Withanolides from Withania aristata and their cytotoxic activity

Article abstract of DOI:10.1016/j.steroids.2010.06.001

Seven new withanolides (1-7), along with three known ones (8-10), were isolated from the leaves of Withania aristata. Their structures were elucidated on the basis of spectroscopic analysis, including 2D NMR experiments and spectrometric techniques, and the absolute configuration of 1 and 2 was established by CD analysis. In the search for new cytotoxic compounds from Withania species, the isolated compounds 1-9, along with two derivatives, were assayed for their cytotoxicity against HeLa, MCF-7 and A-549 human tumor cell lines. Derivative (4S,20R,22R)-27-acetoxy-4-p-bromobenzoyloxy-1-oxo-witha-2,5, 16,24-tetraenolide (13) showed cytotoxicity against all the cell lines assayed with IC₅₀ values ranging from 2.8 to 3.6 μM, and (4S,20R,22R)-4,27-diacetoxy-4-hydroxy-1-oxo-witha-2,5,16,24-tetraenolide (12) exhibited an IC₅₀ value of 5.4 μM on the MCF-7 cell line.

Full text of DOI:10.1016/j.steroids.2010.06.001

Steroids 75 (2010) 974–981
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Withanolides from Withania aristata and their cytotoxic activity
Gabriel G. LLanosa, Liliana M. Araujob, Ignacio A. Jiméneza, Laila M. Moujirb,
Jesús T. Vázqueza, Isabel L. Bazzocchia,∗
a Instituto Universitario de Bio-Orgánica “Antonio González”, Departamento de Química Orgánica, Universidad de La Laguna,
and Instituto Canario de Investigación del Cáncer, Avda. Astrofísico Francisco Sánchez 2, 38206 La Laguna, Tenerife, Spain
b Departamento de Microbiología y Biología Celular, Universidad de La Laguna, Avenida Astrofísico Francisco Sánchez s/n, 38206 La Laguna, Tenerife, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Seven new withanolides (1–7), along with three known ones (8–10), were isolated from the leaves
of Withania aristata. Their structures were elucidated on the basis of spectroscopic analysis, including
2D NMR experiments and spectrometric techniques, and the absolute configuration of 1 and 2 was
established by CD analysis. In the search for new cytotoxic compounds from Withania species, the iso-
lated compounds 1–9, along with two derivatives, were assayed for their cytotoxicity against HeLa,
MCF-7 and A-549 human tumor cell lines. Derivative (4S,20R,22R)-27-acetoxy-4-p-bromobenzoyloxy-
1-oxo-witha-2,5,16,24-tetraenolide (13) showed cytotoxicity against all the cell lines assayed with IC50
values ranging from 2.8 to 3.6 ␮M, and (4S,20R,22R)-4,27-diacetoxy-4-hydroxy-1-oxo-witha-2,5,16,24-
tetraenolide (12) exhibited an IC50 value of 5.4 ␮M on the MCF-7 cell line.
Received 23 April 2010
Received in revised form 28 May 2010
Accepted 1 June 2010
Available online 11 June 2010
Keywords:
Withania aristata
Withanolides
Absolute configuration
Circular dichroism
Cytotoxicity
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
reports in the literature of phytochemical studies on W. aristata are
of the isolation of six withanolides [12–14], and of their cytotoxic
Withania is a small genus of shrubs belonging to the Solanaceaea
family, which are distributed in the East of the Mediterranean area,
Macaronesian region and extend to south Asia [1], and some species
are well known in traditional medicine. In particular, Withania som-
nifera (L.) Dunal, commonly known as “aswagandha”, is one of the
major ingredients of ayurvedic preparations prescribed for pos-
sessing several properties, including antiinflammatory, antitumor,
and antioxidant, and also has been used to treat ulcers, bacte-
rial infections and senile dementia [2]. The therapeutic potential
of Withania species has been attributed to the presence of with-
anolides [3], which are steroidal lactones built on an ergostane
skeleton of 28 carbons functionalized at carbons 1, 22 and 26. In
particular, withaferin A suppresses inflammation [4], and exerts an
immunopotentiating effect [5], in addition to its anti-tumorigenesis
activity, inducing apoptosis [6] in cancer cells and inhibiting angio-
genesis [7]. More recently, it has been reported as a treatment for
central nervous system disorders [8].
[15] and diuretic [14] activities, along with other constituents [16].
As part of an ongoing phytochemical investigation into endemic
species of the Canary Islands, we report herein on the isolation
of seven new withanolides (1–7) from the leaves of W. aristata.
Their structures were determined on the basis of spectrometric and
spectroscopic data by application of 1D and 2D NMR techniques,
including COSY, HSQC, HMBC, and ROESY experiments. The abso-
lute configuration of 1 and 2 was established by analysis of their
CD curves and thus of the p-bromobenzoyl derivative of 1 (13). In
addition, three known withanolides were isolated and identified
as 4␤,17␣,27-trihydroxy-1-oxo-witha-2,5,24-trienolide (8) [17],
4␤,27-dihydroxy-1-oxo-witha-2,5,24-trienolide (9) [18] and 4␤-
hydroxy-1-oxo-witha-2,5,24-trienolide (10) [19] by comparison of
their spectral data with those reported in the literature. Isolated
compounds 1–9 and derivatives 12 and 13 were assayed for their
cytotoxicity against HeLa (carcinoma of the cervix), A-549 (lung
carcinoma), and MCF-7 (breast adenocarcinoma) human cell lines.
The evaluated derivatives 12 and 13 exhibited the highest potency,
followed by compounds 1–4 that showed only weak cytotoxicity.
In the Canary Islands, the genus Withania is represented by three
species: W. somnifera (L.) Dunal, Withania frutescens (L.) Pauqui
and Withania aristata (Aiton) Pauqui. W. aristata, the only endemic
species [9], is widely used in folk medicine as antitumoral, anti-
spasmodic, antirheumatic, for eye and otitis problems, as well as
for insomnia [10] and urinary pathologies [11]. However, the only
2. Experimental
2.1. General methods
∗
Optical rotations were measured on a Perkin Elmer 241 auto-
Corresponding author. Tel.: +34 922 318576; fax: +34 922 318571.
E-mail address: ilopez@ull.es (I.L. Bazzocchi).
matic polarimeter in CHCl3 at 20 ◦C and the [␣D] are given in
0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.06.001

G.G. LLanos et al. / Steroids 75 (2010) 974–981
975
Table 1
1H (400 MHz), and 13C (100 MHz) NMR data (ı, CDCl3, J values in Hz in parentheses) of 1–4.
No.
