Cytotoxic activity of withanolides isolated from Tunisian Datura metel L.

Article abstract of DOI:10.1016/j.phytochem.2011.07.009

Withanolide-type steroids, withametelin Q (1) and 12α- hydroxydaturametelin B (2) along with three known withanolides, were isolated from leaves of Datura metel L. (Solanaceae). The respective structures, characterized mainly by NMR spectroscopy, were identified as (20R,22R,24R)-21,24-epoxy-1α,3β-dihydroxywitha-5,25(27) -dienolide-3-O-β-d-glucopyranoside (1) and (20R,22R,24R)-12α,21,27- trihydroxy-1-oxowitha-2,5,24-trienolide-27-O-β-d-glucopyranoside (2). The cytotoxicity of isolated compounds was evaluated against human lung carcinoma cells (A549) and human colorectal adenocarcinoma cells (DLD-1), respectively. Compound 2 exhibited cytotoxicity against A549 and DLD-1 cell lines, with IC₅₀ values of 7 and 2.0 μM, respectively. However, for compounds 6 and 7, cytotoxicities were higher against DLD-1 cells with IC₅₀ values of 0.6 and 0.7 μM. Both compounds blocked the cell cycle in the S-phase and induced apoptosis. Crown Copyright

Full text of DOI:10.1016/j.phytochem.2011.07.009

Phytochemistry 72 (2011) 2031–2036
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Cytotoxic activity of withanolides isolated from Tunisian Datura metel L.
Amor Bellila a, Cindy Tremblay b, André Pichette b, Brahim Marzouk a, Vakhtang Mshvildadze b,
Serge Lavoie b, Jean Legault b,
⇑
a Aromatic and Medicinal Plants Unit, Biotechnological Center in Borj-Cedria Techno-park, 901 Hammam-Lif, Tunisia
b Chaire de recherche sur les agents anticancéreux d’origine naturelle, Département des sciences fondamentales, Université du Québec à Chicoutimi, Québec, Canada G7H 2B1
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 July 2010
Received in revised form 6 July 2011
Available online 16 August 2011
Withanolide-type steroids, withametelin Q (1) and 12a-hydroxydaturametelin B (2) along with three
known withanolides, were isolated from leaves of Datura metel L. (Solanaceae). The respective structures,
characterized mainly by NMR spectroscopy, were identified as (20R,22R,24R)-21,24-epoxy-1 ,3b-dihydr-
oxywitha-5,25(27)-dienolide-3-O-b- -glucopyranoside (1) and (20R,22R,24R)-12 ,21,27-trihydroxy-1-
oxowitha-2,5,24-trienolide-27-O-b- -glucopyranoside (2). The cytotoxicity of isolated compounds was
a
D
a
D
Keywords:
Datura metel L.
Solanaceae
Cytotoxic activity
Steroids
Withametelin Q
evaluated against human lung carcinoma cells (A549) and human colorectal adenocarcinoma cells
(DLD-1), respectively. Compound 2 exhibited cytotoxicity against A549 and DLD-1 cell lines, with IC50
values of 7 and 2.0 lM, respectively. However, for compounds 6 and 7, cytotoxicities were higher against
DLD-1 cells with IC50 values of 0.6 and 0.7
lM. Both compounds blocked the cell cycle in the S-phase and
induced apoptosis.
Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
12a-Hydroxydaturametelin B
1. Introduction
growth of human cancer cell lines (Ma et al., 2006; Pan et al.,
2007). In the present study, the bioguided fractionation of the
MeOH extract from the leaves of D. metel L. and the isolation and
structure elucidation of two new withanolide-type steroids are
reported.
Datura metel L. is a medicinal herb of the Solanaceae family. It
has been used in traditional medicine, as a fumigant in bronchial
asthma, and for its relaxing effect on the smooth musculature of
the bronchi and bronchioles (Smith et al., 1991; Pretorius and
Marx, 2006). Datura leaves have been used in India and Africa as
a treatment for respiratory disorders which have been demon-
strated to relieve asthma (Lewis and Elvin-Lewis, 1977). In Tunisia,
Datura aerial parts are used in folk medicine for their antiasthmat-
ic, antispasmodic and antiparkinsonian properties (Le Floc’h,
1983). The chemical investigations of Datura species demonstrated
that leaves and seeds especially were rich in alkaloids, including
atropine, hyoscyamine, and scopolamine (Berkov et al., 2006;
Miraldi et al., 2001). These compounds are included in many offi-
cial pharmacopoeias because of their anticholinergic activities.
Recently, several pharmacological investigations have been con-
ducted on Datura. In fact, different extracts obtained from this genus
have been reported to exhibit antimicrobial (Kagale et al., 2004;
Eftekhar et al., 2005; Uzun et al., 2004) and antifungal activities
(Dabur et al., 2004; Rajesh and Sharma, 2002) as well as hypoglyce-
mic (Krishna Murthy et al., 2004) and antimutagenic properties
(Reid et al., 2006).
