Unusually sulfated and oxygenated steroids from Withania somnifera

Article abstract of DOI:10.1016/j.phytochem.2005.10.001

Four (1, 8-10) and six known (2-7) withanolides were isolated from the leaves of Withania somnifera. Among the new compounds, 10 possessed the rare 3-O-sulfate group with the saturation in A ring and 9 contained unusual 1,4-dien-3-one group. Compound 8 did not have usual 2,3 unsaturation in A ring while 1 had the rare C-16 double bond. The structures of all the compounds were elucidated by spectroscopic methods and chemical transformation.

Full text of DOI:10.1016/j.phytochem.2005.10.001

PHYTOCHEMISTRY
Phytochemistry 66 (2005) 2702–2707
www.elsevier.com/locate/phytochem
Unusually sulfated and oxygenated steroids from Withania somnifera
a,
a
a
a
*
Laxminarain Misra , Payare Lal , Rajender S. Sangwan , Neelam S. Sangwan ,
a
b
Girish C. Uniyal , Rakesh Tuli
a
Central Institute of Medicinal and Aromatic Plants, P.O.-CIMAP, Lucknow, UP 226 015, India
b
National Botanical Research Institute, Lucknow, UP 226 001, India
Received 10 August 2005; received in revised form 30 September 2005
Available online 15 November 2005
Abstract
Four (1, 8–10) and six known (2–7) withanolides were isolated from the leaves of Withania somnifera. Among the new compounds, 10
possessed the rare 3-O-sulfate group with the saturation in A ring and 9 contained unusual 1,4-dien-3-one group. Compound 8 did not
have usual 2,3 unsaturation in A ring while 1 had the rare C-16 double bond. The structures of all the compounds were elucidated by
spectroscopic methods and chemical transformation.
ꢀ
2005 Elsevier Ltd. All rights reserved.
Keywords: Withania somnifera L.; Solanaceae; Ashwagandha; Withanolides; Withasteroids; Sulfated; Oxygenated withanolide
1
. Introduction
2. Results and discussion
Withania somnifera L. Dunal (Solanaceae), popularly
Although the leaves of W. Somnifera have been exten-
sively investigated yielding large number of steroidal struc-
tures, our work has, now, afforded 10 steroidal compounds
out of which four are new. Among the six known com-
pounds (2–7), two (2 and 3) have been isolated for the first
time from the leaves. The structure of the known com-
pounds was compared with the spectral data available in
the literature and were identified as: 24,25-dihydrowithan-
olide A (2); withanolide A (3); withanone (4); withaferin A
(5); 27-hydroxy withanone (6) and 17-hydroxy withaferin
A (7). The C NMR data of 2 and 6 have also been
included in Table 1 as they are not traceable in the litera-
ture. The structure elucidation of compounds 1, 8–10 have
been achieved by spectroscopic methods and are discussed
hereunder.
known as ‘‘Ashwagandha’’, is one of the top rank medici-
nal plants of India and is highly valued for its medicinal
and neutraceutical properties (Anonymous, 1962; Sangwan
et al., 2004). It is an annual herb growing in dry and arid
soil as a wild plant (Singh and Kumar, 1998). The plant
is known to synthesize withasteroids (Ray and Gupta,
994) which have shown antioxidant, anti-tumour (Jayap-
rakasam et al., 2003), adaptogenic, anti-stress, anti-convul-
sant, immuno-modulatory and neurological effects
1
1
3
(
Budhiraja et al., 2000; Furmanowa, 2001). The major
source of withanolides in W. somnifera has been reported
to be in its leaves possessing an excellent selective COX-2
inhibitory activity (Jayaprakasam and Nair, 2003; Glotter,
1
991). In this study we report the isolation and character-
ization of four unusual withanolides along with six known
withanolides from its leaves. The structures of new as well
as known compounds were elucidated by spectroscopic
techniques and chemical transformations.
Compound 1 in its IR spectrum showed bands at 3460,
ꢀ1
1710, 1690 and 1120 cm
indicative of hydroxyl, a,b-
unsaturated six membered d-lactone, a,b-unsaturated six
membered ketone and epoxide functionalities, respectively.
+
HRMS showed the [M] at m/z 452.5863 corresponding to
1
*
the molecular formula C H O . The H NMR spectrum
Corresponding author. Tel.: +91 522 2717529; fax: +91 522 2342666.
