Megastigmane glycosides from Urena lobata

Article abstract of DOI:10.1016/j.fitote.2018.02.017

Five new megastigmane glycosides, urenalobasides A–E (1–5), together with 11 known ones (6–16) were isolated from Urena lobata. Their structures were determined by extensive spectroscopic and spectrometric data (1D and 2D NMR, IR, and HRESIMS) and calculated electronic circular dichroism method. Compounds 1 and 2 are two unusual megastigmanes structurally containing a 6/5 fused ring system. Compound 3 exhibits inhibition of nitric oxide production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells with IC₅₀ value of 53.7 ± 1.0 μM (positive control, dexamethasone, IC₅₀ = 16.6 ± 0.8 μM).

Full text of DOI:10.1016/j.fitote.2018.02.017

Fitoterapia xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Megastigmane glycosides from Urena lobata
1
1
Cong Su , Bowen Qi , Juan Wang, Ning Ding, Yun Wu, Xiao-Ping Shi, Zhi-Xiang Zhu, Xiao Liu,
⁎
Xiao-Hui Wang, Jiao Zheng, Peng-Fei Tu, She-Po Shi
Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, People's Republic of
China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Urena lobata L.
Megastigmane glycosides
Urenalobasides A–E
Anti-inflammatory
Nitric oxide production
Five new megastigmane glycosides, urenalobasides A–E (1–5), together with 11 known ones (6–16) were iso-
lated from Urena lobata. Their structures were determined by extensive spectroscopic and spectrometric data (1D
and 2D NMR, IR, and HRESIMS) and calculated electronic circular dichroism method. Compounds 1 and 2 are
two unusual megastigmanes structurally containing a 6/5 fused ring system. Compound 3 exhibits inhibition of
nitric oxide production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells with IC50 value of
53.7 ± 1.0 μM (positive control, dexamethasone, IC50 = 16.6 ± 0.8 μM).
1. Introduction
Nexus 470 FT-IR spectrophotometer (MA, USA) with KBr pellets. UV
spectra were obtained using a Shimadzu UV-2450 spectrophotometer
(Tokyo, Japan). NMR spectra were recorded on a Varian INOVA-500
spectrometer (CA, USA) operating at 500 MHz for H NMR and
Urena lobata, belonging to the family Malvaceae, is widely dis-
1
tributed in Asia, South America, and Africa [1]. In south of China, the
aerial part of U. lobata is commonly used as anti-inflammatory, anti-
pyretic, antalgesic, and antibacterial agents for the treatment of fever,
rheumatism, and diarrhea. In addition, decoctions and preparations
containing U. lobata are also popularly used to treat gynopathies such as
pathological leucorrhea and gonorrhea [2]. Previous investigations led
to the isolation and structural elucidation of flavonoids, coumarins, and
lignans from U. lobata [1,3–6]. However, as a widely used herbal
medicine, the bioactive constituents of U. lobata remain largely un-
known. Therefore, further investigations on the bioactive constituents
of U. lobata are quite essential. In our previous reports, 24 flavonoids
were identified from U. lobata [7,8]. As an ongoing study, five new
megastigmane glycosides, urenalobasides A–E (1–5), together with 11
known ones (6–16) were obtained (Fig. 1). Herein, the isolation and
structural elucidation of the new compounds as well as their inhibitory
effects on nitric oxide (NO) production in LPS-stimulated RAW264.7
macrophage cells are described.
125 MHz for 13C NMR. HRESIMS was recorded on an LCMS-IT-TOF
system, fitted with a Prominence UFLC system and an ESI interface
(Shimadzu, Kyoto, Japan). Silica gel (200–300 mesh, Qingdao Marine
Chemical Inc., Qingdao, China), LiChroprep RP-C18 gel (40–63 μm,
Merck, Germany), D101 macroporous adsorption resin (Qingdao
Marine Chemical Inc., Qingdao, China) and Sephadex LH-20
(Pharmacia) were used for open column chromatography (CC). HPLC
was performed on a Shimadzu LC-20AT pump system (Shimadzu
Corporation, Tokyo, Japan), equipped with a SPD-M20A photodiode
array detector monitoring at 254 nm. A semi-preparative HPLC column
(YMC-Pack C18, 250 × 10 mm, 5 μm) was utilized for compounds se-
paration and purification. TLC was performed using GF254 plates
(Qingdao Marine Chemical Inc., Qingdao, China).
