New monoterpene glycosides from Paeonia suffruticosa Andrews and their inhibition on NO production in LPS-induced RAW 264.7 cells

Article abstract of DOI:10.1016/j.bmcl.2012.09.034

Nine new monoterpene glycosides (1-9), together with 18 known compounds were obtained from the n-butanol soluble fraction of ethanol extract from Paeonia suffruticosa Andrews. Their structures were determined on the basis of chemical methods and spectrosc

Full text of DOI:10.1016/j.bmcl.2012.09.034

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7243–7247
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Bioorganic & Medicinal Chemistry Letters
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New monoterpene glycosides from Paeonia suffruticosa Andrews and their
inhibition on NO production in LPS-induced RAW 264.7 cells
Liqin Ding a,b, Feng Zhao c, Lixia Chen a,b, Zhihu Jiang a,b, Yue Liu a,b, Zhuomin Li a,b, Feng Qiu a,b,
,
⇑
Xinsheng Yao a,b,
⇑
a Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang 110016, People’s Republic of China
b Key Laboratory of Structure-Based Drug Design & Discovery (Ministry of Education), Shenyang Pharmaceutical University, People’s Republic of China
c School of Pharmacy, Yantai University, Laishan District, Yantai 264005, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2012
Revised 31 August 2012
Accepted 11 September 2012
Available online 25 September 2012
Nine new monoterpene glycosides (1–9), together with 18 known compounds were obtained from the
n-butanol soluble fraction of ethanol extract from Paeonia suffruticosa Andrews. Their structures were
determined on the basis of chemical methods and spectroscopic data. The inhibitory effects of these
compounds (except compound 6) on nitric oxide (NO) production in lipopolysaccharide-activated
macrophages were evaluated. Compounds 1, 21, 23, 25, and 27 showed strong inhibitory activity on
NO production with IC50 values of 31.25, 36.13, 12.41, 6.87, and 41.94 lM, respectively.
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Paeonia suffruticosa Andrews
Monoterpene glycosides
Nitric oxide
The root cortex of Paeonia suffruticosa Andrews (Ranunculaceae)
(Chinese name: mudanpi), as one of the most important crude
drugs having anticoagulant, antiinflammatory, analgesic, and sed-
ative activities, has been used as a remedy for cardiovascular,
extravasated blood, stagnated blood and female genital diseases
in Oriental traditional medicine.1–4 ‘Cagelike’ monoterpenes are
one of the major biologically active components existing in Paeonia
species. In the course of searching for bioactive substances from
P. suffruticosa, nine new monoterpenes were isolated and iden-
n-BuOH-soluble part.5 Their structures were elucidated through
physicochemical and spectroscopic methods.
Compound 16 was obtained as a white amorphous powder. The
molecular formula of 1 was determined as C36H42O17 by HR-ESI-
MS at m/z 769.2322 [M+Na]+ (calcd for C36H42O17Na, 769.2314)
and NMR data (Table 1). The 1H NMR spectrum of 1 displayed a
methyl group at dH 1.27 (3H, s), an acetal proton at dH 5.45 (1H,
s), and two anomeric protons at dH 4.66 (1H, d, J = 7.6 Hz) and
4.46 (1H, d, J = 7.8 Hz). The 1H and 13C NMR data as well as acid
hydrolysis and GC comparison with an authentic sample indicated
tified as paeoniflorin
B (1), oxypaeoniflorin sulfonate (2),
4-O-methyloxypaeoniflorin (3), 4-O-methylgalloyloxypaeoniflorin
(4), oxypaeonidanin (5), 9-epi-oxypaeonidanin (6), 9-O-butyloxyp-
aeonidanin (7), 9-O-butylpaeonidanin (8), and 4-O-butyloxypaeon-
iflorin (9), together with 18 known analogs. This Letter herein
describes the isolation and structural elucidation of these new
compounds, and reports the inhibitory effect of these compounds
on NO production in LPS-activated macrophages.
The roots of P. suffruticosa were extracted with EtOH–H2O (7:3,
v/v). The resulting ethanolic extract was concentrated in vacuo,
suspended in H2O, and then successively partitioned with cyclo-
hexane, ethyl acetate and n-BuOH to give the corresponding
extracts. By using combined chromatographic separations, nine
new and eighteen known compounds were isolated from
the presence of a D
-glucopyranosyl moiety.7,8 The b configuration
of the glycosidic linkage was determined from the coupling con-
stant of the anomeric proton. The 13C NMR spectrum displayed
36 carbons including two benzoate moieties, two acetal carbons
at dC 102.3 and 106.3. The 13C NMR spectrum was similar to that
of benzoylpaeoniflorin,9 except for the presence of a glucose moi-
ety, indicating that they had the same molecular skeleton. The
chemical shift of C-40 was downfield to d 81.5, indicating that the
glucose moiety was attached to C-40. This deduction was further
supported by HSQC and HMBC spectra. The HMBC correlations
from H-3 to C-2, C-4, C-5, C-10, from H-7 to C-1, C-2, C-4, C-5, from
H-9 to C-2, C-4, C-6, C-8 and from H-10 to C-1, C-2, C-3 could be
observed. The HMBC correlations from H-10 to C-1, from H-100 to
C-40, from H-8 to C-7000 and from H-60 to C-70000 confirmed that the
glucose moiety (anomeric carbon at dC 100.0) was connected to
C-1, and the other glucose moiety (anomeric carbon at dC 105.0)
was connected to C-40; the benzoyl moiety (carbonyl at dC 168.1)
was connected to C-8, and the other benzoyl moiety (carbonyl at
⇑
Corresponding authors. Tel.: +86 24 23986463; fax: +86 24 23993994 (F.Q.).
