Five withanolides from the leaves of datura metel l. and their inhibitory effects on nitric oxide production

Article abstract of DOI:10.3390/molecules19044548

Four new withanolides named dmetelins A-D (compounds 1-4), along with the known compound 7a,27-dihydroxy-1-oxo-witha-2,5,24-trienolide (5) were isolated from the leaves of Datura metel L. (Solanaceae). Their structures were elucidated on the basis of detailed analysis of 1D and 2D NMR and mass spectrometry data. All the compounds were evaluated for their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells. Compounds 1, 4 and 5 showed significant inhibitory activities, and compounds 2 and 3 showed moderate inhibitory activities with IC50 values of 17.8, 11.6, 14.9, 33.3 and 28.6 μM, respectively.

Full text of DOI:10.3390/molecules19044548

Molecules 2014, 19, 4548-4559; doi:10.3390/molecules19044548
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Five Withanolides from the Leaves of Datura metel L. and Their
Inhibitory Effects on Nitric Oxide Production
†
†
Bing-You Yang , Rui Guo , Ting Li, Yan Liu, Chang-Fu Wang, Zun-Peng Shu, Zhi-Bin Wang,
Jing Zhang, Yong-Gang Xia, Hai Jiang, Qiu-Hong Wang * and Hai-Xue Kuang *
Key Laboratory of Chinese Materia Medica (Ministry of Education), Heilongjiang University of
Chinese Medicine, Ministry of Education, Harbin 150040, China
†
These authors contributed equally to this work.
*
Authors to whom correspondence should be addressed; E-Mails: qhwang668@sina.com (Q.-H.W.);
hxkuang@hotmail.com (H.-X.K.); Tel.: +86-451-8219-3001 (Q.-H.W. & H.-X.K.);
Fax: +86-451-8211-0803 (Q.-H.W. & H.-X.K.).
Received: 7 March 2014; in revised form: 4 April 2014 / Accepted: 8 April 2014 /
Published: 11 April 2014
Abstract: Four new withanolides named dmetelins A–D (compounds 1–4), along with the
known compound 7α,27-dihydroxy-1-oxo-witha-2,5,24-trienolide (5) were isolated from
the leaves of Datura metel L. (Solanaceae). Their structures were elucidated on the basis of
detailed analysis of 1D and 2D NMR and mass spectrometry data. All the compounds were
evaluated for their inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide
(NO) production in RAW264.7 cells. Compounds 1, 4 and 5 showed significant inhibitory
activities, and compounds 2 and 3 showed moderate inhibitory activities with IC50 values
of 17.8, 11.6, 14.9, 33.3 and 28.6 μM, respectively.
Keywords: Datura metel L.; withanolides; dmetelins; NO inhibition
1
. Introduction
Withasteroids are a group of structurally diverse steroidal compounds with a C28 steroidal lactone
skeleton, in which a characteristic feature is the presence of an α,β-unsaturated δ lactone ring in the
side chain. They are presented primarily in the Solanaceae family, which includes Datura, Acnistus,
Dunalia, Jaborosa, Physalis and Withania [1]. The isolation and synthesis of withanolides have

Molecules 2014, 19
4549
received considerable attention due to their significant biological activities, which include antitumor [2],
cytotoxic [3,4], immunosuppressive [4], anti-inflammatory [5,6], and chemoprevention properties [7].
Flos daturae (baimantuoluo in Chinese), the dry flowers of Datura metel L. (Solanaceae), known as
“
Yangjinhua”, have been widely used in Traditional Chinese Medicine for the treatment of coughs,
asthma, rheumatism, pain, and convulsions for centuries [8]. It has also been reported that it displayed
the most promising effects in treatment of psoriasis and were used in a clinical application at the First
Affiliated Hospital of Heilongjiang University of Chinese Medicine (Heilongjiang, China) [9,10].