2.2. Plant material
Urena lobata L. was collected in Guangxi province, China, in
September 2013. The plant material was authenticated by one of the
authors (S.-P. Shi), and a voucher specimen (DTH2013029) is deposited
at the Modern Research Center for Traditional Chinese Medicine,
Beijing University of Chinese Medicine, Beijing, China.
2
. Experimental
2.1. General experimental procedures
Optical rotations were obtained on a Rudolph Autopol IV automatic
polarimeter (NJ, USA). IR spectra were recorded on a Thermo Nicolet
⁎
Corresponding author.
E-mail address: shishepo@163.com (S.-P. Shi).
These two authors contributed equally to this work.
1
https://doi.org/10.1016/j.fitote.2018.02.017
Received 25 November 2017; Received in revised form 5 February 2018; Accepted 10 February 2018
0367-326X/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Su, C., Fitoterapia (2018), https://doi.org/10.1016/j.fitote.2018.02.017

C. Su et al.
Fitoterapia xxx (xxxx) xxx–xxx
Fig. 1. Structures of compounds 1–16.
Table 1
and partitioned with petroleum ether (2 × 6 L), EtOAc (5 × 6 L), and n-
BuOH (3 × 6 L), successively. The n-BuOH extract (128 g) was sub-
jected to D101 macroporous adsorption resin chromatography and
13
3
C NMR (125 MHz) data of compounds 1–5 (δ in ppm, in CD OD).
Position
1a
2a
3a
4a
5a
2
eluted with H O–EtOH (100:0, 90:10, 50:50, 20:80, 0:100) to yield five
δ
C
, type
δ
C
, type
δ
C
, type
δ
C
, type
δ
C
, type
fractions (Fr.1–5). Fr. 2 (20 g) and Fr. 3 (40 g) were combined and
subjected to silica gel chromatography and eluted with a gradient of
EtOAc-MeOH-H O from 30:2:1 to 5:2:1 to give five subfractions (Subfr.
2
A – E). Subfr. C (6.3 g) and subfr. D (7.7 g) were combined and chro-
matographed on Sephadex LH-20 column eluted with MeOH to give
seven subfractions (Fr. C1 – C7). Fr. C4 (4.0 g) was subjected to silica
1
2
3
4
5
6
7
8
9
1
1
1
1
35.2,C
52.4,CH
35.2, C
52.3, CH
202.3, C
39.2, C
54.6, CH
202.3, C
41.9, C
53.7, CH
201.9, C
37.1, C
2
2
2
2
49.0, CH
65.0, CH
41.7, CH
143.7, C
129.2, C
211.7, C
54.4, CH
71.7, CH
20.2, CH
29.4, CH
29.9, CH
20.7, CH
102.5, CH
75.1, CH
77.8, CH
71.6, CH
78.1, CH
2
2
202.3, C
118.0, CH
171.5, C
148.9, C
136.1, CH
60.5, CH
75.4, CH
18.1, CH
27.7, CH
28.4, CH
75.1, CH
102.2, CH
75.0, CH
78.1, CH
71.8, CH
77.9, CH
118.5, CH
171.1, C
149.5, C
137.0, CH
60.9, CH
75.9, CH
17.6, CH
27.8, CH
28.7, CH
75.5, CH
101.9, CH
75.0, CH
77.9, CH
71.8, CH
78.1, CH
125.3, CH
159.6, C
142.6, C
135.4, CH
38.5, CH
74.9, CH
20.2, CH
29.1, CH
29.2, CH
22.9, CH
102.5, CH
75.9, CH
77.0, CH
71.8, CH
78.1, CH
128.8, CH
160.0, C
144.4, C
130.2, CH
38.7, CH
74.9, CH
20.1, CH
28.3, CH
28.4, CH
25.1, CH
102.5, CH
75.9, CH
77.0, CH
71.9, CH
78.1, CH
gel column eluted with a stepwise gradient of petroleum CH
20:1 → 0:1) to afford six fractions (Fr. C4a – C4f). Fr. C4e (1.5 g) was
further separated using ODS C18 column chromatography and eluted
with MeOH–H O (20:1, 15:1, 10:1, v/v) to obtain four fractions (Subfr.
