Potential anti-inflammatory constituents of the stems of Gordonia chrysandra

Article abstract of DOI:10.1021/np200021f

Eight new oleanane-type triterpenoid glycosides, gordonosides I-P (1-8), and two new phenolic glycosides (9 and 10) were isolated from the stems of Gordonia chrysandra. Their structures were elucidated by spectroscopic and chemical methods. In an in vitro bioassay, compound 1 showed a strong inhibitory effect on nitric oxide production in LPS-activated macrophages with an IC ₅₀ value of 0.14 μM. (Chemical Equation Presented).

Full text of DOI:10.1021/np200021f

ARTICLE
pubs.acs.org/jnp
Potential Anti-inflammatory Constituents of the Stems of Gordonia
chrysandra
Hui-Zheng Fu, Chuang-Jun Li, Jing-Zhi Yang, Zhu-Fang Shen, and Dong-Ming Zhang*
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College (Key Laboratory of Bioactive
Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education), Beijing 100050, People’s Republic of China
S Supporting Information
b
ABSTRACT: Eight new oleanane-type triterpenoid glycosides,
gordonosides IꢀP (1ꢀ8), and two new phenolic glycosides (9
and 10) were isolated from the stems of Gordonia chrysandra.
Their structures were elucidated by spectroscopic and chemical
methods. In an in vitro bioassay, compound 1 showed a strong
inhibitory effect on nitric oxide production in LPS-activated
macrophages with an IC50 value of 0.14 μM.
ordonia chrysandra Cowan, belonging to the family Thea-
ceae, is distributed widely in Sichuan, Guizhou, and Yunnan
anomeric protons as β-D-glucuronic acid, R-L-arabinose, β-D-xylose,
and β-D-galactose.
G
Provinces of mainland China. G. chrysandra is used in folk
medicine for treating diarrhea, gastralgia, and arthritis.1 Pre-
viously, two active flavanonol glycosides and eight cytotoxic
acylated triterpenoid saponins from G. chrysandra roots have
been reported.2,3 Until now, no work has been done on the
constituents of the stems of G. chrysandra. As part of a
continuing search for bioactive constituents from G. chrysan-
dra, a 95% EtOH extract of the stems of G. chrysandra has been
investigated with 10 new compounds obtained (1ꢀ10).
Reported herein are the isolation, structure elucidation, and
biological activity of these new compounds.
The sequence of the glycosidic chains in 1 was determined by
the analysis of the 2D NMR spectroscopic data. HMBC correlations
from H-22 to C-10 indicated unambiguously that the angeloyloxy
ester group is attached to C-22 of the aglycone. In addition, a
HMBC correlation from GlcA-H-1 (δH 5.27) to C-3 (δC 89.4)
confirmed that the β-D-glucuronopyranosyl unit is located at C-3.
1
The long-range correlations observed between the H NMR
resonances at δH 6.09 (Gal-H-1) and the 13C NMR resonance at
δC 78.8 (GlcA-C-2), between δH 6.11 (Ara-H-1) and δC 83.7
(GlcA-C-3), and between δH 5.37 (Xyl-H-1) and δC 81.8 (Ara-C-
2) indicated that the tetrasaccharide residue at C-3 of the aglycone is
β-D-xylopyranosyl-(1f2)-R-L-arabinopyranosyl-(1f3)-[β-D-galac-
topyranosyl-(1f2)]-β-D-glucuronopyranoside. Thus, compound 1
was determined as 3-O-β-D-xylopyranosyl-(1f2)-R-L-arabinopyra-
nosyl-(1f3)-[β-D-galactopyranosyl-(1f2)]-β-D-glucuronopyrano-
syl-22R-angeloyloxyolean-12-ene-16R,28-diol and has been named
gordonoside I.
’ RESULTS AND DISCUSSION
Compound 1was obtained as a white, amorphous powder. The IR
spectrum of 1showed hydroxy (3402 cmꢀ1), carbonyl (1678 cmꢀ1),
and R,β-unsaturated carbonyl (1080 cmꢀ1) absorption signals. The
molecular formula, C57H90O24, was determined by HRESIMS (m/z
1181.5703 [M þ Na]þ, calcd for m/z 1181.5714). The NMR data
(Tables1and2) analysisof1indicatedthatithasthesameaglycone,
glucuronic acid (GluA), and arabinose (Ara) moieties as gordono-
side C, which has been reported previously.3 The difference between
1 and gordonoside C is the number of sugars and substituent
Compound 2 was isolated as a white, amorphous powder. The
HRESIMS peak at m/z 1197.5668 [M þ Na]þ indicated the
molecular formula of 2 to be C57H90O25, with one oxygen
atom more than that of 1 (1181 [M þ Na]þ). The IR and
NMR data of 2 were almost identical to those of 1 (Experi-
mental Section; Tables 1 and 2). However, detailed NMR
analysis showed an additional hydroxy group at C-15 (δC 67.5
ppm). These data suggested that 2 is a 15-oxygenated deriva-
tive of 1, which was further confirmed by HMBC and NOESY
experiments on 2. Thus, compound2(gordonoside J) was elucidated
as 3-O-β-D-xylopyranosyl-(1f2)-R-L-arabinopyranosyl-(1f3)-
1
groups. The H NMR spectrum of 1 displayed four anomeric
proton signals at δH 5.27 (1H, d, J = 7.5 Hz), 5.37 (1H, d, J =
8.0 Hz), 6.09 (1H, d, J = 7.5 Hz), and 6.11 (1H, d, J = 7.0 Hz) and
signals for an angeloyl group [δH 2.25 (3H, s), 2.41 (3H, d, J =
7.0 Hz), and 6.24 (1H, q, J = 7.0 Hz); δC 167.8, 136.3, 129.3, 20.7,
and 15.6]. Acid hydrolysis of 1 with 2 M HCl afforded mono-
saccharides, which were identified by GC analysis of their trimethyl-
silyl L-cysteine derivatives4 and the coupling constants of the
Received: January 8, 2011
Published: April 07, 2011
r
Copyright
2011 American Chemical Society and
1066
dx.doi.org/10.1021/np200021f J. Nat. Prod. 2011, 74, 1066–1072
American Society of Pharmacognosy
|

Journal of Natural Products
ARTICLE
the aglycone was established from the following HMBC correla-
tions: H-1 (δH 4.90) of Ara0 with C-2 (δC 81.4) of Ara, H-1 (δH
4.97) of GlcA with C-3 (δC 88.9) of the aglycone, H-1 (δH 5.49)
of Ara with C-3 (δC 86.1) of GlcA, and H-1 (δH 6.32) of Glc with
C-28 (δC 175.7) of the aglycone. Thus, compound 4 (gordono-
side L) was elucidated as 3-O-R-L-arabinopyranosyl(1f2)-R-L-
arabinopyranosyl(1f3)-β-D-glucuronopyranosyl acacic acid
28-O-β-D-glucopyranoside.