1
2
3
4
a
a
a
a
ıH
ıC
ıH
ıC
ıH
ıC
ıH
ıC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
203.5 s
128.7 d
6.76 dd (4.4, 10.1) 143.0 d
203.2 s
128.9 d
142.5 d
69.3 d
138.8 s
130.9 d
31.1 t
30.8 d
42.5 d
49.3 s
22.6 t
34.3 t
46.6 s
57.0 d
30.5 t
124.0 d
155.5 s
16.2 q
22.5 q
35.7 d
16.4 q
78.4 d
32.3 t
148.7 s
121.8 s
166.7 s
12.3 q
20.2 q
201.9 s
140.0 d
138.8 d
187.7 s
139.5 s
137.5 d
30.5 t
30.0 d
42.9 d
51.3 s
22.0 t
31.0 t
46.8 s
56.7 d
34.2 t
124.3 d
155.2 s
16.2 q
23.4 q
35.7 d
16.5 q
78.8 d
32.6 t
152.3 s
125.5 s
166.5 s
57.3 t
203.1 s
128.8 d
142.7 d
69.1 d
138.8 s
130.4 d
30.7 t
31.2 d
42.5 d
49.1 s
22.9 t
5.91 d (10.1)
5.95 d (10.0)
6.77 dd (4.5, 10.0)
4.64 d (4.5)
6.67 d (10.3)
6.73 d (10.3)
5.96 d (10.1)
6.78 dd (4.5, 10.1)
4.64 d (4.5)
4.61 d (4.4)
69.0 d
138.6 s
130.7 d
31.1 t
31.0 d
43.2 d
49.3 s
22.5 t
5.90 br s
1.43b, 2.08 m
1.89 m
5.92 br s
1.68, 2.01 m
1.91 m
6.86 dd (2.2, 10.3)
1.86, 2.32 m
1.72 m
5.95 br s
1.72, 2.13 m
1.66 m
1.64 m
1.65 m
1.87 m
1.65b
m
1.57, 2.21 m
1.50, 1.71m
1.44, 2.23 m
1.52, 1.73 m
1.56, 2.34 m
1.96, 2.15 m
1.67, 2.30 m
1.71, 2.34 m
34.3 t
36.8 t
46.6 s
56.9 d
30.5 t
44.4 s
52.5 d
36.0 t
1.43b
m
1.43 m
1.56, 2.10 m
5.53 br s
1.51 m
1.57, 1.78 m
5.55 br s
1.65b
1.65b
m
m
2.13 m
5.51 br s
124.3 d
155.2 s
16.2 q
22.5 q
35.6 d
16.5 q
78.8 d
32.6 t
4.72 br s
71.5 d
151.3 s
16.3 q
22.5 q
129.2 s
12.0 q
77.8 d
34.6 t
153.7 s
125.0 s
166.8 s
57.2 t
0.81 s
1.44 s
2.52 m
0.85 s
1.48 s
2.53 m
1.11 d (7.0)
4.39 m
0.85 s
1.42 s
0.93 s
1.46 s
2.56b
m
1.10 d (6.9)
4.43 m
1.13 d (7.1)
4.45 m
1.84 s
5.41 dd (3.5, 12.8)
2.26, 2.73 m
2.15, 2.52b
m
2.09, 2.47 m
2.19, 2.56b
m
152.9 s
125.3 s
166.7 s
4.31, 4.36 dAB (12.8) 57.0 t
2.02 s 19.7 q
1.88 s
1.93 s
4.37, 4.42 dAB (11.8)
2.03 s
4.38, 4.43 dAB (12.4)
2.03 s
19.7 q
19.6 q
a
Data are based on DEPT and HSQC experiments.
Overlapping signals.
b
10−1 deg cm2 g−1. UV spectra were obtained on a JASCO V-560
spectrophotometer, and CD spectra on a JASCO J-600 spectropo-
larimeter. IR (film) spectra were measured on a Bruker IFS 55
spectrophotometer. NMR experiments were performed on a Bruker
Avance 400 spectrometer and chemical shifts are shown in ı
(ppm) with tetramethylsilane (TMS) as internal reference. EIMS
and HREIMS were recorded on a Micromass Autospec spectrom-
eter, and ESIMS and HRESIMS (positive mode) were measured on
a LCT Premier XE Micromass Electrospray spectrometer. Silica gel
60 (15–40) ␮M for column chromatography, and silica gel 60 F254
for preparative thin-layer chromatography plates were purchased
from Macherey-Nagel, and Sephadex LH-20 for exclusion chro-
matography was obtained from Pharmacia Biotech.
ity). Preparative thin-layer chromatography developed with
CH2Cl2/acetone (8.5:1.5) was used to purify the new compounds
1 (79.0 mg), 2 (15.0 mg), 3 (8.3 mg), 4 (30.3 mg), 5 (2.3 mg) 6
(2.4 mg) and 7 (3.5 mg), in addition to the known compounds
4␤,17␣,27-trihydroxy-1-oxo-witha-2,5,24-trienolide (8, 9.0 mg),
4␤,27-dihydroxy-1-oxo-witha-2,5,24-trienolide (9, 9.0 mg) and
4␤-hydroxy-1-oxo-witha-2,5,24-trienolide (10, 1.2 mg).
2.3.1. (4S,20S,22R)-4,27-Dihydroxy-1-oxo-witha-2,5,16,24-
tetraenolide (1)
White amorphous solid; [␣]D20 = +70.4◦ (c = 0.50, CHCl3); CD
(MeOH): ꢀext 337 (ꢁε = −0.6), 240 (ꢁε = +2.0) nm; UV (EtOH)
(log ε): ꢀmax 337 (2.1), 235 (3.9) nm; IR (film): ꢂmax 3429, 2970,
2928, 2853, 1689, 1455, 1393, 1013, 755 cm−1 1H and 13C NMR:
;
data are shown in Table 1; EIMS m/z (%): 452 [M]+ (4), 434 (53), 419
(22), 401 (7), 380 (7), 312 (14), 283 (17), 265 (13), 171 (23), 141
(100), 123 (69), 95 (57), 69 (85); HREIMS m/z: 452.2578 (calcd. for
2.2. Plant material
Leaves of W. aristata were collected in Icod de los Vinos, Tener-
ife, Canary Islands (Spain), in May 2005. A voucher specimen
(TFC 48.068) is deposited in the Herbarium of the Department of
Botany, University of La Laguna, Tenerife, and identified by Leticia
Rodríguez-Navarro.
C28H36O5: 452.2563).
2.3.2. (4S,20S,22R)-4-Hydroxy-1-oxo-witha-2,5,16,24-
tetraenolide (2)
White amorphous solid; [␣]D20 = +40.4◦ (c = 1.20, CHCl3); CD
(MeOH): ꢀext 339 (ꢁε = −0.9), 244 (ꢁε = +2.6); UV (EtOH) (log ε):
ꢀmax 335 (2.2), 238 (3.8) nm; IR (film): ꢂmax 3430, 2927, 2856, 1690,
2.3. Extraction and isolation
1455, 1381, 1242, 1212, 1131, 1014, 757 cm−1 1H and 13C NMR:
;
The air-dried powdered leaves of W. aristata (1.65 kg) were
exhaustively extracted with CH2Cl2 in a Soxhlet apparatus and
the solvent was evaporated at reduced pressure. The residue
(71 g) was fractioned by vacuum-liquid chromatography on silica
gel and eluted with hexane/EtOAc mixtures of increasing polar-
ity (from 100:0 to 0:100) affording nine fractions, four of them
(VI, VII, VIII, and IX) containing withanolides by previous 1H
NMR analysis. Each of these fractions was subjected to column
chromatography over Sephadex LH-20 (n-hexane/CHCl3/MeOH,
2:1:1), and silica gel (CH2Cl2/acetone of increasing polar-
data are shown in Table 1; EIMS m/z (%): 436 [M]+ (3), 418 (9), 403
(8), 345 (1), 293 (9), 265 (7), 171 (11), 125 (100), 97 (20); HREIMS
m/z: 436.2643 (calcd. for C28H36O4: 436.2614).