2 2
Cl –MeOH
(
2
2
2
0
1
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
C4e1 – C4e4). Subfr. C4e1 was repeatedly separated and purified by
semi-preparative HPLC (8% aqueous MeCN) to give compounds 1
3
3
(
6.0 mg, t
(2.0 mg, t
48.5 min). Subfr. C4e2 was separated by semi-preparative HPLC (15%
aqueous MeOH) to give compounds 3 (13.0 mg, t 32.0 min), 4
12.4 mg, t 35.0 min), 9 (11.8 mg, t 42.5 min), and 10 (10.0 mg, t
5.5 min). Subfr. C4e3 was subjected to semi-preparative HPLC (12%
aqueous MeCN) to yield compounds 11 (3.0 mg, t 35.0 min), 12
(2.4 mg, t 43.0 min), and 13 (1.8 mg, t 52.5 min). Subfr. C4e4 was
separated by semi-preparative HPLC (30% aqueous MeOH) to afford
compounds 14 (23.0 mg, t 55.0 min), 15 (2.4 mg, t 53.0 min), and 16
(5.8 mg, t 62.5 min).
Urenalobaside A (1): Pale yellow gum, [α]
R
45.0 min), 2 (1.4 mg, t
R
63.0 min), 5 (1.8 mg, t
R
52.5 min), 6
Glu-1
Glu-2
Glu-3
Glu-4
Glu-5
Glu-6
Api-1
Api-2
Api-3
Api-4
Api-5
R
57.0 min), 7 (1.4 mg, t
R
23.0 min), and 8 (1.5 mg, t
R
R
(
4
R
R
R
63.0, CH
2
62.9, CH
2
69.1, CH
2
69.1, CH
2
62.9, CH
2
111.0, CH
78.0, CH
80.4, C
75.0, CH
65.4, CH
111.0, CH
78.0, CH
80.5, C
75.1, CH
65.5, CH
R
R
R
2
2
2
2
R
R
a
Assignments were carried out based on HSQC and HMBC experiments.
R
25: −55.4 (c 0.05,
D
MeOH); UV (MeOH) λmax (log ε): 206 (3.09), 289 (3.55); IR (KBr) νmax
3
1
:
2
.3. Extraction and isolation
−
1
1
13
389, 2940, 1655, 1653, 1082 cm ; H and C NMR data, see Tables
−
and 2; negative-ion HRESIMS: m/z 419.1469 [M + Cl] (calcd for
The air-dried U. lobata (13 kg) was refluxed with 95% EtOH for
C
19
H
28
O
8
Cl, 419.1478).
Urenalobaside B (2): Pale yellow gum, [α]
MeOH); UV (MeOH) λmax (log ε): 206 (3.08), 289 (3.55); IR (KBr) νmax
three times (3 × 180 L, each for 1 h). After removal of solvent under
reduced pressure, the residue (1.35 kg) was suspended in water (6 L),
25: −14.1 (c 0.05,
D
:
2

C. Su et al.
Fitoterapia xxx (xxxx) xxx–xxx
Table 2
1
3
H NMR (500 MHz) data of compounds 1–5 (δ in ppm, in CD OD).
No.
1a
2a
3a
4a
5a
2
2.26, d, (16.0)
2.27, d, (16.0)
2.38, d, (16.0)
2.34, s
2.30, s
1.68, dd, (12.5, 2.5)
1.41, t, (12.5)
2
.38, d, (16.0)
3
4
3.94, m
2.29, dd, (17.5, 6.0)
5.91, s
5.92, s
5.87, s
5.89, s
1
.93, m
7
8
6.37, br s
2.83, m
6.25, br s
2.91, m
6.36, t, (6.5)
2.68, dd, (12.0, 6.5)
5.99, t, (7.0)
2.63, m
3.19, dd, (19.0, 6.5)
2.65, dd, (19.0, 6.5)
4.42, dd, (12.5, 6.5)
1.26, d, (6.5)
1.18, s
2
.53, m
9
4.07, m
1.31, d, (6.0)
1.27, s
1.16, s
4.80, m
4.03, m
1.26, br s
1.27, s
3.16, dd, (12.0, 6.0)
1.26, d, (6.0)
1.30, br s
1.30, br s
2.14, s
3.14, dd, (12.0, 6.0)
1.23, d, (6.0)
1.19, br s
1.18, br s
2.27, s
10
