Inhibitory constituents from the aerial parts of Polygala tenuifolia on LPS-induced NO production in BV2 microglia cells in commemoration of Professor Xin-Sheng Yao's 80th Birthday

Article abstract of DOI:10.1016/j.bmcl.2013.08.085

Five new phenolic glycosides, tenuisides A-E (1-5), and a new megastigmane glycoside, tenuiside F (6), along with seventeen known compounds (7-23) were isolated from the aerial parts of Polygala tenuifolia Willd. Their structures were established by detailed analysis of NMR and HRESIMS spectroscopic data, and the absolute configurations of compounds 5 and 6 were determined by CD spectra and in-NMR-tube Mosher's method. The inhibitory effects of these compounds were evaluated on NO production in LPS-activated BV-2 microglia cells. Compound 17 showed the strongest activity, with an IC₅₀ value of 7.4 μM, while compounds 1, 8, 14, and 18 showed the moderate activities, with IC₅₀ values of 16.2-38.5 μM. And their primary structure-activity relationships (SARs) of NO inhibitory effects were also briefly discussed.

Full text of DOI:10.1016/j.bmcl.2013.08.085

Bioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibitory constituents from the aerial parts of Polygala tenuifolia
on LPS-induced NO production in BV2 microglia cells
Tian-Xing Shi a, Shu Wang b, Ke-Wu Zeng a, Peng-Fei Tu a, Yong Jiang a,
⇑
a State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, People’s Republic of China
b Department of Medicinal Chemistry and Pharmaceutical Analysis, Logistics College of Chinese People’s Armed Police Forces, Tianjin 300162, 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:
Five new phenolic glycosides, tenuisides A–E (1ꢀ5), and a new megastigmane glycoside, tenuiside F (6),
along with seventeen known compounds (7–23) were isolated from the aerial parts of Polygala tenuifolia
Willd. Their structures were established by detailed analysis of NMR and HRESIMS spectroscopic data,
and the absolute configurations of compounds 5 and 6 were determined by CD spectra and in-NMR-tube
Mosher’s method. The inhibitory effects of these compounds were evaluated on NO production in LPS-
activated BV-2 microglia cells. Compound 17 showed the strongest activity, with an IC50 value of
Received 3 July 2013
Revised 15 August 2013
Accepted 21 August 2013
Available online xxxx
Keywords:
Polygala tenuifolia
Glycosides
Absolute configuration
NO inhibition
7.4
38.5
briefly discussed.
l
M, while compounds 1, 8, 14, and 18 showed the moderate activities, with IC50 values of 16.2–
lM. And their primary structure–activity relationships (SARs) of NO inhibitory effects were also
Ó 2013 Elsevier Ltd. All rights reserved.
The root of Polygala tenuifolia Willd. (Yuanzhi), a well-known tra-
ditional Chinese medicine, is used as an expectorant, tonic and sed-
ative agent.1 In previous studies, various xanthones, saponins, and
oligosaccharide esters were isolated from the roots or the cortexes
of this plant.2–7 In addition, the aerial parts of this plant are also used
in China as a folk medicine called ‘‘Xiaocao’’, for its expectorant, sed-
ative, and anti-inflammatory properties.8 In order to search for new
bioactive chemical components from the aerial parts and to compre-
hensively use the source of P. tenuifolia, a systematic phytochemical
investigation was carried out on the aerial parts of P. tenuifolia, as a
part of our ongoing research programs on Polygala genus plants.3–
1607 and 1489 cmꢀ1 (aromatic group). The 1H NMR data of 1
showed the presence of a characteristic hydrogen-bonded hydroxy
proton at dH 12.20 (C-1-OH), a group of ABX coupled aromatic sig-
nals at dH 7.59 (1H, d, J = 9.0 Hz), 7.45 (1H, d, J = 3.0 Hz), and 7.38
(1H, dd, J = 9.0, 3.0 Hz), two ortho-coupled aromatic protons at dH
7.61 (1H, d, J = 10.0 Hz) and 6.70 (1H, d, J = 10.0 Hz), and an ano-
meric proton at dH 4.90 (1H, d, J = 7.5 Hz). The 13C NMR spectrum
of 1 presented 19 carbon signals, including 12 aromatic carbons,
a carbonyl group, and a hexosyl moiety. All these above data sug-
gested 1 to be a xanthone glycoside with a monosubstituted A ring
and a disubstituted B ring.13
On acid hydrolysis, 1 yielded D-glucose (Supplementary data).
Comparing with 1,4,7-trihydroxyxanthone and 1,4,8-trihydroxyx-
anthone,14,15 glycosidation was found at C-4 position of 1, for the
upfield shifts of C-4, C-2 and C-8b, and the downfield shifts of C-3,
C-4a and C-1. The glycosidation position can be further deduced from
the HMBC correlations of H-10/C-4, H-3/C-1, C-4, C-4a, H-2/C-4, C-8b,
C-1, and the NOE correlations of H-3 with H-10. In addition, correla-
tions of H-8/C-7, C-9, C-4b, H-6/C-4b, C-7, and H-5/C-7, C-8a in HMBC
spectrumsuggestedthatthehydroxylwaslocatedatC-7position. The
J value (7.5 Hz) of the anomeric proton at dH 4.90 indicted a b config-
uration for the glucose moiety. Thus, compound 1 was determined to
6,9,10
In the current study, five new phenolic glycosides, tenuisides
A-E (1–5), and a new megastigmane glycoside, tenuiside F (6), along
with seventeen known compounds (7–23) (Figure 1) were isolated
from the aerial parts of P. tenuifolia.11 Their structures were estab-
lished by analysis of NMR, HRESIMS and CD spectroscopic data, in-
NMR-tube Mosher’s method, as well as chemical transformations.