E-mail addresses: fengqiu20070118@163.com (F. Qiu), tyaoxs@jnu.edu.cn
(X. Yao).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.034

7244
L. Ding et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7243–7247
Table 1
1H and 13C NMR spectroscopic data for compound 1a
No.
1Hb
13Cc
89.5
No.
1Hb
13Cc
1
2
3
4
5
6
7
8
100
4.46 (d, 7.8)
3.41 (m)
3.41 (m)
3.36 (m)
105.0
74.7
78.4
71.5
78.0
87.2
44.5
106.3
43.9
72.1
23.1
61.7
102.3
19.7
100.0
74.0
76.4
81.5
75.0
64.7
200
1.73 (d, 10.9) 1.89 (d, 12.5)
2.57 (d, 6.4)
300
400
500
3.41 (m)
600
3.72 (dd, 11.8, 6.2) 3.95 (dd, 11.8, 1.8)
62.7
1.73 (d, 10.9) 2.52 (m)
4.76 (dd, 17.4, 12.2)
5.45 (s)
1.27 (s)
4.66 (d, 7.6)
3.83 (t, 7.3)
3.61 (t, 8.8)
3.66 (t, 8.8)
3.33 (m)
1000
131.4
130.8
129.9
134.6
168.1
131.3
130.7
129.8
134.5
167.7
2000/6000
3000/5000
4000
8.08 (d, 7.3)
7.53 (t, 7.3)
7.65 (t, 7.3)
9
10
10
20
30
40
50
60
7000
10000
20000/60000
30000/50000
40000
8.08 (d, 7.3)
7.53 (t, 7.3)
7.65 (t, 7.3)
4.66 (dd, 13.9, 4.4) 4.89 (m)
70000
a
b
c
The data were measured in CD3OD.
600 MHz for 1H NMR; the coupling constants (J) are in parentheses and reported in Hz; chemical shifts are given in ppm.
75 MHz for 13C NMR.
Table 2
1H NMR spectroscopic data for compounds 2–6c
No.
3
2a
3b
4a
5b
6b
2.73 (d, 12.8)
2.92 (m)
3.94 (m)
2.45 (d, 10.7)
2.92 (m)
5.06 (m)
2.13 (d, 12.0)
1.93 (br d, 12.0)
2.75 (d, 6.6)
1.90 (d, 10.2)
2.47 (dd,10.2, 6.6)
4.70 (s)
2.16 (d, 11.1)
2.18 (d, 12.4)
2.98 (d, 6.5)
2.10 (d, 10.7)
2.75 (dd, 10.7, 7.0)
4.97 (d, 12.2)
5.07 (d, 5.7)
5.83 (s)
2.47 (d, 18.2)
2.81 (d, 18.2)
2.91 (m)
2.14 (d, 9.8)
2.91 (m)
4.66 (d, 11.9)
4.76 (d, 11.9)
5.00 (s)
3.05 (m)
5
7
2.98 (s)
2.39 (m)
3.01 (m)
4.58 (d, 11.3)
4.80 (d, 13.2)
4.78 (s)
8
9
5.90 (s)
5.43 (s)
10
10
20
30
1.56 (s)
1.37 (s)
1.66 (s)
5.07 (d, 6.8)
3.97 (m)
1.42 (s)
4.55 (d, 7.6)
3.25 (m)
1.45 (s)
4.54 (d, 7.6)
3.25 (m)
4.55 (d, 7.6)
3.96 (m)
4.13 (m)
4.53 (d, 7.2)
3.20 (d, 9.0)
3.22 (m)
3.23 (m)
4.18 (m)
3.35 (m)
3.25 (m)
40
50
60
4.13 (m)
3.83 (m)
4.29 (m)
4.44 (d, 10.0)
8.19 (d, 8.5)
7.08 (d, 8.5)
4.18 (m)
4.05 (m)
3.25 (m)
3.25 (m)
3.85 (dd, 11.7, 6.9)
3.60 (dd, 11.7, 2.4)
7.87 (d, 8.7)
6.82 (d, 8.7)
3.25 (m)
3.35 (m)
3.61 (dd, 10.8, 5.6)
3.85 (d, 10.8)
7.83 (d, 8.7)
3.30 (m)
3.84 (d, 12)
3.60 (d, 9.0)
6.83 (d, 8.4)
7.90 (d, 8.4)
4.95 (d, 6.4)
5.10 (d, 10.6)
8.19 (d, 8.8)
7.09 (d, 8.8)
7.90 (s)
200/600
300/500
6.81 (d, 8.7)
2000/6000
–OCH3
3.37 (s)
3.41 (s)
3.28 (s)
3.35 (s)
a
b
c
C5D5N was used as solvent.