Withanolides have been studied for treating psoriasis as the main constituents of the effective part of
flowers of D. metel [11,12]. Baimantuoluolines A–J, and baimantuoluosides A–H were also isolated
and reported [8,13–18]. However, the dry flowers of D. metel have the disadvantages of long
florescence and low yield in comparison to its leaves. At the same time, the leaves of this herb, which
can be regenerated every year, were typically discarded. In order to expand the available resources, our
group found that the leaves of D. metel have some similarities in chemical constituents with its flowers
and significant advantages in terms of high and stable yield. As a result, four new withanolides were
isolated and named dmetelins A–D (compounds 1–4), together with one known withanolide,
7
α,27-dihydroxy-1-oxowitha-2,5,24-trienolide (5) [19] (Figure 1). The structures of compounds 1–5
were determined by the interpretation of spectroscopic analysis, including 1D and 2D NMR
spectroscopy. All isolates were identified as major active constituents having inhibitory effects of NO
production in LPS-activated macrophage cell line, RAW 264.7 murine macrophages. Herein, we report
the isolation, structural elucidation, and NO inhibitory effects of these isolates.
Figure 1. Structures of compounds 1–5.
2
. Results and Discussion
Compound 1 was isolated as a white amorphous powder, and its molecular formula was determined
+
to be C28
H
40
O
5
by HRESIMS (m/z 479.2779 [M+Na] ). The UV spectrum showed an absorption
maximum at 224 nm, suggesting the presence of an α,β-unsaturated δ lactone, and carbonyl groups,
1
respectively [20]. The H-NMR spectrum of 1 (Table 1) displayed the characteristic signals of the
common withanolide steroid. Three tertiary methyl groups at δ 0.78 (3H, s), 1.28 (3H, s), 2.10 (3H, s),
and a secondary methyl group at 1.04 (3H, d, J = 6.6 Hz) were attributed to Me-18, Me-19, Me-28, and
Me-21, respectively. The Me-27 signal was replaced by one set of oxygen-bearing methylene protons δ

Molecules 2014, 19
4550
4
1
.37 (1H, d, J = 11.7 Hz) and 4.29 (1H, d, J = 11.7 Hz). An olefinic proton at δ 5.70 (1H, dd, J = 5.3,
1
1
.2 Hz) showed H- H COSY correlation peaks with H-7 δ 3.77 (1H, t, J = 3.8 Hz) (Figure 2), which
1
3
was assigned to the vinylic protons H-6. The C-NMR spectrum of 1 (Table 2) revealed 28 carbons. It
was composed of four methyl groups at δ 12.1 (C-18), 18.7 (C-19), 13.8 (C-21), and 20.2 (C-28). The
presence of four olefin carbons at δ 146.6, 125.9, 157.9, and 126.4 were attributed to C-5, C-6, C-24,
and C-25, respectively. The characteristic downfield at δ 215.9 and 168.6 were due to a carbonyl group
C-1 and a lactone carbonyl group C-26. The signals at δ 65.1, 80.2, and 56.4 were assigned to the three
oxygen-bearing carbons at C-7, C-22, and C-27. Assignments of all function groups of 1 were
1
1
achieved by H- HCOSY, HMBC and HSQC (Figure 2). Thus, the planar structure of 1 was determined
as shown.
1
Table 1. H-NMR data (400 MHz) of the aglycones of 1–4 (in CD
3
OD, δ in ppm).
No.