2. Results and discussion
D. metel leaves were extracted using MeOH and MeOH–H2O
(80:20) with a yield of 18%. The cytotoxicity of the MeOH–H2O
extract was assessed against human lung carcinoma (A549), colo-
rectal adenocarcinoma (DLD-1) and normal skin fibroblast (WS1)
cell lines. The results were obtained using a resazurin assay and
are expressed as the concentration inhibiting 50 percent of cell
growth (IC50) (data not shown). The MeOH–H2O extract was cyto-
toxic against A549, DLD-1 and WS1 cells with IC50 values of 11.9,
3.1 and 7.9 l
g mLꢀ1, respectively. A bioguided fractionation of
the MeOH–H2O extract was conducted in order to isolate and iden-
tify the compounds responsible for the activity. The MeOH–H2O
extract was successively partitioned with n-hexane, dichlorometh-
ane and EtOAc. The cytotoxicity of each fraction was again tested.
The results showed that the three partitions were cytotoxic against
cancer and normal cell lines with IC50 values ranging from 1.8 to
Cytotoxic activity studies of D. metel L. have established the
presence of several withanolide-type steroids which inhibit cell
11.9
The EtOAc partition (IC50 3.9–9.5
l
g mLꢀ1 (data not shown).
lg/mL) was selected for fur-
ther separation because the extraction yield was higher (13.2 g)
in comparison with the n-hexane (1.4 g) and the dichloromethane
partitions (1.1 g). The EtOAc partition was then fractionated on
⇑
Corresponding author. Tel.: +1 418 545 5011; fax: +1 418 545 5012.
E-mail address: jean_legault@uqac.ca (J. Legault).
0031-9422/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.07.009

2032
A. Bellila et al. / Phytochemistry 72 (2011) 2031–2036
Diaion HP-20 resulting in five new fractions named A, B, C, D and E
and all were tested for their biological activities. The results
showed that fraction B exerted highest cytotoxicity against A549
bond at dC 139.1 (C-5), 125.4 (C-6) and dH 5.52 (1H, m, H-6), a ter-
minal olefin at dH 6.09 and 6.67 (each 1H, s, H2-27) and three ter-
tiary methyl groups at dH 0.73 (3H, s, H3-18), 1.02 (3H, s, H3-19)
and 1.43 (3H, s, H3-28), almost identical to those of withametelin
(7) (Shingu et al., 1987; Oshima et al., 1987). The difference be-
tween compounds 1 and 7 was established by 1H–1H COSY cou-
pling of two oxymethine protons at dH 3.81 (1H, br s, H-1) and
4.05 (1H, tt, J = 4.8 and 11.1 Hz) with a methylene group at dH
1.83 (1H, m, H-2ax) and 2.13 (1H, m, H-2eq) suggesting the presence
of 1,3-dihydroxy groups. The latter was located in ring A from the
long-range correlations between H3-19 and dC 73.6 (C-1), 42.6
(C-9), 42.8 (C-10) and 139.1 (C-5). The sugar moiety, characterized
by COSY, was determined to be b-glucose as supported by the large
coupling constant of the anomeric proton at dH 4.37 (1H, d,
J = 7.8 Hz, H-10) (Agrawal, 1992). Furthermore, long-range correla-
tions between H-10 and C-3 and between H-3 and C-10 indicated
that the b-glucosyl unit was attached to C-3.
(IC50 = 5.3 l l
g mLꢀ1) and DLD-1 (IC50 = 2 g mLꢀ1) cells, compared
to fractions A, C, D and E. Fraction B (3.5 g) was separated by
repeated column chromatography to afford 9 fractions (B1–B9).
Fractions B1–B6 showed highest cytotoxicity with IC50 values
ranging from 0.5 to 5.5 l
g mLꢀ1 (data not shown). These bioactive
fractions were submitted to fractionation by combining column
chromatography and preparative reversed-phase HPLC to give five
pure compounds. Three known compounds daturametelin B (6),
withametelin (7) and daturametelin A (8), were identified by
NMR spectroscopic analysis and comparison with literature data
(Shingu et al., 1987). Unknown compounds 1 and 2 were identified
using NMR (Tables 1 and 2) and mass spectroscopy as described
below. The structures of compounds were presented in Fig. 1.
Compound 1 was obtained as a white amorphous solid with a
molecular formula of C34H50O10 as determined from its HRMS
pseudomolecular ion peak at m/z 641.3295 (calcd. 641.3302 for
[M+Na]+). IR absorption bands were observed at 3383, 2938,
1725, 1223 and 1066 cmꢀ1 suggesting the presence of an alcohol
The 1H and 13C NMR chemical shift of ring C and D and of the
lactonic side-chain are almost identical to withametelin (7)
(Oshima et al., 1987), also isolated in this work. This comparison
is important since the absolute stereochemistry of compound 7
was indirectly determined by a X-ray crystallographic analysis
(Shingu et al., 1990). Thus, the relative configuration of the side-
chain, which was difficult to establish from NOESY data, could be
deduced to be the same as the compound 7. Furthermore,
and an a
,b-unsaturated lactone. The 13C and DEPT-135 NMR spec-
tra of 1 showed peaks attributed to six quaternary, 14 methine, 11
methylene, and three methyl carbons. Detailed analysis of 1H and
HSQC spectra confirmed the presence of a trisubstituted double
Table 1
1H NMR spectroscopic data of compounds 1–5.