The inhibitory effects of the isolated compounds on LPS-induced
NO production were evaluated in BV-2 microglia cells. Detailed
information of the isolation, structure elucidation, and biological
activities of these isolates was reported herein.
Tenuiside A (1)12a was obtained as a yellow powder. Its molec-
ular formula was deduced as C19H18O10 from HRESIMS (m/z
405.0835 [MꢀH]ꢀ). The IR spectrum of 1 displayed absorption
bands at 3431 cmꢀ1 (hydroxy group), 1651 cmꢀ1 (carbonyl group),
be 4-O-b-D-glucopyranosyl-1,7-dihydroxyxanthone.
Tenuiside B (2)12b was obtained as a pale yellow powder. Its
molecular formula was determined as C20H22O9 from HRESIMS
spectrum. The IR spectrum of 2 displayed absorption bands at
3399, 2909, 1651, 1611, and 1462 cmꢀ1 for hydroxyl, methyl,
carbonyl, and aromatic groups, respectively. The 1H NMR data of
2 (Table 1) showed the presence of a phenolic hydroxyl signal at
⇑
Corresponding author. Tel./fax: +86 10 82802719.
E-mail addresses: yongjiang@bjmu.edu.cn, jiangy02@163.com (Y. Jiang)
In commemoration of Professor Xin-Sheng Yao’s 80th Birthday.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.085
Please cite this article in press as: Shi, T.-X.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.08.085

2
T.-X. Shi et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Figure 1. Chemical structures of the isolated compounds from the aerial parts of P. tenuifolia.
Table 1
1H and 13C NMR data of compounds 2–4 (in DMSO-d6, d in ppm)
No.
2
3
4
dH mult. (J in Hz)
dC
dH mult. (J in Hz)
dC
dH mult. (J in Hz)
dC
1
2
3
4
110.0
156.1
96.4
159.8
91.7
109.5
156.2
95.4
160.5
93.3
109.3
156.5
94.3
159.6
96.7
6.23 d (2.0)
6.31 d (2.0)
6.29d (1.5)
6.18 d (1.5)
6.11 d (2.0)
6.04 d (2.0)
5
6
7
158.3
194.4
137.8
128.8
128.6
133.0
128.5
128.7
100.4
73.2
158.1
193.9
138.1
128.9
128.4
132.9
128.8
129.4
100.7
73.1
156.7
193.2
130.3
111.7
147.5
151.8
125.3
114.8
100.9
73.4
10
20
30
40
50
60
Glc-10
20
30
40
50
60
7.69 d (7.5)
7.48 t (7.5)
7.60 t (7.5)
7.48 t (7.5)
7.69 d (7.5)
4.85 d (7.5)
3.23 m
3.28 m
3.17 m
3.31 m
7.69 d (7.5)
7.45 t (7.5)
7.57 t (7.5)
7.45 t (7.5)
7.69 d (7.5)
4.76 d (8.0)
3.04 t (8.0)
3.16 t (8.0)
2.87 t (8.0)
3.21 m
7.33 d (1.5)
6.76 d (8.5)
7.13dd(8.5, 1.5)
4.71 d (8.0)
2.90 t (8.0)
3.17 t (8.0)
3.05 t (8.0)
3.21 m
3.66 d (11.5)
3.45 dd (11.5, 5.5)
3.77 s
76.7
69.7
77.3
60.7
76.6
69.4
77.1
60.6
76.6
69.7
77.2
60.8
3.72 d (11.0)
3.49 dt (11.0, 6.0)
3.59 s
3.65 d (11.5)
3.44 dd (11.5, 5.5)
3.55 s
–OMe
–OH
55.5
55.5
55.7
9.82 s
9.62 br s
dH 9.82 (1H, s), five protons of mono-substituted phenyl at dH 7.69
(2H, d, J = 7.5 Hz), 7.60 (1H, t, J = 7.5 Hz), and 7.48 (2H, d,
J = 7.5 Hz), two meta-coupled aromatic protons at dH 6.31 (1H, d,
J = 2.0 Hz) and 6.23 (1H, d, J = 2.0 Hz), a methoxy group at dH 3.59
(3H, s), and an anomeric proton at dH 4.85 (1H, d, J = 7.5 Hz). The
H-100 of glucose with C-4 further proved the linkage positions of the
methoxy group and the glucose. Thus, compound 2 was deter-
mined to be 4-O-b-D-glucopyranosyl-2-hydroxy-6-methoxybenzo-
phenone.
Tenuiside C (3)12c was obtained as a yellow powder, which had
a molecular formula of C20H22O9 elucidated by HRESIMS spectrum.