28 36
5
E-mail address: laxmisra@hotmail.com (L. Misra).
of 1 showed signals for four tertiary methyls at d 0.80,
0
031-9422/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2005.10.001

L. Misra et al. / Phytochemistry 66 (2005) 2702–2707
2703
Table 1
13
C NMR spectral data for compound 1, 2, 8–10 (75 MHz)
Carbon
1(CDCl
3
)
2(CDCl
3
)
6(CDCl
3
)
8(CDCl
3
)
9(CDCl
3
)
3
10(CD OD)
1
2
3
4
5
6
7
8
9
202.4 s
131.9 d
143.0 d
69.4 d
63.0 s
60.0 d
30.6 t
28.0 d
44.2 d
46.3 s
20.5 t
33.9 t
48.0 s
56.6 d
32.2 t
124.0 d
154.8 s
15.5 q
16.2 q
35.5 d
15.7 q
78.6 d
30.6 t
149.9 s
121.0 s
167.3 s
11.6 q
19.7 q
–
202.0 s
128.3 d
138.5 d
37.0 t
72.5 s
56.5 d
55.5 d
35.3 d
35.8 d
50.4 s
21.0 t
203.4 s
129.4 d
140.4 d
37.7 t
73.8 s
56.7 d
212.0 s
41.3 t
154.9 d
129.2 d
186.7 s
123.5 d
147.8 s
210.6 s
39.7 t
73.0 d
45.7 t
73.5 s
57.3 d
57.0 d
36.9 d
44.6 d
53.0 s
22.2 t
a
66.0 d
a
47.0 t
73.2 s
56.6 d
56.8 d
35.3 d
44.9 d
51.6 s
21.2 t
a
29.4 t
a
56.7 d
29.3 t
a
36.0 d
30.1 d
42.6 d
47.0 s
21.7 t
39.3 t
43.0 s
56.6 d
24.7 t
27.6 t
51.9 d
11.9 q
19.6 q
39.2 d
13.3 q
78.2 d
31.7 t
156.9 s
122.9 s
166.8 s
a
36.9 d
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
51.8 s
22.4 t
37.7 t
40.9 t
36.6 t
37.9 t
b
b
b
b
43.4 s
49.3 s
48.3 s
50.0
46.7 d
23.8 t
33.9 t
85.0 s
10.2 q
16.6 q
36.6 d
15.6 q
80.5 q
33.6 t
153.2 s
121.7 s
168.8 s
12.6 q
20.7 q
–
s
b
b
b
54.9
d
46.5 d
45.6 d
23.5 t
21.0 t
23.3 t
34.0 t
84.6 s
10.0 q
15.3 q
44.0 d
14.9 q
79.6 d
33.3 t
154.8 s
126.9 s
166.6 s
56.7 t
19.9 q
–
22.6 t
32.6 t
84.9 s
9.3 q
52.3 d
13.5 q
13.9 q
75.8 s
19.9 q
79.7 d
31.6 t
15.9 q
35.6 d
15.0 q
78.9 d
32.2 t
151.6 s
120.0 s
168.0 s
12.1 q
20.7 q
–
b
31.4 d
b
40.6
d
b
176.0 s
12.9 q
20.5 q
–
58.0 t
a
20.5 q
OAC
171.7 s
a
2
0.8 q
a
Values interchangeable for substance.
Assignment by comparison to the analogous steroids (Su et al., 2002; Choudhary et al., 2004).
b
1
.44, 1.86, 1.95. The doublet at d 1.09 (J = 7.0 Hz) sug-
22 also showed the vicinal coupling with H-23. These data
supported that a double bond is unusually present at C-16
along with the usual unsaturation at C-2 and C-24 (Jayap-
rakasam and Nair, 2003).
gested that C-21 is a secondary methyl having the usual
a-orientation. The down field chemical shifts of the C-27
and C-28 methyl singlets at d 1.86 and 1.95 supported that
both of them are located on a double bond. The relatively
more downfield signals and their typical coupling constants
at d 6.21 (d, J = 10.0 Hz, H-2) and 7.01 (dd, J = 10.0,
1
3
The C NMR spectrum indicated the presence of 28
carbon resonances including five methyl, five methylene,
10 methine and eight quaternary carbons. The character-
istic down field signals at d 202.4 and 167.3 were due to
a,b-unsaturated ketone and lactone carbonyls, respec-
tively, along with the characteristic doublets at d 143.0
and 131.9 for the vinylic carbons at C-3 and C-2, respec-
tively, in ring A. The singlets at d 149.9 and 121.0 were
attributed to the quaternary vinylic carbons at C-24 and
C-25, respectively (Fuska et al., 1982), while a doublet
at d 124.0 and a singlet at d 154.8 suggested that an addi-
tional CH@C is present in the molecule which has been
placed at C-16, 17 as H-16 correlated with the H-15 in
6
.0 Hz, H-3), clearly indicated that 1 belongs to the withaf-
erin A type of carbon skeleton with C-4, 5 and 6 function-
alities as b-oriented owing to the cis-fusion of A/B ring as
evidenced by the downfield chemical shift (d 1.44) of H-19
methyl in the H NMR spectrum (Kirson et al., 1971; Lavie
et al., 1966; Anjaneyulu and Rao, 1997a,b; Fuska et al.,
1
1
982). A broad singlet at d 5.52 suggested that the com-
pound contains another double bond with a proton on
1
1
only one of the carbons (CH@C). H H COSY showed
that this singlet has correlation with H-15 which clearly
indicated that the double bond is present at C-16. The dou-
blet at d 3.70 (J = 6.0 Hz) and a broad singlet at d 3.20
were assigned to 4b-hydroxy-5b,6b-epoxy system. The typ-
ical downfield methine double triplet at d 4.39 (J = 12.0,
1
1
H H COSY (Chen et al., 1990). The typical signals at
d 78.6, 69.4, 63.0 and 60.0 were assigned to the oxygen-
ated carbons at C-22, C-4, C-5 and C-6, respectively.