CD3OD was used as solvent.
300 MHz for 1H NMR; the coupling constants (J) are in parentheses and reported in Hz; chemical shifts are given in ppm.
dC 167.7) was connected to C-60. The relative configuration of 1 was
determined by the NOE correlations between H-8 and both H-7
and H-9, and between H-10 and H-3. On the basis of the 1H, 13C
and 2D NMR (HSQC, HMBC, NOESY) data, the structure of 1 was
unambiguously established and named paeoniflorin B.
to C-8. The HMBC correlations from H-3 to C-2, C-4, C-5, from
H-7 to C-2, C-4, C-5, from H-9 to C-2, C-4, C-5, C-6, C-8, from
H-10 to C-1, C-2, C-3, and from H-8 to C-1, C-5, C-6, C-9 could be
observed. The HMBC correlations from H-10 to C-1 confirmed that
the glucose moiety was connected to C-1. From these spectroscopic
data, the structure of 2 was unambiguously deduced to be oxypae-
oniflorin sulfonate.
The HR-ESI-MS of compound 210 showed a quasimolecular ion
peak at m/z 583.1094 [M+H+Na]+, corresponding to the elemental
composition of the quasimolecular ion
C
23H28O14NaS+ (calcd
The molecular formula of compound 3,12 C24H30O12, was estab-
lished by HR-ESI-MS (m/z 533.1634 [M+Na]+) and NMR data. The
1H NMR spectrum displayed a methyl group at dH 1.37 (3H, s), a
methoxy group at dH 3.37 (3H, s), an acetal proton at dH 5.43
(1H, s), and an anomeric proton at dH 4.53 (1H, d, J = 7.2 Hz). The
583.1092). The 1H NMR spectrum of 2 revealed a methyl group
at dH 1.56 (3H, s), an acetal proton at dH 5.90 (1H, s), and an ano-
meric proton at dH 4.55 (1H, d, J = 7.6 Hz). The sugar unit was iden-
tified to be b-D
-glucopyranose as that in 1. The 13C NMR spectrum
displayed 23 carbons including a p-hydroxybenzoyl moiety, two
acetal carbons at dC 95.4 and 105.1. The NMR spectra were similar
to those of sodium paeoniflorin sulfonate,11 except for the presence
of a p-hydroxybenzoyl moiety in 2 instead of a benzoyl moiety in
sodium paeoniflorin sulfonate. The HMBC cross-peak from H-8 to
C-700 confirmed that the p-hydroxybenzoyl moiety was connected
sugar unit was identified to be b-D-glucopyranose as that in 1.
The 13C NMR spectrum displayed 24 carbons including a p-hydrox-
ybenzoyl moiety, and two acetal carbons at dC 101.6 and 108.5. The
NMR spectra were closely similar to those of oxypaeoniflorin,13 ex-
cept for the presence of a methoxy group. The HMBC correlations
from the methoxy protons to C-4 confirmed that the methoxy

L. Ding et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7243–7247
7245
Table 3
between H-8 and H-9, and between H-3 and both H-3 and the
methoxy protons. From these spectroscopic data, compound 3
was identified as 4-O-methyloxypaeoniflorin.
13C NMR spectroscopic data for compounds 2–6c
No.
2a
3b
4a
5b
88.7
87.7
50.0
209.8
48.5
65.3
27.7
63.5
107.8
20.8
100.1
75.2
78.2
71.9
78.2
63.0
122.1
133.1
116.4
163.9
168.1
6b
85.4
88.4
51.2
211.6
49.4
65.1
30.3
62.1
106.2
21.1
100.4
75.1
78.2
71.9
78.1
63.0
122.0
133.0
116.5
164.0
168.0
The HR-ESI-MS of compound 414 exhibited a unique [M+Na]+
ion peak at m/z 685.1744, corresponding to the molecular formula
of C31H34O16 (calcd for C31H34O16Na, 685.1745). The 1H and 13C
NMR spectra of 4 were similar to those of compound 3 except
the presence of the galloyl moiety (Tables 2 and 3). The chemical
shift of C-60 was downfield for 2.7 ppm, and a galloyl moiety was
observed in the 13C NMR spectrum of 4. The HMBC correlation
from H-60 to C-7000 (d 167.2) confirmed that the galloyl moiety
was connected to C-60. On the basis of the 1H, 13C, and 2D NMR
(HSQC, HMBC, NOESY) data, the structure of compound 4 was
determined as 4-O-methylgalloyloxypaeoniflorin.