1
1
2
3
4
3.88 br s
2
2.32 m, 2.67 m
2.00 m, 1.62 m
2.22 m, 2.63 m
1.85 m, 2.24 m
4.11 t (7.4)
5.48 br s
5.86 dd (10.0, 2.4)
5.84 dd (10.0, 2.5)
3
6.93 ddd (10.0, 4.8, 2.4) 6.91 ddd (10.0, 4.8, 2.5)
4
3.44 m
3.40 m
2.93 dd (21.4, 4.8)
5.48 br s
2.96 dd (21.5, 4.8)
6
7
8
9
5.70 dd (5.3, 1.2)
3.77 t (3.8)
1.43 m
5.98 dd (10.0, 2.3)
5.63 br d (10.0)
2.07 m
5.80 dd (5.8, 1.5)
3.80 t (5.5)
1.46 m
3.71 d (8.4)
1.42 m
1.99 m
1.54 m
2.00 m
1.68 m
1
1
1.50 m, 1.73 m
1.29 m, 1.97 m
1.54 m
1.44 m, 1.66 m
1.32 m, 2.03 m
1.24 m
1.59 m, 2.23 m
1.34 m, 2.03 m
1.32 m
1.53 m, 2.24 m
1.32 m, 2.04 m
1.24 m
1
1
1
1
1
1
1
2
2
2
2
2
4
5
6
7
8
9
0
1
2
3
1.78 m, 1.19 m
1.79 m, 1.41 m
1.24 m
1.85 m, 1.37 m
1.83 m, 1.43 m
1.30 m
1.84 m, 1.22 m
1.83 m, 1.41 m
1.28 m
1.92 m, 1.53 m
1.78 m, 1.37 m
1.22 m
0.78 s
0.82 s
0.79 s
0.80 s
1.28 s
1.00 s
1.24 s
1.28 s
1.95 m
1.98 m
1.98 m
1.94 m
1.04 d (6.6)
4.47 dt (13.2, 3.4)
1.03 d (6.7)
4.47 dt (13.3, 3.4)
1.05 d (6.6)
4.50 dt (13.5, 3.6)
2.58 dd (17.9, 13.6)
2.22 dd (17.9, 3.0)
4.47 d (11.2)
4.62 d (11.2)
2.14 s
1.04 d (6.6)
4.48 dt (13.2, 3.4)
2.54 dd (18.0, 13.5)
2.20 dd (18.0, 3.1)
4.37 d (11.7)
4.29 d (11.7)
2.10 s
2.56 dd (17.8, 13.7) 2.54 dd (18.0, 13.4)
.18 dd (17.8, 3.4) 2.21 dd (18.0, 3.3)
2
2
2
7
8
4.29 d (11.7)
4.36 d (11.7)
4.29 d (11.7)
2.10 s
4
.37 d (11.7)
2.10 s
OCH
3
3.38 s

Molecules 2014, 19
Figure 2. H, H-COSY and Selected HMBC correlations of compounds 1–4.
4551
1
1
1
3
3
Table 2. C-NMR (100 MHz) data of the aglycones of 1–4 (in CD OD, δ in ppm).
No.
1
2
3
4
5
6
7
8
1
2
3
4
215.9
39.0
26.2
31.9
146.6
125.9
65.1
38.6
36.2
55.5
23.3
40.5
43.7
50.7
25.1
28.3
53.2
12.1
18.7
40.5
13.8
80.2
30.7
157.9
126.4
168.6
56.4
20.2
72.5
33.0
75.4
123.4
143.3
130.1
132.1
38.5
45.3
40.4
21.4
40.9
45.1
55.2
25.0
28.3
53.2
12.2
19.9
40.4
13.7
80.1
30.7
157.9
126.4
168.5
56.4
20.2
55.8
205.7
128.4
147.7
34.4
141.7
127.5
64.8
39.5
36.4
52.3
24.6
40.8
43.6
51.0
25.0
28.2
53.2
12.2
18.8
40.5
13.8
80.2
30.8
160.4
123.6
168.6
63.6
20.9
205.9
128.4
147.6
33.8
138.2
130.8
72.1
42.2
42.6
51.4
24.9
41.0
44.4
57.4
27.7
28.5
52.7
12.4
19.2
40.4
13.8
80.2
30.7
157.9
126.4
168.6
56.4
20.2
9
1
1
0
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
OCH
3

Molecules 2014, 19
4552
The configuration of compound 1 (Figure 3) was established by NOE correlations. Me-18/Me-21,
H-20, H-16β, H-20/H-16β, and H-16/H-22 determined that C-20 of 1 had an S configuration.