2.3.3. (20S,22R)-27-Hydroxy-1,4-dioxo-witha-2,5,16,24-
tetraenolide (3)
White amorphous solid; [␣]D20 = +26.6 (c = 0.83, CHCl3); UV
(EtOH) (log ε): ꢀmax 218 (4.3) nm; IR (film): ꢂmax 3446, 2927, 1694,
1625, 1458, 1393, 1271, 1129, 1022, 755 cm−1 1H and 13C NMR:
;

976
G.G. LLanos et al. / Steroids 75 (2010) 974–981
Table 2
1H (400 MHz), and 13C (100 MHz) NMR data (ı, CDCl3, J values in Hz in parentheses) of 5–8.
No.
5
6
7
8
a
a
a
a
ıH
ıC
ıH
ıC
ıH
ıC
ıH
ıC
1
2
3
4
204.2 s
127.9 d
145.2 d
33.4 t
202.1 s
140.2 d
139.0 d
187.8 s
203.7 s
127.6 d
147.5 d
67.0 d
203.2 s
128.8 d
142.7 d
69.1 d
5.88 dt (2.6, 10.0)
6.78 dd (2.4, 5.0, 10.0)
2.86 dd (5.0, 21.0)
3.32 dd (2.4, 21.0)
5.90 dd (2.4, 10.2)
6.79 dd (2.1, 10.2)
5.05 br s
5.95 d (10.0)
6.77 dd (4.4, 10.0)
4.64 d (4.6)
6.67 d (10.3)
6.73 d (10.3)
5
6
7
8
9
136.0 s
124.4 d
30.5 t
31.8 d
42.8 d
50.4 s
23.5 t
37.0 t
44.5 s
52.7 d
36.1 t
71.7 d
151.6 s
16.4 q
18.8 q
129.3 s
12.1 q
77.9 d
139.6 s
137.4 d
30.7 t
140.2 s
121.2 d
30.1 t
32.7 d
42.8 d
50.5 s
22.7 t
31.9 t
47.7 s
50.2 d
23.4 t
36.2 t
84.8 s
14.7 c
19.5 c
42.4 d
9.1 c
138.6 s
130.7 d
30.8 t
5.58 d (8.0)
1.65, 2.00 m
6.87 dd (2.0, 5.7)
6.05 d (6.1)
1.65, 2.13 m
1.68 m
5.93 s
1.68, 2.13 m
1.56 m
1.85,2.32b
1.62 m
m
1.70b
1.72b
m
m
30.1 d
42.7 d
51.5 s
23.5 t
37.1 t
44.9 s
52.6 d
36.0 t
71.7 d
151.4 s
16.6 q
23.7 q
129.5 s
12.1 q
77.8 d
32.7 d
42.2 d
49.0 s
1.87 m
1.66 m
1.59 m
10
11
12
13
14
15
16
17
18
19
20
21
22
1.72b, 2.37m
1.70b, 2.33m
1.67, 2.31 m
1.69, 2.32b
1.57, 2.31 m
1.62, 1.72 m
1.33, 1.60 m
1.61, 1.70 m
22.6b
t
m
31.8 t
47.6 s
50.2 d
23.5 t
36.1 t
84.9 s
14.7 c
1.64 m
1.85, 2.64 m
4.72 br s
1.69b
m
1.69 m
1.27, 1.76 m
1.70, 2.00 m
1.69b
m
1.75, 2.36 m
4.76 br s
1.25, 1.75 m
1.69b, 1.99 m
0.96 s
1.27 s
0.97 s
1.42 s
0.87 s
1.29 s
2.37 m
1.07 d (7.0)
4.70 dt (2.6, 8.8)
0.84 s
1.45 s
2.34 m
1.05 d (7.0)
4.68 dt (2.8, 8.1)
22.6b
42.4 d
9.1 c
c
1.88 s
5.41 dd (3.6, 12.9)
1.88 s
5.40 dd (3.5, 13.0)
79.0 d
79.0 d
23
24
25
26
27
2.26, 2.73 m
34.7 t
153.7 s
125.1 s
166.9 s
57.4 t
2.26, 2.75 m
34.8 t
153.8 s
125.1 s
166.7 s
57.4 t
1.47, 2.57 m
32.6 t
154.1 s
124.9 s
166.9 s
57.2 t
2.51 d (7.8)
32.6 t
154.1 s
125. 0 s
166.9 s
57.2 t
4.39, 4.44
4.37, 4.42
4.37, 4.42
4.35, 4.40
dAB (12.6)
2.06 s
dAB (12.2)
2.06 s
dAB (12.5)
2.05 s
dAB (12.6)
2.03 s
28
19.7 q
19.7 q
19.8 c
19.7 c
a
Data are based on DEPT and HSQC experiments.
Overlapping signals.
b
data are shown in Table 1; EIMS m/z (%): 450 [M]+ (15), 435 (14),
417 (12), 380 (3), 310 (18), 281 (29), 187 (14), 141 (100), 123 (91),
95 (35), 69 (32); HREIMS m/z: 450.2406 [M]+ (calcd. for C28H34O5:
450.2406).
2.3.7. (22R)-4˛,17˛,
27-Trihydroxy-1-oxo-witha-2,5,24-trienolide (7)
White amorphous solid; [␣]D20 = −7.3 (c = 0.6, CHCl3); UV
(EtOH) (log ε): ꢀmax 217 (4.0) nm; IR (film): ꢂmax 3428, 2930, 2859,
1690, 1463, 1396, 1216, 1133, 1027, 759 cm−1 1H and 13C NMR:
;
data are shown in Table 2; ESIMS (positive) m/z (%): 493 [M+Na]+
(100); HRESIMS m/z: 493.2557 [M+Na]+ (calcd. for C28H38O6Na:
493.2566).
2.3.4. (4S,22R)-4,16ˇ,27-Trihydroxy-1-oxo-witha-
2,5,17(20),24-tetraenolide (4)
White amorphous solid; [␣]D20 = +14.3 (c = 0.80, CHCl3); UV
(EtOH) (log ε): ꢀmax 212 (4.4) nm; IR (film): ꢂmax 3420, 2932, 1688,
1454, 1385, 1292, 1181, 1136, 1016, 755 cm−1 1H and 13C NMR:
;
2.3.8. Acetylation of 1
data are shown in Table 1; EIMS m/z (%): 468 [M]+ (1), 432 (25),
417 (12), 399 (42), 300 (12), 263 (28), 249 (34), 183 (33), 171 (71),
129 (75), 91 (100), 67 (45); HREIMS m/z: 468.2512 [M]+ (calcd. for
C28H36O6: 468.2502).
A mixture of Ac2O (4 drops), compound 1 (10.0 mg), Et3N
(0.1 mL) in dry Cl2CH2 (1.5 mL) was stirred at 0 ◦C for 2 h. The
mixture was evaporated to dryness, and the residue was purified
by preparative TLC (n-hexane/dioxane, 6:4) to give derivatives 11
(2.5 mg) and 12 (9.5 mg).
