-
4–C-8 (1, 2) and at the C-8–C-9 (3–7) positions of C18 chain, confirm
the position the double bond at C-4–C-8 (1, 2) and C-8–C-9 (3–7)
in the long chain bases. The two double bonds in cerebrosides 1
and 2, one at C-4/C-5 and another between C-8/C-9 are trans ori-
ented in 1 owing to the values of chemical shifts of allylic methylene
ıC > 30 and the J values (Su et al., 1999), while in cerebroside 2 the
double bond between C-8/C-9 is cis oriented by the upfield shifted
carbon chemical shifts of C-7 (ıC 27.63) and C-10 (ıC 27.36). The
ꢀ8 double bond in 3–7 was determined to be cis (Z) by the upfield
shifted carbon chemical shifts of C-7 (ıC 27.81) and C-10 (ıC 27.70)
(Kang et al., 2001a,b) and the relative smart coupling constant of
H-8 at ıH 5.40 (dt, J = 10.8, 7.0 Hz) and H-9 at ıH 5.45 (dt, J = 10.8,
6.8 Hz). 1D and 2D 1H NMR spectroscopy, DQF-COSY and HMQC
indicated that the head group consists of a single glucose residue
in the ␤ configuration. The glucose configuration was determined
by the characteristic chemical shifts, the spin–spin splitting and the
multiplicity of the characteristic resonance of the H-4ꢀꢀ proton, as
well as by the splittings of the other ring protons. The absolute con-
figuration of the glucopyranose moiety was determined to be the
d-form using the Hara method (Hara et al., 1987).
Consideration of the biogenesis and steric hinderance of sphin-
golipids, generally were acknowledged to determine the absolute
stereochemistry of the phytosphingosines moiety. On the basis of
the 13C NMR spectral data, the relative stereochemistries at C-2 (ıC
54.6) and C-3 (ıC 72.4, 72.3) were deduced to be 2S and 3S (Tao
et al., 2010) for the cerebrosides 1 and 2. The relative stereochem-
istry of 3–7 at C-2, C-3, C-4 was proposed as 2S, 3S, 4R, identical to
that of d-sphingosine on the basis of 13C NMR spectral data, since
the chemical shifts of C-2 (ıC 51.4, 51.2, 51.9, 51.4, 51.9), C-3 (ıC
76.1, 76.2, 76.09, 76.1) and C-4 (76.2, 72.5) were in agreement with
those reported in the literature (Yang et al., 2009).
ꢀ
ꢁ
(Abs control − Abs sample)
FRS% =
× 100.
Abs control
The analyses were carried out in triplicate and results are
expressed as mean SE. IC50 values were calculated by using Graph
Pad Prism 4.0.
4. Results and discussion
A phytochemical study on E. helioscopia revealed a complex mix-
ture of cerebrosides (1–7), together with glucoclionasterol (8), a
digalactosyl diacyl glycerol (9) and a monogalactosyldiacylglycerol
(10).
The fresh leaves of E. helioscopia were extracted with methanol
at room temperature. After evaporation of the extract, the residue
obtained was subjected to flash column chromatography to give
five fractions. Further chromatographic purifications of fraction 5
gave a complex mixture of cerebrosides which was then submit-
ted to reversed phase column ‘flash chromatography’ to give the
cerebrosides (1–7) (Fig. 1).
The IR spectra of 1–7 showed bands at 3315, 1635, 1075,
1036 cm−1 indicating the presence of hydroxyl, amide and C
functional groups.
O
The 1H and 13C NMR spectra of 1–7 (Table 1) indicated the pres-
ence of a sugar moiety (ıH 4.95–4.90, 1H, d, J = 8.0 Hz, anomeric
proton; ıC 105.7–105.3), an amide function (ıH 8.4–8.5, 1H, d, J = 8.0,
9.0 Hz, NH; ıc 175.8–175.3) and a long chain aliphatic and olefinic
functions (ıH 0.90–0.84, t, J = 6.0, 7.0, 7.1 Hz, CH3; ıH 1.26–1.24, CH2,
ıH 5.45–5.39, 1H each, both t, J = 4.9 Hz, ıc 129.5–132.3). These data
are suggestive of a glycosphingolipid structure.
The 13C NMR spectra of 1–7 (Table 1) were assigned by a
combination of DEPT, HMQC and HMBC experiments. Important
long-range correlations were observed between C-1ꢀꢀ and Hab
-
1; C-1 and H-1ꢀꢀ, H-2, H-3; C-2 and NH and C-1ꢀ and NH and
H-2ꢀ. These results again supported the glycosphingolipid struc-
ture (Fig. 2). Methanolysis (Gaver and Sweely, 1965) of 1–7 yield
methyl glucosides, fatty acid methyl esters (FAMs) and dihydroxy
(1–2) and trihydroxy long chain bases (3–7) (LCBs). The fatty
acid methylesters were identified by EI-MS analysis as methyl
2-hydroxypalmitate (FAMs-1,2), methyl 2-hydroxytetracosenoate
(FAM-3), methyl 2-hydroxyhexacosenoate (FAM-4), methyl 2-
hydroxytetracosanoate (FAM-5), methyl 2-hydroxyoctacosenoate
(FAM-6) and methyl 2-hydroxyhexacosanoate (FAM-7).