1
2
3
4
5
6
7
8
89.0
88.2
88.7
88.0
40.4
86.2
41.3
86.1
41.5
95.4
43.2
108.5
40.0
108.6
41.1
72.5
70.9
71.0
24.8
22.5
22.6
61.0
60.2
60.6
9
105.1
20.2
101.6
18.6
102.0
19.8
10
10
100.6
75.1
99.2
74.0
100.3
74.8
20
30
78.7
77.0
78.3
Compound 514 was obtained as a white amorphous powder. The
molecular formula of 5 was determined to be C24H30O12 on the ba-
sis of HR-ESI-MS (m/z 533.1639 [M+Na]+) and NMR data. Compar-
ison of the 1H and 13C NMR data with those of paeonidanin9
showed many similarities except for the absence of a benzoyl moi-
ety and the appearance of a p-hydroxybenzoyl moiety, suggesting
that the p-hydroxybenzoyl moiety was located at C-8. This deduc-
tion was confirmed by HSQC and HMBC spectra. The HMBC corre-
lation from H-8 to C-700 was observed. The NOE correlations
between H-8 and both H-7 and H-9, between H-10 and both H-3
and the methoxy protons established the relative configuration
of compound 5. On the basis of these data, compound 5 was con-
cluded to be oxypaeonidanin.
40
71.9
70.7
71.9
50
78.5
77.0
75.2
60
63.0
61.9
64.6
100
121.6
132.7
116.4
163.8
166.7
121.0
131.9
115.3
162.7
167.0
121.3
132.5
116.2
163.7
166.6
121.3
110.3
147.7
141.1
167.2
51.3
200/600
300/500
400
700
1000
2000/6000
3000/5000
4000
7000
–OCH3
50.4
56.0
57.4
a
b
c
C5D5N was used as solvent.
CD3OD was used as solvent.
75 MHz for 13C NMR.
The HR-ESI-MS of 614 showed a quasimolecular ion [M+Na]+ at
m/z 533.1622. Taking into account the 1H and 13C NMR spectra, the
molecular formula was established as C24H30O12. The NMR spectra
of 6 were similar to those of 9-epi-paeonidanin,9 except that the
benzoyl moiety connected to C-8 was absent in 6 and replaced
group was connected to C-4. The relative configuration of 3 was
determined by the NOE correlations between H-7 and H-8,
Table 4
1H and 13C NMR spectroscopic data for compounds 7–9a
No.
7
8
9
1Hb
13Cc
88.7
1Hb
13Cc
88.7
1Hb
13Cc
89.3
1
2
3
87.5
49.9
87.5
50.0
87.4
43.0
2.48 (d, 18.2)
2.90 (m)
2.50 (d, 18.2)
2.90 (m)
1.93 (m)
2.14 (d, 12.5)
4
5
6
7
209.7
48.8
65.5
27.7
209.6
48.8
65.5
27.6
109.5
41.5
71.9
23.6
3.00 (m)
3.00 (m)
2.74 (d, 6.4)
2.16 (d, 10.4)
2.93 (m)
4.66 (d, 11.9)
4.82 (m)
2.19 (d, 10.6)
2.95 (m)
4.73 (d, 11.9)
4.90 (m)
2.46 (dd, 10.9, 7.0)
1.90 (d, 10.9)
4.70 (s)
8
63.6
64.1
61.4
9
5.16 (s)
1.42 (s)
4.56 (d, 7.5)
3.27 (m)
3.39 (m)
3.27 (m)
3.27 (m)
3.85 (d, 10.4)
3.63 (m)
106.8
20.8
100.1
75.2
78.2
71.9
78.2
63.0
5.21 (s)
1.44 (s)
106.8
20.9
100.1
75.2
78.2
71.9
78.2
63.0
5.42 (s)
1.37 (s)
102.7
19.8
100.1
75.2
78.2
72.0
78.1
63.0
10
10
20
30
40
50
60
4.59 (d, 7.6)
3.29 (m)
3.40 (m)
3.29 (m)
3.31 (m)
3.84 (m)
3.64 (m)
4.53 (d, 7.5)
3.21 (m)
3.32 (m)
3.24 (m)
3.24 (m)
3.85 (m)
3.61 (m)
100
122.1
133.2
116.3
163.9
168.1
69.4
131.3
130.9
129.7
134.6
168.0
69.4
122.1
133.1
116.4
164.0
168.2
64.5
200/600
300/500
400
7.89 (d, 8.6)
6.82 (d, 8.6)
8.05 (d, 7.2)
7.49 (t, 7.4)
7.63 (t, 7.4)
7.90 (d, 8.6)
6.84 (d, 8.6)
700
1000
3.33 (m)
3.65 (m)
1.41 (m)
1.24 (m)
0.82 (t, 7.3)
3.33 (m)
3.65 (m)
1.41 (m)
1.23 (m)
0.82 (t, 7.3)
3.60 (m)
2000
3000
4000
32.7
20.4
14.3
32.7
20.4
14.3
1.52 (m)
1.51 (m)
0.89 (t, 7.2)
33.2
20.3
14.3
a
b
c
The data were measured in CD3OD.