2.3.5. (22R)-16ˇ,27-Dihydroxy-1-oxo-witha-
2,5,17(20),24-tetraenolide (5)
2.3.8.1. (4S,20S,22R)-27-Acetoxy-4-hydroxy-1-oxo-witha-2,5,16,24-
tetraenolide (11). White amorphous solid; [␣]D25 = +80.1◦ (c = 0.20,
CHCl3); UV (EtOH) (log ε): ꢀmax 338 (2.6), 237 (3.2) nm; IR (film):
ꢂmax 3854, 3748, 3673, 2929, 2362, 1701, 1540, 1458, 1397, 1258,
White amorphous solid; [␣]D20 = +11.7 (c = 0.23, CHCl3); UV
(EtOH) (log ε): ꢀmax 219 (3.5) nm; IR (film): ꢂmax 3443, 2923, 2854,
1698, 1457, 1381, 1287, 1135, 1015, 755 cm−1 1H and 13C NMR:
;
data are shown in Table 2; ESIMS (positive) m/z (%): 475 [M+Na]+
(22); HRESIMS m/z: 475.2462 [M+Na]+ (calcd. for C28H36O5Na:
475.2460).
1066, 838 cm−1 1H NMR (CDCl3, 400 MHz): ı 0.87 (3H, s), 1.15
;
(3H, d, J = 7.0 Hz), 1.46 (1H, m), 1.50 (3H, s), 1.52 (1H, m), 1.56 (1H,
m), 1.67 (1H, m), 1.70 (1H, m), 1.73 (1H, m), 1.75 (1H, m), 1.84
(1H, m), 2.08 (3H, s, OAc), 2.10 (3H, s), 2.16 (1H, m), 2.17 (1H, m),
2.18 (1H, m), 2.26 (1H, m), 2.58 (1H, m), 2.60 (1H, m), 4.48 (1H, br
d, J = 11.2 Hz), 4.67 (1H, br s), 4.89, 4.94 (2H, dAB, J = 12.3 Hz), 5.57
(1H, s), 5.98 (1H, d, J = 10.2 Hz), 5.99 (1H, br s), 6.79 (1H, dd, J = 4.2,
10.2 Hz); 13C NMR (CDCl3, 100 MHz): ı 16.4 (q), 16.8 (q), 20.5 (q),
22.7 (q), 22.9 (t), 30.7 (t), 31.3 (d), 31.3 (t), 33.1 (t), 34.6 (t), 35.8
(d), 43.5 (d), 46.9 (s), 49.5 (s), 57.2 (d), 58.0 (t), 69.5 (d), 78.6 (d),
121.9 (s), 124.5 (d), 129.1 (d), 130.9 (d), 139.1 (s), 142.6 (d), 155.5
(s), 156.7 (s), 165.0 (s), 203.3 (s), OAc [22.8 (q), 170.9 (s)]; EIMS m/z
2.3.6. (22R)-16ˇ,27-Dihydroxy-1,4-dioxo-witha-
2,5,17(20),24-tetraenolide (6)
White amorphous solid; [␣]D20 = +15.1 (c = 0.07, CHCl3); UV
(EtOH) (log ε): ꢀmax 218 (4.0) nm; IR (film): ꢂmax 3447, 2926, 2859,
1700, 1452, 1385, 1282, 1137, 1017, 756 cm−1 1H and 13C NMR:
;
data are shown in Table 2; ESIMS (positive) m/z (%): 489 [M+Na]+
(13); HRESIMS m/z: 489.2266 [M+Na]+ (calcd. for C28H34O6Na:
489.2277).

G.G. LLanos et al. / Steroids 75 (2010) 974–981
977
Fig. 1. Structure of natural compounds 1–10 and derivatives 11–13.
(%): 476 [M+−18] (18), 416 (13), 401 (17), 293 (50), 249 (19), 209
(7), 183 (22), 172 (22), 123 (100), 95 (40); HREIMS m/z: 476.2578
[M+−18] (calcd. for C30H36O5: 476.2563).
IR (film): ꢂmax 2926, 2856, 1717, 1590, 1457, 1379, 1264, 1096,
1013, 848, 755, 610, 525 cm−1 1H NMR (CDCl3): ı 0.86 (3H, s),
;
1.15 (3H, d, J = 6.9 Hz), 1.49 (1H, m), 1.55 (3H, s), 1.57 (1H, m), 1.67
(1H, m), 1.72 (1H, m), 1.75 (1H, m), 1.78 (1H, m), 1.79 (1H, m), 1.93
(1H, m), 2.08 (3H, s, OAc), 2.09 (3H, s), 2.14 (1H, m), 2.19 (1H, m),
2.23 (1H, m), 2.33 (1H, m), 2.58 (1H, m), 2.60 (1H, m), 4.47 (1H, br
d, J = 11.6 Hz), 4.89, 4.94 (2H, dAB, J = 12.3 Hz), 5.56 (1H, br s), 6.07
(1H, d, J = 4.8 Hz), 6.11 (1H, d, J = 9.8 Hz), 6.24 (1H, d, J = 5.0 Hz), 6.83
(1H, dd, J = 4.8, 9.8 Hz), p-Br-OBz [7.61 (2H, d, J = 8.3 Hz), 7.90 (2H,
d, J = 8.3 Hz)]; 13C NMR (CDCl3): ı 16.4 (q), 16.8 (q), 20.5 (q), 22.0
(q), 22.9 (t), 30.8 (t), 31.1 (d), 31.3 (t), 33.1 (t), 34.6 (t), 35.8 (d), 43.6
(d), 46.9 (s), 49.4 (s), 57.0 (d), 58.0 (t), 70.9 (d), 78.5 (d), 121.9 (s),
124.5 (d), 131.3 (d), 134.4 (s), 134.5 (d), 139.5 (d), 155.5 (s), 156.6
(s), 165.0 (s), 202.9 (s), OAc [20.9 (q), 170.9 (s)], p-Br-OBz [128.4
(s), 128.9 (s); 131.2 (2 × d), 131.8 (2 × d), 165.1 (s)]; EIMS m/z (%):
476 [M+−p-Br-OBz] (5), 416 (7), 401 (8), 293 (23), 249 (7), 201 (73),
182 (100), 171 (33), 123 (34), 91 (13), 75 (18); HRESIMS (positive)
m/z: 701.1914 [M+Na]+ (calcd for C37H41O7NaBr: 701.1913).