According to the literature [3,18], an α-oriented H-22 atom gives rise to H-22/H-23 values of 0.5–4.0
and 9.0–13.8 Hz, whereas the β-oriented form shows values of 2.5-7.0 and 2.0-5.0 Hz. Thus, the
configuration of 1 at C-22 was determined as R due to the H-22 coupling constants (J = 3.4, 13.2 Hz).
The orientation of hydroxyl group at C-7 was also deduced from the NOESY spectrum (Figure 3). The
NOE correlations of Me-18/H-8β and H-8β/H-7β suggested the β-orientation of H-7 and α orientation
of the 7-OH group. On the basis of the spectroscopic studies, the structure of 1 was fully established,
and determined to be 7α,27-dihydroxy-(20S,22R)-1-oxo-witha-5,24-dienolide and given the trivial
name dmetelin A.
Figure 3. Key NOESY contacts of compounds 1–4.
Compound 2 was obtained as a white amorphous powder with the molecular formula C29
H
42
O
5
,
+
established by positive HRESIMS from the [M+Na] signal at m/z 493.2921. The NMR data of 2
exhibited a close resemblance to those of 1, and differences between them were observed only in their
A/B rings. The NMR data of 1 showed a conjugated diene system composed of a disubstituted double
bond (δ 5.98 and 130.1, δ 5.63 and 132.1), together with one trisubstituted double bond (δ 5.48, 143.3,
and 123.4). The presence of an oxygenated methine at δ 72.5 (C-1) instead of the keto carbon in 1
implied that a hydroxyl group was attached to C-1. The HMBC correlations of δ 2.24 (H-2)/123.4,
2
.07 (H-8)/130.1, and 1.54 (H-9)/132.1 suggested that the conjugated diene system was located at C-4,
C-5, C-6, and C-7 (Figure 2). A three proton singlet at δ 3.38 (3H, s) was due to -OCH group, which
3
was located at C-3 on the basis of δ 3.38/75.4 (C-3) observed in its HMBC spectrum (Figure 2). The
orientation of the hydroxyl group at C-1 was assigned as α on the basis of the small coupling constants
of H-1 δ 3.88 (1H, br s)/H-2. Furthermore, the NOESY correlation of Me-19/H-1β also supported the
α orientation of 1-OH. In addition, NOEs between Me-19/H-2β and H-2α/H-3α suggested a β
3
orientation of 3-OCH . The configuration of C-20 and C-22 were in good agreement with that of 1.
Consequently, compound 2 was identified as 1α,3β,27-trihydroxy-(20S,22R)-1-oxo-witha-4,6,24-
trienolide and given the common name dmetelin B.

Molecules 2014, 19
4553
Compound 3 was isolated as a white amorphous powder. The HRESIMS spectrum showed peaks at
+
8
01.3694 [M+Na] , corresponding to the molecular formula C40
H
58
O15. The NMR spectra (Tables 1
and 2) of 3 showed four methyl groups at δ 0.79 (3H, s, Me-18), 1.24 (3H, s, Me-19), 1.05 (3H, d,
J = 6.6 Hz, Me-21), and 2.14 (3H, s, Me-28), two α, β-unsaturated olefinic protons at δ 5.86 (1H, dd,
J = 10.0, 2.4 Hz, H-2) and 6.93 (1H, ddd, J = 10.0, 4.8, 2.4 Hz, H-3), along with one olefinic proton at
δ 5.80 (1H, dd, J = 5.8, 1.5 Hz, H-6), which supported a withanolide possessing a 2, 5-dien-1-one
system in rings A/B [21]. Apart from the glycosidic signals, compound 3 displayed 28 carbons. The
signals at δ 160.4, 123.6 and 168.6 were assigned to C-24, C-25 and C-26 of the α, β-unsaturation δ
lactone ring respectively, in the six-member ring E. Two anomeric protons doublets at δ 4.34 (1H, d,
J = 7.8 Hz, H-1′) and 4.41 (1H, d, J = 7.8 Hz, H-1″) were correlated to two anomeric carbons at δ
1
03.9 (C-1′) and 104.9 (C-1″), in its HSQC spectrum (Figure 2), which suggested that 3 contained a
