Cytotoxic activity of diterpenoids isolated from the aerial parts of Elaeoselinum asclepium subsp. meoides

Article abstract of DOI:10.1055/s-2008-1074579

The phytochemical investigation of the acetone extract of the aerial parts of Elaeoselinum asclepium (L.) Bertol. subsp. meoides (Desf.) Fiori afforded several known diterpenoids as well as meoidic acid (5), new in the literature. The cytotoxic activities

Full text of DOI:10.1055/s-2008-1074579

Letter 1285
Cytotoxic Activity of Diterpenoids
Isolated from the Aerial Parts of
Elaeoselinum asclepium
subsp. meoides
Sergio Rosselli1, Antonella Maggio1, Cristina Eiroa1,
Carmen Formisano2, Maurizio Bruno1, Carlo Irace3,
Carmen Maffettone3, Nicola Mascolo3
1 Dipartimento di Chimica Organica, Università degli Studi di
Palermo, Palermo, Italy
2 Dipartimento di Chimica delle Sostanze Naturali, Università degli
Studi di Napoli “Federico II”, Napoli, Italy
3 Dipartimento di Farmacologia Sperimentale, Università degli
Studi di Napoli “Federico II”, Napoli, Italy
Abstract
!
The phytochemical investigation of the acetone extract of the
aerial parts of Elaeoselinum asclepium (L.) Bertol. subsp. meoides
(Desf.) Fiori afforded several known diterpenoids as well as
meoidic acid (5), new in the literature. The cytotoxic activities
of elasclepic acid (1), ent-atis-16-en-19-oic acid (2), ent-beyer-
15-en-19-oic acid (3), ent-kaur-16-en-19-oic acid (4) and meoi-
dic acid (5) were investigated on rat glioma C6 cells by evalua-
tion of cell growth inhibition.
were in agreement with a formula of C27H38O6. The 1H- and 13C-
NMR spectra showed signals for an exocyclic double bond
(δH = 5.00, 1H, brs, H-17a; δH = 4.90, 1H, brs, H-17b; δC = 111.5,
CH2, C-17; δC = 151.6, C, C-16), a diagnostic quartet of quartets of
an angeloxy ester at δH = 6.01, 1H, qq, H-3′, whose presence was
confirmed by the signals of the two vinylic methyl groups
(δH = 2.01, 3H, dq, CH3 –4'; δC = 15.8, CH3, CH3 –4′; δH = 1.92, 3H,
quint, CH3 –5′; δC = 21.0 CH3, CH3 –5′) and of the two quaternary
carbons at δC = 167.0 C, C-1′ and δC = 128.8 C, C-2′. Furthermore,
there was clear evidence for the presence of two tertiary methyl
groups (δH = 1.18, 3H, s, Me-18; δC = 20.6, CH3, C-18; δH = 0.98, 3H,
s, Me-20; δC = 12.5 CH3, C-20), a carboxylic group (δC = 182.7, C, C-
19), an acetyl and two methines carrying the two esters
(δH = 5.36, 1H, brs, H-15; δC = 75.1, CH, C-15; δH = 5.07, 1H, t, H-7;
δC = 74.8 CH, C-7). Extensive 2 D NMR experiments (1H-1H COSY,
HMBC, HSQC) clearly indicated the presence of the atisane skel-
eton as well as the position of the substituents and allowed to
Key words
!
Elaeoselinum asclepium subsp. meoides · Umbelliferae · diterpe-
noids · meoidic acid · cytotoxic activity
The former genus Elaeoselinum W. D. J. Koch ex DC., (Umbellifer-
ae) in sensu latu [1] included two taxa, Distichoselinum and
Margotia [2], [3], that are now segregated as monotypic genera.
Only seven species belong to the genus Elaeoselinum s.l. [4] and
all are confined to the Mediterranean region, mainly in western
regions and Morocco.
E. asclepium (L.) Bertol. s.l. is the only taxon present in Italy and
no phytochemical investigations have been published on this
isotype, in fact, all the species studied so far have a Spanish ori-
gin [5].
E. asclepium subsp. meoides is a rare species growing in the
mountains of Sicily. Investigation of the root of this subspecies,
collected in Spain, resulted in the isolation of two ent-beyerane
diterpenoids: elasclepiol and elasclepic acid (1) [6]. In this work,
the results of the investigation on the phytochemical composi-
"
assign all the protons and carbons (● Table 1).
A survey of the literature showed that this compound was the
de-methyl derivative of margotianin (6), an atisane diterpenoid
from Elaeoselinum gummiferum [9], isolated several years ago.
This information allowed the structure assignment as depicted
in formula 5 and the trivial name of meoidic acid was given to
it. Furthermore, treatment of 5 with diazomethane afforded
margotianin (6) [9] confirming the structure and the absolute
configuration of meoidic acid.
tion of the aerial parts, not previously studied, and the cytotoxic
activity of some diterpenes (1–5) are reported.●
"
■
Examination of the acetone extract of the aerial parts of
Elaeoselinum asclepium (L.) Bertol. subsp. meoides (Desf.) Fiori
yielded four known diterpenoids: elasclepic acid (1) [6], ent-
atis-16-en-19-oic acid (2) [7], ent-beyer-15-en-19-oic acid (3)
[7] and ent-kaur-16-en-19-oic acid (4) [8], and the new atisane
derivative meoidic acid (5).
The structure of compound 5 was determined by spectral meth-
ods: positive ESI-MS and homo- and heteronuclear 1 D and 2 D
NMR experiments. In fact, its elemental analysis and ESI-MS
"
In ● Table 1, the full NMR signal assignments of compound 1 are
also reported. These data were not reported previously.
The biological activity of the isolated compounds was investiga-
ted on the rat glioma cell line (C6 line) by evaluation of cell
growth and viability [10]. Since gliomas show a poor response
to cytotoxic drugs, they are very resistant to most chemothera-
peutic agents. Although many advances in antineoplastic thera-
Letter… Planta Med 2008; 74: 1285–1287