The NMR data of 3 (Table 1) were similar to those of 2 except for
the obvious absence of a hydrogen-bonded hydroxy signal. The
same molecular formula and the similar NMR data suggested that
compounds 2 and 3 were a pair of isomers. The HMBC correlations
of H-100/C-2, –OMe/C-6, H-3/C-2, C-4, C-5, and H-5/C-1, C-3, C-4,
C-6 indicated that the glycosidation position in 3 was C-2 position.
b-D-glucose moiety was determined by the same method as de-
scribed for compound 1 (Supplementary data). The 13C NMR spec-
trum of 2 (Table 1) gave 20 carbon signals, including a carbonyl
signal (dC 194.4), two groups of phenyl signals, a glucose moiety,
and a methoxy signal. The NMR data of 2 were very similar to those
of tenuiphenone A6 except that a group of rhamnose signals were
missing. HMBC correlations between methoxy group with C-6, and
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T.-X. Shi et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
3
Table 2
Thus, compound 3 was determined to be 2-O-b-D-glucopyranosyl-
NMR data of compounds 6 and 6b (in CD3OD, d in ppm)
4-hydroxy-6-methoxybenzophenone.
Tenuiside D (4)12d was obtained as a yellow powder, with a
molecular formula of C20H22O11 deduced from HRESIMS spectrum.
The 1H NMR data of 4 (Table 1) showed the presence of a set of
characteristic ABX aromatic protons at dH 7.33 (1H, d, J = 1.5 Hz),
7.13 (1H, dd, J = 8.5, 1.5 Hz), and 6.76 (1H, d, J = 8.5 Hz), two
meta-coupled aromatic protons at dH 6.11 (1H, d, J = 2.0 Hz) and
6.04 (1H, d, J = 2.0 Hz), a methoxy group at dH 3.77 (3H, s), and
an anomeric proton at dH 4.71 (1H, d, J = 7.5 Hz). The large coupling
constant of the anomeric proton and acid hydrolysis result
No.
6
6b
dHmult. (J in Hz)
dC
40.8
dC
1
2
40.7
46.5
1.62 t (12.5)
1.42 dd (12.5, 3.0)
4.03 m
46.5
3
4
5
6
7
65.4
45.7
79.2
65.3
45.7
77.8
78.9
131.2
136.1
69.6
24.2
27.1
26.2
27.5
1.75 m
77.8
6.07 d (16.0)
5.84 dt (16.0, 7.5)
4.38 m
1.28 d (6.5)
0.81 s
133.3
134.8 (ꢀ1.3)
78.8 (+9.2)a
22.0 (ꢀ2.2)a
27.6
26.4
27.8
101.3
79.0
79.0
(Supplementary data) identified the sugar as b-D
-glucose. The 13C
a
8
9
NMR data (Table 1) suggested that 4 was also a phenone glycoside.
The HMBC correlations of H-20 and H-60 to C-7 indicated that car-
bonyl group was in ortho-position to H-20 and H-60; correlations
of –OMe to C-30, H-20 to C-30, C-10, C-60 and C-40, H-60 to C-20 and
C-40, H-50 to C-10,C-30 and C-40 suggested that –OMe and –OH were
linked to C-30 and C-40 positions, respectively; correlations of H-100
to C-2, H-3 to C-1, C-5, C-2 and C-4, H-5 to C-1, C-3, C-4 and C-6
confirmed the linkages of glucose and hydroxyls were at C-2,
C-4 and C-6, respectively. In addition, a strong NOE effect was
observed between the anomeric proton of glucose and H-3 of agly-
cone in 1D-NOESY experiment, which further proved that the
glycosidation was in C-2 of the aglycone. Thus, compound 4 was
10
11
12
13
Glc-10
20
1.13 s
1.15 s
4.39 d (8.0)
3.38 t (8.0)
3.46 t (8.0)
3.19 ob
30
40
72.1
77.9
62.9
50
3.20 ob
60
3.87 d (11.5)
3.57 dd (11.5, 5.5)
5.44 d (1.5)
3.95 d (1.5)
Api-100
200
300
400
110.4
78.4
79.3
75.6
determined to be 2-O-b-D
-glucopyranosyl-4,40,6-trihydroxy-30-
4.19 d (10)
3.83 d (10.0)
4.35 d (11.5)
4.31 d (11.5)
methoxybenzophenone.
500
68.6
Tenuiside E (5)12e was obtained as a white powder. Its molecu-
lar formula was indicated as C26H32O11 by HRESIMS spectrum,
indicating 11 degrees of unsaturation. The 1H NMR data of 5
showed the presence of a set of characteristic ABX aromatic proton
signals at dH 7.07 (1H, d, J = 8.5 Hz), 6.97 (1H, d, J = 1.5 Hz), and 6.85
(1H, dd, J = 8.5, 1.5 Hz), two aromatic protons at dH 6.76 (1H, s) and
6.75 (1H, s), a pair of cis-coupled olefinic protons at dH 6.36 (1H, d,
J = 12.0 Hz) and 5.65 (1H, dt, J = 12.0, 5.5 Hz), and two methoxy
protons at dH 3.79 (3H, s) and 3.74 (3H, s). The anomeric proton
at dH 4.88 (1H, d, J = 7.0 Hz) revealed the presence of one sugar
Sin-1000
2000, 6000
3000, 5000
4000
126.7
107.1
149.6
139.9
147.7
115.6
169.1
57.0
6.91 s
7000
7.66 d (16.0)
6.43 d (16.0)
8000
9000
–OMe
3.88 s
a
D
d6-6b.
b
Overlapped.
unit, which was further identified as b-D-glucose by the same
method as described above (Supplementary data). The 13C NMR
exhibited 26 signals, indicating a lignan glycoside skeleton of 5.