Position
1a
2a
3b
4b
5b
1
2a
3.81, br s
1.83, m
3.85, br s
1.75, m
7.12, d (7.6)
6.98, t (7.5)
5.82, ddd (1.0, 3.0, 9.9)
5.88, ddd (0.8, 2.9, 9.9)
2b
3
4a
4b
6a
6b
7a
7b
8
9
2.13, m
4.05, tt (4.8, 11.1)
2.31, m
2.47, m
5.52, m
2.09, m
3.99, tt (4.6, 11.5)
2.30, m
2.39, m
5.60, br d (5.4)
6.91, ddd (2.4, 4.9, 9.9)
2.89, dd (4.8, 21.3)
3.35, m
6.82, ddd (2.4, 4.9, 9.9)
2.86, dd (5.1, 21.5)
3.30, m
6.93, d (6.8)
5.63, br d (6.0)
5.59, br d (5.7)
2.62, m
2.77, m
1.38, m
2.00, m
1.36, m
2.27, m
1.51, m
2.34, m
1.57, m
2.09, m
1.35, m
1.31, m
1.82, m
1.40, m
1.85, m
1.87, m
0.75, s
1.61, m
1.97, m
1.50, m
1.74, m
1.47, m
1.57, m
1.42, m
1.96, m
1.19, m
1.19, m
1.72, m
1.42, m
1.83, m
1.81, m
0.73, s
1.02, s
1.63, m
2.02, m
1.48, m
2.00, m
1.81, m
2.46, dt (3.3, 14.2)
4.05, br s
1.62, m
2.01, m
1.51, m
1.65, m
1.47, m
1.53, m
1.45, m
1.95, m
1.17, m
1.18, m
1.71, m
1.40, m
1.75, m
1.77, m
0.69, s
1.64, m
2.00, m
1.46, m
2.03, m
1.80, m
2.42, dt (3.2, 14.5)
4.12, br s
11a
11b
12a
12b
14
15a
15b
16a
16b
17
18
19
20
21a
21b
22
23a
23b
27a
27b
28
10
1.72, m
1.24, m
1.75, m
1.46, m
1.79, m
2.15, m
0.82, s
1.25, s
1.77, m
1.22, m
1.72, m
1.43, m
1.72, m
2.18, m
0.76, s
1.22, s
1.85, m
3.85, dd (3.0, 12.6)
4.10, m
4.45, m
2.19, m
3.21, dd (16.9, 18.6)
4.35, d (12.1)
4.42, d (12.1)
2.03, br s
1.03, s
2.18, s
1.81, m
1.83, m
3.86, m
1.86, m
3.73, dd (3.0, 13.2)
3.91, br d (13.2)
4.66, br s
1.90, m
2.05, m
6.02, s
1.85, m
3.70, dd (1.8, 13.3)
3.97, d (13.3)
4.72, br s
1.97, m
2.16, m
6.10, s
3.67, dd (1.6, 13.2)
3.94, d (13.2)
4.70, br s
1.96, m
2.13, m
6.09, s
6.67, s
1.43, s
4.37, d (7.8)
3.14, dd (7.8, 9.1)
3.35, m
4.05, d (12.0)
4.51, dt (3.4, 13.0)
2.35, dd (3.2, 18.5)
3.06, dd (13.0, 18.5)
4.47, d (11.3)
4.64, d (11.3)
2.13, s
4.32, d (7.8)
3.16, dd (8.0, 8.9)
3.35, m
6.76, s
1.44, s
6.68, s
1.45, s
20
30
40
3.26, m
3.26, m
3.85, dd (1.4, 11.8)
3.64, m
3.28, m
3.27, m
3.67, dd (5.1, 11.6)
3.86, m
50
60
a
Spectra recorded in CD3OD.
Spectra recorded in CDCl3.
b

A. Bellila et al. / Phytochemistry 72 (2011) 2031–2036
2033
Table 2
Acid hydrolysis of compound 1 allowed isolation of (+)-
D
-glu-
13C NMR spectroscopic data of compounds 1–5a.
cose (as measured by optical polarimetry from the aqueous frac-
tion) and the rearranged aglycone 5 from the organic phase NMR
spectra of compound 5 showed the presence of an aromatic ring
which was located in ring A from the HMBC correlations between
dH 2.18 (3H, s, H3-19) and dC 137.1 (C-4), 135.9 (C-5) and 128.2 (C-
3) and between dH 7.12 (1H, d, J = 7.6 Hz, H-1) and dC 45.8 (C-9).