1286 Letter
Table 1 Spectroscopic data of compounds 1 and 5 in CDCl3
1
5
H (J Hz)
C
H (J Hz)
C
HMBC
1
1.72, 0.96
1.46, 1.26
2.18, 1.06
40.0 t
1.72, 1.05
1.87, 1.49
2.24, 1.09
39.9 t
H-3b, Me-20
2
19.1 t
37.7 t
43.6 s
56.2 d
20.6 t
32.1 t
53.2 s
52.0 d
37.9 s
19.8 t
32.4 t
48.2 s
94.3 d
132.1 d
133.5 d
18.7 t
38.0 t
43.2 s
49.2 d
26.3 t
74.8 d
38.9 s
39.9 d
37.9 s
26.0 t
35.4 d
26.4 t
26.6 t
75.1 d
151.6 s
111.5 t
3
Me-18
4
H-5, H-3b, Me-18
Me-18, Me-20
H-7, H-5
5
1.13
1.67
6
1.85, 0.94
1.82, 1.19
2.28, 1.92
5.07 t (3.0)
7
H-15
8
H-5
9
1.20
2.22
H-15, Me-20
10
11
1.45, 1.16
1.40 (2 H)
1.72, 1.49
2.36
12
H-15, H-17a, H-17b
13
1.57, 1.24
1.19 (2 H)
5.36 brs
14
4.54 brs
15
5.71 d (5.7)
5.44 d (5.7)
H-17a, H-17b
H-15, H-17a, H-17b
H-15
16
17
5.00 brs, 4.90 brs
1.18 s
Me-17
Me-18
19
0.95 s
1.24 s
19.2 q
29.0 q
183.1 s
14.1 q
168.4 s
128.8 s
136.7 d
14.3 q
12.1 q
28.6 q
182.7 s
12.5 q
167.0 s
128.8 s
136.5 d
15.8 q
21.0 q
170.4 s
21.7 q
H-3b
H-5
Me-20
1′
2′
0.70 s
0.98 s
H-5, Me-18, H-3b, H-9, H-1a
H-7, Me-4′, Me-5′
Me-4′, Me-5′
Me-4′, Me-5′
3′
6.83 qq (7.0, 1.0)
1.78 brd (7.0)
1.81 brd (1.0)
6.01 qq (7.0, 1.0)
2.01 dq (7.0, 1.0)
1.92 quint (1.0)
Me-4′
Me-5′
Ac
H-3′
H-15
Me-Ac
1.95 s
py have taken place, the rat C6 glioma cell line, like most malig-
nant gliomas encountered clinically, is resistant to many phar-
macological treatments [11]. Compared to primary neural cells,
the C6 cell line offers some major advantages like easy accessi-
bility and culture as well as a good availability of high cell num-
bers. Thus, the rat C6 glioma-derived cell line is widely used in
biological research.
For bioactivity experiments, the C6 cells were continuously trea-
ted for 48 h with various concentrations of each compound and,
after cell counting, the MTT assay was performed [12]. No cell
growth inhibitory activity was observed for compounds 1, 2, 4
and 5 at a concentration below 150 μM, whereas compound 3
exhibited an IC50 value of 77 16 (results are expressed as
mean S.D., n = 24). The positive control for cytotoxicity was
performed using digitonin (Calbiochem), a non-ionic lytic deter-
gent, which showed an IC50 value lower than 1 μM on C6 glioma
cells.
Plant material: The aerial parts of Elaeoselinum asclepium (L.)
Bertol. subsp. meoides (Desf.) Fiori were collected in June 2006
near Dingoli, Piana degli Albanesi (Palermo) in Sicily, Italy and
voucher specimens (PAL 06–597, authenticated by Prof. F. Rai-
mondo) were deposited in the Herbarium of the Botanical Gar-
den of Palermo, Italy.
Extraction and isolation: Dried and finally powdered aerial parts
(671 g) of E. asclepium subsp. meoides were extracted with
Me2CO (5 L) at room temperature for a week. After filtration,
the solvent was evaporated to give a gum (22 g) which was chro-
matographed on silica gel (Merck No. 7734, deactivated with
15 % H2O, 400 g) column (5×20 cm) eluted with petroleum ether,
petroleum ether-EtOAc mixtures, EtOAc and EtOAc-MeOH mix-
tures. 500 mL fractions were collected as follows: 1–12, petrole-
um ether; 13–18, petroleum ether-EtOAc (9:1); 19–22, petrole-
um ether-EtOAc (4:1); 23–26, petroleum ether-EtOAc (7:3);
27–30, petroleum ether-EtOAc (3:2); 31–34, petroleum ether-
EtOAc (1:1); 35–38, petroleum ether-EtOAc (2:3); 39–42, pe-
troleum ether-EtOAc (3:7); 43–46, petroleum ether-EtOAc
(1:4); 47–50, petroleum ether-EtOAc (1:9); 51–54, EtOAc;
55–58, EtOAc-MeOH (9:1); 59–62, EtOAc-MeOH (4:1). Frac-
tions 23–26 were re-chromatographed on a silica gel (150 g)
column (2×20 cm). Elution with n-hexane-EtOAc (4:1, 2 L),
100 mL fractions being collected, gave a mixture of three com-
pounds (400 mg). Fractions 27–34 were re-chromatographed
on silica gel (150 g) column (2×20 cm). Elution with n-hexane-
EtOAc (7:3, 2 L),100 mL fractions being collected, gave a mixture
of three compounds (1 g) and pure elasclepic acid (1).
Materials and Methods
!
General: Optical rotations were determined on a JASCO P-1010
1
digital polarimeter. H- and 13C-NMR spectra were recorded in
CDCl3 on a Bruker AVANCE 300 MHz NMR spectrometer, using
the residual solvent signal (δ = 7.27 in 1H and δ = 77.00 in 13C) as
reference. 1H-NMR and 13C-NMR assignments were determined
by DEPT, 1H-1H COSY, HMBC, HSQC spectra. ESI-MS was obtained
with an Applied Biosystem API-2000 mass spectrometer (Ap-
plied Biosystem). Elemental analysis was carried out with a Per-
kin-Elmer 240 apparatus.
Letter… Planta Med 2008; 74: 1285–1287