The molecular formula of 5, C53H84O23, was indicated from
the HRESIMS peak at m/z 1111.5297 [M þ Na]þ. The NMR
spectroscopic data of compound 5 were almost identical with
those of 4 except that the arabinose in 4 was replaced by a rhamnose
moiety in 5 (Tables 1 and 2). The connectivity for the sugar residues
was further confirmed from the following HMBC correlations: GlcA-
H-1 (δH 4.98)/aglycone-C-3 (δC 89.2), Ara-H-1 (δH 5.21)/GlcA-
C-3 (δC 86.5), Rha-H-1 (δH 5.99)/Ara-C-2 (δC 80.5), and Glc-H-1
(δH 6.33)/aglycone-C-28 (δC 175.9). Thus, compound 5 (gor-
donoside M) was determined as 3-O-R-L-rhamnopyranosyl(1f2)-
R-L-arabinopyranosyl(1f3)-β-D-glucuronopyranosylacacic acid 28-
O-β-D-glucopyranoside.
Compound 6 had the molecular formula C58H92O28, as
indicated from the HRESIMS (m/z 1259.5672 [M þ Na]þ,
calcd for 1259.5667). The NMR spectroscopic data of compound 6
resembled those of 3, except that 6 has one additional xylose and
glucose units when compared with 3 (Tables 1 and 2). The linkages
of the sugars and the sugars with the aglycone were established from
the following HMBC correlations: GlcA-H-1 (δH 4.90)/aglycone-C-
3 (δC 89.6), Xyl-H-1 (δH 4.99)/Ara-C-2 (δC 82.2), Ara-H-1 (δH
5.77)/GlcA-C-3 (δC 83.8), Glc0-H-1 (δH 5.90)/GlcA-C-2 (δC
78.2), and Glc-H-1 (δH 6.32)/aglycone-C-28 (δC 175.9). Thus,
compound 6 (gordonoside N) was determined as 3-O-β-D-xylo-
pyranosyl-(1f2)-R-L-arabinopyranosyl-(1f3)-[β-D-glucopyrano-
syl-(1f2)]-β-D-glucuronopyranosylacacic acid 28-O-β-D-glucopy-
ranoside.
Compound 7 gave the same molecular formula as 6, namely,
C58H92O28, as deduced by HRESIMS (m/z 1259.5672 [M þ
Na]þ, calcd for 1259.5667). The NMR spectroscopic data of 7
matched quite well with those for 6. Further analysis indicated that
the glucose in 6 was replaced by a galactose in 7 (Tables 1 and 2). In
addition, the D-galactose in 7 was assigned to C-2 of GlcA from the
HMBC correlation between the H-1 (δH 5.74) of galactose and
C-2 (δC 83.9) of GlcA. Thus, compound 7 (gordonoside O) was
determined as 3-O-β-D-xylopyranosyl-(1f2)-R-L-arabinopyrano-
syl-(1f3)-[β-D-galactopyranosyl-(1f2)]-β-D-glucuronopyranosy-
lacacic acid 28-O-β-D-glucopyranoside.
Compound 8 was obtained as a white, amorphous powder,
with the molecular formula C59H94O28, as deduced from the
[Mþ Na]þ peak at m/z1273.5827 by HRESIMS. The NMR spectro-
scopic data of compounds 8 and 6 were almost identical, except that
the arabinose in 6 was replaced by a rhamnose in 8 (Tables 1 and2).
In addition, the connectivity for the sugar residues was confirmed
from the following HMBC correlations: GlcA-H-1 (δH 4.94)/
aglycone-C-3 (δC 89.6), Glc0-H-1 (δH 5.29)/GlcA-C-2 (δC 78.8),
Xyl-H-1 (δH 5.61)/GlcA-C-3 (δC 86.1), and Rha-H-1 (δH 5.88)/
Glc0-C-3 (δC 81.3). Thus, compound 8 (gordonoside P) was
determined as 3-O-R-L-rhamnopyranosyl-(1f3)-β-D-glucopyrano-
syl-(1f2)-[β-D-xylopyranosyl-(1f3)]-β-D-glucuronopyranosylacacic
acid 28-O-β-D-glucopyranoside.