300 MHz for 1H NMR; the coupling constants (J) are in parentheses and reported in Hz; chemical shifts are given in ppm.
75 MHz for 13C NMR.

7246
L. Ding et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7243–7247
Figure 1. Structures of compounds 1–27.
Table 5
Inhibitory effect of compounds isolated from Paeonia suffruticosa Andrews on NO production induced by LPS in macrophages (IC50: l
M)a
Compound
IC50 SD
Compound
IC50 SD
Compound
IC50 SD
1
2
3
4
5
7
8
9
10
31.25 1.78
>100
>100
>100
90.88 5.73
>100
>100
>100
>100
11
12
13
14
15
16
17
18
19
>100
>100
>100
>100
>100
>100
74.22 4.89
>100
>100
20
21
22
23
24
25
26
27
>100
36.13 2.10
>100
12.41 1.09
>100
6.87 0.41
>100
41.94 2.35
40.64 3.22
Hydrocortisoneb
a
NO concentration of control group: 4.38 0.81
Positive control.
lM. NO concentration of LPS treated group: 45.71 10.95 lM.
b
by a p-hydroxybenzoyl moiety, indicating that the p-hydrox-
ybenzoyl moiety was connected to C-8. The HMBC cross-peak from
H-8 to C-700 confirmed the above presumption. The relative config-
uration of 6 was established by the NOE correlations observed be-
tween H-9 and H-8, between H-7 and H-8, and between H-10 and
H-3, while the correlation between the methoxy protons and H-10
was not observed. On the basis of the 1H, 13C, and 2D NMR (HSQC,
except that the benzoyl moiety connected to C-8 was absent in 9
and replaced by a p-hydroxybenzoyl moiety, indicating that the
p-hydroxybenzoyl moiety was connected to C-8. The HMBC corre-
lations from H-8 to C-700 confirmed that the p-hydroxybenzoyl
moiety was connected to C-8. On the basis of all the evidences,
compound 9 was elucidated as 4-O-butyloxypaeoniflorin.
In addition to nine new monoterpene glycosides (1–9), 18
known ones (Fig. 1), paeoniflorin (10),11,16 paeonidanin (11),9
4-O-methylpaeoniflorin (12),16 mudanpioside E (13),13 oxypaeoni-
florin (14),13 benzoylpaeoniflorin (15),9 mudanpioside D (16),13 8-
O-debenzoylpaeoniflorin (17),17 galloyloxypaeoniflorin (18),18
mudanpioside H (19),19 mudanpioside J (20),20 galloylpaeoniflorin
(21)21, moudanpioside B (22),13 moudanpioside F (23),13 4-O-
butylpaeoniflorin (24),15 paeoniflorigenone (25),22 paeonidanin A
(26),7 and 4-O-methylbenzoylpaeoniflorin (27)7 were isolated
and identified by comparison of their spectroscopic data with
those reported in the literatures.
HMBC, NOESY) data, the structure of
9-epi-oxypaeonidanin.
6 was identified as
Analyses of the 1H and 13C NMR spectra of compounds 714 and
814 (Table 4), revealed that 7 and 8 had the same characteristic
molecular skeleton as compound 5 and paeonidanin,9 respectively.
The main difference among compounds 7, 8 and compound 5 and
paeonidanin is that the presence of an n-butyl group at C-9 in 7 and
8 instead of a methyl group in compound 5 and paeonidanin,
respectively. The HMBC correlation from H-9 to C-1000 (d 69.4) con-
firmed that the n-butyl group was connected to C-9. On the basis of
these data, the structures of 7 and 8 were determined as 9-O-
butyloxypaeonidanin and 9-O-butylpaeonidanin, respectively.
The HR-ESI-MS of 914 gave a quasimolecular ion [M+Na]+ at m/z
575.2098. Taking into account the 1H and 13C NMR spectra, the
molecular formula was determined as C27H36O12. The 13C NMR
spectrum was closely similar to that of 4-O-butylpaeoniflorin,15
All isolated compounds except compound 6 (due to limit
amount of sample) were evaluated for their inhibitory effects on
NO production induced by LPS in macrophages (Table 5). In addi-
tion, the cell viability in the present experiment was determined
by the MTT method in order to discover whether the inhibitory ef-
fects on NO production were due to the cytotoxicity of the test

L. Ding et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7243–7247
7247
compounds.23 As shown in Table 5, comparing with positive con-
trol hydrocortisone (IC50 40.64 3.22 M), compounds 1, 21, 23,
from 100 °C up to 140 °C at a rate of 4 °C/min in 10 min, then increasing up to
170 °C at a rate of 13 °C/min, maintained for 10 min, finally up to 200 °C at a
l
rate of 5 °C/min in 6 min. The peaks of authentic samples of
-glucose after treatment in the same manner were detected at 21.58 and
21.66 min.