diglycosidic moiety. The attachment of one glucose unit to C-27 of the aglycone was confirmed by the
HMBC correlations (Figure 2) observed from H-1′ to C-27. Other HMBC correlations to glucose
linkages (Figure 2) were H-1″/C-6′ and H-6′/C-1″. The orientation of 7-OH could be deduced by
Me-18/H-8β and H-8β/H-7β observed in the NOESY spectrum, which suggested that 7-OH has an
α orientation. The configuration of C-20 and C-22 of 3 were established to be identical to those of 1 by
their similar NOESY correlations, as depicted on the 3D structure (Figure 3). Hence, 3 was identified
as 7α,27-dihydroxy-(20S,22R)-1-oxo-witha-2,5,24-trienolide-27-O-β-D-glucopyranosy-(6′→1″)-β-D-
glucopyranoside and assigned the common name dmetelin C.
Compound 4 was obtained as a white amorphous powder. The molecular formula C28
H
38
O
5
was
+
deduced from the HRESIMS with [M+H] at m/z 455.2715 (calcd. for C28
H
39
O
5
, 455.2797). The NMR
data of 4 (Tables 1 and 2) were very similar to those of 7α,27-dihydroxy-1-oxowitha-2,5,24-trienolide [19],
1
3
also isolated as compound 5 during this work. A comparison of the C-NMR data of these two
compounds indicated that they have the same planar structure. The key differences were the signals for
carbons around C-7, which suggested that these two compounds should have different orientations of
the hydroxyl group at C-7. The orientation of 7-OH was deduced by the NOE observed for Me-18/H-12β,
H-12α/H-9α, and H-9α/H-7α in the NOESY experiment (Figure 3), indicating the α-orientation of H-7
and β-orientation of the 7-OH group. The configuration of C-20 and C-22 were also akin to those of
compound 1. Therefore, the structure of 4 was identified as 7β,27-dihydroxy-(20S,22R)-1-oxo-witha-
2
,5,24-trienolide and given the common name dmetelin D.
Since NO acts as an inflammatory mediator, inhibitors of NO production may exhibit therapeutic
potential for the treatment of inflammation with overproduction of NO. Withanolides display great
anti-inflammatory activity [22–24], so the compounds were evaluated for their effects on the inhibition
of NO production in LPS-activated RAW264.7 cells [25]. In our experiment, the assayed compounds
were first dissolved in DMSO, and the final concentration 0.2% (v/v) in cell culture supernatants did
not show any effect on the assay systems. The effect of compounds 1–5 on cell viability was evaluated
by the MTT assay to ascertain the absence of cytotoxicity (over 90% cell survival) to macrophage cells
at the concentration of 100 μM. The inhibitory effects of withanolides (Figure 4A) were evaluated by
LPS-induced NO generation in RAW 264.7 cells. Compounds 1, 4 and 5 presented the significant
anti-inflammatory activity, with IC50 values of 17.8, 11.6 and 14.9 μM, respectively. L-NMMA
(IC50 = 13.1 μM) was used as positive control. Compounds 2 and 3 revealed moderate anti-inflammatory
activity, with IC50 values of 33.3 and 28.6 μM (Figure 4B).

Molecules 2014, 19
4554
Figure 4. Inhibitory activities of compounds 1–5 on NO production in LPS-induced RAW
64.7 cells. (A) Inhibitory effects on NO production of compounds 1–5 and positive control
L-NMMA). All results are expressed as the mean ± S.D. of three independent experiments,
2
(
each one performed in triplicate. (B) IC50s of compounds 1–5 and the positive control.