Therefore the structures of 1–7 were proposed to be 1-O-
(␤-d-glucopyranosyl)-(2S,3S,4E,8E)-2-[(2ꢀR)-2ꢀ-hydroxyhexadeca-
noylamino)-4(E),8(E)-octadiene-1,3-diol (1), 1-O-(␤-d-gluco-
pyranosyl)-(2S,3S,4E,8Z)-2-[(2ꢀR)-2ꢀ-hydroxyhexadecanoylamino)-
4(E),8(Z)-octadiene-1,3-diol (2), 1-O-(␤-d-glucopyranosyl)-(2S,
3S,4R,8Z)-2-[(2ꢀR)-2ꢀ-hydroxytetracosenoilamino]-8(Z)-octadece-
ne-1,3,4-triol
[(2ꢀR)-2ꢀ-hydroxyhexacosenoilamino]-8(Z)-octadecene-1,3,4-triol
(4),
1-O-(␤-d-glucopyranosyl)-(2S,3S,4R,8Z)-2-[(2ꢀR)-2ꢀ-hydro-
xytetracosanoilamino]-8(Z)-octadecene-1,3,4-triol (5), 1-O-
(3),
1-O-(␤-d-glucopyranosyl)-(2S,3S,4R,8Z)-2-
The MS spectra of 1–7 showed molecular ions [M+H]+ peaks at
m/z 714, 842, 870, 844, 898, 872 and fragments ions at m/z 552 [714-
glc]+, 680 [842-glc]+, 708 [870-glc]+, 682 [844-glc]+, 736 [898-glc]+
and 710 [872-glc]+. The absolute configuration at C-2 the 2-hydroxy
fatty acid was presumed to be R from the specific rotation of the
fatty acids methyl esters ([␣]D-110) (Higuchi et al., 1990; Shibuya
et al., 1990a,b; Kang et al., 2001a,b).
The FAMs obtained from the methanolysis of the cerebrosides
3, 4 and 6 exhibited 13C NMR signals at about 175.8, 131.2 and
130.4 expected for monounsaturated fatty acid methyl esters. The
resonance at about 27.7, 27.8 ppm confirms the Z geometry of
the double bonds in the long-chain fatty acids (Table 1). In order
to establish the position of the double bond, the monounsatu-
rated fatty acid methyl esters were treated with dimethyl disulfide
(DMDS) and I2 and the products subjected to electron impact
(EI)-MS analysis (Scribe et al., 1988). The characteristic fragments
at m/z = 317, 345, 359 and 173, obtained between the cleavage
between the sulphide carbons, indicate the position of the double
bonds in FAMs moiety of 3, 4 and 6 (Fig. 3).
(␤-d-glucopyranosyl)-(2S,3S,4R,8Z)-2-[(2ꢀR)-2ꢀ-hydroxyoctacose-
noilamino]-8(Z)-octadecene-1,3,4-triol(6) and 1-O-(␤-d-gluco-
pyranosyl)-(2S,3S,4R,8Z)-2-[(2ꢀR)-2ꢀ-hydroxyhexacosanoilamino]-
8(Z)-octadecene-1,3,4-triol (7). The cerebrosides 1–7 isolated
and identified are not new. For example, cerebrosides 1 and 2
are identical to soya-cerebrosides I and II isolated from soybean
(Shibuya et al., 1990a,b) and cerebroside 5 is identical to poke-
weed cerebroside isolated from Phytolaccae Radix (Kang et al.,
2001a,b).
Although, we investigated different species of Euphorbiaceae
and complex mixtures of cerebrosides were isolated and identi-
fied (Cateni et al., 2003), to our knowledge this is the first report
on the isolation and structure elucidation of cerebrosides from E.
helioscopia L.
From fraction 3 successive chromatographic purifications have
led to the isolation and structure elucidation of glucoclionas-
terol (8). Glucoclionasterol was previously isolated from Euphorbia
wulfenii Hoppe ex Koch (Falsone et al., 1997).
On the other hand, on the basis of mass spectrome-
try analysis the LCB components were suggested to be

F. Cateni et al. / Chemistry and Physics of Lipids 181 (2014) 90–98
95
all the 1H and 13C NMR signals for the sugar and glycerol moieties.
The results suggested that two residues of linolenic acid were
connected to C-1 and C-2 and ␤-d-galactopyranose to C-3 of
glycerol, respectively. Compound 9 was thus characterized as (2S)-
1,2-O-di-linolenoyl-3-O-␤-d-galactopyranosylglycerol (9), which
From fraction 4 the compounds 9 and 10 have been isolated and
identified (Fig. 1). The spectral data of 9 indicated the presence of
a sugar and long-chain unsaturated aliphatic system strongly sug-
gesting a glycolipid. The 1H and 13C NMR spectra of 9 together with
1H–1H COSY, HMQC and HMBC spectra, led to the assignments of
Fig. 1. Chemical structures of compounds 1–10 isolated from E. helioscopia L.