D-glucose and
25, and 27 displayed strong inhibitory activities on NO production.
However, compounds 5 and 17 exhibited weak activities. Interest-
ingly, compound 27 isolated from Paeonia albiflora also signifi-
cantly suppressed NO production in LPS-activated N9 microglia
in our previous study on Paeonia albiflora.7 Analogs with paeonid-
anin, such as compounds 5, 7, 8, 11, and 26 displayed no inhibitory
activity on NO production, only compound 5 showed weak activity.
L
9. Okasaka, M.; Kashiwada, Y.; Kodzhimatov, O. K.; Ashurmetov, O.; Takaishi, Y.
Phytochemistry 2008, 69, 1767.
10. Oxypaeoniflorin sulfonate (2): White, amorphous powder; ½a D25
ꢁ13.6 (c, 0.11,
ꢀ
MeOH); UV(MeOH) kmax (loge): 258 (3.87) nm; IR (KBr) mmax: 3432, 1706, 1609,
1515, 1450, 1278, 1169, 1079, 650 cmꢁ1; HR-ESI-MS m/z 583.1094 [M+H+Na]+
(calcd for C23H28O14NaS, 583.1097); 1H NMR (300 MHz, C5D5N), see Table 2;
13C NMR (75 MHz, C5D5N), see Table 3.
11. Hayes, P. Y.; Lehmann, R.; Penman, K.; Kitching, W.; De Voss, J. J. Tetrahedron
Lett. 2005, 46, 2615.
References and notes
12. 4-O-Methyloxypaeoniflorin (3): White, amorphous powder; ½a D25
ꢀ
ꢁ9.7 (c, 0.13,
MeOH); UV (MeOH) kmax (loge): 258 (5.23) nm; IR (KBr) mmax: 3422, 1705,
1. Kubo, M.; Tani, T.; Kosoto, H.; Kimura, Y.; Arichi, S. Shoyakugaku Zasshi 1979, 33,
155.
2. Li, Q. Chin. Trad. Herb. Drugs 1988, 19, 276.
3. Hirai, A.; Terano, T.; Hamazaki, T.; Sajiki, J.; Saito, H.; Tahara, K.; Tamura, Y.;
Kumagai, A. Thromb. Res. 1983, 31, 29.
4. Okubo, T.; Nagai, F.; Seto, T.; Satoh, K.; Ushiyama, K.; Kano, I. Biol. Pharm. Bull.
2000, 23, 199.
1609, 1515, 1451, 1279, 1167, 1078 cmꢁ1; HR-ESI-MS m/z 533.1634 [M+Na]+
(calcd for C24H30O12Na, 533.1635); 1H NMR (300 MHz, CD3OD), see Table 2; 13
NMR (75 MHz, CD3OD), see Table 3.
C
13. Lin, H. C.; Ding, H. Y.; Wu, T. S.; Wu, P. L. Phytochemistry 1996, 41, 237.
14. 4-O-Methylgalloyloxypaeoniflorin (4): white, amorphous powder; ½a D25
ꢁ42.9 (c,
ꢀ
0.08, MeOH); UV(MeOH) kmax (loge): 258 (2.83) nm; IR (KBr) mmax: 3417, 1703,
1608, 1515, 1452, 1279, 1077, 770 cmꢁ1; HR-ESI-MS m/z 685.1744 [M+Na]+
5. The n-BuOH layer (450 g) was subjected to D101 macroporous adsorptive resin
with H2O, 20%, 40%, 60% and 95% EtOH to obtain 5 fractions (A–E). Fraction A
(200.0 g) was rechromatographed on D101 macroporous adsorptive resin and
eluted with H2O, 10%, 20%, 30% and 95% EtOH to obtain 5 fractions (A1–A5).