3
. Experimental Section
3
.1. General
Optical rotations were measured using a Perkin-Elmer 341 polarimeter. UV spectra were obtained
on a Shimadzu UV-1601 instrument. IR spectra were recorded on a Shimadzu FTIR-8400S. NMR
spectra were recorded in MeOH using TMS as an internal standard on Bruker DPX 400 instrument
1
13
(
400 MHz for H NMR and 100 MHz for C NMR). Chemical shifts (δ) were expressed in ppm with
reference to the solvent signals. HRESIMS was recorded on IonSpec Ultima 7.0 FTICR; GC analysis
was performed on Agilent 7890A-5975C gas chromatograph equipped with a DB-1701 column
(
60 m × 0.25 mm × 0.25 m, film thickness); detection Triple-Axis Detector; Preparative HPLC
Waters, Delta 600-2487) was measured by Hypersil-ODS II (10 μm, 20 × 300 mm, Yilite, Da Lian,
(
China); Column chromatography (CC) was carried out using silica gel of 200–300 mesh size (Qingdao
Marine Chemical Ltd, Qingdao, China); All the solvents were of analytical grade and were purchased
from TianJinFuYu Company Ltd. (Tianjin, China)
3
.2. Plant Material
The leaves of D. metel were collected at Lingao County, Hainan Province, People’s Republic of
China, in September 2012, and was identified by Prof. Zhenyue Wang (Department of Chinese
Medicine Resources, Heilongjiang University of Chinese Medicine). A voucher specimen (2012184)
has been deposited at the Herbarium of Heilongjiang University of Chinese Medicine, P. R. China.
3
.3. Extraction and Isolation
The air-dried leaves of D. metel (16 kg) were extracted with 75% EtOH (100 L × 3.0 h × 3) at room
temperature. The 75% EtOH extract was evaporated under vacuum (45 °C) to yield a residue (1.75 kg),

Molecules 2014, 19
4555
which was suspended in water and then sequentially extracted with petroleum ether (PE) and EtOAc.
The EtOAc portion of the extract (226 g) was chromatographed on silica gel with CH Cl /MeOH
30:1 → 0:1, v/v) to afford Fractions A–E. Fraction B (36.2 g) was rechromatographed on a silica gel
column (CH Cl /MeOH 25:1 → 0:1, v/v) to give five subfractions (B1-B5). Compound 5 (22 mg) was
2
2
(
2
2
obtained from fraction B2 after purification by CC using (CH
Subfraction B3 was further purified by ODS column, eluting with MeOH/H
yield 4 (28 mg). Subfraction B4 was chromatographed over a ODS column (MeOH/H
and semi-preparative HPLC (66% MeOH/H O, flow rate 3 mL/min) to give 1 (18 mg, t
and 2 (15 mg, t = 45.3 min). Fraction E (11.6 g) was purified by CC and eluted with CH
10:1 → 0:1, v/v) to afford three subfractions (E1–E3), and Subfraction E3 was further purified by
semi-preparative HPLC (40% MeOH/H O, flow rate 3 mL/min) to yield 3 (15 mg, t = 25.6 min).
2
Cl
2
/MeOH 20:1 → 0:1, v/v),
O (1:9 → 1:0, v/v) to
O 2:8 → 1:0, v/v)
= 39.1 min),
Cl /MeOH
2
2
2
R
R
2
2
(
2
R
2
6
Dmetelin A (1). [α] + 28.0 (c = 0.10, MeOH); UV (MeOH) λmax (logε): 224 (4.37) nm; IR (KBr) vmax
:
D
−
1
1
13
3
401, 3169, 2933, 2688, 1701, 1689, 1262, 1068, 1023, 821 cm ; H and C-NMR data, see Tables 1
+
and 2; HRESIMS m/z: 479.2779 [M+Na] (calcd. for C28
H
40
O
5
Na, 479.2773).