2.3.8.2. (4S,20S,22R)-4,27-Diacetoxy-1-oxo-witha-2,5,16,24-
tetraenolide (12). White amorphous solid; [␣]D25 = +22.4◦ (c = 0.30,
CHCl3); UV (EtOH) (log ε): ꢀmax 342 (2.0), 239 (3.7) nm; IR (film):
ꢂmax 2926, 2856, 1737, 1514, 1462, 1379, 1235, 1152, 1027, 962,
810, 634; 1H NMR (CDCl3, 400 MHz): ı 0.87 (3H, s), 1.15 (3H, d,
J = 7.0 Hz), 1.44 (3H, s), 1.47 (1H, m), 1.55 (1H, m), 1.62 (1H, m), 1.70
(1H, m), 1.71 (1H, m), 1.72 (1H, m), 1.76 (1H, m), 1.94 (1H, m), 2.08
(3H, s, OAc), 2.09 (3H, s), 2.10 (3H, s, OAc), 2.15 (1H, m), 2.17 (1H,
m), 2.21 (1H, m), 2.27 (1H, m), 2.56 (1H, m), 2.59 (1H, m), 4.47 (1H,
br d, J = 11.2 Hz), 4.94, 4.89 (2H, dAB, J = 11.8 Hz), 5.56 (1H, br s), 5.81
(1H, d, J = 4.6 Hz), 6.04 (1H, d, J = 10.0 Hz), 6.13 (1H, d, J = 5.0 Hz),
6.72 (1H, dd, J = 4.6, 10.0 Hz); 13C NMR (CDCl3, 100 MHz): ı 16.2
(q), 16.5 (q), 20.4 (q), 21.8 (q), 22.8 (t), 30.7 (t), 31.0 (d), 31.2 (t),
33.0 (t), 34.6 (t), 35.8 (d), 43.5 (d), 46.9 (s), 49.3 (s), 57.1 (d), 58.0
(t), 70.2 (d), 78.8 (d), 121.9 (s), 124.5 (d), 130.9 (d), 133.9 (s), 134.6
(d), 139.8 (d), 155.5 (s), 156.6 (s), 165.0 (s), 203.0 (s), OAc [21.7 (q),
170.2 (s)], OAc [21.8 (q), 170.9 (s)]; EIMS m/z (%): 536 [M+] (1), 476
(17), 416 (13), 401 (24), 327 (7), 293 (47), 249 (19), 197 (5), 171
(38), 159 (17), 123 (100), 95 (41); HREIMS m/z: 536.2750 (calcd.
for C32H40O7: 536.2774).
2.4. Cytotoxicity assay
HeLa (human carcinoma of the cervix), A-549 (human lung
carcinoma), and MCF-7 (human breast adenocarcinoma) cell
lines were each grown as a monolayer in Dulbecco’s modified
Eagle’s medium, DMEM (Sigma), supplemented with 5% fetal
bovine serum (Gibco), and 1% of penicillin–streptomycin mixture
(10,000 UI/mL). The cells were maintained at 37 ◦C in 5% CO2 and
98% humidity. Cytotoxicity was assessed using the colorimetric
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide] reduction assay [20]. Exponentially growing cell suspensions
(0.1 ml of 2 × 104 cells/well) were incubated in a microtiter well
plate (96-well Iwaki) with the compounds at different concentra-
tions pre-dissolved in DMSO. After 48 h the optical density was
measured using a microELISA reader (Multiskan Plus II) at 550 nm
after dissolving the MTT formazan with DMSO (150 ␮L). The per-
centage viability was plotted against the compound concentration,
2.3.9. p-Bromobenzoylation of 11
A mixture of p-bromobenzoyl chloride (4.0 mg), compound 11
(2.0 mg), Et3N (0.05 mL) and DMAP (2.0 mg) in dichloromethane
(0.5 mL) was stirred at room temperature for 2 h. The mixture was
purified by preparative TLC using dichloromethane/acetone (9:1)
to give 13 (2.2 mg).
2.3.9.1. (4S,20S,22R)-27-Acetoxy-4-p-bromobenzoyloxy-1-oxo-
witha-2,5,16,24-tetraenolide
(13). White
amorphous
solid;
[␣]D25 = +118.2◦ (c = 0.2, CHCl3); CD (MeOH): ꢀext 345 (ꢁε = −1.5),
243 (ꢁε = +25.1); UV (EtOH) (log ε): ꢀmax 320 (2.7), 240 (4.3) nm;

978
G.G. LLanos et al. / Steroids 75 (2010) 974–981
Fig. 2. Selected key HMBC (
) and ROESY (
) correlations of 1.
and the 50% cell viability (IC50) was calculated from the curve. Cyto-
toxic assays were run in triplicate. The variations were less than 10%
and the average value was estimated.
blets at ıC 130.7 and 124.3 and two singlets at ıC 138.6 and 155.2
suggested that two additional CH C are present in the molecule
[21].
These data supported that compound 1 is an oxo-witha-
tetraenolide, which presents along with the usual unsaturations at
C-2 and C-24, a double bond at C-5, unlike in the more widespread
5␤,6␤-epoxy withanolide group [21], and an additional one at C-
16, which is also unusual in the withanolide skeleton [23]. The
3. Results and discussion
Repeated chromatography of the CH2Cl2 extract of the leaves of
W. aristata on silica gel and Sephadex LH-20 afforded seven new
withanolides (1–7, Fig. 1).
ꢁ
16-withanolides have been proposed as precursors not only for
17␣-hydroxy- but also for the important group of 17␤-hydroxy-
withanolides [24].
Compound 1 was assigned the molecular formula C28H36O5
by positive high resolution electron ionization mass spectrome-
try (HREIMS). The IR spectrum revealed the presence of hydroxyl
(3429 cm−1) and ␣,␤-unsaturated carbonyl (1689 cm−1) function-
alities, and the UV spectrum showed absorption bands at 235
and 337 nm, characteristic of the ␣,␤-unsaturated ketone and
␣,␤-unsaturated ␦-lactone systems, respectively, present in most
withanolides [21]. The EIMS displayed peaks at m/z 452 [M]+, 434
[M−H2O]+, 312 [M−side chain]+, and a characteristic fragment at
m/z 141, corresponding to a hydroxy-substituted ␣,␤-unsaturated
␦-lactone which originated by the cleavage of the C-20/C-22 bond
[22].
The regiosubstitution of the different functions was determined
by an HMBC experiment (Fig. 2), showing as the most relevant
three-bond correlations those of the vinylic proton resonance at ıH
5.90 (H-6) with the signals at ıC 69.0 (C-4), 31.0 (C-8) and 49.3 (C-
10), and correlation of the proton resonances at ıH 5.51 (H-16), 0.81
(Me-18), and 1.10 (Me-21) with the signal at ıC 155.2 (C-17), which
confirmed the additional double bonds were sited at C-5 and C-16.
Moreover, the oxymethine proton at ıH 4.61 (H-4) showed corre-
lation with the carbon signals at ıC 128.7 (C-2), 130.7 (C-6) and
49.3 (C-10), whereas the ␣,␤-unsaturated ␦-lactone was located
on the side chain due to the cross-peak of the signals at ıH 1.10
(H-21)/ıC 78.8 (C-22), ıH 2.02 (H-28)/ıC 125.3 (C-25), and ıH 4.31,
4.36 (H-27)/ıC 152.9 (C-24) and 166.7 (C-26). The configuration
of 1 was established on the basis of the coupling constants, and
comparison with values reported in the literature [21], and con-
firmed by a ROESY experiment (Fig. 2). Accordingly, the structure
of 1 was established as 4␤,27-dihydroxy-1-oxo-witha-2,5,16,24-
tetraenolide.