The signals appearing at d 19.7, 16.2, 15.7, 15.5 and
11.6 were assigned to the C-28, C-19, C-21, C-18 and
C-27 methyls, respectively (Fuska et al., 1982). On acety-
5
.5, 3.5 Hz) was assigned to the H-22 of the lactone moiety.
1
1
In H H COSY spectrum of 1, the H-3 correlated with the
H-2 and H-4, whereas the H-6 interacted with H-7. The H-
1
lation it gave an acetate which in its H NMR spectrum

2704
L. Misra et al. / Phytochemistry 66 (2005) 2702–2707
showed shifting of the doublet to d 4.65 for H-4 along
with the additional singlet at d 2.01 for the acetate. These
data clearly established the structure of compound 1 as
(4) and withanolide-A (3) (Kirson et al., 1971; Anjaneyulu
and Rao, 1997a,b). On acetylation 8 gave an acetate with
its multiplet for H-3 shifting to d 5.02 along with an addi-
tional singlet at d 2.06 for the acetate. These data clearly
established the structure of compound 8 as 2,3-dihydro-
3b-hydroxy withanone.
2
7-deoxy-16-en-withaferin A.
Compound 8 in its IR spectrum showed bands at 3420,
ꢀ
1
1
720, 1700 and 1120 cm
indicative of hydroxyl, a,b-
unsaturated six membered d-lactone, a,b-unsaturated six
Compound 9a was obtained on separation from the
acetylated complex fraction 8. The IR spectrum of 9a
showed bands at 1765, 1710 and 1665 cm indicative of
membered ketone and epoxide functionalities, respectively.
+
ꢀ1
HRMS showed the [M] at m/z 488.6090 corresponding to
1
the molecular formula C H O . The H NMR spectrum
ester, a,b-unsaturated six membered d-lactone and cross
2
8
40
7
of 8 showed four quartets at d 0.83, 1.23, 1.85, 1.92 for
methyls at H-19, H-18, H-28 and H-27, respectively. The
doublet at d 1.02 (J = 7.0 Hz) suggested that C-21 is a sec-
conjugated cyclohexadienone functionalities, respectively.
+
HRMS showed the [M] at m/z 480.3856 corresponding
1
to the molecular formula C H O . The H NMR spec-
3
0
40
5
1
ondary methyl having the usual a-orientation. In the H
trum of 9a showed quartet for three tertiary methyls of a
withasteroid at d 0.78, 1.25, 2.06. The doublet at d 1.03
(J = 7.0 Hz) suggested that C-21 is a secondary methyl
with the usual a-orientation. The singlets for H-28
appeared at d 2.06 and for H-27 (2H) at d 4.89 along with
NMR, the typical vinylic signals for the H-2 and H-3, were
absent while an additional signal resonated at d 4.32 which
showed correlation with H-2 and H-4 at d 2.68, 2.92 and
1
1
2
.28 in the H H COSY. These observations clearly indi-
cated that C-2,3 double bond is saturated and C-3 is oxy-
a singlet at d 2.07 for OCOCH suggesting it to possess an
3
genated to a hydroxy (W1/2 = 17.5 Hz). The doublet
acetate at C-27 (Furmanowa et al., 2001). The unusual
downfield signals at d 7.60 (d, J = 5.5 Hz), 5.96 (d,
J = 5.5 Hz) and 5.80 (br s) were attributed to vinylic pro-
tons at H-1, H-2 and H-4, respectively, typical to the 1,4-
dien-3-one minabeolide steroids earlier isolated from a soft
coral Minabea sp. (Ksebati and Schmitz, 1988). The char-
acteristic down field double triplet at d 4.43 (J = 12.0,
5.5, 3.0 Hz) was assigned to H-22 of the lactone moiety.
(
3
J = 3.2 Hz) at d 3.03 for H-6 and a double doublet at d
.30 (J = 3.2, 2.1 Hz) for H-7 along with a broad singlet
overlapped with H-7 at d 3.27 for 5-OH were also present.
The oxygenation at 5,6,7 carbons were comparable to the
well established withanone type of carbon skeleton having
a-orientation owing to the trans-junction of A/B ring as
evidenced by the 0.26 ppm upfield chemical shift of H-19
1
1
1
protons in the H NMR spectrum (Kirson et al., 1971).