[β-D-galactopyranosyl-(1f2)]-β-D-glucuronopyranosyl-22R-angel-
oyloxyolean-12-ene-15R,16R,28-triol.
Compound 3 was isolated as a white, amorphous powder. The
molecular formula, C47H74O19, was determined by HRESIMS
(m/z 965.4697 [M þ Na]þ, calcd for 965.4717). The NMR
spectroscopic data of 3 resembled those of 1, indicating the two
compounds have a similar structure (Tables 1 and 2). However,
comparison of the NMR data of 3 with those of acacic acid5
indicated that 3 is a 3,28-bidesmoside of acacic acid. In addition,
1
the H and 13C NMR signals for 3 showed three anomeric
protons at δH 4.99 (1H, d, J = 6.5 Hz), 5.36 (1H, d, J = 6.0 Hz),
and 6.32 (1H, d, J = 8.0 Hz), with the corresponding carbon
resonances at δC 106.5, 105.6, and 95.7, respectively. The
linkages of the sugars and the sugars with the aglycone were
established from the following HMBC correlations: GlcA-H-1
(δH 4.99)/aglycone-C-3 (δC 89.0), Ara-H-1 (δH 5.36)/GlcA-C-
3 (δC 85.5), and Glc-H-1 (δH 6.32)/aglycone-C-28 (δC 175.7).
Thus, compound 3 (gordonoside K) was elucidated as 3-O-R-
L-arabinopyranosyl(1f3)-β-D-glucuronopyranosyl acacic acid
28-O-β-D-glucopyranoside.
Compound 4 was found to possess the molecular formula
C52H82O23 according to the HRESIMS peak at m/z 1097.5125
[M þ Na]þ. The NMR spectroscopic data were almost identical
to those of 3, except for an additional set of R-L-arabinose
resonances with an anomeric proton signal at δH 4.90 (1H, d,
J = 7.0 Hz) and a downfield shift of C-2 (from δC 74.5 to δC 81.4)
of the L-arabinopyranosyl unit due to a glycosidation shift
(Tables 1 and 2). The linkage position of the sugar units with
Compound 9 was isolated as a white, amorphous powder. The
IR spectrum of 9 indicated the presence of hydroxy (3423 cmꢀ1),
carbonyl (1670 cmꢀ1), and aromatic ring signals (1611 and
1510 cmꢀ1). The HRESIMS of 9 showed a quasimolecular ion
1067
dx.doi.org/10.1021/np200021f |J. Nat. Prod. 2011, 74, 1066–1072

Journal of Natural Products
ARTICLE
Table 1. H NMR Spectroscopic Data for Compounds 1ꢀ8a
1
position
1
2
3
4
5
6
7
8
3
3.59 dd
(11.5, 4.0)
3.25 m
3.35 brd
(11.0)
0.77 m
1.79 m
1.92 m
5.57 brs
1.77 m
2.42 m
5.31 brs
3.49 m
2.79 m
1.34 m
1.79 m
1.25 m
2.39 m
2.10 m
1.25 s
3.34 brd
(13.0)
3.36 dd
(11.0, 4.0)
0.82 m
3.22 dd
(11.5, 4.5)
3.24 dd
(10.5, 4.0)
3.25 dd
(12.0, 4.0)
0.75 m
5
9
1.05 m
2.04 m
2.12 m
5.69 brs
1.93 m
2.25 m
4.82 m
3.37 m
2.92 m
1.74 m
3.23 m
2.05 m
6.55 m
0.77 m
1.71 m
1.82 m
5.45 brs
4.25 m
0.72 m
1.40 m
2.00 m
5.57 brs
1.24 m
0.74 m
1.76 m
1.91 m
5.56 brs
1.25 m
0.74 m
1.76 m
1.91 m
5.56 brs
1.24 m
1.76 m
1.93 m
5.58 brs
1.23 m
2.39 m
5.31 brs
3.49 brd (11.0)
2.79 m
1.37 m
2.57 m
1.77 m
2.36 m
2.06 m
1.27 s
1.74 m
1.91 m
5.56 brs
1.25 m
2.39 m
5.29 brs
3.46 m
2.78 m
1.36 m
1.76 m
2.54 m
2.08 m
2.39 m
1.16 s
11
12
15
16
4.58 m
5.30 brs
3.44 brd (10.5)
2.76 m
1.37 m
2.55 m
1.79 m
2.42 m
2.12 m
1.24 s
5.56 brs
3.46 m
2.78 m
1.42 m
1.76 m
2.54 m
2.39 m
2.09 m