Compound 2 was isolated as a white amorphous solid with
the molecular formula C28H36O4 (HREIMS), and analysis of its
spectral data (Table 1) revealed a structure of a 4␤-hydroxy-1-
oxo-witha-tetraenolide. Therefore, its 1H NMR spectrum showed
signals assignable to an enone group in ring A [ı 5.95 (1H, d,
J = 10.0 Hz, H-2) and ı 6.77 (1H, dd, J = 4.5, 10.0 Hz, H-3)], an ␣,␤-
unsaturated ␦-lactone system (ı 1.88, Me-27 and ı 1.93, M-28),
two vinylic protons at ı 5.92 (1H, br s, H-6) and ı 5.53 (1H, br s,
H-16), and two oxymethine protons at ı 4.64 (1H, d, J = 4.5 Hz, H-4)
and ı 4.39 (1H, m, H-22). The 13C NMR spectrum (Table 1) indi-
cated the presence of 28 carbons resonances, including five methyl,
five methylene, ten methine and eight quaternary carbons. These
data indicated that compound 2 was related to 1, the most striking
difference observed was the disappearance of the signals for the
hydroxymethyl group at C-27 (ıH 4.31, 4.36, ıC 57.0) and the pres-
ence of an additional methyl group (ıH 1.88, ıC 12.3), suggesting
compound 2 is the 27-deoxy derivative of 1. The stereochemistry
and regionsubstitution partners were determined by analysis of the
ROESY, and HMBC experiments, respectively (Fig. 2). The structure
The 1H NMR spectrum of 1 (Table 1) showed signals for two
tertiary methyls at ı 0.81 (Me-18) and ı 1.44 (Me-19), a doublet
at ı 1.10 (J = 6.9 Hz), suggesting that the secondary methyl at C-21
has the usual ␣-orientation, and the downfield chemical shift of
the C-28 methyl (ı 2.02) indicates that it is located on a double
bond. In addition, signals at ı 5.91 (1H, d, J = 10.1 Hz, H-2) and ı
6.76 (1H, dd, J = 4.4, 10.1 Hz, H-3) could be attributed to the most
common enone system of withanolides [21]. Moreover, signals of
two vinylic protons at ı 5.90 (br s, H-6) and ı 5.51 (br s, H-16), two
oxymethine protons at ı 4.61 (d, J = 4.4 Hz, H-4) and ı 4.43 (m, H-22),
and a hydroxymethyl group at ı 4.31, 4.36 (dAB, J = 12.8 Hz, H-27)
were observed. In the 1H–1H COSY experiment of 1, H-3 correlated
with H-2 and the oxymethine proton H-4, indicating the presence
of a 4-hydroxy-2-en-1-one unit in the molecule, whereas the H-20
interacted with H-21 and H-22, which in turn is linked to H-23.
The H-6 and H-16 also showed vicinal coupling with H-7 and H-15,
respectively.
The 13C NMR (Table 1) spectrum indicated the presence of
28 carbon resonances, including four methyl, six methylene, ten
methine and eight quaternary carbons. The characteristic down-
field signals at ıC 203.5 and 166.7 were due to the ␣,␤-unsaturated
ketone and lactone carbonyl, respectively, and the doublets at ıC
143.0 and 128.7 were assigned to the vinylic carbons at C-3 and C-
2, respectively. The singlets at ıC 152.9 and 125.3 were attributed to
the quaternary vinylic carbons at C-24 and C-25, respectively, and
the signals at ıC 78.8, 69.0 and 57.0 correspond to the oxygenated
carbons at C-22, C-4 and C-27, respectively. Moreover, the two dou-

G.G. LLanos et al. / Steroids 75 (2010) 974–981
979
The structure of compound 3 was defined by spectroscopic data
and compared with those of the previously described compound
1. The HREIMS gave a molecular ion peak at m/z 450.2406 provid-
ing the molecular formula C28H34O5, which indicated an additional
degree of unsaturation with respect to 1. Its NMR spectra closely
matched that of 1, the main difference being the presence in com-
pound 3 of a ketone signal at ıC 187.7 (s), as well as the downfield
shifts of the signals corresponding to C-2 (ıC 140.0, d), C-3 (ıC 138.8,
d), C-5 (ıC 139.5, s) and C-6 (ıC 137.5, d), together with the absence
of the oximethine (ıH 4.61, d, J = 4.4 Hz; ıC 69.0, d) signals in 1. Fur-
thermore, a complete set of 2D NMR spectra (COSY, ROESY, HSQC,
HMBC) was acquired in order to gain the complete and unam-
biguous assignment of the 1H and 13C NMR resonances as listed
in Table 1, and revealed that 3 was 27-hydroxy-1,4-dioxo-witha-
2,5,16,24-tetraenolide.
Fig. 3. CD positive pairwise interactions involving the chromophore at C-4, the
enone chromophore and the double bond at C-5 for derivative 13.
The structure of compound 4 was established as 4␤,16␤,27-
trihydroxy-1-oxo-witha-2,5,17(20),24-tetraenolide on the basis
of spectroscopic data interpretation. Its IR spectrum displayed
absorption bands of hydroxyl (3420 cm−1), ␣,␤-unsaturated ␦-
lactone and ␣,␤-unsaturated ketone (1688 cm−1) functions, these
last two moieties are supported by an absorption at ꢀmax 212 nm
in the UV spectrum. The molecular formula of 4 was determined
to be C28H36O6 by HREIMS at m/z 468.2512 (M+, calcd 468.2502).
The NMR spectral data (Table 1) showed four methyl singlets (ıH
0.93, 1.46, 1.84 and 2.03), three oxygenated methines [ıH (4.64,
d, J = 4.5 Hz, H-4; 4.72, br s, H-16; 5.41, dd, J = 3.5, 12.8 Hz, H-22)],
an oxygenated methylene (ıH 4.38, 4.43, dAB, J = 12.4 Hz, H-27),
a trisubstituted double bond [ıH 5.95, br s, H-6; ıC 138.8 (C-5),
130.4 (C-6)], a tetrasubstituted double bond [ıC 151.3 (C-17), 129.2
(C-20)], an ␣,␤-unsaturated ␦-lactone [ıC 153.7 (C-24), 125.0 (C-
25), 166.8 (C-26)], and an enone [ıH 5.96, d, J = 10.1 Hz, H-2; ıH
6.78, dd, J = 4.5, 10.1 Hz, H-3; ıC 203.1 (C-1), 128.8 (C-2), 142.7
(C-3)] moiety. The regiosubstitution was established by an HMBC
experiment, and the relative stereochemistry, was defined on the
basis of a ROESY experiment. Thus, a cross-peak of H-4 with H-
9␣ and one of H-16 with H-14␣ confirmed the ␤ stereochemistry
of the hydroxyl groups at C-4 and C-16, while correlation of Me-
21 with Me-18 and H-12 indicated a Z configuration of the ꢁ17
(Fig. 4).
of compound 2 was therefore established as 4␤-hydroxy-1-oxo-
witha-2,5,16,24-tetraenolide.
The absolute configuration of compounds 1 and 2 was deter-
mined by means of circular dichroism (CD). Both compounds
exhibited a broad negative Cotton effect around 335 nm, corre-
sponding to the n–␲* transition of the enone chromophore, as
well as a broad positive Cotton effect around 240 nm, assigned
to the overlapping of the ␲–␲* transition of the enone and the
n–␲* transition of the ␣,␤-unsaturated ␦-lactone. In addition, the
stereochemistry of the lactone ring was determined from the pos-
itive Cotton effect around 240 nm (1, ꢁε = +2.0; 2, ꢁε = +2.6). This
positive Cotton effect is in agreement with a 22R absolute configu-
ration, as occurred with jaborosolactone A, which was determined
by comparison with parasorbic acid, all four exhibiting almost
identical intensity at this wavelength [25–27]. In general, with-
anolides show positive Cotton effects for the n–␲* transition of
the enone chromophore around 340 nm. However, compounds 1
and 2 exhibits the opposite sign. This could be due to the absence
of the 5␤,6␤-epoxy function present in many withanolides [28].