The HMBC correlations of H-2 and H-6 to C-7 and H-20 and H-60
to C-70 indicated that C-7 and C-70 were linked to C-1 and C-10,
respectively; correlations of H-7 to C-40, H-8 to C-50 and C-40
confirmed the skeleton of benzo[b]furan lignan of 5. In addition,
the correlation of H-100 to C-4 in HMBC spectrum showed the
linkage position of glucose.
d, J = 8.0 Hz), two methoxy protons at dH 3.89 (6H, s), one doublet
methyl proton at dH 1.28 (3H, d, J = 6.5 Hz), and three singlet
methyl protons at dH 0.81, 1.13, 1.15 (each 3H, s). The 13C NMR
spectrum of 6 displayed 11 signals assignable to a glucose and an
apiose moieties, 11 signals to a sinapoyl group, and the left 13
carbons were attributed to a megastigmane skeleton,22,23 by com-
binative analysis of 1H–1H COSY, HSQC and HMBC spectra. From
the above evidence, the structure of 6 was deduced to be a megas-
timane diglycoside with a sinapoyl group.
The NMR spectroscopic data of megastigmane moiety of 6 were
essentially similar to those of (3S,5R,6R,7E,9S)-megastiman-7-ene-
3,5,6,9-tetrolor or kiwiionol23 (they are a pair of epimers at the
9-position with almost the same NMR data), except that a glycos-
idation shift was observed in C-9 position (a downfield shift of
9.6 ppm for C-9, along with upfield shifts of 2.0 and 1.1 ppm for
C-10 and C-8, respectively). The HMBC correlations of H-10 to
C-9, H-100 to C-20, as well as H-500 to C-9000 further proved the linkage
positions and sequences of the sugar moiety and the sinapoyl
group.
From the coupling patterns of the adjacent well-resolved proton
signals (J2ax-3 and J2eq-3 appeared to be 12.5 and 3.0 Hz, respec-
tively) in the 1H NMR spectrum, the hydroxy group at the C-3 posi-
tion was suggested to be an equatorial orientation. In addition, in
comparison of the chemical shift values in the 1H and 13C NMR
spectra, the relative structure of the ring portion of 6 was assumed
to be the same as that of (3S,5R,6R,7E,9S)-megastiman-7-ene-
3,5,6,9-tetrolor or kiwiionol (10).23 However, the chirality of the
The relative configuration of H-7 and H-8 was deduced to be
trans according to their coupling constant of 6.5 Hz.16,17 The CD
spectrum of 5 showed a negative Cotton effect at 281 nm, indicat-
ing a 7R,8S configuration.18–20 It should be noted that the NMR and
CD data of 5 were similar to (7R,8S)-dehydrodiconiferyl alcohol
4-O-b-D
-glucopyranoside,21 except that the configuration of double
band was changed from E- to Z-geometry, which further supported
the structure assignment. Thus, compound 5 was determined to
be (ꢀ)-(7R,8S)-70Z-dehydrodiconiferyl alcohol 4-O-b-
D-glucopyra
noside.
Tenuiside F (6)12f was obtained as a white amorphous powder.
Its molecular formula was determined as C35H52O17 from HRESIMS
(m/z 743.3127 [MꢀH]ꢀ). The IR spectrum displayed absorption
bands at 3435 cm–1 (hydroxyl group), 2927 cm–1 (methyl group),
1701 cm–1 (carbonyl group), 1633 cm–1 (double bond), 1516,
1460 cm–1 (aromatic group), and 1070, 1028 cm–1 (C–O bond).
The 1H NMR spectrum data of 6 (Table 2) showed the presence
of two aromatic proton signals at dH 6.91 (2H, s), two couples of
olefinic protons at dH 7.66 (1H, d, J = 16.0 Hz), 6.43 (1H, d,
J = 16.0 Hz)/6.07 (1H, d, J = 16.0 Hz), 5.84 (1H, dt, J = 16.0, 7.5 Hz),
two anomeric protons at dH 5.44 (1H, d, J = 1.5 Hz) and 4.39 (1H,
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4
T.-X. Shi et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Scheme 1. Chemical transformations of 6.
Figure 2.
D
d values (dSꢀdR) obtained from the 1H NMR spectra of the MTPA esters of 6b.
C-9 position must be determined independently, which was ex-
pected to be R on the basis of the glucose-induced shift-trend
(Table 2).24 This assumption was further confirmed by means of
in-NMR-tube Mosher’s method.25 On mild alkaline and enzymatic
anthone (21),39 1,3,7-trihydroxy-2-methoxyxanthone (22),40 and
1,3,6-trihydroxy-7-methoxyxanthone (23).41
All the isolated xanthones and new compounds were evaluated
for their inhibitory effects against LPS-induced NO production in
hydrolysis of 6 (Scheme 1), the aglycone (6b = 10),
-apiose were obtained (Supplementary data). Then, the liberated
aglycone 6b, was esterified with (R)- and (S)-MTPA-Cl, respec-
tively. The d values of the MTPA esters are shown in Figure 2,
D-glucose, and
BV2 microglia cells.42 When the 50
lM initial concentration was
D
tested, all the xanthones showed good inhibitory effects (with
inhibitory rates more than 80%), but compounds 15, 16, 20-22 also
presented cytotoxicities under the tested concentration. So in the
following step, only the compounds with good inhibitory effects
and low cytotoxities were further determined for their IC50 values
against NO production and the results are presented in Table 3.