1.17 s
5.29 brs
3.47 m
2.78 m
1.35 m
1.79, m
2.54, m
2.10 m
2.39 m
1.24 s
18
3.00 brd (12.0)
2.77 m
19a
19b
21
2.03 m
2.86 m
1.29 m
22
6.19 dd (12.0, 6.5)
23
24
25
26
27
1.61 s
1.42 s
1.13 s
1.19 s
2.20 s
1.25 s
1.11 s
0.79 s
1.00 s
1.86 s
0.95 s
0.95 s
0.96 s
1.03 s
1.09 s
1.05 s
0.80 s
0.83 s
0.81 s
0.81 s
0.81 s
0.80 s
1.10 s
1.10 s
1.07 s
1.10 s
1.09 s
1.09 s
1.85 s
1.85 s
1.86 s
1.85 s
1.82 s
1.83 s
28a
28b
29
30
30
4.02 d (10.5)
3.88 d (10.5)
1.37 s
3.72 d (10.5)
3.62 d (10.5)
1.05 s
0.98 s
1.02 s
0.99 s
1.02 s
0.99 s
1.02 s
0.98 s
1.02 s
0.98 s
1.02 s
0.98 s
1.02 s
1.61 s
1.27 s
6.24 q (7.0)
2.41 d (7.0)
2.25 s
5.83 q (7.0)
2.02 d (7.5)
1.82 s
40
50
Sugar (C-28)
Glc 1
6.32 d (8.0)
4.17 m
6.32 d (8.0)
4.13 m
6.33 d (8.0)
4.17 m
6.32 d (8.0)
4.14 m
6.32 d (8.0)
4.15 m
6.32 d (8.0)
4.13 m
2
3
4
5
6
4.02 m
4.02 m
4.03 m
4.09 m
4.01 m
4.01 m
4.30 m
4.27 m
4.26 m
4.57 m
4.57 m
4.33 m
4.26 m
4.25 m
4.28 m
4.26 m
4.64 m
4.17 m
4.38 m
4.38 m
4.35 m
4.33 m
4.34 m
4.37 m
4.43 m
4.43 m
4.46 m
4.37 m
4.45 m
4.54 m
Sugar (C-3)
GlcA 1
5.27 d (7.5)
4.96 m
4.93 d (7.5)
4.66 m
4.99 d (6.5)
4.18 m
4.97 d (7.5)
4.12 m
4.98 d (7.5)
4.06 m
4.90 d (7.5)
4.34 m
4.93 d (7.0)
3.99 m
4.94 d (7.0)
4.41 m
2
3
4.75 m
4.43 m
4.38 m
4.22 m
4.32 m
4.40 m
4.43 m
4.36 m
4
4.85 m
4.33 m
4.46 m
4.51 m
4.40 m
4.30 m
4.30 m
4.41 m
5
4.87 m
4.58 m
4.26 m
4.62 m
4.68 m
4.20 m
4.55 m
4.39 m
6
Ara (1f3) GlcA
1
2
3
4
5
6.11 d (7.0)
4.89 m
4.83 m
4.58 m
4.05 m
5.75 d (6.0)
4.58 m
4.32 m
4.22 m
3.59 t (13.5)
4.33 m
5.36 d (6.0)
4.13 m
4.46 m
4.30 m
3.79 d (11.5)
4.38 m
5.49 d (5.0)
4.56 m
4.49 m
4.36 m
3.64 d (12.0)
4.37 m
5.21 d (7.5)
4.26 m
4.68 m
4.60 m
3.77 d (7.5)
4.26 m
5.77 d (6.5)
4.56 m
4.46 m
4.32 m
3.76 m
5.77 d (5.5)
4.55 m
4.39 m
4.30 m
3.72 m
4.66 m
4.35 m
4.43 m
Xyl (1f2) Ara
1
2
3
4
5
5.37 d (8.0)
4.48 m
4.36 m
4.44 m
3.82 m
5.04 d (7.0)
4.17 m
4.01 m
4.15 m
3.48 t (9.5)
4.32 m
4.99 d (8.0)
4.24 m
4.67 m
4.26 m
3.46 m
5.02 d (7.5)
4.15 m
4.25 m
4.24 m
3.24 m
5.61 d (7.5)
4.05 m
3.83 m
4.13 m
3.75 m
4.65 m
4.37 m
4.30 m
4.21 m
Gal (1f2) GlcA
1
2
3
4
5
6
6.09 d (7.5)
4.85 m
4.65 m
4.96 m
4.60 m
5.74 d (7.5)
4.49 m
4.32 m
4.52 m
4.25 m
5.74 d (8.0)
4.48 m
4.32 m
4.55 m
4.25 m
4.04 m
3.76 m
4.34 m
4.74 m
4.43 m
4.45 m
1068
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Journal of Natural Products
ARTICLE
Table 1. Continued
position
1
2
3
4
5
6
7
8
Ara0 (1f2) Ara
1
2
3
4
5
4.90 d (7.0)
4.13 m
4.62 m
4.22 m
3.73 t (11.0)
4.38 m
Rha (1f2) Ara
1
5.99 brs
2
4.71 m
3
4.54 m
4
4.28 m
5
4.39 m
6
1.65 d (6.0)
Glc0 (1f2) GlcA
1
2
3
4
5
6
5.90 d (7.5)
4.13 m
4.57 m
4.11 m
4.01 m
5.29 d (7.0)
4.43 m
4.39 m
4.45 m
4.26 m
4.32 m
4.30 m
4.38 m
4.45 m
Rha (1f2) Glc0
1
2
3
4
5
6
5.88 brs
4.61 m
4.51 m
4.30 m
4.59 m
1.63 d (6.0)
a 1H NMR data (δ) were measured in C5D5N at 500 MHz for proton. Coupling constants (J) in Hz are given in parentheses. The assignments were
based on TOCSY, HSQC, and HMBC experiments.