11
12
13
1.16, s
1.03, s
1.64, s
b
4.80
Glu-1
Glu-2
Glu-3
Glu-4
Glu-5
Glu-6
4.36, d, (8.0)
3.14, dd, (10.0, 8.0)
3.36, m
3.27, m
3.27, m
4.38, d, (8.0)
3.11, dd, (9.0, 8.0)
3.35, m
3.26, m
3.26, m
4.34, d, (8.0)
3.99, m
3.42, m
3.25, t, (9.5)
3.36, m
3.99, m
3.59, dd, (11.0, 6.5)
4.99, d, (2.5)
3.88, d, (2.5)
4.33, d, (8.0)
3.99, m
3.40, m
3.25, t, (9.0)
3.35, m
3.97, m
3.58, dd, (11.5, 6.5)
4.99, d, (2.5)
3.88, d, (2.0)
3.95, m
4.36, d, (7.5)
3.11, dd, (12.0, 7.5)
3.35, m
3.26, m
3.26, m
3.65, dd, (12.0, 6.0)
3.65, dd, (12.5, 4.5)
3.87, d, (12.5)
3.84, dd, (12.0, 1.0)
3.65, dd, (12.0, 5.5)
3
.88, dd, (12.0, 2.0)
Api-1
Api-2
3
.95, d, (9.5)
Api-4
Api-5
3.75, dd, (9.5, 2.0)
3.55, m
3.75, d, (9.5)
3.55, m
a
Assignments were carried out based on HSQC and HMBC experiments.
Overlapped with H O signal.
2
b
−
3
1
C
400, 2935, 1653, 1650, 1076 cm 1; 1H and 13C NMR data, see Tables
2
analyzed using silica plate developed with EtOAc−MeOH−H O
(7:5:1). Glucose was detected from the hydrolysate of 1, Glucose and
apiose were detected from the hydrolysates of 3 and 4.
−
and 2; negative-ion HRESIMS: m/z 419.1483 [M + Cl] (calcd for
19
H
28
O
8
Cl, 419.1478).
Urenalobaside C (3): Colorless gum, [α]
D
25: −52.4 (c 0.05, MeOH);
UV (MeOH) λmax (log ε): 206 (3.66), 229 (3.48), 282 (3.78); IR (KBr)
2.6. Enzymatic hydrolysis of compounds 1, 3, and 4
−
1
1
13
ν
max: 3410, 2935, 1653, 1650, 1056 cm
;
H and C NMR data, see
−
Tables 1 and 2; negative-ion HRESIMS: m/z 537.2092 [M + Cl] (calcd
Each compound (1, 3, and 4) 2.0 mg was dissolved in citric acid
for C24
38
H O11Cl, 537.2108).
buffer solution (pH 4.5, 60 mL), and incubated with 4.5 mg cellulase at
7 °C for 16 h. After stopped the reaction by the addition of MeOH, the
Urenalobaside D (4): Colorless gum, [α]
D
25: −54.0 (c 0.05, MeOH);
3
UV (MeOH) λmax (log ε): 206 (3.52), 230 (3.33), 288 (3.62); IR (KBr)
reaction mixture was extracted with EtOAc. Residue obtained from the
organic layer was purified by HPLC (35% MeCN) to obtain the aglycons
A1 (0.3 mg, t 13.0 min), A3 (0.8 mg, t 12.0 min), and A4 (0.6 mg, t
R R R
−
1
1
13
ν
max: 3375, 2930, 1655, 1650, 1082 cm
;
H and C NMR data, see
−
Tables 1 and 2; negative-ion HRESIMS: m/z 537.2106 [M + Cl] (calcd
for C24
38
H O11Cl, 537.2108).
1
4.0 min).
.7. ECD calculations
A preliminary conformational analysis was performed with the
Urenalobaside E (5): Colorless gum, [α]
D
25: −24.0 (c 0.05, MeOH);
UV (MeOH) λmax (log ε): 202 (2.86), 227 (2.84); IR (KBr) νmax: 3397,
2
−1
1
13
2
2
C
935, 1653, 1650, 1056 cm ; H and C NMR data, see Tables 1 and
−
;
negative-ion HRESIMS: m/z 423.1770 [M + Cl]
Cl, 423.1791).