Among the screened compounds, 17 was the most potent NO pro-
D
which indicated the 3S and 9R configurations of 6. Meanwhile,
the absolute configurations of the sugar components were deter-
mined by GC analysis as described for compound 1 (Supplemen-
tary data). Therefore, the structure of compound
6 was
established as (3S,5R,6S,7E,9R)-7-megastigmene-3,6,9-triol 3-O-b-
D-[20-O-b-D-(600-O-sinapoyl) apiofuranosyl] glucopyranoside.
By comparing the spectroscopic data with literature values, the
18 known compounds isolated in this investigation were identified
as: 30,4,6 -trihydroxy- benzophenone-2-O-b-D-glucopyranoside
(7),26 lancerin (8),27 neolancerin (9),28 kiwiionol (10),23 cis-syringin
(11),29 (+)-syringaresionl-40-O-b-D-glucoside (12),30 liriodendrin
(13),31 mangiferin (14),32 7-O-methylmangiferin (15),33 1,6-dihy-
droxyxanthone (16),34 1,7-dihydroxy-4-methoxyxanthone (17),35
1,7-dihydroxy-2,3-methylenedioxyxanthone (18),36 (+)-syringaresi-
nol (19),37 euxanthone (20),38 1,6,7-trihydroxy-2,3-dimethoxyx-
duction inhibitor, with an IC50 value of 7.4
ble to the positive control indomethacin. Through analysis the
results obtained, preliminary structure-activity relationship
lM, which is compara-
a
(SAR) of xanthones for NO inhibition was summarized as follows.
7-Hydroxy group seems to have significant contribution for the
inhibitory activity, for example all the active compounds have such
a group in their structures, and when 7-hydroxy group change to
7-methoxyl (23), the inhibition decreases obviously. Moreover,
compounds 8 and 14 showed stronger activity than compound 9,
suggesting that glycosidation in C-4 is better than in C-2, and the
existence of ortho-disubstituted hydroxyls could improve the
activity.
Acknowledgements
Table 3
NO inhibitory activity of the isolated compounds
This work was financially supported by National Science Fund
for Excellent Young Scholars (No. 81222051), and National Key
Technology R&D Program ‘New Drug Innovation’ of China (Nos.
2012ZX09301002-002-002 and 2012ZX09304-005).
Compound
IC50 (lM)
1
8
9
14
17
18
38.4
28.6
79.2
38.5
7.4
Supplementary data
20.6
23
>100
7.1
Supplementary data (Experimental details and NMR, HRMS, CD
spectra for compounds 1–6) associated with this article can
be found, in the online version, at http://dx.doi.org/10.1016/
Indomethacina
a
Positive control.
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T.-X. Shi et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
5
437.1089). (e) Tenuiside E (5): colorless powder, ½a D20
ꢁ
ꢀ20.0 (c 0.3, MeOH); UV
j.bmcl.2013.08.085. These data include MOL files and InChiKeys of
the most important compounds described in this article.
(MeOH) kmax (loge) 308 (3.89) nm; IR (KBr) mmax 3746, 2915, 1650, 1592,
1468 cmꢀ1; CD (MeOH,
D
e
) 213 (ꢀ2.23), 281 (ꢀ2.04); 1H NMR (DMSO-d6,
500 MHz): dH 12.20 (1H, s, 1-OH), 7.07 (1H, d, J = 8.5 Hz, H-5), 6.97 (1H, d,
J = 1.5 Hz, H-2), 6.85 (1H, 1H, dd, J = 8.5, 1.5 Hz, H-6), 6.75 (1H, s, H-60), 6.74
(1H, s, H-20), 6.36 (1H, d, J = 12.0 Hz, H-70), 5.65 (1H, dt, J = 12.0, 5.5 Hz, H-80),
5.52 (1H, d, J = 6.5 Hz, H-7), 4.88 (1H, d, J = 7.0 Hz, Glc-H-100), 4.23 (2H, t,
J = 5.5 Hz, H2-90), 3.79 (3H, s, 3-OMe), 3.74 (3H, s, 50-OMe), 3.72 (1H, m, H-9a),
3.64 (1H, m, H-9b), 3.65, 3.44 (each 1H, m, H-600), 3.45 (1H, m, H-8), 3.26 (1H,
m, H-300), 3. 25 (1H, m, H-200), 3.15 (1H, m, H-400), 3.03 (1H, m, H-500); 13C NMR
(DMSO-d6, 125 MHz): dC 149.0 (C-3), 146.7 (C-40), 146.2 (C-4), 143.3 (C-50),
135.1 (C-1), 131.5 (C-80), 130.2 (C-10), 129.1 (C-30), 128.7 (C-70), 118.0 (C-6),
117.5 (C-60), 115.3 (C-5), 113.1 (C-20), 110.4 (C-2), 100.0 (C-100), 86.9 (C-7), 77.0
(C-500), 76.9 (C-300), 73.2 (C-200), 69.7 (C-400), 63.0 (C-9), 60.6 (C-600), 58.2 (C-90),
55.7 (–OMe ꢂ 2), 53.2 (C-8). HRESIMS [M+CI]ꢀ m/z 555.1620 (calcd for
References and notes
1. Chinese Pharmacopoeia Commission In Chinese Pharmacopoeia; China Medical
Science Press: Beijing, 2010; Vol. 1, pp 146–147.