peak at m/z 511.1461 [M ꢀ H]ꢀ, supporting a molecular
formula of C23H28O13 (calcd for C23H27O13, 511.1457). The
1H NMR spectrum of 9 in DMSO-d6 showed a singlet at δH 7.18
(2H, s), two aromatic protons at δH 6.12 (1H, d, J = 2.0 Hz) and
6.15 (1H, d, J = 2.0 Hz), four aromatic methoxy singlets at δH
3.56, 3.59, 3.76, and 3.76, and an anomeric signal of a sugar unit at
δH 4.82 (1H, d, J = 7.5 Hz). The above information, together
with the observation of an ester carbonyl signal at δC 165.6,
indicated the presence of a 3,5-O-dimethoxygalloyl unit in the
molecule.6,7 Acid hydrolysis of 9 afforded D-glucose, which was
determined by measuring its specific rotation.3 A series of
HMBC correlations from H-10 to C-1 and from H-60 to C-700
indicated that C-1 and C-700 are attached to the glucose C-10
and C-60, respectively. Also, HMBC correlations from H-7 to
C-3; H-8 to C-4; H-2 to C-1, C-3, C-4, and C-6; and H-6 to
C-1, C-2, C-4, and C-5 demonstrated that a hydroxy group was
present at C-5. Thus, compound 9 was determined as 1-O-3,
4-dimethoxy-5-hydroxyphenyl-(6-O-3,5-dimethoxygalloyl)-β-D-
glucopyranoside.
In an in vitro bioassay, compounds 1ꢀ10 at 5 μM inhibited nitric
oxide production in rat polymorphonuclear leukocytes (PMNS)
induced by the LPS by 96.6 ( 3.7, 9.1 ( 2.7, 6.4 ( 3.6, 14.0 ( 2.3,
2.9 ( 2.2, 8.9 ( 2.5, 12.6 ( 0.5, 5.9 ( 1.2, 4.7 ( 2.0, and 6.5 ( 2.5%
(n = 3), respectively. Dexamethasone, the positive control, gave a
74.7 ( 2.9% (n = 3) inhibition at the same concentration. The most
active compound, 1, showed a strong inhibitory effect on nitric
oxide production in LPS-activated macrophages with an IC50
value of 0.14 μM, suggesting that 1 may serve as a potential anti-
inflammatory lead.
’ EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured on a Perkin-Elmer 241 automatic digital polarimeter. UV
spectra were recorded using a Shimadzu UV-300 spectrophotometer. IR
spectra were recorded on a Nicolet 5700 FT-IR spectrometer by a
transmission microscope method. 1D and 2D NMR spectra were
1
obtained at 500 and 125 MHz for H and 13C, respectively, on an
Compound 10 exhibited a quasimolecular ion peak at m/z 481
[M ꢀ H]ꢀ in its ESIMS. The molecular formula C22H26O12 was
indicated by HRESIMS (m/z 481.1354 [M ꢀ H]ꢀ, calcd for
481.1351). The IR and NMR spectra of 10 resembled those of 9
except that the NMR signals of the symmetrically tetrasubsti-
tuted benzene moiety and two methoxy groups of 9 were
replaced by the signals attributed to a 1,2,4-trisubstituted ben-
zene moiety and one methoxy group in 10 (Table 3). These data
indicated that 10 is a demethoxy derivative of 9, which was con-
firmed by HMBC analysis of 10. Thus, compound 10 was deter-
mined as 1-O-3,4-dimethoxy-5-hydroxyphenyl-(6-O-vanilloyl)-β-D-
glucopyranoside.
INOVA 500 MHz spectrometer in DMSO or pyridine-d5 with solvent
peaks as references. ESIMS and HRESIMS were obtained using an
Agilent 1100 series LC/MSD Trap SL mass spectrometer. GC was
conducted using an Agilent Technologies 7890A instrument (Agilent).
Preparative HPLC was carried out on a Shimadazu LC-6AD instrument
with a SPD-20A detector, using a YMC-Pack ODS-A column (250 ꢁ
20 mm, 5 μm). Column chromatography was performed with silica gel
(200ꢀ300 mesh, Qingdao Marine Chemical Inc., Qingdao, People’s
Republic of China) and ODS (50 μm, YMC, Japan). TLC was carried
out with glass precoated silica gel GF254 plates. Spots were visualized
under UV light or by spraying with 10% sulfuric acid in EtOH followed
by heating.