In H H COSY spectrum, the H-1 at d 7.60 showed corre-
lation with the H-2 at d 5.96 while H-4 did not show any
correlation supporting the placement of double bonds at
C-1 and C-4. The possibility of linear dienone systems, like
2,4-dien-1-one or 4,6-dien-1-one, is ruled out by the pres-
ence of a band at 240 nm in the UV spectrum of 9a. This
fact was further supported by the typical signals for the
protons at C-1 and C-2 for the a,b-unsaturated ketone at
d 7.60 and 5.96 and a single singlet at d 5.80 for second con-
jugated double bond which clearly supported the place-
ment of the second conjugated double bond at C-4,5.
The typical down field double triplet at d 4.63 (J = 11.0,
5
.5, 3.0 Hz) was assigned to the H-22 of a withasteroidal
lactone (Jayaprakasam and Nair, 2003; Abou-Douh,
002; Anjaneyulu and Rao, 1997a,b).
2
1
3
The C NMR spectrum indicated the presence of 28
carbon resonances including five methyl, seven methylene,
eight methine and eight quaternary carbons. The down
field signals at d 212.0 and 168.0 supported the presence
of a saturated ketone in A ring and an unsaturated d-lac-
tone carbonyl of E ring, respectively. The singlets at d
1
3
1
51.7 and 120.0 were attributed to the quaternary vinylic
The C NMR spectrum also supported above structure
by indicating the presence of 28 carbon resonances includ-
ing four methyl, eight methylene, nine methine and seven
quaternary carbons. The down field signals at d 186.7
and 166.8 were due to the ketone and lactone carbonyls,
respectively. The signals at d 154.9, 123.5, 129.2 and
147.8 were assigned to the vinylic carbons at C-1, C-2, C-
4 and C-5, respectively, in ring A while the typical singlet
at d 156.9 and 122.9 were attributed to the quaternary viny-
lic carbons at C-24 and C-25, respectively. The characteris-
tic signals at d 78.2 and 58.0 were assigned to oxygenated
carbons at C-22 and C-27, respectively. The signals for ace-
tate at C-27 appeared at d 20.8 and 171.7. The quartets at d
20.5, 19.6, 13.3 and 11.9 were assigned to the C-28, C-19,
C-21 and C-18 methyls, respectively (Ksebati and Schmitz,
1988; Ray and Gupta, 1994). These data clearly suggested
that compound 9a is 27-acetoxy-3-oxo-witha-1,4,24-trieno-
lide, which was isolated after the acetylation of the original
hydroxy compound (9).
carbons at C-24 and C-25, respectively. Further character-
istic signals at d 84.9, 78.9, 73.9, 66.0, 56.8 and 56.6
supported the presence of the oxygenation at C-17, C-22,
C-5, C-3, C-7 and C-6, respectively. Rest of the signals
appearing at d 20.7, 15.9, 15.0, 12.1 and 9.3 were assigned
to the C-28, C-19, C-21, C-27 and C-18 methyls, respec-
tively (Anjaneyulu and Rao, 1997a,b; Shingu et al., 1989).
The HSQC spectrum of 8 showed that the carbon signal
resonating at d 66.6 had a correlation with H-3 at d 4.32
while in the HMBC, this signal showed correlations with
H-2 at d 2.68 and 2.92 as well as H-4 at d 2.28 supporting
that a hydroxyl is present at C-3 without the typical unsat-
uration at C-2,3. The stereochemistry was assigned as b
since the multiplet at d 4.32 showed W1/2 = 17.0 Hz (Cho-
udhary et al., 2004). However, the stereochemistry at C-5,
1
C-6 and C-7 was assigned as a by comparing the
H
NMR chemical shift for H-19 at d 1.23 and the typical cou-
pling constants in the known compounds like withanone

L. Misra et al. / Phytochemistry 66 (2005) 2702–2707
2705
Compound 10 in its IR spectrum showed bands at 3420,
720, 1710, 1230 and 1120 cm indicative of hydroxyl, sat-
urated ketone, a,b-unsaturated six membered d-lactone,
lower polarity (Rf 0.38, CHCl :EtOAc:MeOH:C H ,
3 6 6
ꢀ
1
1
1
70:2:4:24) on the TLC (Shingu et al., 1989). In the H
NMR spectrum of 10, the multiplet at d 4.93 for H-3 got
also shifted to upfield at d 4.32 when converted to 8. In
sulfate and epoxide functionalities, respectively. HRMS
+
1
1
showed the [M] at m/z 568.6905 corresponding to the
the H H COSY spectrum of 10, the H-3 showed correla-
1
molecular formula C H O S. The H NMR spectrum
tion with H-2 and H-4 supporting the placement of sulfate
2
8
40 10
1
13
of 10 showed almost similar signals as in case of 8 whose
structure elucidation has already been described above.