96
F. Cateni et al. / Chemistry and Physics of Lipids 181 (2014) 90–98
Fig. 1. (Continued ).
was previously isolated from Euphorbia nicaensis All. (Cateni et al.,
2004). Also compound 10, identified as (2S)-1,2-O-di-linolenoyl-
glyceryl-6-O-(␣-d-galactopyranosyl)-␤-d-galactopyranoside was
proposed in an our preliminary paper (Cateni et al., 2004). The
galactose configuration was determined by the characteristic
chemical shifts, the spin–spin splitting and the multiplicity of the
characteristic resonance of the H-4ꢀ proton, as well as by the split-
tings of the other ring protons. Methyl glycosides obtained from
methanolysis of 1–7, 9 and 10 were converted to trimethylsilyl
derivatives and the silylated derivatives obtained were analyzed
by gas chromatograph–mass spectrometer (GC–MS) system. Iden-
tification of galactose was carried out by study of the MS spectra,
comparison with members of the NBS library and with retention
times of methyl ␣- and ␤-d-galactopyranoside and methyl ␣- and
␤-d-glucopyranoside standard TMS derivatives (Cateni et al., 2008).
The free-radical scavenging activity (FRS) of E. helioscopia
methanolic leaves extract, determined by using DPPH assay, was
initially evaluated at a stock concentration of 150 ␮g/mL. Suc-
cessively, stock solutions were serially diluted into a range of
75–7.5 ␮g/mL, and their corresponding FRS activity was deter-
mined as percentage values. An interesting FRS activity % was
evidenced (Fig. 4), since E. helioscopia reached a maximum effect
(Emax) of 60% at 15 ␮g/mL and with an EC50 value of 6,9 ␮g/mL. This
result would be considered of high interest, taking into account that
the EC50 value of the commercially available standardized green tea
extract (Green Select®), used as reference standard, is very close
(4.6 ␮g/mL) to that found.
Antioxidant potential of E. helioscopia whole plant was exten-
sively demonstrated by different authors (Nikolova et al., 2011;
Uzair et al., 2009), while only one paper focused on the activity of

F. Cateni et al. / Chemistry and Physics of Lipids 181 (2014) 90–98
97
O
H
N
OH
HO
OH
1H-1H COSY
HMBC
O
NH
OH
HO
(CH2)7CH3
2
3
4
1
OH
C
H
Fig. 2. Selected 1H–1H COSY (bold lines) and HMBC (full-line arrows) correlations of ceramide of 1 and sphingosine of 3.
O
H3CS
SCH3
CH2
6
O
CH2
m
OH
m/z 173
FAM-DMDS-3: m=11, m/z=317 (M-173); m/z=173 (C10H21S)+
FAM-DMDS-4: m=13, m/z=345 (M-173); m/z=173 (C10H21S)+
FAM-DMDS-6: m=14, m/z=359 (M-173); m/z=173 (C10H21S)+
NH2
SCH3
SCH3
HO
CH2
8
OH
SCH3
SCH3
m/z=187
m/z=149
LCB-DMDS-1,2
NH2
OH
HO
CH2
2
CH2
7
OH
SCH3
H3CS
m/z=187
m/z=221
LCB-DMDS-3-7
Fig. 3. FAM-DMDS and LCB-DMDS derivatives of cerebrosides 1–7.

98
F. Cateni et al. / Chemistry and Physics of Lipids 181 (2014) 90–98
Fig. 4. Free radical scavenging activity % of E. helioscopia methanolic extract together with the active fraction (fraction 4) and compound 10.
different parts of the plant (flowers, stem and leaves) in correlation
with the phenolics and flavonoids content (Nikolova et al., 2011).
Our results are in line with those reported by Maoulainine et al.
(2012) in terms of free radical potential of the methanolic leaves
extract, taking into account the variability related to time and site
of collecting plant. Moreover our work focused on the lipid-soluble
fraction of the methanolic leaves extract. Basing on the promising
results in terms of FRS activity obtained for the crude extract, the
FRS screening was extended to the fractions isolated during the
purification process, in order to identify the components responsi-
ble for the activity observed. In this view, each fraction was assayed
by using the DPPH method, in the same conditions described for the
crude extract.
Fraction 4 resulted to be the only one active, exerting a FRS
effect similar to that of the crude extract, which Emax was higher 67
at 15 ␮g/mL (Ec50 = 5.7 ␮g/mL) (Fig. 4). Since the structural study
revealed that this fraction is a mixture of compounds 9 and 10, we
isolated and tested both compounds. Interestingly FRS activity was
associated only with compound 10 with an Emax of 69% at 15 ␮g/mL
(EC50 = 5.2 ␮g/mL). Therefore, bioactivity-guided fractionation was
successful in leading the identification of the compound responsi-
ble for the FRS activity of E. helioscopia methanolic leaves extract.
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