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Journal of Natural Products
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Table 2. 13C NMR Spectroscopic Data for Compounds 1ꢀ8a
Table 2. Continued
position
1
2
3
4
5
6
7
8
position
1
2
3
4
5
6
7
8
Ara0 (1f2) Ara
1
2
3
4
5
Rha (1f2) Ara
1
2
3
1
2
3
4
5
6
7
8
38.5 38.9 38.4 38.5 38.7 38.7 38.7 38.7
26.3 26.6 26.3 26.3 26.6 26.5 26.5 26.5
89.4 89.5 89.0 88.9 89.2 89.6 89.6 89.6
39.4 39.5 39.3 39.3 39.5 39.5 39.6 39.6
55.5 55.5 55.5 55.6 55.8 55.8 55.9 55.8
18.2 18.8 18.2 18.3 18.6 18.5 18.5 18.5
32.9 36.7 33.2 33.2 33.4 33.4 33.5 33.4
39.9 41.5 39.8 39.9 40.1 40.1 40.1 40.0
46.7 47.0 46.9 46.9 47.1 47.1 47.1 47.1
36.6 36.9 36.7 36.8 37.0 36.9 37.0 36.9
23.6 23.9 23.5 23.6 23.8 23.8 23.8 23.7
123.1b 124.8 123.1b 122.5b 122.7b 123.0 123.1b 123.1
143.5 144.5 144.2 144.2 144.4 144.4 144.4 144.4
41.4 47.7 41.8 41.9 42.1 42.1 42.1 42.0
34.9 67.5 35.7 35.7 36.0 35.9 36.0 35.9
69.9 75.0 74.2 74.3 74.4 74.4 74.4 74.4
44.6 45.2 48.9 48.9 49.1 49.1 49.1 49.1
40.7 41.7 41.0 41.1 41.3 41.3 41.3 41.3
47.2 47.1 46.9 46.9 47.1 47.1 47.1 47.2
31.9 32.0 30.6 30.6 30.8 30.8 30.8 30.8
41.5 41.5 35.9 35.9 36.2 36.1 36.2 36.1
72.8 72.7 32.0 32.1 32.3 32.2 32.2 32.2
27.8 28.0 27.8 27.0 28.0 27.9 28.0 27.9
16.6 16.8 16.7 16.7 16.9 16.7 16.7 16.7
15.4 15.8 15.4 15.4 15.6 15.6 15.6 15.6
16.5 17.5 17.3 17.3 17.5 17.5 17.5 17.5
27.4 21.3 27.0 27.8 27.3 27.2 27.2 27.2
63.4 63.0 175.7 175.7 175.9 175.9 175.9 175.9
33.3 33.4 33.0 33.0 33.2 33.2 33.2 33.2
25.0 25.1 24.3 24.4 24.6 24.6 24.6 24.5
167.8 167.9
106.6
72.0
74.2
69.2
65.2
103.8
72.3
72.5
74.1
70.1
18.6
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
10
4
5
6
Glc0 (1f2) GlcA
1
2
3
4
5
6
102.7
72.7
77.4
76.4
78.2
63.5
105.0
72.5
81.3
77.2
78.8
63.4
Rha (1f3) Glc0
1
2
3
4
5
6
104.1
71.9
72.1
74.0
70.1
18.5
a 13C NMR data (δ) were measured in C5D5N at 125 MHz for carbon.
The assignments were based on TOCSY, HSQC, and HMBC experi-
ments. b The data were overlapped by solvent peaks.
20
129.3 129.5
136.3 136.3
15.6 15.8
20.7 20.8
Plant Material. The stems of Gordonia chrysandra were collected at
Xishuangbanna, Yunnan Province, China, in May 2009 and identified by
Prof. Jingyun Cui (Xishuangbanna Tropical Botanical Garden, Chinese
Academy of Sciences). A voucher specimen (No. 21790) was deposited at
the Institute of Materia Medica, Chinese Academy of Medical Sciences,
Beijing.
30
40
50
Sugar (C-28)
Glc 1
2
3
4
5
6
95.7 95.7 95.9 95.9 95.9 95.9
73.9 74.1 74.2 74.2 74.2 74.2
79.1 79.2 79.4 79.4 79.4 79.4
70.9 70.9 71.1 71.1 71.1 71.1
78.6 78.7 78.9 78.9 79.1 78.5
61.9 62.0 62.2 62.2 62.5 62.1
Extraction and Isolation. The air-dried stems of G. chrysandra
(4.8 kg) were extracted with 95% EtOH (3 ꢁ 14 L) at reflux for 3 ꢁ 3 h,
and the extract was evaporated under reduced pressure to yield a dark brown
residue (200.5 g). The residue was suspended in water (2000 mL) and
then partitioned with CHCl3 (5 ꢁ 2000 mL), EtOAc (5 ꢁ 2100 mL), and
n-BuOH (5 ꢁ 1800 mL), successively. After removing solvent, the n-BuOH-
soluble portion (15.1 g) was fractionated via silica gel column chromatog-
raphy, eluting with CHCl3ꢀMeOHꢀH2O (7:3:0.5), to afford 10 fractions,
A1ꢀA10, on the basis of TLC analysis. Fraction A6 (1.8 g) was subjected to
separation over an ODS column (50 μm, 20ꢀ70% MeOHꢀH2O) to afford
seven fractions. Fraction 3 (520 mg) was purified by preparative HPLC
(YMC-ODS-A 5 μm, 250 mm ꢁ 20 mm, detection at 210 nm) using 36%
CH3CNꢀH2O (7 mL/min) containing 0.05% TFA as mobile phase to yield
compounds 1 (15 mg), 2 (10 mg), and 3 (30 mg). Fraction 5 (620 mg) was
subjected to preparative HPLC (YMC-ODS-A 5 μm, 250 mm ꢁ 20 mm,
detection at 210 nm) using 38% CH3CNꢀH2O (7 mL/min) containing
0.05% TFA as mobile phase to yield compounds 4(10mg), 5(18mg), and6
(60 mg). Fraction 7 (530 mg) was separated by reversed-phase HPLC with
36% CH3CNꢀH2O containing 0.05% TFA as mobile phase to yield com-
pounds 7 (20 mg) and 8 (22 mg). Fraction 2 (140 mg) was purified further
by preparative HPLC (YMC-ODS-A 5 μm, 250 mm ꢁ 20 mm, detection at
280 nm) using 14% CH3CNꢀH2O (7 mL/min) as mobile phase to yield
compounds 9 (22 mg) and 10 (24 mg).