However, when subjected to acetylation by acetic anhy-
dride in the presence of pyridine, it yielded withanone (4)
which is a typical conversion for 3-O-sulfates to a 2-ene ste-
roids (Shingu et al., 1989). Compound 10 when refluxed
with dioxane and pyridine, it got converted to 8. This reac-
tion is again typical for conversion of sulfate group into the
group at C-3. The rest of the H NMR and C NMR sig-
nals of 10 (see Section 3) also supported its structure as 2,3-
dihydro withanone-3b-O-sulfate.
The configuration at C-22 in all the isolated steroids as
1
been assigned as R, since in their H NMR spectra, the
H-22 has appeared as double triplet by having couplings
with the protons of C-23 whereas in the case of S config-
uration, it shows negligible coupling with H-23 (Rahman
et al., 1998) which is a normal configuration for the ste-
roids isolated from W. somnifera, so far (Ray and Gupta,
1994) and has got the biogenetic support also (Glotter,
1991). However, the presence of sulfate group in steroids
is quite rare and has once been reported from Datura
metel (Shingu et al., 1989). Further isolation of such
molecules and the studies on their biogenetic aspects, will
certainly help reveal the role of sulfated steroids in
W. somnifera.
1
corresponding hydroxy group (Shingu et al., 1989). The H
1
3
NMR and C NMR data of the product (8) matched well
with the authentic sample of 3b-hydroxy-2,3-dihydro-
withanone available with us. The compound 8 thus formed,
was acetylated to match with 8a confirming the proposed
structure. The presence of sulfate group in the molecule
was further supported by the fact that 10 showed high
polarity (R 0.25, CHCl :EtOAc:MeOH:C H 70:2:8:20)
f
3
6
6
as compared to its corresponding hydroxy compound with
2
8
2
4
27
2
3
2
2
5
6
R'
2
1
2
2
1
8
20
O
O
O
O
1
2
R''
1
1
1
7
O
O
1
9
13
14
1
6
9
2
3
1
15
1
0
8
5
7
6
4
O
O
R
R
1
1
, R= OH
a, R= OAc
5, R= R'= OH, R''= H
5a, R= R'= OAc, R''= H
7
7
, R= R'= R''= OH
a, R= R'= OAc, R''= OH
R'''
R''
O
O
R
R'
O
O
O
R
OH
O
O
2
3
4
6
6
8
8
1
, 2-ene, R= R'= R'''= H, R''= OH, 24,25-dihydro
, 2-ene, R= R'= R'''= H, R''= OH
, 2-ene, R= R''= R'''= H, R'= OH
, 2-ene, R= R''= H, R'= R'''= OH
a, 2-ene, R= R''= H, R'= OH, R'''= OAc
, R''= R'''= H, R= R'= OH
9
9
, R= OH
a, R= OAc
a, R''= R'''= H, R= OAc, R'= OH
0, R''= R'''= H, R= OSO H, R'= OH
3

2706
L. Misra et al. / Phytochemistry 66 (2005) 2702–2707
1
3
. Experimental
327 (24), 285 (22), 125 (100); H NMR spectral data
300 MHz, CDCl ) d H: 6.21 (1H, d (J = 10.0 Hz), H-2),
(
3
3
.1. General
7.01 (1H, dd (J = 10.0, 6.0 Hz), H-3), 3.70 (1H, d
J = 6.0 Hz), H-4), 3.20 (1H, br s, H-6), 2.20 (2H, dd
(
Melting point of compounds was recorded on Fisher
(J = 6.0, 4.0 Hz), H-15), 5.52 (1H, br s, H-16), 0.80 (3H,
s, H-18), 1.44 (3H, s, H-19), 1.09 (3H, d (J = 7.0 Hz), H-
21), 4.39 (1H, dt (J = 12.0, 5.5, 3.5 Hz), H-22), 2.49 (2H,
Johns melting point apparatus. NMR spectra were recorded
on a 300 MHz Bruker AV-300 FTNMR spectrometer using
TMS as internal standard and FT-IR on a Perkin–Elmer
1
3
br s, H-23), 1.86 (3H, s, H-27), 1.95 (3H, s, H-28);
C
1
710B instruments. FAB HRMS spectra were recorded on
NMR spectral data (75 MHz, CDCl ) Table 1.
3
JEOL SX 102/DA-6000 mass spectrometer using Argon/
Xenon (6 kV, 100 mA) as the FAB gas.