Fraction A3 was further chromatographed on a C-18 reversed-phase open
column and purified by Prep-HPLC eluted with CH3CN–H2O (15:85) to afford
compounds 2 (tR 26.8 min, 25 mg) and 23 (tR 21.4 min, 12 mg). Fraction A4 was
subjected to a C-18 reversed-phase open column and further purified by Prep-
HPLC eluted with MeOH–H2O (3:7) to obtain compounds 4 (tR 44.8 min, 20 mg)
and 6 (tR 46.8 min, 2 mg). Fraction B (85.0 g) was subjected to silica gel column
chromatography with a CHCl3–MeOH gradient system to give nine fractions
(B1–B9). Fraction B3 was isolated and purified by ODS open column
chromatography and Prep-HPLC eluted with MeOH–H2O (4:6) to give
compounds 11 (tR 64.5 min, 80 mg), 12 (tR 94.6 min, 190 mg), 19 (tR
48.5 min, 30 mg), and 20 (tR 73.2 min, 60 mg). Fraction B5 was then
fractionated by chromatography on Sephadex LH-20 eluted with MeOH, and
then purified by Prep-HPLC using MeOH–H2O (3:7) as eluent to give
compounds 3 (tR 57.8 min, 120 mg), 5 (tR 37.8 min, 80 mg), 10 (tR 84.6 min,
190 mg), 13 (tR 114.8 min, 30 mg), and 14 (tR 46.8 min, 60 mg). Fraction C
(150.0 g) was chromatographed on silica gel column chromatography, eluted
with a gradient of CHCl3–MeOH to obtain eight fractions (C1–C8). Fraction C4
was chromatographed on Sephadex LH-20 eluted with MeOH and further
subjected to a C-18 reversed-phase open column and purified by Prep-HPLC
eluted with MeOH–H2O (3:7) to obtain compounds 1 (tR 85.8 min, 36 mg), 7 (tR
65.3 min, 80 mg), 9 (tR 72.8 min, 120 mg), 16 (tR 57.8 min, 45 mg) and 22 (tR
43.8 min, 14 mg). Fraction C6 afforded compounds 17 (tR 42.8 min, 12 mg), 18
(tR 55.7 min, 35 mg) and 21 (tR 96.4 min, 120 mg) using the same methods with
(calcd for C31H34O16Na, 685.1745); 1H NMR (300 MHz, C5D5N), see Table 2; 13
C
NMR (75 MHz, C5D5N), see Table 3. Oxypaeonidanin (5): white, amorphous
powder; ½a 2D5
ꢁ33.3 (c, 0.16, MeOH); UV(MeOH) kmax (loge): 258 (4.18) nm; IR
ꢀ
(KBr) m
max: 3421, 1708, 1609, 1515, 1452, 1280, 1167, 1077, 771 cmꢁ1; HR-ESI-
MS m/z 533.1639 [M+Na]+ (calcd for C24H30O12Na, 533.1635); 1H NMR
(300 MHz, CD3OD), see Table 2; 13C NMR (75 MHz, CD3OD), see Table 3. 9-
Epi-oxypaeonidanin (6): white, amorphous powder;
½
a 2D5
ꢀ
ꢁ21.3 (c, 0.15,
MeOH); UV(MeOH) kmax (log
e
): 258 (2.83) nm; IR (KBr)
m
max: 3420, 1648,
1516, 1403, 1278, 1077, 772 cmꢁ1; HR-ESI-MS m/z 533.1622 [M+Na]+ (calcd
for C24H30O12Na, 533.1629); 1H NMR (300 MHz, CD3OD), see Table 2; 13C NMR
(75 MHz, CD3OD), see Table 3. 9-O-Butyloxypaeonidanin (7): white, amorphous
powder; ½a 2D5
ꢁ35.0 (c, 0.2, MeOH), UV(MeOH) kmax (loge): 258 (3.99) nm; IR
ꢀ
(KBr) m
max: 3414, 1712, 1609, 1515, 1454, 1280, 1167, 1078, 771 cmꢁ1; HR-ESI-
MS m/z 575.2101 [M+Na]+ (calcd for C27H36O12Na, 575.2105); 1H NMR
(300 MHz, CD3OD); 13C NMR (75 MHz, CD3OD), see Table 4. 9-O-
Butylpaeonidanin (8): white, amorphous powder; ½a D25
ꢁ16.6 (c, 0.16, MeOH);
ꢀ
UV(MeOH) kmax (loge): 230 (4.06) nm; IR (KBr) mmax: 3419, 1714, 1604, 1585,
1452, 1384, 1280, 1099, 1076, 714 cmꢁ1; HR-ESI-MS m/z 559.2149 [M+Na]+
(calcd for C27H36O11Na, 559.2155); 1H NMR (300 MHz, CD3OD); 13C NMR
(75 MHz, CD3OD), see Table 4. 4-O-Butyloxypaeoniflorin (9): white, amorphous
powder; ½a 2D5
ꢁ16.8 (c, 0.13, MeOH); UV(MeOH) kmax (loge): 258 (4.03) nm; IR
ꢀ
(KBr) m ;
max: 3421, 1708, 1692, 1609, 1515, 1446, 1350, 1279, 1078, 772 cmꢁ1
HR-ESI-MS m/z 575.2098 [M+Na]+ (calcd for C27H36O12Na, 575.2104); 1H NMR
(300 MHz, CD3OD); 13C NMR (75 MHz, CD3OD), see Table 4.