This result could be explained by acid-catalyzed dehydration of 1
followed by rearrangement of ring A. A similar rearrangement
Position
1a
2a
3b
4b
5b
1
2
3
4
5
6
7
8
9
73.6
206.5
128.4
148.2
34.4
137.4
125.7
31.7
34.5
38.6
51.3
31.3
74.6
47.4
49.1
24.7
26.9
39.7
13.4
19.3
46.0
59.8
79.3
33.7
161.0
123.5
168.6
63.6
20.8
104.0
75.1
78.1
71.6
78.1
62.8
72.9
204.6
127.8
145.9
33.4
135.5
124.7
30.3
33.1
36.7
49.7
31.3
73.5
45.8
47.7
23.5
25.9
37.8
13.1
18.9
44.7
59.4
78.2
32.7
154.5
125.2
167.2
57.5
20.0
124.0
126.3
128.2
137.1
135.9
28.1
29.0
39.6
45.8
141.2
28.1
41.2
44.2
56.5
24.8
27.7
49.0
13.0
19.9
41.6
61.7
77.4
34.0
70.9
140.9
167.6
130.6
25.7
37.7
74.9
39.1
139.1
125.4
32.9
33.3
42.6
42.8
21.4
40.8
44.0
57.6
25.3
27.6
48.9
12.9
20.0
41.6
61.6
77.5
33.9
70.9
140.9
167.6
130.6
25.7
102.7
75.2
78.1
71.8
78.0
63.0
38.3
66.4
41.4
137.4
125.3
31.7
31.9
41.5
41.6
20.3
39.4
42.8
56.1
24.1
26.6
47.5
12.6
19.4
39.8
60.5
75.6
33.2
69.4
138.9
165.3
130.0
25.6
was observed when
5a,6a-epoxy-3b-methanesulfonoxyandro-
stan-17-one was treated with HBr in acetic acid (Hanson and
Reese, 1983). To the best of our knowledge, aromatic compound
5 has never been described elsewhere. The native aglycone 3 was
finally obtained after enzymatic hydrolysis at 37 °C. From the
above data, the structure of 1 was identified as (20R,22R,24R)-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
10
21,24-epoxy-1
a,3b-dihydroxywitha-5,25(27)-dienolide-3-O-b-D-
glucopy ranoside.
Compound 2 was isolated as a white amorphous solid with a
molecular formula of C34H48O11 as determined from its HRESIMS
pseudomolecular ion at m/z 655.3091 (calcd. 655.3094 for
[M+Na]+). Strong bands were observed in IR spectrum at 3369,
2915, 1682, 1663, 1076 cmꢀ1 suggesting the presence of alcohols
and
a
,b-unsaturated ketones. The 1H and 13C NMR spectra were
found to be almost identical to those of daturametelin B (6), also
isolated during this work. Careful analysis of COSY, HSQC and
HMBC spectra allowed determination of an additional alcohol at
C-12. This was supported by HMBC correlation between H3-18
and C-12 at dC 74.6. The stereochemistry at position 12 was deter-
20
30
mined to be
hydrolysis of compound 2 afforded (+)-
a
since H-12 at dH 4.05 was a broad singlet. Acid
-glucose (as measured by
40
D
50
optical polarimetry from the aqueous fraction) and the aglycone
4, which has never been described in the literature. Furthermore,
long-range correlation between H-10 and C-26 and between H2-
27 and C-10 indicated that the b-glucosyl unit was attached to C-
27. Therefore, the structure of 2 was elucidated as (20R,22R,24R)-
60
a
Spectra recorded in CD3OD.
Spectra recorded in CDCl3.
b
12a,21,27-trihydroxy-1-oxowitha-2,5,24-trienolide-27-O-b-D-gluc
unambiguous NOESY correlations between H3-18 and H-20 and H-
21 confirmed the b-configuration of C-17. Stereochemistry in rings
A and B were linked to rings C and D through NOESY correlation of
H3-19 and H3-18 with H-8. The configurations of the alcohol
opyranoside.
The cytotoxicity of compounds 1, 2, 6–8 was evaluated against
A549, DLD-1 and WS1 cell lines and the results are displayed in Ta-
ble 3. Etoposide and camptothecin were used as positive controls
groups were determined to be 1a and 3b from the multiplicity of
with IC50 values respectively of 7 and 0.11
lM for A549 and 4
their associated proton signal (broad singlet for H-1 and triplet of
and 0.31 M for DLD-1. The results showed that the isolated wit-
l
triplet for H-3) which was further confirmed from NOESY correla-
hanolides were significantly more active against DLD-1 than
tion between H3-19 and H-1 and between H-3 and H-2
a
and H-4a.
A549 cells. However, no significant selectivity for cancer cells
28
26
O
O
OH
OR
25
27
21
22
OH
18
O
O
O
O
O
O
20H
17
H
H
OH19
O
H
H
H
9
1
H
H
H
H
H
H
3
RO
2
4
5
1
3
R = β-
D
-Glc
R = β-
D
-Glc
R = H
R = H
O
OH
OR
OR
O
O
O
O
O
O
H
H
H
O
O
O
H
H
H
H
H
H
H
H
H
6
7
8
R = β-
D
-Glc
R = β-D-Glc
Fig. 1. Structures of compounds 1–8.

2034
A. Bellila et al. / Phytochemistry 72 (2011) 2031–2036
Table 3
G0/G1
S
G2/M
Cytotoxic activities of compounds 1, 2 and 6–8 against A549, DLD-1 and WS1.