Acetylation of 1. Compound 1 (5 mg) was taken in a
flask and to it acetic anhydride (1 ml) was added in pres-
ence of 1–2 drops of pyridine which after usual work up
1
3
.2. Plant material
gave 1a (5 mg). H NMR spectral data (300 MHz,
CDCl ) d H: 6.25 (1H, d (J = 10.0 Hz), H-2), 7.05 (1H,
3
The W. somnifera (Ashwagandha) leaves were collected
dd (J = 10.0, 6.0 Hz), H-3), 4.65 (1H, d (J = 6.0 Hz), H-
4), 3.23 (1H, br s, H-6), 2.20 (2H, dd (J = 6.0, 4.0 Hz),
H-15), 5.53 (1H, br s, H-16), 0.81 (3H, s, H-18), 1.42
(3H, s, H-19), 1.09 (3H, d (J = 7.0 Hz), H-21), 4.37 (1H,
dt (J = 12.0, 5.5, 3.5 Hz), H-22), 2.50 (2H, br s, H-23),
1.87 (3H, s, H-27), 1.93 (3H, s, H-28), 2.05 (3H, s,
OCOCH3).
from Lucknow, India in July 2001 and identification of the
plant material was done by the taxonomists of CIMAP and
the accession is being maintained by us in the institute
farm. The plant genotype is deposited (No. RS-NMITLI-
II.A) in the National Gene Bank at CIMAP, Lucknow
for the field conservation and maintenance.
3
.3. Extraction and isolation of compounds
3.3.2. 6a,7a-epoxy-3b,5a,17a-trihydroxy-1-oxo-witha-24-
enolide (8)
30
The shade dried leaves (850 g) were ground and defatted
M.p.: 258 ꢁC; ½aꢁ : +66.00ꢁ MeOH, c = 0.25); IR (KBr)
D
ꢀ1
three times with n-hexane by keeping at RT overnight. The
spent material was further extracted with MeOH (3· 1 l) at
RT overnight. The methanol extract (108 g) was chromato-
graphed over a column of silica gel (720 g) with n-hexane as
mobile phase and then elution was carried out in n-hexane
and EtOAc with solvent gradient. The polarity was
increased by sequentially adding 5%, 25%, 50%, 75% ethyl
acetate, pure ethyl acetate and finally 5%, 10%, 15% and
cm : 3420 (OH), 1710, 1690, 1120; HRMS: 488.6080
(Calc. for C H O 488.6170); FABMS m/z (rel. int.):
2
8
40
7
+
+
+
488 (2.5) [M] , 470 (2.5) [M ꢀ H O] , 452 (5) [Mꢀ2H O] ,
2
2
1
345 (70), 125 (100); H NMR spectral data (300 MHz,
CDCl ) d H: 2.68 and 2.92 (2H, d (J = 8.5 Hz), H-2),
3
4.32 (1H,
m
(W1/2 = 17 Hz), H-3), 2.28 (2H,
d
(J = 8.0 Hz), H-4), 3.27 (1H, br s, OH at C-5), 3.03 (1H,
d (J = 3.2 Hz), H-6), 3.30 (1H, dd (J = 3.2, 2.1 Hz), H-7),
0.83 (3H, s, H-18), 1.23 (3H, s, H-19), 1.02 (3H, d
(J = 7.0 Hz), H-21), 4.63 (1H, dt (J = 11.0, 5.5, 3.0 Hz),
H-22), 2.52 (2H, d (J = 4.5 Hz), H-23), 1.85 (3H, s, H-
2
0% methanol was added in the ethyl acetate. The fractions
(
150 ml each) of CC were collected and pooled into nine
major fractions based on their TLC pattern. Fr. 1 yielded
oleic, linoleic and palmitic acids while fr. 2 and 3 were dis-
carded as they contained chlorophyll and other pigments.
Fr. 4 yielded compound 1 (320 mg, Rf 0.72, CHCl3:
EtOAc:MeOH:C H 70:2:4:24), 2 (70 mg) and 3 (52 mg).
1
3
27), 1.92 (3H, s, H-28); C NMR spectral data (75 MHz,
CDCl ) Table 1.
3
Acetylation of 8. Compound 8 (5 mg) was taken in a
flask and to it acetic anhydride (1 ml) was added in the
6
6
Fr. 5 on further CC and crystallization yielded mainly com-
pound 4 (1.95 g) and 5 (450 mg). Fr. 6 after further CC
gave 4 (675 mg), 5 (2.5 g), 6 (155.0 mg) and 7 (263 mg).
Fr. 7 after CC afforded 8 (135 mg, R 0.38, CHCl :EtOAc:-
presence of pyridine and after usual work up 8a (5 mg)
1
was obtained. H NMR spectral data (300 MHz, CDCl )
3
d H: 2.65 and 2.90 (2H, d (J = 8.5 Hz), H-2), 5.02 (1H, m
(W1/2 = 17 Hz), H-3), 2.24 (2H, d (J = 8.0 Hz), H-4), 3.27
(1H, br s, OH at C-5), 3.03 (1H, d (J = 3.2, Hz), H-6),
3.30 (1H, dd (J = 3.2, 2.1 Hz), H-7), 0.83 (3H, s, H-18),
1.23 (3H, s, H-19), 1.02 (3H, d (J = 7.0 Hz), H-21), 4.63
(1H, dt (J = 11.0, 5.5, 3.0 Hz), H-22), 2.52 (2H, d
(J = 4.5 Hz), H-23), 1.85 (3H, s, H-27), 1.94 (3H, s, H-
28), 2.06 (3H, s, OCOCH3).
f
3
MeOH:C H 70:2:4:24) while fr. 8 after further CC yielded
6
6
sucrose (315 mg), and 10 (335 mg, R 0.25, CHCl :EtOAc:-
f
3
MeOH:C H 70:2:8:20). Fr. 9 after acetylation and CC
6
6
yielded 9a (25 mg, R 0.30, CHCl :EtOAc:MeOH:C H
6
f
3
6
7
0:2:4:24) glucose penta acetate (310 mg) and sucrose octa
acetate (265 mg).