(calcd for
19
H
32
O
8
SYBYL-X 2.0 software package using the random search method with
the MMFF94s force field. The conformers were further optimized by
using the TDDFT method at the B3LYP/6-31G(d) level, and the fre-
quency was calculated at the same level of theory. The stable con-
formers without imaginary frequencies were subjected to ECD calcu-
lation by the TDDFT method at the B3LYP/6-31 + G(d) level with the
CPCM model in MeOH. ECD spectra of different conformers were si-
mulated using SpecDis v1.51with a half-band width of 0.3 eV, and the
final ECD spectra were obtained according to the Boltzmann calculated
contribution of each conformer (S38). All calculations were performed
with the Gaussian 09 program package [12,13].
2
.4. Biological assays
The murine macrophage RAW264.7 cells were purchased from
Peking Union Medical College (PUMC) Cell Bank (Beijing, China). Cell
maintenance, experimental procedures, and data determination for the
inhibition of NO production and the viability assay are the same as
previously described [9–11]. The IC50 values were determined using
GraphPad Prism 5 software from experiments performed in triplicate
(
GraphPad Software, Inc., San Diego, CA, USA). Dexamethasone was
used as a positive control (16.6 ± 0.8 μM). All the compounds were
prepared as stock solutions in DMSO (final solvent concentration less
than 0.2% in all assays).
3. Results and discussion
2
The 95% EtOH extract of the U. lobata was suspended in H O and
2
.5. Acid hydrolysis of compounds 1, 3, and 4
extracted successively with petroleum ether, EtOAc, and n-BuOH. The
n-BuOH soluble fraction was fractionized by D101 macroporous ad-
sorption resin, and repeatedly separated by silica gel and Sephadex LH-
20 column chromatography, and purified by semi-preparative HPLC to
afford five new megastigmane glycosides (1–5) together with 11 known
Each compound (1, 3, and 4) 2.0 mg was dissolved in 1 M HCl
(
3.0 mL) and heated at 90 °C for 3 h. The reaction mixture was cooled to
room temperature and extracted with EtOAc. The aqueous layer was
3

C. Su et al.
Fitoterapia xxx (xxxx) xxx–xxx
ones (Fig. 1).
Compound 1 was obtained as a pale yellow gum, [α]
28 8
.05, MeOH). Its molecular formula was assigned as C19H O
D
25–55.4 (c
by the
M + Cl] ion peak at m/z 419.1469 in the HRESIMS spectrum, which
0
[
−
1
3
1
was supported by the C NMR data (Table 1). The H NMR spectrum of
1
H
showed the presence of three methyls at δ
-10), 1.27 (3H, s, H -11), and 1.16 (3H, s, H
2.38 (1H, d, J = 16.0 Hz, H-2), 2.26 (1H, d, J = 16.0 Hz,
H-2), two oxygen-bearing methines at δ 4.80 (1H, m, H-13) and 4.07
1H, m, H-9), one methine at δ 2.83 (1H, m, H-8), and two olefinic
5.91 (1H, s, H-4), respectively.
H
1.31 (3H, d, J = 6.0 Hz,
3
3
3
-12), one methylene
signals at δ
H
H
(
H
protons at δ
H
6.37 (1H, br s, H-7) and δ
H
In addition, protons tentatively assigned to a glucopyranosyl moiety
1
were observed in the H NMR spectrum of 1, the anomeric proton was
presented at δ
H
4.36 (1H, d, J = 8.0 Hz), corresponding to the carbon
102.2 in the HSQC spectrum. Acid hydrolysis of 1 with
presented at δ
C
1
M HCl afforded glucose, revealing the occurrence of a glucopyranosyl
moiety in 1. Irrespective of the carbons due to the glycopyranosyl
1
3
moiety, the C NMR spectrum of 1 showed the presence of 13 carbons
including four conjugated olefinic carbons, one carbonyl carbon, three
methyls, three methines, one methylene, and one quaternary carbon,
which suggested that the aglycon of 1 might be a megastigmane-type
Fig. 3. Exprimental and calculated ECD spectra of 1, A1, and 2 (in MeOH).
invalidity of the glycosyl moiety to the ECD absorption. The presence of
a typically negative Cotton effect at 295 nm in the experimental ECD
spectrum of A1 allowed establishing the S configuration of C-13. In the
NOESY spectrum of 1, the NOE correlations of H-8/H-13 revealed a cis-
orientation of H-8 and H-13. Accordingly, the absolute configuration of
C-8 was assigned as R. The absolute configuration of C-9 should be
resolved using Mosher's method. Unfortunately, the preparation of (R/
S)-MTPA esters were failed due to limited amount of A1. Therefore, the
structure of 1 was elucidated as shown in Fig. 1, named as urenaloba-
side A. As far as our knowledge, megastigmanes with a 6/5 fused ring
system have never been reported.