2
6
Dmetelin B (2). [α] + 16.0 (c = 0.10, MeOH); UV (MeOH) λmax (logε): 225 (5.60) nm; IR (KBr) vmax
:
D
−
1
1
13
3
4
367, 2963, 2869, 1685, 1388, 1075 cm ; H and C-NMR data, see Tables 1 and 2; HRESIMS m/z
+
93.2921 [M+Na] (calcd. for C29
H
42
O
5
Na, 493.2930).
2
6
Dmetelin C (3). [α] + 44.0 (c = 0.10, MeOH); UV (MeOH) λmax (logε): 222 (5.28) nm; IR (KBr) vmax
:
D
−
1
1
13
3
1
400, 2937, 2360, 2349, 1716, 1695, 1681, 1219, 1126, 1028 cm ; H and C-NMR data, see Tables
+
–3; HRESIMS m/z 801.3694 [M+Na] (calcd. for C40
H
58
O
15Na, 801.3673).
2
6
Dmetelin D (4). [α] + 20.0 (c = 0.10, MeOH); UV (MeOH) λmax (logε): 221 (6.52) nm; IR (KBr) vmax
:
D
−
1
1
13
3
402, 3367, 3129, 2963, 2915, 2869, 1685, 1075, 799 cm ; H and C-NMR data, see Tables 1 and 2;
+
HRESIMS m/z 455.2715 [M+H] (calcd. for C28
H
39
O
5
, 455.2797).
1
13
Table 3. H- and C-NMR data (400/100 MHz) data of the glycosyl groups for 3 (in
CD
3
OD, δ in ppm).
NO.
δ
H
δ
C
NO.
1″
δ
H
δ
C
1
2
3
4
5
6
′
′
′
′
′
′
4.34 d (7.8)
3.18 t (8.9)
3.41 m
103.9
75.2
77.9
71.7
77.2
4.41 d (7.8)
3.36 t (9.2)
3.30 m
104.9
75.0
78.0
71.5
78.0
2″
3″
3.28 m
4″
3.36 m
3.43 m
5″
3.30 m
3.78 dd (11.6, 5.8) 69.9
.16 dd (11.6, 1.8)
6″ 3.68 dd (11.8, 5.0) 62.8
4
3.86 dd (11.8, 2.0)
3
.4. Acid Hydrolysis of Compound 3 and GC Analysis
Compound 3 (2.0 mg) was refluxed with H
water bath for 3 h. After that time, the reaction mixture was extracted with ethyl acetate (3 × 5 mL).
The aqueous layer was neutralized with 2 M NaHCO and then evaporated to dryness. The residue of
2
O (2 mL) and 2 N aqueous HCl (1 mL) at 80 °C on a
3

Molecules 2014, 19
4556
sugar was dissolved in 1 mL anhydrous pyridine and treated with L-cysteine methyl ester hydrochloride
1.5 mg) stirred at 60 °C for 1 h. HMDS-TMCS (150 μL hexamethyldisilazane-trimethylchlorosilane,
:1) was added and the mixture was kept at 60 °C for another 30 min. The precipitate was centrifuged
off, and the supernatant was concentrated under a N stream. The mixture was partitioned between
(
3
2
2
n-hexane and H O (0.1 mL each), and the hexane extracted (1 μL) was analyzed on an Agilent
7
890A-5975C gas chromatograph equipped with a DB-1701 column (60 m × 0.25 mm × 0.25 m, film
thickness) under the following conditions: firstly temperature was maintained at 50 °C, secondly
raising to 190 °C at the rate of 40 °C/min, thirdly raising the temperature to 200 °C at the rate of
0
.5 °C/min, fourthly raising the temperature to 210 °C at the rate of 1 °C/min, finally, raising the
temperature to 280 °C at the rate of 20 °C/min. The carrier gas was He (1.0 mL/min), injector
temperature: 250 °C; and split ratio: 1/20. By comparison of the retention times of authentic samples of
D-glucose, the absolute configurations of the sugar residues were gave to be D-glucose (t
.5. Cell Culture
Raw 264.7, a murine macrophage cell line was purchased from the Cell Bank of the Chinese
R
= 17 min).