2. Li, J.; Jiang, Y.; Tu, P. F. J. Asian Nat. Prod. Res. 2006, 6, 499.
3. Jiang, Y.; Tu, P. F. J. Asian Nat. Prod. Res. 2003, 5, 279.
4. Jiang, Y.; Tu, P. F. Phytochemistry 2002, 60, 813.
5. Jiang, Y.; Zhang, W.; Xu, X. J.; Tu, P. F. J. Nat. Prod. 2005, 68, 875.
6. Jiang, Y.; Tu, P. F. Chem. Pharm. Bull. 2005, 53, 1164.
7. Jiang, Y. Y.; Duan, Y. Y.; Liu, Y.; Fang, M.; Shi, R. B. J. Beijing Univ. Tradit. Chin.
Med. 2011, 34, 122.
8. Jiangsu New Medical College Dictionary of Chinese Herb Medicines; Shanghai
Scientific and Technologic Press: Shanghai, 1977. pp 242.
9. Zhou, Y. H.; Jiang, Y.; Shi, H. M.; Chen, Y. P.; Tu, P. F. Helv. Chim. Acta 2008, 91,
897.
C
26H32O11Cl, 555.1633). (f) Tenuiside F (6): colorless powder, ½a D20
ꢀ20.0 (c
ꢁ
0.1, MeOH); UV (MeOH) kmax (loge) 328 (3.18) nm; IR (KBr) mmax 3435, 2927,
1701, 1604, 1516, 1460 cmꢀ1 1H and 13C NMR data, see Table 2; HRESIMS
.
[MꢀH]ꢀ m/z 743.3129 (calcd for C35H51O17, 744.3126).
13. Jiang, Y.; Tu, P. F. J. Peking Univ. Health Sci. 2004, 36, 94.
14. Iinuma, M.; Tetsuroito, H.; Tnanaka, T.; Madulid, D. A. Phytochemistrv 1996, 42,
1195.
10. Li, J.; Jiang, Y.; Tu, P. F. J. Nat. Prod. 2005, 68, 1082.
11. Dried aerial parts (20 kg) of P. tenuifolia were cut and extracted for three times
with 70% aqueous EtOH. After removing the solvent in vacuo, the residue was
suspended in water and extracted with EtOAc for three times. The aqueous
solution was subjected to adsorptive macroporous resins D101 column
chromatography (CC), eluted with water, 10% aqueous EtOH, 30% aqueous
EtOH, 50% aqueous EtOH, and 70% aqueous EtOH, successively. The EtOAc
extract (300 g) was subjected to silica gel CC and eluted with gradient CHCl3–
MeOH (12:1–1:1, v/v) to yield 3 fractions. Compounds 2 (19 mg) and 3 (20 mg)
were obtained by CC on silica gel (CHCl3–MeOH, 10:1–1:1, v/v), Sephadex LH-
20 (MeOH), and semi-preparative HPLC (CH3CN:H2O, 21:79, v/v) from Fr.1. Fr.2
was purified by silica gel CC (CHCl3–MeOH, 80:1–1:1, v/v) and Sephadex LH-20
CC (MeOH), followed by semi-preparative HPLC with 21% CH3CN as mobile
phase to provide compound 1 (4 mg). Compounds 8 (2 mg) and 9 (7 mg) were
obtained by chromatography on Sephadex LH-20 (MeOH), ODS (MeOH:H2O,
1:1, v/v), and semi-preparative HPLC (MeOH:H2O, 52:48, v/v) from Fr. 3. The
10% EtOH elute (8 g) was subjected to ODS CC (MeOH:H2O, 1:9–1:0, v/v) and
silica gel CC, eluting with CHCl3–MeOH–H2O (80:20:1, v/v) followed by
recrystallisation to yield compound 10 (9.8 mg). The 30% EtOH elute (50 g) was
subjected to silica gel CC eluting with CHCl3–MeOH–H2O (6:1:0.1–1:1:0.1, v/v)
in gradient to yield 4 fractions. Compound 11 (19 mg) was obtained by ODS
(MeOH:H2O, 1:9–1:0, v/v) from Fr.1 and Fr.2 was filtered to yield compound 3
(10 mg). The filtrate was purified by ODS (MeOH:H2O, 1:9–1:0, v/v), Sephadex
LH-20 (MeOH) followed by HPLC, using 23% MeOH as eluent to provide
compound 7 (5 mg). Compound 4 (8 mg) was obtained by ODS (MeOH:H2O,
1:9–1:1, v/v) and semi-preparative HPLC (MeOH:H2O, 1:4, v/v) from Fr.3. Fr.4
was filtered to yield compound 14 (3.5 g). The 50% EtOH elute (110 g) was
subjected to silica gel CC eluting with CHCl3–MeOH (9:1–1:1, v/v) in gradient
to yield 2 fractions. Compound 12 (10 mg) was obtained by ODS (MeOH:H2O,
1:9–1:1, v/v) from Fr.1. Fr.2 was subjected to Sephadex LH-20 CC (MeOH) to
yield 5 subfractions. Subfraction 1 purified by Sephadex LH-20 (MeOH) and
ODS (MeOH:H2O, 1:9–1:0, v/v) CC provided 2 portions. Compound 5 (1.5 mg)
was obtained by semi-preparative HPLC (MeOH:H2O, 35:65, v/v) from portion
1. Compound 6 (8 mg) was obtained by silica gel CC (EtOAc:MeOH, 7:1, v/v)
from portion 2. Compounds 15 (260 mg), 16 (8 mg), 17 (2.5 mg), and 18 (7 mg)
were obtained by recystallisation from subfractions 2, 3, 4, and 5, respectively.