Although NMR data indicate the 4␤ configuration at C-4, this was
easily confirmed applying the CD exciton chirality method [29] to
the p-bromobenzoyl derivative of 1 (13). Its CD spectrum showed
a positive Cotton effect at 243 nm (ꢁε = +25.1) in addition to the
expected negative Cotton effect at 345 nm (ꢁε = −1.5). The pos-
itive exciton pairwise interactions involving the 1La band of the
p-bromobenzoate chromophore and the ␲–␲* transitions of the
double bond and the enone chromophores (Fig. 3) are in agree-
ment with a 4S absolute configuration. The absolute configuration
of withanolides 1 and 2 determined by CD analysis is in agreement
with the absolute configuration of withaferin A as determined by
X-ray analysis [30].
The molecular formula of 5 was determined to be C28H36O5
through ESIMSandNMR data. Thestrong IRabsorption peaks(3443,
1698 cm−1) indicated the presence of hydroxyl, ketone, and lac-
tone groups. The NMR spectroscopic data were similar to those of
4, except for the presence of a methylene assigned to H2-4 (ıH 2.86,
J = 5.0, 21.0 Hz and 3.32, J = 2.4, 21.0 Hz; ıC 33.4) instead of a methine
as in 4 (ıH 4.64, J = 4.5 Hz; ıC 69.1), suggesting 5 is the 4-dehydroxy
derivative of 4. This conclusion was supported by COSY, HSQC,
Fig. 4. Selected key HMBC (
) and ROESY (
) correlations of 4.

980
G.G. LLanos et al. / Steroids 75 (2010) 974–981
Fig. 5. Selected key HMBC (
) and ROESY (
) correlations of 7.
Table 3
HMBC and ROESY experiments, and the full assignments of the NMR
data are listed in Table 2. Hence, compound 5 was determined to
be 16␤,27-dihydroxy-1-oxo-witha-2,5,17(20),24-tetraenolide.
Compound 6 was found to have the molecular formula C28H34O6
as determined through HRESIMS and NMR experiments. Its NMR
spectroscopic data (Table 2) were similar to those of 4, except for
the presence of a ketone signal at ıC 187.8, and the absence of the
signals of the OH-4 (ıH 4.64, J = 4.5 Hz; ıC 69.1) present in 4. This
suggests 6 is the 4-oxo-derivative of 4; a conclusion supported
by 2D experiments. Accordingly, the structure of compound 6
was established as 16␤,27-dihydroxy-1-oxo-witha-2,5,17(20),24-
trienolide.
Compound 7 showed spectral data resembling those of the
known isolated compound 8 [17], and the same molecular formula
(C28H38O6) determined by HREISM. The 13C NMR and DEPT spec-
tra (Table 2) were almost identical to those exhibited by 8, with
small differences observed in the chemical shifts of C-2, C-3, C-4,
C-6, C-10 and Me-19. Several additional pieces of evidence sug-
gested that 7 was the C-4 epimer of the known isolated 8. Thus,
the 1H NMR spectrum showed the following differences: (i) the
absence of the characteristic signals assigned to H-3 (ıH 6.77) and
H-4 (ıH 4.64) as double doublet and doublet, respectively (ii) the
presence of a broad singlet at ıH 5.05 (H-4) and a double doublet
at ıH 6.79 (H-3); (iii) an upfield shift of the Me-19 (ıH 1.29 in 7
and ıH 1.45 in 8), spectroscopic data that are in agreement with
data reported by Hirayama et al. [31] for regioisomeric 4-hydroxy-
methylnaphthalen-1(4H)-ones. The ROESY experiment (Fig. 5) of 7
confirmed the ␣-orientation of the 4-hydroxyl group, showing ROE
correlation between H-4 and Me-19. Thus, compound 7 was deter-
mined to be 4␣,17␣,27-trihydroxy-1-oxo-witha-2,5,24-trienolide.
The occurrence of natural 4␣-hydroxy-withanolides is scarce, and
there are only a few examples in the literature [32], such as the
reports of pubescenol from Physalis pubescence [33]. Furthermore,
toour knowledge, compound 7 is thefirstexample ofa4␣-hydroxy-
2-en-1-one withanolide compound.
Cytotoxic activity (IC50, ␮M) of compounds 1–9, 12 and 13.
Compounds
HeLa
A-549
MCF-7
1
2
3
4
5
6
7
8
10.8
11.0
15.3
>40
>40
>40
>40
24.0
19.6
14.7
3.5
25.2
28.4
35.2
>40
>40
>40
>40
>40
35.7
17.0
3.6
13.5
21.6
13.9
10.3
>40
>40
26.6
27.0
32.4
5.4
9
12
13
C1a
C2a
2.8
3.2 × 10−3
0.8 × 10−2
4.8 × 10−2
4.1
49.4
5.8
a
C1 (actinomycine) and C2 (mercaptopurine) used as positive controls.
(IC50 > 20 ␮M). These results are in line with previous reports on
the relevance of both the 4␤-hydroxy-2-en-1-one and the 5␤,6␤-
epoxy moieties for the expression of cytotoxicity [34] in this type
of compounds. However, the esterification of the hydroxyl groups
at C-4 and C-27 increases the cytotoxicity of the ꢁ5-withanolides
(1 versus 12 and 13).
Acknowledgements
We are indebted to the Agencia Canaria de Investigación,
Innovación y Sociedad de la Información (C200801000049), and
FICIC-GI (2009) projects for financial assistance. GGLL thanks to
the Gobierno de Canarias for a fellowship. The authors thank to
Leticia Rodríguez-Navarro (Department of Botany, University of La
Laguna) for the identification of the plant.
Appendix A. Supplementary data
The detailed 1H and 13C NMR (Table 2) assignments of
the one known withanolide 4␤,17␣,27-trihydroxy-1-oxo-witha-
2,5,24-trienolide (8) [17], which have not previously reported, were
achieved by 1D and 2D techniques including DEPT, HMBC, HSQC,
COSY, and ROESY.
The isolated steroids 1–9 and derivatives 12 and 13 were tested
against a small panel of human cancer cell lines: HeLa (carci-
noma of the cervix), MCF-7 (breast adenocarcinoma) and A549
(lung carcinoma) for their in vitro cytotoxicity. As summarized in
Table 3, derivative 13 showed cytotoxic effect against the tested
tumor cell lines (IC50 values ranging from 2.8 to 3.6 ␮M), and 12
exhibited an IC50 value of 5.4 ␮M on the MCF-7 cell line. Fur-
thermore, compounds 1–4 showed some degree of activity against
HeLa and/or MCF-7 cell lines, with IC50 values ranging from 10.3 to
13.9 ␮M, the other assayed natural compounds (5–9) being inactive
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.steroids.2010.06.001.