28 8
sesquiterpene. The molecular formula (C19H O ) of 1 revealed that the
index of hydrogen deficiency (IHD) of 1 was six, four of which were
contributed to two double bonds, one carbonyl group, and one gluco-
pyranosyl moiety. The remaining two IHD suggested the presence of
two carbocyclic rings in 1. All the protons and carbons were un-
ambiguously assigned (Tables 1 and 2) by 2D NMR experiments, in-
1
1
1
1
cluding H- H COSY, HSQC, HMBC, and NOESY. In the H- H COSY
spectrum, the correlation between H-8 and H-13 strongly suggested
that a C-C bond was formed between C-8 and C-13, which was con-
firmed by HMBC correlations of H-7/C-13 and H-9/C-13 (Fig. 2). The
linkage of the aglycon and the glucopyranosyl moiety was established
by the long range correlation between the anomeric proton Glu-H-1 and
C-9, which was further confirmed by the NOESY correlation between H-
2
5
Compound 2 was obtained as a pale yellow gum, [α]
D
–14.1 (c
28 8
H O by the
0.05, MeOH). Its molecular formula was assigned as C19
−
[M + Cl] ion peak at m/z 419.1483 in the HRESIMS spectrum.
Comparison of the NMR data of 2 with those of 1 revealed that com-
pound 2 shared a very similar skeleton with that of 1. Unambiguous
assignment of the protons and carbons of 2 by 2D NMR experiments
allowed establishing the planar structure of 2, which was completely
same to that of 1. However, the quite different retention time (S9)
suggested that 1 and 2 may be stereoisomers. In the experimental ECD
spectrum of 2, the presence of a positive Cotton effect at 295 nm sug-
gested that the absolute configuration of C-13 was R. Considering that
the NOE correlation of H-8/H-13 could not be found in the NOESY
spectrum of 2, trans-oritentation of H-8 and H-13 was arbitrarily as-
signed, resulting in a tentatively determination of the absolute config-
uration of C-8 as R. Therefore, the structure of 2 was elucidated as
shown in Fig. 1, named as urenalobaside B.
9
and Glu-H-1.The relatively large coupling constant of the anomeric
proton (J = 8.0 Hz) suggested that the anomeric carbon of the gluco-
pyranoyl moiety was in β configuration.
In order to resolve the absolute configuration of 1, the electronic
circular dichroism (ECD) spectra of the aglycon of 1 (A1) were calcu-
lated using TDDFT at the B3LYP/6-31G level with the CPCM model in
MeOH. Analysis of the calculated ECD spectra of eight isomers (S38)
revealed that the ECD spectrum of A1 was crucially depended on the
absolute configuration of C-13 but little concern with the configurations
of C-8 and C-9. When the configuration of C-13 was R, positive Cotton
effect could be found at 295 nm in the calculated ECD spectrum of A1.
In contrast, negative Cotton effect was presented at 295 nm, corre-
sponding to the S configuration of C-13. To obtain the experimental
ECD spectrum of A1, we have to prepare the aglycon by hydrolysis of 1.
However, analysis of the hydrolysate by LC-MS revealed that acid hy-
drolysis produced a degradation product but not the expected aglycon
A1. Therefore, enzymatic hydrolysis of 1 by cellulase was performed,
leading to the generation of the proto-aglycon A1 with a molecular
Compound 3 was obtained as a colorless gum, [α]
D
25–52.4 (c 0.05,
MeOH). Its molecular formula was assigned as C24
H
38
O
11 by the
1
−
[M + Cl] ion peak at m/z 537.2092 in the HRESIMS spectrum. The H
NMR spectrum exhibited the presence of two quaternary methyls at
δ
H
1.30 (6H, br s, H
13), a secondary methyl at δ
methylenes at δ 2.34 (2H, s, H
-8), one oxygen-bearing methines at δ
3
-11, H
3
-12), a vinyl methyl at δ
1.26 (1H, d, J = 6.0 Hz, H
–2) and 2.68 (2H, dd, J = 12.0, 6.5 Hz,
3.16 (1H, dd, J = 12.0,
H
2.14 (3H, s, H
3
-
formula of C13
A1 by HPLC, the experimental ECD spectra of A1 and 1 were collected
Fig.3). The superposable ECD spectra of 1 and A1 revealed the
H
18
O
3
assigned by HRESIMS (S8). After purification of
H
3
-10), two
H
2
(
H
2
H
Fig. 2. Key HMBC correlations of compounds1, 3, and 5.