Sugar (C-3)
GlcA 1
2
3
4
5
6
105.4 105.6 106.5 106.1 106.7 105.6 105.6 105.2
78.8 79.1 74.2 74.0 74.6 78.2 78.2 78.8
83.7 83.8 85.5 86.1 86.5 83.8 83.9 86.1
70.9 71.1 71.2 71.1 71.4 71.1 71.0 69.3
77.1 77.3 78.6 77.2 77.5 77.4 77.3 77.2
171.9 172.3 172.0 172.0 172.3 172.0 172.1 172.2
Ara (1f3) GlcA
1
2
3
4
101.5 101.8 105.6 102.7 106.5 101.8 101.8
81.8 81.9 74.5 81.4 80.5 82.2 81.9
73.1 73.4 72.6 73.2 72.3 73.3 73.3
68.1 68.3 69.0 72.8 69.4 68.3 68.3
65.7 65.9 66.9 67.5 68.0 66.0 65.9
5
Xyl (1f2) Ara
1
2
3
4
106.6 106.7
75.5 75.6
78.0 78.2
70.5 70.7
67.3 67.5
107.0 106.8 103.7
75.6 75.7 76.3
78.6 78.9 77.7
70.7 70.7 71.5
67.5 67.5 67.9
5
Gal (1f2) GlcA
1
2
3
4
5
6
103.3 103.5
73.6 73.8
74.9 75.0
69.9 70.1
76.2 76.4
61.7 61.9
103.5
73.8
75.1
70.1
76.4
61.9
Gordonoside I (1): white, amorphous powder; mp 241ꢀ242 °C;
[R]20D ꢀ2.7 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 206 (4.31) nm;
IR νmax 3402, 2950, 1783, 1678, 1437, 1375, 1203, 1162, 1080,
1047 cmꢀ1
;
1H NMR (pyridine-d5, 500 MHz) and 13C NMR
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Journal of Natural Products
ARTICLE
Table 3. NMR Spectroscopic Data (500 MHz, DMSO-d6) for
NMR (pyridine-d5, 125 MHz) are given in Tables 1 and 2, respectively;
(þ)-ESIMS m/z 1111 [M þ Na]þ; HRESIMS m/z 1111.5297
[M þ Na]þ (calcd for C53H84O23Na, 1111.5296).
Compounds 9 and 10a
9
10
Gordonoside N (6): white, amorphous powder; mp 238ꢀ239 °C;
[R]20D ꢀ4.8 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 204 (4.32) nm;
IR νmax 3396, 2939, 1731, 1674, 1434, 1365, 1223, 1160, 1077, 1047 cmꢀ1
;
position
δC
δH (J in Hz)
δC
δH (J in Hz)
1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz)
are given in Tables 1 and 2, respectively; (þ)-ESIMS m/z 1259
[M þ Na]þ; (ꢀ)-ESIMS m/z 1235 [M ꢀ H]ꢀ; HRESIMS m/z 1259.5672
[M þ Na]þ (calcd for C58H92O28Na, 1259.5667).
1
153.7
92.8
153.6
92.9
2
6.12 d (2.0)
6.13 d (3.0)
3
153.3
131.6
150.8
97.2
153.3
131.6
150.8
97.2
4
Gordonoside O (7): white, amorphous powder; mp 232ꢀ233 °C;
[R]20D ꢀ7.3 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 204 (4.28),
276 (2.94) nm; IR νmax 3372, 2936, 1728, 1673, 1430, 1366, 1201, 1141,
5
6
6.15 d (2.0)
3.56 s
6.18 d (3.0)
3.57 s
1075, 1044 cmꢀ1; H NMR (pyridine-d5, 500 MHz) and 13C NMR
1
OMe-7
55.5
55.6
(pyridine-d5, 125 MHz) are given in Tables 1 and 2, respectively; (þ)-
ESIMS m/z 1259 [M þ Na]þ; HRESIMS m/z 1259.5667 [M þ Na]þ
(calcd for C58H92O28Na, 1259.5667).
OMe-8
60.1
3.59 s
60.1
3.61 s
10
20
30
40
50
60
100.6
73.2
4.82 d (7.5)
3.23 m
100.5
73.2
4.82 d (8.0)
3.21 m
Gordonoside P (8): white, amorphous powder; mp 227ꢀ228 °C;
76.3
3.32 m
76.3
3.30 m
[R]20D ꢀ12.5 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 204 (4.29) nm;
IR νmax 3390, 2942, 1727, 1676, 1434, 1365, 1203, 1141, 1075, 1045 cmꢀ1
;
70.2
3.25 m
70.1
3.23 m
1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz)
are given in Tables 1 and 2, respectively; (þ)-ESIMS m/z 1273
[M þ Na]þ; (ꢀ)-ESIMS m/z 1249 [M ꢀ H]ꢀ; HRESIMS m/z 1273.5827
[M þ Na]þ (calcd for C59H94O28 Na, 1273.5824).
73.9
3.71 m
73.9
3.69 m
64.2
4.20 m
64.1
4.15 dd (12.0, 7.0)
4.57 d (12.0)
4.62 d (11.0)
70
100
200
300
400
500
600
165.6
119.3
107.0
147.6
140.9
147.6
107.0
56.1
165.6
120.4
112.6
147.5
151.8
115.3
123.7
55.6
1-O-3,4-Dimethoxy-5-hydroxyphenyl-(6-O-3,5-dimethoxygalloyl)-
β-D-glucopyranoside (9): white, amorphous powder; mp 132ꢀ133 °C;
[R]20D ꢀ50.7 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 207 (3.10),
277 (2.44) nm; IR νmax 3423, 3005, 2942, 2845, 1700, 1611, 1510, 1463,
1427, 1337, 1228, 1188, 1110, 1076, 766 cmꢀ1; 1H NMR (pyridine-d5, 500
MHz) and 13C NMR (pyridine-d5, 125 MHz) are given in Table 3; (þ)-
ESIMS m/z535 [M þ Na]þ;(ꢀ)-ESIMSm/z511 [M ꢀ H]ꢀ;HRESIMS
m/z 511.1461 [M ꢀ H] ꢀ (calcd for C23H27O13, 511.1457).