Letter 1287
Radial chromatography of 100 mg of the mixture, using silica gel
(45 g, Merck 1.07749.1000) impregnated with AgNO3 (5% w/w),
eluted with n-hexane-EtOAc (4:1, 800 mL), 20 mL fractions be-
ing collected, gave in order of elution ent-atis-16-en-19-oic acid
(2), ent-beyer-15-en-19-oic acid (3) and ent-kaur-16-ene-19-oic
acid (4).
received
revised
accepted
January 28, 2008
May 15, 2008
May 18, 2008
Bibliography
DOI 10.1055/s-2008-1074579
Planta Med 2008; 74: 1285–1287
© Georg Thieme Verlag KG Stuttgart · New York
Published online July 4, 2008
ISSN 0032-0943
Fractions 35–38 were re-chromatographed on a silica gel col-
umn. Elution with n-hexane-EtOAc (1:1,1 L), 50 mL fractions be-
ing collected, gave meoidic acid (5, 100 mg). The physical and
spectroscopical data of compounds 1–4 and 6 were in complete
agreement with those reported in the literature.
Elasclepic acid (1): 300 mg; [α]2D5: –85.2 (c 0.28, EtOH) [6].
ent-Atis-16-en-19-oic acid (2): 13 mg; [α]2D5: –68.8 (c 0.41, CHCl3)
[13].
Correspondence
Prof. Maurizio Bruno
Dipartimento di Chimica Organica
Università degli Studi di Palermo
Viale delle Scienze
Parco d'Orleans II
90128 Palermo
ent-Beyer-15-en-19-oic acid (3): 30 mg; [α]2D5: + 7.3 (c 0.60,
CHCl3) [13].
Italy
ent-Kaur-16-en-19-oic acid (4): 20 mg; [α]2D5: –111.4 (c 0.19,
CHCl3) [14].
Phone: +39/091/596/905
Fax: +39/091/596/825
bruno@dicpm.unipa.it
Meoidic acid (5): 100 mg; oil; [α]2D2: –47.5: (c 0.6, CHCl3); ESI-MS
(positive mode): m/z = 497 [M + K] (6), 481 [M + Na] (56), 459
[M + H] (100); anal. calcd. for C27H38O6 (458.59): C 70.71, H
13
8.35; found: C 70.68, H 8.37; 1H-NMR and C-NMR: see ● Ta-
"
ble 1.
Margotianin (6): 20 mg; obtained after treatment of compound 5
(20 mg) with CH2N2; [α]2D5: –80.2 (c 0.17, CHCl3) [11].
Cell culture and cytotoxic assay : The cytotoxic assays were per-
formed according to literature procedures [12].
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