References
[1] Eich E. Solanaceae and Convolvulaceae: secondary metabolites. Biosynthe-
sis, chemotaxonomy. Biological and economic significance (a handbook).
Berlin/Heidelberg: Springer-Verlag; 2008. p. 16–21.
[2] Gupta GL, Rana AC. Withania somnifera (Ashwagandha): a review. PHCOG Rev
2007;1:129–36.
[3] Mirjalili MH, Moyano E, Bonfill M, Cusido RM, Palazon J. Steroidal lactones
from Withania somnifera, an ancient plant for novel medicines. Molecules
2009;14:2373–93.
[4] Kaileh M, Berghe WV, Heyerick A, Horion J, Piette J, Libert C, et al. A strongly
elicits I␬B kinase ␤ hyperphosphorylation concomitant with potent inhibition
of its kinase activity. J Biol Chem 2007;282:4253–64.
[5] Patwardhan B, Gautam M. Botanical immunodrugs: scope and opportunities.
DDT 2005;10:495–502.

G.G. LLanos et al. / Steroids 75 (2010) 974–981
981
[6] Mandal Ch, Dutta A, Mallick A, Chandra S, Misra L, Sangwan RS, et al. With-
aferin A induces apoptosis by activating p38 mitogen-activated protein kinase
signaling cascade in leukemic cells of lymphoid and myeloid origin through
mitochondrial death cascade. Apoptosis 2008;13:1450–64.
[7] Prasanna KS, Shilpa P, Bharathi SP. Withaferin A suppresses the expression
of vascular endothelial growth factor in Ehrlich ascites tumour cells via Sp1
transcription factor. Curr Trends Biotechnol Pharm 2009;3:138–48.
[8] Bargagna-Mohan P, Paranthan RR, Hamza A, Dimova N, Trucchi B, Srinivasan C,
et al. Withaferin A targets intermediate filaments glial fibrillary acidic protein
and vimentin in a model of retinal gliosis. J Biol Chem 2010;285:7657–69.
[9] Anderson GJ, Bernardello G, Opel MR, Santos-Guerra A, Anderson M. Repro-
ductive biology of the dioecious Canary Islands endemic Whithania aristata
(Solanaceae). Am J Bot 2006;93:1295–305.
[21] Glotter E. Withanolides and related ergostane-type steroids. Nat Prod Rep
1991:415–41.
[22] Ramaiah PA, Lavie D, Budhiraja RD, Sudhir S, Garg KN. Spectroscopic studies on
a withanolide from Withania coagulans. Phytochemistry 1984;23:143–9.
[23] Misra L, Lal P, Sangwan RS, Sangwan NS, Uniyal GC, Tuli R. Unusually
sulfated and oxygenated steroids from Withania somnifera. Phytochemistry
2005;66:2702–7.
[24] Velde VV, Lavie DA. ꢁ16-Withanolide in Withania somnifera as a possible pre-
cursor for ␣-side-chains. Phytochemistry 1982;21:731.
[25] Lavie D, Kirson I, Glotter E, Snatzke G. Conformational studies on certain 6-
membered ring lactones. Tetrahedron 1970;26:2221–8.
[26] Snatzke G. Circular dichroism and optical rotatory dispersion – principles and
application to the investigation of the stereochemistry of natural products.
Angew Chem Int Ed 1968;7:14–25.
[27] Snatzke G, Snatzke F. In: Ciardelli F, Salvadori P, editors. The carbonyl chro-
mophore: unsaturated ketones and lactones in fundamental aspects and recent
developments in optical rotatory dispersion and circular dichroism. New York:
Heyden & Son Ltd.; 1973.
[10] Cruz SJ. Más de 100 Plantas Medicinales. Medicina Popular Canaria Mono-
grafías. Las Palmas de Gran Canaria: Imprenta Pérez Galdós; 2007.
[11] Martín-Herrera D, Abdala S, Benjumea D, Pérez-Paz P. Diuretic activity
of Withania aristata: an endemic Canary Island species. J Ethnopharmacol
2007;113:487–91.
[12] González AG, Bretón JL, Trujillo J. Withanolidos de las Withania aristata Pauq.
Y Withania frutescens Pauq. An Quím 1972;68:107–8.
[28] Kuroyanagi M, Shibata K, Umehara K. Cell differentiation inducing steroids from
Withania somnifera L. (DUN.). Chem Pharm Bull 1999;47:1646–9.
[29] Nakanishi K, Berova N. In: Nakanishi K, Berova N, Woody RW, editors. The exci-
ton chirality method in circular dichroism: principles and applications. New
York: VCH Publishers; 1994.
[30] McPhail AT, Sim GA. Stable carbonium ions, LIV. Protonation of and hydride
ion abstraction from cycloalkanes and polycycloalkanes in fluorosulfonic acid-
antimony pentafluoride. J Am Chem Soc 1968.
[31] Hirayama M, Fukatsu S, Ikekawa N. Synthetic studies of withanolides.
Part 2. Synthesis of 4a␤,5␤-epoxy-4a,5,6,7,8,8a-hexahydro-4␤-hydroxy-8a␤-
methylnaphthalen-1(4H)-one and related compounds. J Chem Soc Perkin Trans
1981;1:88.
[32] Zhu X, Ikeda T, Nohara T, Ando J, Midzuki K, Yoshimitsu H. Withanolide
type from Solanum cilistum. Tennen Yuki Kagobutsu Toronkai Koen Yoshishu
2000;42:439–44.
[13] González AG, Bretón JL, Trujillo JM. Esteroides de Withanias III. Lactonas
esteroidales de la Withania frutescens Pauq. An Quím 1974;70:64–8.
[14] Benjumea D, Martín-Herrera D, Abdala S, Gutiérrez-Luis J, Quin˜ones W, Car-
dona D, et al. Withanolides from Whitania aristata and their diuretic activity. J
Ethnopharmacol 2009;123:351–5.
[15] González AG, Darias V, Martín-Herrera DA, Suárez MC. Cytostatic activity of
natural withanolides from Spanish withanias. Fitoterapia 1982;53:85–8.
[16] Valera MA, Santos GA. Investigaciones fitoquímicas en plantas canarias.
Madrid: Centro de estudios Fundación Ramón Areces; 2002. p. 197.
[17] González AG, Bretón JL, Trujillo J. Withania steroids. III. Steroidal lactones of
Withania frutescens. J An Quím 1974;70:69–73.
[18] Nittala SS, Lavie D. Withanolides of Acnistus breviflorus. Phytochemistry
1981;20:2735–9.
[19] Hirayama M, Gamoh K, Ikekawa N. Synthetic studies of withanolides. Part 6.
Stereoselective synthesis of withaferin A and 27-deoxywithaferin A. Tetrahe-
dron Lett 1982;23:4725–8.
[33] Row L, Ramachandra RK, Sambi DK, Matsuura T. Pubescenol, a withanolide from
Physalis pubescence. Phytochemistry 1984;23:427–30.
[34] Damu AG, Kuo P-C, Su C-R, Kuo T-H, Chen T-H, Bastow KF, et al. Isolation,
structures, and structure-cytotoxic activity relationships of withanolides and
physalins from Physalis angulata. J Nat Prod 2007;70:1146–52.
[20] Mosmann T. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays.
1983;65:55–63.
J Immunol Methods