7.18 s
7.40 brs
6.85 d (8.0)
7.46 dd (8.0, 1.5)
3.77 s
7.18 s
3.76 s
3.76 s
OMe
1-O-3,4-Dimethoxy-5-hydroxyphenyl-(6-O-vanilloyl)-β-D-glucopyra-
noside (10): white, amorphous powder; mp 127ꢀ128 °C; [R]20D ꢀ46.7
(c 0.04, MeOH); UV (MeOH) λmax (log ε) 208 (3.27), 263 (2.72), 292
(2.41) nm; IR νmax 3405, 3005, 2941, 2845, 1696, 1602, 1510, 1461, 1431,
1286, 1224, 1175, 1105, 1073, 765 cmꢀ1; 1H NMR (pyridine-d5, 500 MHz)
and 13C NMR (pyridine-d5, 125 MHz) are given in Table 3; (þ)-ESIMS
m/z 505 [M þ Na]þ; (ꢀ)-ESIMS m/z 481 [M ꢀ H]ꢀ; HRESIMS m/z
481.1354 [M ꢀ H]ꢀ (calcd for C22H25O12, 481.1351).
56.1
a Data assignments were based on HSQC and HMBC experiments.
(pyridine-d5, 125 MHz) are given in Tables 1 and 2, respectively; (þ)-
ESIMS m/z 1181 [M þ Na]þ; (ꢀ)-ESIMS m/z 1157 [M ꢀ H]ꢀ;
HRESIMS m/z 1181.5703 [M þ Na]þ (calcd for C57H90O24Na,
1181.5714).
Acid Hydrolysis and Sugar Analysis. The determination of the
absolute configuration of the sugars in compounds 1ꢀ10 was conducted
as described previously.3,4
Gordonoside J (2): white, amorphous powder; mp 239ꢀ240 °C;
[R]20D ꢀ4.8 (c 0.06 MeOH); UV (MeOH) λmax (log ε) 204 (4.28) nm;
IR νmax 3389, 2940, 1781, 1727, 1675, 1455, 1365, 1204, 1169, 1078,
1049 cmꢀ1; 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-
d5, 125 MHz) are given in Tables 1 and 2, respectively; (þ)-ESIMS m/z
1197 [M þ Na]þ; (ꢀ)-ESIMS m/z 1173 [M ꢀ H]ꢀ; HRESIMS m/z
1197.5668 [M þ Na]þ (calcd for C57H90O25Na, 1197.5663).
Gordonoside K (3): white, amorphous powder; mp 226ꢀ227 °C;
[R]20D ꢀ7.0 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 204 (4.33) nm;
IR νmax 3383, 2939, 1730, 1673, 1445, 1363, 1256, 1204, 1142, 1073,
1047 cmꢀ1; 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-
d5, 125 MHz) are given in Tables 1 and 2, respectively; (þ)-ESIMS m/z
965 [M þ Na]þ; (ꢀ)-ESIMS m/z 941 [M ꢀ H]ꢀ; HRESIMS m/z
965.4697 [M þ Na]þ (calcd for C47H74O19Na, 965.4717).
Inhibitory Effects on Nitric Oxide Production in LPS-Activated
Macrophages. The procedure for NO determination was based on the
Griess reaction.8 One hundred microliters of culture supernatant or sodium
nitrite standard (5.2ꢀ103.6 μM) was mixed with an equal volume of Griess
reagent [a mixture of 0.1% (w/v) N-(1-naphthyl)ethylenediamine dihy-
drochloride and 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid; the
two parts being mixed together within 1 h of use] using a 96-well plate. After
20 min at room temperature, the absorbance at 540 nm was measured by a
microtitration plate reader.
’ ASSOCIATED CONTENT
Gordonoside L (4): white, amorphous powder; mp 234ꢀ235 °C;
[R]20D ꢀ9.3 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 204 (4.27) nm;
IR νmax 3404, 2937, 1728, 1674, 1434, 1366, 1202, 1142, 1075, 1026 cmꢀ1;1H
NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz) are
given in Tables 1 and 2, respectively; (þ)-ESIMS m/z 1097 [M þ Na]þ;
HRESIMS m/z1097.5125 [M þ Na]þ (calcdforC52H82O23Na, 1097.5139).
Gordonoside M (5): white, amorphous powder; mp 235ꢀ236 °C;
S
Supporting Information. MS and 1D and 2D NMR
b
spectra of compounds 1ꢀ10. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
[R]20 ꢀ18.0 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 204
Corresponding Author
*Tel: þ86-10-63165227. Fax: þ86-10-63165227. E-mail: zhangdm@
D
(4.32) nm; IR νmax 3404, 2943, 1728, 1675, 1435, 1388, 1365, 1203,
1142, 1074, 1049 cmꢀ1; H NMR (pyridine-d5, 500 MHz) and 13C
1
imm.ac.cn.
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Journal of Natural Products
ARTICLE
’ ACKNOWLEDGMENT
Financial support was provided by the National Natural
Science Foundation of China (NSFC, Grant No. 20972191).
We thank our colleagues of our institute: Prof. X. J. Jing for NMR
measurements, Prof. J. B. Li for MS measurements, and Profs. X.
G. Chen, Q. Hou, and Z. F. Shen for bioassays.
’ REFERENCES
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