15. Ha, D. T.; Ngoc, T. M.; Lee, I. S.; Lee, Y. M.; Kim, J. S.; Jung, H. J.; Lee, S. M.; Na, M.
K.; Bae, K. H. J. Nat. Prod. 2009, 72, 1465.
16. Braca, A.; Kiem, P. V.; Yen, P. H.; Nhiem, N. X.; Quang, T. H.; Cuong, N. X.; Minh,
C. V. Fitoterapia 2008, 79, 117.
17. Stavri, M.; Mathew, K. T.; Bucar, F.; Gibbons, S. Planta Med. 2003, 69, 956.
18. Yoshikawa, M.; Uchida, E.; Kawaguchi, A.; Kitagawa, I.; Yamahara, J. Chem.
Pharm. Bull. 1992, 40, 2248.
19. Ding, H. Y.; Wu, Y. C.; Lin, H. C.; Chan, Y. Y.; Wu, P. L.; Wu, T. S. Chem. Pharm.
Bull. 1999, 47, 652.
20. Ding, H. Y.; Lin, H. C.; Teng, C. M.; Wu, Y. C. J. Chin. Chem. Soc. 2000, 47, 381.
21. Zhang, X. Y.; Wang, J. H.; Li, X. Shenyang Yaoke Da. Xue. 2001, 18, 30.
22. Shimizu, M.; Hayashi, T.; Morita, N.; Kiuchi, F.; Noguchi, H.; Iitaka, Y.; Sankawa,
U. Chem. Pharm. Bull. 1983, 31, 577.
fraction C4. In the same way, compounds
8 (tR 37.0 min, 20 mg), 15 (tR
72.4 min, 150 mg), 24 (tR 56.0 min, 40 mg), 25 (tR 25.6 min, 20 mg), 26 (tR
37.8 min, 30 mg) and 27 (tR 61.3 min, 30 mg) were isolated from fraction D
(16.0 g) by silica gel column and Prep-HPLC eluted with CHCl3–MeOH (100:0–
0:100) and MeOH–H2O (55:45), respectively.
6. Paeoniflorin B (1): white, amorphous powder; ½a D25
ꢁ15.4 (c 0.13, MeOH);
ꢀ
UV(MeOH) kmax (loge): 229 (4.40) nm; IR (KBr) mmax: 3422, 1716, 1602, 1540,
1452, 1278, 1072, 1026, 714 cmꢁ1; HR-ESI-MS m/z 769.2322 [M+Na]+ (calcd for
C
36H42O17Na, 769.2314); 1H NMR (600 MHz, CD3OD); 13C NMR (75 MHz,
CD3OD), see Table 1.
7. Duan, W. J.; Yang, J. Y.; Chen, L. X.; Zhang, L. J.; Jiang, Z. H.; Cai, X. D.; Zhang, X.;
Qiu, F. J. Nat. Prod. 2009, 72, 1579.
23. Determination of NO production and cell viability assay: The nitrite concentration
in the medium was measured as an indicator of NO production according to
the Griess reaction. Briefly, RAW264.7 cells were seeded into 96-well tissue
8. Hydrolysis of compounds 1–9 and determination of the absolute configuration of
sugars. A solution of each compound (2.0 mg) in 2 N HCl (2 mL) was stirred at
90 °C in a stoppered vial for 2 h. After cooling, the solution was extracted with
cyclohexane for three times, the combined H2O-soluble part was evaporated
under a stream of N2. Anhydrous pyridine solutions (2 mL) of each residue and
culture plates at a density of 1 ꢂ 105 cells/well, and stimulated with 1
lg/mL of
LPS in the presence or absence of compounds. After incubation at 37 °C for
24 h, 100 lL of cell-free supernatant was mixed with 100 lL of Griess
containing equal volumes of 2% (w/v) sulfanilamide in 5% (w/v) phosphoric
acid and 0.2% (w/v) of N-(1-naphthyl)ethylenediamine solution to determine
nitrite production. Absorbance was measured in a microplate reader at 550 nm
against a calibration curve with sodium nitrite standards. Experiments were
performed in triplicate, and data are expressed as the mean SD of three
independent experiments,24 and there is no cytotoxicity was observed in this
experiment.
L-cysteine methyl ester hydrochloride (2.0 mg) were mixed and warmed at
60 °C for 2 h. The trimethylsilylation reagent trimethylsilylimidazole (0.15 mL)
was added, followed by warming at 60 °C for another 1 h. After drying the
solution, the residue was partitioned between H2O and cyclohexane. The
cyclohexane layer was concentrated, then dissolved in 200
and analyzed by GC using an Agilent DB-1701 column (30 m ꢂ 320
0.25 m). The temperatures of the injector and detector were 325 and 280 °C,
respectively. A temperature gradient system was used for the oven, increasing
l
L of cyclohexane,
lm,
l
24. Dirsch, V. M.; Stuppner, H.; Vollmar, A. M. Planta Med. 1998, 64, 423.