4

C. Su et al.
Fitoterapia xxx (xxxx) xxx–xxx
6
.0 Hz, H-9), and two olefinic protons at δ
t, J = 6.5 Hz, H-7). In addition, two anomeric protons were observed at
4.34 (1H, d, J = 8.0 Hz) and 4.99 (1H, d, J = 2.5 Hz), corresponding
to the carbons at δ 102.5 and 111.0 in the HSQC spectrum, respec-
tively. Acid hydrolysis of 3 with 1 M HCl afforded glucose and apiose.
H
5.87 (1H, s, H-4), 6.36 (1H,
apiofuranosyl-(l → 6)-β-D-glucopyranoside (14) [19], (6S,7E,9ξ)-6,9,10-
trihydroxy-4,7-megastigmadien-3-one10-O-β-D-glucopyranoside (15)
[23], bridelionoside A (16) [24], respectively.
Compounds 1, 3–16 were evaluated for their inhibitory effects on
the NO production in LPS-stimulated RAW264.7 macrophage cells.
Compounds 3 exhibited weak inhibition of NO production with IC50
value of 53.7 ± 1.0 μM.
δ
H
C
1
3
The C NMR spectrum showed the presence of 24 carbons, 13 of which
were assigned to the aglycon, and the other 11 carbons were assigned to
glucopyranosyl and apiofuranosyl moieties. All the protons and carbons
were unambiguously assigned by analysis of the 1D and 2D NMR
spectra. In the HMBC spectrum, the correlations of H-4/C-6, H-7/C-5,
Conflict of interest
4
,5
6,7
C-6, C-8, and H
3
-13/C-4, C-5, C-6 established the Δ and Δ con-
The authors declare no conflict of interest.
Acknowledgments
jugated double bonds, the HMBC correlations of H-2/C-3 determined
the carbonyl group was at C-3. Additionally, the HMBC correlation of
Glu-H-1/C-9 established that the glucopyranosyl moiety was linked at
C-9 of the aglycon, the HMBC correlation of Api-H-1/Glu-C-6 estab-
lished that the apiofuranosyl miety was linked at C-6 of the glucopyr-
anosyl moiety (Fig. 2). In the NOESY spectrum, the correlation of H-7/
H-13 suggested a E configuration of Δ double bond. Enzymatic hy-
drolysis of 3 by cellulase afforded the known compound (6E,9S)-9-hy-
droxy-4,6-megastigmadien-3-one (S22–23) [14,15]. Accordingly, the
structure of 3 was determined as shown in Fig. 1, named as ur-
enalobaside C.
This work was financially supported by the National Natural Science
Foundation of China (No.81573312), the National Key Technology R&D
Program “New Drug Innovation” of China (No. 2018ZX09711001-006-
009).
6
,7
Appendix A. Supplementary data
The NMR and HRESIMS spectra for compounds 1–5, the aglycon of
compounds 3 and 4 (A3, A4), and the calculated ECD spectra of the
eight stereoisomers of 1 are available in the online version.
Compound 4 was obtained as a colorless gum, [α]
D
25–54.0 (c 0.05,
MeOH). The HRESIMS spectrum showed the presence of a quasimole-
cular ion peak [M + Cl] at m/z 537.2106, consistent with an em-
−
Supplementary data associated with this article can be found in the
pirical molecular formula of C24
H
38
O
11. Comparison of the NMR data of
online version, at https://doi.org/10.1016/j.fitote.2018.02.017
4
with those of 3 revealed that 4 shared a similar skeleton to 3. The only
6
,7
difference was that the E configuration of Δ double bound in 3 was
changed into Z configuration in 4, which was confirmed by the NOESY
correlation of H-7/H-11. Enzymatic hydrolysis of 4 by cellulase af-
forded the known compound (6Z,9S)-9-hydroxy-4,6-megastigmadien-3-
one (S30–31) [14,15]. Accordingly, the structure of 3 was determined
as shown in Fig. 1, named as urenalobaside D.
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