The 70% EtOH elute (38 g) was subjected to silica gel CC eluting with CHCl3–
MeOH–H2O in gradient (6:1:0.1–1:1:0.1, v/v) and Sephadex LH-20 (MeOH) to
yield 4 fractions. Fr.1 was purified by ODS (MeOH:H2O, 3:7–7:3, v/v) to provide
compound 19 (3 mg). Compound 20 (3 mg) was obtained by semi-preparative
HPLC (CH3CN:H2O, 40:60, v/v) from Fr.2. Compounds 21 (4 mg) and 22 (5 mg)
were obtained by semi-preparative HPLC (CH3CN:H2O, 36:64, v/v) from Fr.3.
Compound 23 (2 mg) was obtained by semi-preparative HPLC (CH3CN:H2O,
42:58, v/v) from Fr. 4.
15. Duan, Y. H.; Dai, Y.; Wang, G. H.; Zhang, X.; Chen, H. F.; Chen, J. B.; Yao, X. S.;
Zhang, X. K. J. Nat. Prod. 2010, 73, 1283.
16. Matsuda, N.; Sato, H.; Yaoita, Y.; Kikuchi, M. Chem. Pharm. Bull. 1996, 44, 1122.
17. Seca, A. M.; Silva, A. M.; Silvestre, A. J.; Cavaleiro, J. A.; Domingues, F. M.;
Pascoal-Neto, C. Phytochemistry 2001, 58, 1219.
18. Antus, S.; Kurtán, T.; Juhász, L.; Kiss, L.; Hollósi, M.; Májer, Z. Chirality 2001, 13,
493.
19. Yoshikawa, M.; Morikawa, T.; Xu, F.; Ando, S.; Matsuda, H. Heterocycles 2003,
60, 1787.
20. Yuen, M. S. M.; Xue, F.; Mak, T. C. W.; Wong, H. N. C. Tetrahedron 1998, 54,
12429.
21. Attoumbre, J.; Hano, C.; Mesnard, F.; Lamblin, F.; Bensaddek, L.; Raynaud-Le
Grandic, S.; Laine, E.; Fliniaux, M. A.; Baltora-Rosset, S. C.R. Chim. 2006, 9, 420.
22. Cam, T. I.; Tran, H. Q.; Hoang, T. H.; Chau, V. M.; Phan, V. K. Tap Chi Hoa Hoc
2009, 47, 81.
23. Otsuka, H.; Hiratab, E.; Shinzatoc, T.; Takeda, Y. Phytochemistry 2003, 62, 763.
24. Kasai, R.; Suzuno, M.; Asakawa, I.; Tanaka, O. Tetrahedron Lett. 1977, 18, 175.
25. Su, B. N.; Park, E. J.; Mlbwambo, Z. H.; Santarsiero, B. D.; Mesecar, A. D.; Fong, H.
H. S.; Pezzuto, J. M.; Kinghorn, A. D. J. Nat. Prod. 2002, 65, 1278.
26. Kaya, D.; Yalcn, N. F.; Bedir, E.; Calis, I.; Steinhauser, L.; Albert, K.; Ersoz, T.
Phytochemistry Lett. 2011, 4, 459.
27. Miyase, T.; Noguchi, H.; Chen, X. M. J. Nat. Prod. 1999, 62, 993.
28. Zhang, W. M.; Shan, W. H. Chin. Tradit. Herb Drugs 2005, 36, 1767.
29. Alike, D.; Gisela, V.; Vincenz, G.; Jorg, L.; Axel, Z. Phytochemistry 1997, 45, 51.
30. Guo, H. L.; Zhou, J. Y. China J. Chin. Mater. Med. 2005, 30, 44.
31. Huang, X. F.; Luo, J.; Zhang, Y. Chin. Pharm. J. 2007, 42, 339.
32. Li, C. J.; Zhang, D. M.; Yu, S. S. Chin. Tradit. Herb Drugs 2007, 38, 1146.
33. Masao, F. J.; Takao, I. Chem. Pharm. Bull. 1982, 30, 2342.
34. Xuan, W. D.; Bian, J.; Yuan, B.; Qian, X. R.; Li, W. H.; Xiong, Z. Chin. Tradit. Herb
Drugs 2008, 39, 1616.
35. Kijjoa, A.; Jose, M.; Gonzalez, T. G.; Pinto, M. M. M.; Damas, A. M.;
Mondranondra, I.; Silva, A. M. S.; Herz, W. Phytochemistry 1998, 49, 2159.
36. Mao, S. L.; Liao, S. X.; Wu, J. H.; Liang, H. Q.; Chen, H. S.; Zhang, C. K. Acta Pharm.
Sin. 1996, 31, 118.
37. Yu, Y.; Gao, H.; Dai, Y.; Li, X. X.; Li, J. M.; Yao, X. S. Chin. Tradit. Herb. Drugs 2010,
41, 509.