3
.3.1. 5b,6b-epoxy-4b-hydroxy-1-oxo-witha-2,16,24-
trienolide (1)
M.p.: 268 ꢁC; ½aꢁ : +92.60ꢁ CHCl , c = 0.25); IR (KBr)
3.3.3. 27-acetoxy-3-oxo-witha-1,4,24-trienolide (9a)
30
M.p.: 213–15 ꢁ C; ½aꢁ : +24.60ꢁ MeOH, c = 0.25); UV
D
3
D
0
MeOH
max
ꢀ1
3
k
nm (loge): 240 (2.72); IR (KBr) cm : 1765, 1710,
ꢀ
1
cm : 3460 (OH), 1710, 1690, 1120; HRMS: 452.5863
1665; HRMS: 480.3856 (Calc. for C H O 480.3862);
30 40 5
+
(
Calc. for C H O 452.5874); FABMS m/z (rel. int.):
FABMS m/z (rel. int.): 480 (15) [M] , 438 (10)
2
8
36
5
+
+
+
+
4
52 (10) [M] , 434 (5) [M ꢀ H O] , 416 (5) [M ꢀ 2H O] ,
[M ꢀ COCH ] , 420 (15), 313 (25), 141 (100), 123 (55);
2
2
2

L. Misra et al. / Phytochemistry 66 (2005) 2702–2707
Anjaneyulu, A.S.R., Rao, D.S., 1997a.
2707
1
H NMR spectral data (300 MHz, CDCl ) d H: 7.60 (1H, d
A
new withanolides from
3
the leaves of Withania somnifera. Indian J. Chem. 36B, 161–
65.
Anjaneyulu, A.S.R., Rao, D.S., 1997b. New withanolides from the roots
of Withania somnifera. Indian J. Chem. 36B, 424–433.
Anonymous, 1962. The Wealth of India, vol. 8. PID, CSIR, New Delhi,
pp. 37–38.
(
(
(
J = 5.5 Hz), H-1), 5.96 (1H, d (J = 5.5 Hz), H-2), 5.80
1H, s, H-4), 0.78 (3H, s, H-18), 1.25 (3H, s, H-19), 1.03
3H, d (J = 7.0 Hz), H-21), 4.43 (1H, dt (J = 12.0, 5.5,
1
3
.0 Hz), H-22), 2.50 (2H, d (J = 4.5 Hz), H-23), 4.89 (2H,
br s, H-27), 2.06 (3H, s, H-28), 2.07 (3H, s, OCOCH );
3
1
3
Budhiraja, R.D., Krishan, P., Sudhir, S., 2000. Biological activity of
withanolides. J. Sci. Ind. Res. 59, 904–911.
C NMR spectral data (75 MHz, CDCl ) Table 1.
3
Chen, C.M., Chen, Z.T., Hsieh, C.H., Li, W.S., Wen, S.Y., 1990.
Withangulatin A, a new withanolide from Physalis angulata. Hetero-
cycles 31, 1371–1375.
Choudhary, M.I., Yousuf, S., Nawaz, S.A., Ahmed, S., Atta-ur-Rah-
aman, 2004. Cholinesterase inhibiting withanolides from Withania
somnifera. Chem. Pharm. Bull. 52, 1358–1361.
Furmanowa, M., Gajdzis-Kuls, D., Ruszkowska, J., Czarnocki, Z.,
Obidoska, G., Sadowska, A., Rani, R., Upadhyay, S.N., 2001. In
vitro propagation of Withania somnifera with immunosuppressive
activity. Planta Med. 67, 146–149.
Fuska, J., Prousek, J., Rosazaa, J., Budesinsky, M., 1982. Microbial
transformations of natural anti-tumor agents. 23. Conversion of
withaferin-A to 12b- and 15b-hydroxy derivatives of withaferin-A.
Steroids 40, 157–169.