A 100
Compounds
IC50 (lM)
A549
DLD-1
17
2.0 0.3
0.6 0.2
0.7 0.1
WS1
22
1.3 0.2
1.0 0.1
1.0 0.2
80
60
40
20
0
Daturamalakoside A (1)
Daturamalakoside B (2)
Daturametelin B (6)
Withametelin (7)
Daturametelin A (8)
Etoposidea
92
7
4.2 0.4
3.5 0.2
2
1
3
2
52
7.4 0.4
0.11 0.01
3
24
4.0 0.9
0.31 0.01
2
24
21
3
3
Camptothecina
0.49 0.04
a
Control.
was observed in comparison with normal cells. Compounds 6 and 7
were found to be strongly cytotoxic against DLD-1 cells (IC50
0.6–0.7 M) and their activities were significantly higher than
Ctr
5
CPT 0.1 0.5
1
0.1 0.5
1
5
=
l
Compound 6
Compound 7
etoposide and compounds 1, 2, 8 and slightly lower than the cam-
ptothecin. The results showed that new withanolides 1 and known
compound 8 were moderately active against DLD-1 with IC50 val-
Viable
Apoptosis
Necrosis
B 100
ues of 17 and 24
pound 2 was also found to be strongly active against DLD-1
(IC50: 2 M) with a cytotoxicity higher than etoposide (IC50
M). Unfortunately, cytotoxicities of the aglycone of glycosides
lM, respectively. Interestingly, the new com-
l
:
80
4
l
1, 2, 6 and 8 were not assessed because isolated quantities were
not sufficient. The presence of a hydroxyl group on the carbon 21
of compound 6 increased significantly (about 40-fold) the cytotox-
icity against DLD-1 cells in comparison with compound 8. How-
ever, the presence of another hydroxyl group on the carbon 12 of
compound 2 reduced significantly (about 3-fold) the cytotoxicity
in comparison with compound 6.
60
40
20
0
As compounds 6 and 7 were strongly cytotoxic against DLD-1
cells, experiments were conducted to evaluate their mode of ac-
tion. Several studies have shown that withanolides extracted from
Whitania somnifera induce accumulation of cells in sub-G1 (Senthil
et al., 2007) or in G0/G1 after treatment of a new withanolide,
tubocapsenolide A, in MDA-MB-231 cells (Chen et al., 2008). There-
fore, the effect of compounds 6 and 7 was evaluated on the cell cy-
cle distribution using flow cytometry. Camptothecin used as
positive control induces a cell cycle arrest in S-phase and early
G2 (Del Bino et al., 1992). The results are presented in Fig. 2A. After
24 h, the viability of cells treated with both withanolides was
greater than 85% (data not shown). Surprisingly, the results show
after 24 h that, as camptothecin, both withanolides induce an
accumulation of DLD-1 cells in the S-phase (43–77% of cells) in
comparison with 33% for untreated cells. To our knowledge, this
is the first report of an induction of cell cycle arrest in the S-phase
by withanolides. A possible explanation is that withanolides pos-
sess a lactone ring which could inhibit topoisomerase I activity
as camptothecin (Ulukan and Swaan, 2002). However, this
assumption will be verified. On the other hand, some withanolides
have been shown to induce apoptosis by activating caspases (Sen-
5
5
Ctr
0.1 0.5
1
0.1 0.5
1
CPT
Compound 6
Compound 7
Fig. 2. Withanolide effects on cell cycle after 24 h (A) and on the induction of
apoptosis or necrosis after 48 h (B) in DLD-1 cells by flow cytometry analysis.
Camptothecin (CPT) 1 lM was used as positive control.
3. Concluding remarks
In conclusion, eight withanolides were isolated from leaves ofD.
metel L. including two new structures (1 and 2). Two compounds (6
and 7) were found to be strongly active against colorectal cancer
cell line, DLD-1. In contrast to others withanolides previously re-
ported in the literature, compounds 6 and 7 blocked the cell in S-
phase as camptothecin, a topoisomerase I inhibitor.
4. Experimental
thil et al., 2007) or potentiate apoptosis by inhibiting NF-jB activa-
tion (Ichikawa et al., 2006). Moreover, S-phase blockage induced by
camptothecin results in apoptosis (Del Bino et al., 1992). Therefore,
the effect of compounds 6 and 7 on the induction of apoptosis in
DLD-1 cells was assessed using flow cytometry. After 48 h, over
90% of cells treated with compounds 6 and 7 were not viable.
The results presented in Fig. 2B shows that both compounds induce
4.1. General experimental procedures
Reversed phase-high performance liquid chromatography anal-
yses were carried out using an analytical Agilent 1100 series with
diode array detection and equipped with an Inertsil ODS-C18 col-
umn (4.6 mm ꢁ 250 mm,
5 lm) at room temperature, with
preferentially apoptosis at a low dose of 0.1
pound 6) and 67% (compound 7) of cells in sub-G1. In contrast,
at a high dose of 5 M, compounds 6 and 7 induce necrosis with
lM with 93% (com-
MeOH–H2O (85:15) used as mobile phase. The preparative HPLC
instrument used in this study was a preparative Agilent 1100 HPLC
Series. Separation was carried out on this system with an Inerstil
l
a percentage of cells larger than 80%. Altogether, these results
showed that withanolides 6 and 7 induce DLD-1 cell blockage in
the S-phase and promoted programmed cell death at low dose
and necrosis at high dose.