38. Pan, M. D.; Mao, Q. Acta Pharm. Sin. 1984, 19, 899.
39. Lin, L. L.; Huang, F.; Chen, S. B.; Yang, D. J.; Chen, S. L.; Yang, J. S.; Xiao, P. G.
China J. Chin. Materia Med. 2005, 30, 827.
40. Wang, H.; Ye, G.; Ma, C. H.; Tang, Y. H.; Fan, M. S.; Li, Z. X.; Huang, C. G. J. Pharm.
Biomed. Anal. 2007, 45, 793.
41. Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P. L.; Wishnok, J. S.;
Tannenbaum, S. R. Anal. Biochem. 1982, 126, 131.
42. Determination of NO production and cell viability assay: BV-2 cells were obtained
from Peking Union Medical College (PUMC) Cell Bank. LPS (Escherichia coli
0111:B4), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) and indomethacin (P99%) were obtained from Sigma–Aldrich (MO,
USA). Griess reagent was purchased from Nanjing Jiancheng Bioengineering
Institute (Nanjing, China). The purity of the isolated compounds (>95%) used
for the biological assay in vitro was determined by 1H NMR and HPLC
techniques. BV-2 cells were seeded at the density of 5 ꢂ 104 cells per well in
48-well culture plate, then treated with various concentrations of each
12. Physical and spectroscopic data of new compounds: (a) tenuiside A (1): yellow
powder; ½a 2D0
ꢁ
ꢀ6.0 (c 0.1, MeOH); UV (MeOH) kmax (log
e
) 392 (3.39), 317
(3.23), 286 (3.40) nm; IR (KBr) mmax 3431, 1651, 1607, 1489 cmꢀ1
;
1H NMR
(DMSO-d6, 500 MHz): dH 7.61 (1H, d, J = 10.0 Hz, H-3), 7.59 (1H, d, J = 9.0 Hz, H-
5), 7.45 (1H, d, J = 3.0 Hz, H-8), 7.38 (1H, dd, J = 9.0, 3.0 Hz, H-6), 6.70 (1H, d,
J = 10.0 Hz, H-2), 4.90 (1H, d, J = 7.5 Hz, Glc-H-10), 3.68 (1H, d, J = 12.5 Hz, H-
60a), 3.48 (1H, m, H-60b), 3.35 (1H, overlapped, H-20), 3.29 (1H, overlapped, H-
50), 3.27 (1H, overlapped, H-30), 3.19 (1H, t, J = 9.0 Hz, H-40); 13C NMR (DMSO-
d6, 125 MHz): dC 181.5 (C-9), 154.9 (C-1), 154.2 (C-7), 149.2 (C-4b), 145.9 (C-
4a), 137.3 (C-4), 125.7 (C-6), 125.2 (C-3), 120.4 (C-8a), 119.7 (C-5), 108.2 (C-
8b), 108.1 (C-2), 107.7 (C-8), 102.2 (C-10ꢀ), 77.1 (C-50), 76.6 (C-30), 73.4 (C-20),
compound and LPS (1.0 lg/mL, Escherichia coli 0111:B4, Sigma–Aldrish). The
cell viability of the cultured cells was detected by MTT method. The NO
concentration was detected by the Griess reagent. Briefly, BV-2 cells were
69.7 (C-40), 60.7 (C-60); HRESIMS [M-H] m/z 405.0835 (calcd for C19H17O10
,
405.0822). (b) Tenuiside B (2): yellow powder; ½a D20
ꢀ82.0 (c 0.1, MeOH); UV
ꢁ
(MeOH) kmax (loge) 306 (3.74) nm; IR (KBr) mmax 3399, 2908, 1650, 1610,
1426 cmꢀ1 1H and 13C NMR data, see Table 1; HRESIMS [M+Na]+ m/z 429.1167
.
treated with LPS (1.0
l
g/mL) and compounds for 24 h. After that, 400
lL of
(calcd for C20H22O9Na, 406.1156). (c) Tenuiside C (3): yellow powder; ½a D20
ꢁ
culture supernatant was allowed to react with 100
lL of Griess reagent (1%
sulfanilamide/0.1% naphthylethylene diamine dihydrochloride/2% phosphoric
acid) for 10 min at room temperature in the dark. Then, the optical density
(100 lL per well) was measured at 540 nm using a microplate reader (Tecan
Trading AG, Switzerland). Sodium nitrite in medium was used to calculate a
standard curve in the assay. The experiments were performed in triplicates.
Indomethacin was used as a positive control.
ꢀ33.8 (c 0.065, MeOH); UV (MeOH) kmax (loge) 295 (3.49) nm; IR (KBr) mmax
3434, 2913, 1656, 1620, 1438 cmꢀ1 1H and 13C NMR data, see Table 1;
;
HRESIMS [MꢀH]ꢀ m/z 405.1185 (calcd for C20H21O9, 405.1191). (d) Tenuiside D
(4): yellow powder; ½a D20
ꢁ
ꢀ16.6 (c 0.3, MeOH); UV (MeOH) kmax (log
e
) 295
(4.04) nm; IR (KBr) mmax 3396, 2914, 1712, 1613, 1467 cmꢀ1
.
1H and 13C NMR
data, see Table 1; HRESIMS [MꢀH]ꢀ m/z 437.1109 (calcd for C20H22O11
,
Please cite this article in press as: Shi, T.-X.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.08.085