3
witha-24-enolide (10)
.3.4. 5a,17a-dihydroxy-6a,7a-epoxy-1-oxo-3b-O-sulfate-
30
M.p.: 158 ꢁC; ½aꢁ : +59.40ꢁ MeOH, c = 0.25); IR (KBr)
D
ꢀ
1
cm : 3420 (OH), 1720, 1710, 1230, 1120; HRMS: 568.6905
(
5
4
Calc. for C H O S 568.6900); FABMS m/z (rel. int.):
28 40 10
+
+
+
68 (2) [M] , 488 (5) [M ꢀ SO ] , 470 (5) [M ꢀ H SO ] ,
3
2
4
1
52 (5), 345 (70), 325 (25), 125 (100), 123 (40); H NMR
spectral data (300 MHz, CD OD) d H: 2.74 and 2.99
3
(
2H, dd (J = 12.5, 5.5 Hz), H-2), 4.93 (1H, m (W1/
=
17 Hz), H-3), 2.54 and 2.38 (2H, d (J = 7.5 Hz), H-4),
2
3
.30 (1H, br s, OH at C-5), 3.06 (1H, d (J = 3.2, Hz), H-
), 3.24 (1H, dd (J = 3.2, 2.1 Hz), H-7), 0.88 (3H, s, H-
8), 1.25 (3H, s, H-19), 1.05 (3H, d (J = 7.0 Hz), H-21),
.63 (1H, br dt (J = 11.0, 5.5, 3.0 Hz), H-22), 2.52 (2H, d
6
1
4
Glotter, E., 1991. Withanolides and related ergostane type steroids. Nat.
Prod. Rep., 415–441.
Jayaprakasam, B., Nair, M.G., 2003. Cyclooxygenase-2 enzyme inhib-
itory withanolides from Withania somnifera leaves. Tetrahedron 59,
(
2
J = 4.5 Hz), H-23), 1.84 (3H, s, H-27), 2.06 (3H, s, H-
8); C NMR spectral data (75 MHz, CD OD) Table 1.
8
41–849.
1
3
3
Jayaprakasam, B., Zhang, Y., Seeram, N.P., Nair, M.G., 2003. Growth
inhibition of human tumor cell lines by withanolides from Withania
somnifera leaves. Life Sci. 74, 125–132.
Kirson, I., Glotter, E., Lavie, D., Abraham, A., 1971. Constituents of
Withania somnifera Dun. Part XII. The withanolides of an Indian
Chemotype. J. Chem. Soc. (C), 2032–2044.
Solvolysis of 10. A solution of 10 (10 mg) in pyridine–
dioxane (4:1, v/v, 3.0 ml) was heated on a water bath at
0 ꢁC for 5 h. The reaction mixture was dried completely
under reduced pressure and the residue was purified by pre-
parative TLC on silica gel (CHCl :EtOAc:MeOH:C H
8
3
6
6
Ksebati, M.B., Schmitz, F.J., 1988. Minabeolides: A group of
1
7
0:2:10:18) to afford the hydrolysate 8 (5 mg). H NMR spec-
withanolides from a soft coral, Minabea sp. J. Org. Chem. 53, 3926–
3929.
Lavie, D., Greenfield, S., Glotter, E., 1966. Constituents of Withania
somnifera Dun. Part VI. The stereochemistry of withaferin A.. J.
Chem. Soc. (C)., 1753–1767.
Rahman, A., Choudhary, M.I., Yousuf, M., Gul, W., Qureshi, S., 1998.
New withanolides from Withania coagulans. Chem. Pharm. Bull. 46,
1853–1856.
tral data (300 MHz, CDCl ) were similar to that of 8. The
3
product 8, thus, obtained was further acetylated by the usual
method affording its acetate which was similar to 8a.
Elimination of sulfate from 10. A solution of 10 (5 mg) in
pyridine–acetic anhydride (1:3 v/v, 3.0 ml) was heated on a
water bath at 80 ꢁC for 5 h and reaction mixture was dried
completely under reduced pressure. The residue was puri-
Ray, A.B., Gupta, M., 1994. Withasteroids,
a growing group of
naturally occurring steroidal lactones. In: Progress in the Chemistry
of Organic Natural Products, vol. 63. Springer, New York, pp. 1–
fied by crystallization (EtOAc–CHCl ) to afford a com-
pound identical with withanone (4).
3
1
06.
Sangwan, R.S., Chaurasiya, N.D., Misra, L.N., Lal, P., Uniyal, G.C.,
Sharma, R., Sangwan, N.S., Suri, K.A., Qazi, G.N., Tuli, R., 2004.
Phytochemical variability in commercial herbal products and prep-
arations of Withania somnifera (Ashwagandha). Curr. Sci. 86, 461–
Acknowledgements
4
65.
Shingu, K., Furusawa, Y., Nohara, T., 1989. New withanolides,
daturametelins C, D, E, and G- Ac from Datura metel
L. (Solanaceous studies. XIV). Chem. Pharm. Bull. 37, 2132–
135.
We are thankful to CSIR, New Delhi for a research
grant under the NMITLI sponsored research project and
the Director CIMAP, Lucknow for providing the research
facilities.
F
2
Singh, S., Kumar, S., 1998. The Indian Ginseng Ashwagandha. CIMAP,
Lucknow, India, pp. 1–293.
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