Prep ODS column (20 mm ꢁ 250 mm, 10
lm). MeOH–H2O
(85:15) was used as a mobile phase with flow rate 2 mL minꢀ1
.
Analytical thin-layer chromatography (TLC) was performed with
silica gel 60 F254, 0.25 mm pre-coated TLC plates (Silicycle, Québec,

A. Bellila et al. / Phytochemistry 72 (2011) 2031–2036
2035
Canada) using CHCl3/MeOH/H2O as the mobile phase. Compounds
were visualized under visible light after spraying the plates with
20% H2SO4 solution in EtOH followed by 1% vanillin in EtOH and
heated at 110 °C for 5 min. The 230–400 mesh silica gel (Silicycle,
Québec, Canada) was used for column chromatography (CC).
Nuclear magnetic resonance (NMR) spectra were recorded on a
Bruker Avance spectrometer at 400 MHz (1H) and 100 MHz (13C),
equipped with a 5 mm QNP probe. Elucidations of chemical struc-
tures were based on analysis of 1H, 13C, COSY, TOCSY, HMBC, HSQC
and DEPT-135 experiments. Signals are reported as m (multiplet), s
(singlet), d (doublet), t (triplet), dd (doublet of doublet), dt (doublet
of triplet), ddd (doublet of doublet of doublet), br s (broad singlet)
and coupling constants are reported in hertz (Hz). The chemical
shifts are reported in ppm (d) relative to tetramethylsilane (TMS).
The exchangeable OH NMR signals appearing sometimes were
not listed. Optical rotations were obtained using the sodium D line
at ambient temperature on a Rudolph Research Analytical Autopol
IV automatic polarimeter. Accurate mass measurements (HRMS)
were performed on an LC-MSD-TOF instrument from Agilent Tech-
nologies in positive electrospray mode. FTIR spectra were obtained
using KBr disks on a Perkin–Elmer Spectrum One Instrument.
Chemical reagents were purchased from Sigma–Aldrich Co. Canada
or Alfa Aesar Co. and were used as received. The solvents were ob-
tained from VWR International Co. and were used as received.
isolation was completed by preparative reversed-phase HPLC.
New compounds 1 (26.2 mg) and 2 (35.3 mg) were isolated from
B4. Daturametelin B (6) (100.6 mg) was isolated from B1 and B2
and daturametelin D (7) (50.9 mg) was isolated from B3. Datura-
metelin A (8) (16.8 mg) was isolated from B5. All known com-
pounds were identified by comparing spectroscopic data with
reported values.
4.4.1. Daturamalakoside A (20R,22R,24R)-21,24-epoxy-1
a
,3b-
dihydroxywitha 5,25(27)-dienolide-3-O-b-
D
-glucopyranoside (1)
White amorphous solid. ½a D25
ꢂ
ꢀ35.96 (c = 1, MeOH). IR mmax
:
3383, 2938, 2906, 2868, 1725, 1382, 1223, 1066, 1016 cmꢀ1; HRE-
SIMS m/z: 641.3295. Calcd. for C34H50O10Na: 641.3302. For 1H and
13C NMR spectroscopic data (see Tables 2 and 3).
4.4.2. Daturamalakoside B (20R,22R,24R)-12
a,21,27-trihydroxy-1-oxo
witha-2,5,24-trienolide-27-O-b-
D
-glucopyranoside (2)
Light yellow amorphous solid. ½a D25
ꢂ
+11.90 (c = 1, MeOH). IR
m
max: 3369, 2915, 1682, 1663, 1398, 1076, 1038 cmꢀ1; HRESIMS
m/z: 655.3092. Calcd. for C34H48O11Na: 655.3094. For 1H and 13C
NMR spectroscopic data (see Tables 2 and 3).
4.4.3. Daturamalakin A (20R,22R,24R)-21,24-epoxy-1
a
,3bꢀdihydroxy
witha-5,25(27)-dienolide (3)
White amorphous solid. ½a D25
ꢂ
ꢀ39.9 (c = 0.4, CHCl3). IR mmax
:
4.2. Plant material
3400, 2917, 1718, 1565, 1223, 756 cmꢀ1
; HRESIMS m/z:
457.2952. Calcd. for C28H40O5: 457.2954. For 1H and 13C NMR spec-
troscopic data (see Tables 2 and 3).
D. metel L. was collected in August 2008 from its natural habitat
in south Tunisia. Leaves were air-dried in darkness at ambient tem-
perature and ground to a fine powder with an electrical mortar.
Botanical identification of this species was carried out by Prof. A.
Smaoui (Biotechnologic Center in Borj-Cedria Technopark, Tunisia),
according to the Tunisian flora (Pottier-Alapetite, 1979). A voucher
specimen was deposited in the Aromatic and Medicinal Plants Unit,
Biotechnological Center in Borj-Cedria Techno-park, Hammam-Lif,
Tunisia (D.m.08003).