3
Academic of Sciences, (Shanghai, China) and maintained in supplemented with 10% Fetal Bovine
Serum (Hyclone, Logan City, UT, USA), penicillin (100 U/mL) and streptomycin (100 μg/mL) at
3
2
7 °C in a humidified incubator with 5% CO .
3
.6. Cell Viability Assay
Cell viability was assessed using the MTT assay as described previously [26,27]. In brief, RAW
5
2
3
64.7 cells were seeded into a 96-well plate at a density of 1.0 × 10 cells per well and incubated at
7 °C for 24 h. The cells were then treated with various concentrations of the samples. The maximum
concentration of vehicle (dimethylsulfoxide, DMSO) in the culture media was 0.2% (v/v). After an
additional 24 h incubation at 37 °C, 100 μL of MTT (0.5 mg/mL in PBS) was added to the wells, and
the incubation continued for another 2 h. The medium was then discarded and 150 μL DMSO was added.
The resulting color was assayed at 540 nm using a microplate reader (Molecular Devices, San
Francisco, CA, USA).
3
.7. The Determination of NO Production from RAW 264.7
To evaluate the effective of the tested materials on LPS-induced NO production, Raw 264.7
5
macrophages in 10% FBS-DMEM were plated at densities of 1.0 × 10 cell/wells in 96-well plates and
grew overnight, after incubation, 20 μL serially diluted drugs (DMSO+serum-free DMEM as solvent)
were applied to the cells for 4 h, and incubated in the medium with 20 μL LPS (lipopolysaccharide) of
1
μg/mL in the presence or absence of test samples for 24 h. Then, cells were dispensed into 96-well
plates. One hundred μL of each supernatant was mixed with the same volume of Griess reagent [28]
1% sulfanilamide in 5% H PO and 0.1% N-1-naphthyletylenediamide dihydrochloride) and
(
3
4
incubated at room temperature, away from the light, for 10 min. The absorbance was measured at 540
nm using an ELISA reader (PerkinElmer, Waltham, MA, USA), and the concentration of nitrite was
calculated by comparison with a sodium nitrite standard curve. For this assay, N-monomethyl-L-

Molecules 2014, 19
4557
arginine (L-NMMA) was used as positive control. Three independent experiments were performed,
with each one in triplicate.
3
.8. Statistical Analysis
The IC50 values, the sample concentrations resulting in 50% inhibition of NO production, were
determined by using nonlinear regression analysis (Sigma Plot 8.0; SPSS Inc. Chicago, IL, USA). The
data are presented as mean ± S.D. of more than three independent experiments.
4
. Conclusions
This study demonstrated that the leaves of D. metel were rich in withanolides, showing a similar
structure as compounds found in its flowers. Moreover, five withanolides, including four new
compounds 1–4, were isolated and identified, and these compounds were found to be responsible for
the ability to inhibit NO production by activated macrophages. According to our results, further
phytochemical and pharmacological studies of leaves D. metel are clearly worthwhile.
Acknowledgments
This work was financed by the Outstanding Young Scientists Program of Heilongjiang Province
(
No. JC201105).
Author Contributions
Prof. Q.-H. W. and Prof. H.-X. K. were responsible for guarantor of integrity of entire study.
Prof. B.-Y. Y. is responsible for study concepts. R. G. and L. T. performed research. G. R. and Y. L.
analyzed data. C.-F. W., Z.-P. S. and Z.-B. W. are responsible for Statistical analysis. J. Z., Y.-G. X.
and H. J. are responsible for manuscript preparation. Prof. B.-Y. Y. and R. G. wrote the paper.
Prof. Q.-H. W. and H.-X. K. approved for manuscript final version.
Conflicts of Interest
The authors declare no conflict of interest.
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2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
(