4.4.4. Daturamalakin B (20R,22R,24R)-12
a
,21,27-trihydroxy-1-oxow
itha-2,5,24-trienolide (4)
Light yellow amorphous solid. ½a D25
ꢂ
+18.1 (c = 0.4, CHCl3). IR
m
max: 3386, 2922, 1663, 1381, 1078, 754 cmꢀ1; HRESIMS m/z:
493.2564. Calcd. for C28H38O6Na: 493.2566. For 1H and 13C NMR
spectroscopic data (see Tables 2 and 3).
4.3. Extraction and isolation
4.4.5. Phenowithanolide (5)
White amorphous solid. ½a D25
ꢂ
ꢀ3.61 (c = 0.5, CHCl3). IR mmax
:
Dried and powdered leaves (0.5 kg) of the plant were extracted
with MeOH (3 ꢁ 3 L) followed by MeOH–H2O (80:20) (2 ꢁ 2 L), un-
der reflux for 8 h. A sample of 10 mL of the solution of MeOH–H2O
extract was evaporated to dryness to assess the extraction yield to
18% (w,w). The organic phase of the MeOH–H2O solution was
evaporated. The aqueous residue was treated with n-hexane
(3 ꢁ 500 mL) and with CH2Cl2 (3 ꢁ 500 mL) to remove chlorophyll
and other lipophilic constituents. The steroids were finally ex-
tracted from the aqueous residue with EtOAc (7 ꢁ 500 mL). The
EtOAc phase was concentrated to produce a brown powder
(13.2 g).
2938, 2868, 1720, 1381, 1220, 753, 666 cmꢀ1; HRESIMS m/z:
420.2731. Calcd. for C28H36O3: 420.2737. The 1H and 13C NMR
spectroscopic data (see Tables 2 and 3).
4.5. Acid hydrolysis
In screwed cap vials, 10% of HCl (10 mL) was individually added
to compounds 1 and 2 (10 mg each). The resulting solutions heated
at 100 °C for 4 h. The aglycones were extracted with CHCl3
(3 ꢁ 10 mL), and the aqueous phases containing sugars were neu-
tralized with 10% N-methyl-N,N-di-n-octylamine. Monosaccha-
rides were analyzed by TLC with authentic samples using
CH2Cl2–MeOH–H2O (50:25:5), spots were visualized using naph-
thoresorcinol solution in EtOH.
4.4. Bioguided fractionation
The EtOAc fraction was subjected to Diaion HP-20 (Supelco,
USA) CC eluting with H2O–EtOH under increasing percentages of
EtOH (from 30% to 100%, v,v) to give five fractions: A (0.82 g), B
(3.51 g); C (2.90 g); D (0.26 g); E (0.25 g). Cytotoxicity of all frac-
tions was tested against two cancer cell lines and one normal cell
line. The most active fraction B was subjected to open on silica gel
CC, eluted with a solvent mixture in various proportions of CHCl3–
MeOH–H2O (55:6:0.5); (50:7:0.5); (50:10:0.5) (50:12:0.5);
(50:15:0.5) and finally eluted with MeOH, affording 9 fractions
(B1–B9). The B1–B6 fractions showed greatest in vitro cytotoxicity
against both cancer cell lines. These fractions were further applied
4.6. Enzymatic hydrolysis
Compounds 1 and 2 (20 mg) were dissolved in 50 mL acetate
buffer (10 g sodium acetate anhydrous, 1.6 mL glacial acetic acid,
500 mL H2O and pH 5) and 200 mg of b-glycosidase from almonds
(7–8 IU mgꢀ1) was added. The mixture was incubated at 40 °C for
22 h. After the hydrolysis, the reaction mixture was extracted with
CHCl3 (3 ꢁ 50 mL) (Moreau and Hicks, 2004). The residue (agly-
cone), after evaporating and drying, was analyzed by NMR
spectroscopy.
to
a series of silica gel CC eluting with CHCl3–MeOH–H2O
(50:10:0.5) and with CHCl3–MeOH (80:1), (50:1), (25:1). Finally,

2036
A. Bellila et al. / Phytochemistry 72 (2011) 2031–2036
Chen, W.Y., Chang, F.R., Huang, Z.Y., Chen, J.H., Wu, Y.C., Wu, C.C., 2008.
4.7. Cell culture
Tubocapsenolide A, a novel withanolide, inhibits proliferation and induces
apoptosis in MDA-MB-231 cells by thiol oxidation of heat shock proteins. J. Biol.
Chem. 25, 17184–17193.
The human lung carcinoma A549 (#CCL-185), colorectal adeno-
carcinoma DLD-1 (#CCl-221) and skin fibroblast WS1 (#CRL-1502)
cell lines were obtained from the American Type Culture Collection
(ATCC, Manassas, USA). Cells lines were grown in minimum essen-
tial medium containing Earle’s salts (Mediatech CellgroÒ, Herndon,
USA), supplemented with 10% fetal calf serum (Hyclone, Logan,
USA), 1ꢁ solution of vitamins, 1ꢁ sodium pyruvate, 1ꢁ non-
Dabur, R., Singh, H., Chhillar, A.K., Ali, M., Sharma, G.L., 2004. Antifungal potential of
Indian medicinal plants. Fitoterapia 75, 389–391.
Del Bino, G., Bruno, S., Yi, P.N., Darzynkiewicz, Z., 1992. Apoptotic cell death
triggered by camptothecin or teniposide. The cell cycle specificity and effects of
ionizing radiation. Cell Prolif. 25, 537–548.
Eftekhar, F., Yousefzadi, M., Tafakori, V., 2005. Antimicrobial activity of Datura
innoxia and Datura stramonium. Fitoterapia 76, 118–120.
Hanson, J.R., Reese, P.B., 1983. A deuterium NMR study of 4-methyloestratriene
formation. Tetrahedron 24, 3405–3408.
essential amino acids, 100 IU of penicillin and 100 l
g mLꢀ1 of
streptomycin (Mediatech CellgroÒ). Cells were cultured at 37 °C
Ichikawa, H., Takada, Y., Shishodia, S., Jayaprakasam, B., Nair, M.G., Aggarwal, B.B.,
2006. Withanolides potentiate apoptosis, inhibit invasion, and abolish
in a humidified atmosphere containing 5% CO2.
osteoclastogenesis through suppression of nuclear factor-
jB
(NF-
jB)
activation and NF-
1445.
jB-regulated gene expression. Mol. Cancer Ther. 5, 1434–
4.8. Cytotoxicity assay
Kagale, S., Marimuthu, T., Thayumanvan, B., Nandakumar, R., Samiyappan, R., 2004.
Antimicrobial activity and induction of systemic resistance in rice by leaf
extract of Datura metel against Rhizoctonia solani and Xanthomonas oryzae pv
Oryzae. Physiol. Mol. Plant Pathol. 65, 91–100.
Krishna Murthy, B., Nammi, S., Kota, M.K., Krishna Rao, R.V., Koteswara Rao, N.,
Annapurna, A., 2004. Evaluation of hypoglycemic and antihyperglycemic effects
of Datura metel (Linn.) seeds in normal and alloxan-induced diabetic rats. J.
Ethnopharmacol. 91, 95–98.
Le Floc’h, E., 1983. Contribution à une étude ethnobotanique de la flore tunisienne.
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Health. John Wiley and Sons, New York, pp. 90–93.
Ma, L., Xie, C.M., Li, J., Lou, F.C., Hu, L.H., 2006. Daturametelins H, I, and J: three new
withanolide glycosides from Datura metel L. Chem. Biodiv. 3, 180–186.
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and scopolamine in Datura stramonium. Fitoterapia 72, 644–648.
Moreau, R.A., Hicks, K.B., 2004. The in vitro rates of hydrolysis of phytosterol
conjugates and other plant lipids by mammalian digestive enzymes, compared
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O’Brien, J., Wilson, I., Orton, T., Pognan, F., 2000. Investigation of the Alamar Blue
(resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity.
Eur. J. Biochem. 267, 5421–5426.
Exponentially growing cells were plated at a density of 5 ꢁ 103
cells per well in 96-well microplates (BD Falcon) in culture med-
ium (100
ment. Then, cells were incubated for 48 h in the presence or
absence of 100 L of increasing concentrations of extract, fraction
lL) and were allowed to adhere for 16 h before treat-
l
or pure compounds dissolved in culture medium and DMSO. The
final concentration of DMSO in the culture medium was main-
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using the resazurin reduction test (O’Brien et al., 2000). Fluores-
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Fl™ plate reader (Labsystems) using an excitation wavelength of
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4.9. Flow cytometry
Oshima, Y., Bagchi, A., Hikino, H., Sinha, S.C., Sahai, M., Ray, B., 1987. Withametelin,
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plated in petri dishes and were allowed to adhere for 24 h. After 24
or 48 h of treatment with selected concentration of products, cell
2027.
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pellet resuspended in 50
um iodide (20
g mLꢀ1). After 15 min of incubation on ice, cells
were fixed in 25% ethanol in PBS. After addition of Hoechst reagent
(112
g mLꢀ1), cells were analyzed using an EPICS ELITE ESP
lL PBS was supplemented with propidi-
l
l
(Beckman-Coulter, Québec, Canada) flow cytometer. Cell cycle
analysis was performed with the MulticycleAV software (Phoenix
flow system, San Diego, CA).
Rajesh, G., Sharma, L., 2002. Studies on antimycotic properties of Datura metel. J.
Ethnopharmacol. 80, 193–197.
Reid, K.A., Maes, J., Maes, A., Van Staden, J., De Kimpe, N., Mulholland, D.A.,
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South African plants. J. Ethnopharmacol. 106, 44–50.
Acknowledgements
Senthil, V., Ramadevi, S., Venkatakrishnan, V., Giridharan, P., Lakshmi, B.S.,
Vishwakarma, R.A., Balakrishnan, A., 2007. Withanolide induces apoptosis in
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The authors wish to kindly thank C. Dussault for its technical
assistance. This study was supported by the Tunisian Ministry for
high education, research and technology and by the ‘‘Chaire de
recherche sur les agents anticancéreux d’origine naturelle’’ of the
Université du Québec à Chicoutimi, Canada.
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