Dammarane-type triterpenoids from Gentianella azurea

Article abstract of DOI:10.1021/np500077z

Thirteen new dammarane-type triterpenoids (1-13) and four known analogues, gentirigenic acid (14) and the gentirigeosides A, B, and E (15-17), were isolated from Gentianella azurea. Their structures were elucidated by detailed analysis of the NMR, MS, and X-ray crystallographic data. This is the first report of dammarane-type triterpenoids in the Gentianella genus. In addition, the known structures of gentirigenic acid (14) and the gentirigeosides A, B, and E (15-17) were revised based on the X-ray diffraction analysis. Gentirigeoside A (15) was found to inhibit nitric oxide production in RAW 264.7 macrophages with an IC₅₀ value of 6.6 ± 2.1 μM.

Full text of DOI:10.1021/np500077z

Article
pubs.acs.org/jnp
Dammarane-Type Triterpenoids from Gentianella azurea
Yu-jie Huang, Hui Lu, Xue-li Yu, Shu-wei Zhang, Wen-qiong Wang, Lin-yin Fen, and Li-jiang Xuan*
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road,
Zhangjiang Hi-Tech Park, Shanghai 201203, People’s Republic of China
*
S Supporting Information
ABSTRACT: Thirteen new dammarane-type triterpenoids
(
1−13) and four known analogues, gentirigenic acid (14)
and the gentirigeosides A, B, and E (15−17), were isolated
from Gentianella azurea. Their structures were elucidated by
detailed analysis of the NMR, MS, and X-ray crystallographic
data. This is the first report of dammarane-type triterpenoids
in the Gentianella genus. In addition, the known structures of
gentirigenic acid (14) and the gentirigeosides A, B, and E (15−17) were revised based on the X-ray diffraction analysis.
Gentirigeoside A (15) was found to inhibit nitric oxide production in RAW 264.7 macrophages with an IC value of 6.6 ± 2.1
50
μM.
Gentianella azurea, a traditional Tibetan medicinal plant, is
widely distributed in west China, including Tibet, Sichuan,
Yunnan, and Xinjiang Provinces, and has been used to trea1t
inflammation resulting from hepatitis, icterus, and rheumatism.
Previous studies on plant constituents from the Gentianaceae
family have demonstrated their high anti-inflammatory
2
activities.
Nitric oxide (NO) is a signaling molecule that plays an
3
important role in inflammation and immunity. Several studies
have shown that NO plays a crucial role in the modulation of
4
multiform acute and chronic inflammatory disorders. There-
fore, inflammatory conditions can be treated by inhibiting NO
5
production.
To identify anti-inflammatory natural products from G.
azurea, its crude extract was investigated and led to the isolation
and identification of 13 new dammarane-type triterpenoids (1−
1
3) and four known analogues, gentirigenic acid (14) and
6
gentirigeosides A, B, and E (15−17). Their inhibitory activities
against lipopolysaccharide-induced NO production in RAW
2
2
64.7 macrophages were assessed, and an IC50 value of 6.6 ±
.1 μM was observed for gentirigeoside A (15). In addition,
these results add to the sparse literature concerning G.
1
,7
azurea.
RESULTS AND DISCUSSION
■
Compound 1 was obtained as an amorphous powder. The
molecular formula of 1 was determined to be C H O based
on the quasi-molecular ion at m/z 673.3917 [M + Na] (calcd
lactone carbonyl carbon (δ 179.8), one trisubstituted double
C
3
6
58 10
+
bond (δ 138.9 and 125.7), and one hexosyl (δ 106.6, 79.2,
C
C
1
3
79.1, 76.0, 72.2, and 63.3) moiety. These moieties accounted
for four indices of hydrogen deficiency, suggesting four
additional rings in the structure of 1; thus, compound 1 was
likely a tetracyclic triterpenoid glycoside. Acid hydrolysis of 1
yielded D-glucose, and the coupling constant of the anomeric
6
73.3922) in the HRESI(+)MS analysis and C NMR
spectroscopic data. The IR spectrum showed absorption
−1
−1
bands for hydroxy (3423 cm ) and γ-lactone (1764 cm )
groups. The 13C NMR data (Table 1) exhibited 36 carbon
resonances, which were classified by a DEPT experiment into
six tertiary methyl, 11 methylene, 12 methine, and seven
quaternary carbons. The NMR spectroscopic data (Tables 1
and 2) showed characteristic resonances of carbons from one
Received: January 28, 2014
Published: May 7, 2014
©
2014 American Chemical Society and
American Society of Pharmacognosy
1201
dx.doi.org/10.1021/np500077z | J. Nat. Prod. 2014, 77, 1201−1209

Journal of Natural Products
Article
Table 1. 13C NMR Data for Compounds 1−13 in Pyridine-d5 (δ in ppm)
a
C
b
no.
1
2
3
4
5
6
7
8
9
10
11
12
13
1
2
3
4
5
6
7
8
9
39.4
27.3
89.2
44.9
57.0
19.1
36.2
41.0
51.3
37.1
22.3
27.6
43.6
51.0
32.0
26.2
46.1
15.8
16.5
79.3
179.8
41.2
74.5
125.7
138.9
26.0
18.5
23.7
63.6
16.8
106.6
76.0
79.1
72.2
79.2
63.3
39.4
27.4
89.2
44.9
57.0
19.1
36.3
41.0
51.4
37.1
22.4
28.4
45.3
50.5
32.0
26.6
45.6
15.9
16.5
81.5
178.7
39.5
75.6
124.0
139.1
26.0
18.5
23.7
63.6
16.5
106.6
76.0
79.1
72.2
79.1
63.3
39.4
27.3
89.2
44.9
56.9
19.1
36.2
41.2
51.3
37.0
22.3
27.6
43.6
51.0
32.1
26.2
46.1
15.8
16.4
79.2
179.8
41.2
74.3
123.8
143.1
14.5
67.0
23.7
63.5
16.8
106.6
76.0
79.2
72.3
79.2
63.3
39.3
27.1
91.0
44.2
57.0
18.8
36.0
40.9
51.2
36.9
22.3
27.6
43.5
50.9
32.0
26.1
46.1
15.7
16.6
79.3
179.8
41.1
74.4
125.7
138.8
25.9
18.5
22.9
63.7
16.8
104.9
82.8
78.6
70.2
78.8
61.9
105.5
76.1
79.2
71.5
79.0
63.1
39.3
27.1
90.9
44.2
56.9
18.8
36.9
40.8
51.2
36.9
22.3
28.3
45.2
50.3
32.0
26.5
45.6
15.7
16.5
81.4
178.6
39.4
75.5
124.2
139.8
26.0
18.5
22.9
63.7
16.6
104.9
82.7
78.5
70.2
78.7
61.9
105.4
76.1
79.2
71.4
78.9
63.0
39.6
27.2
89.3
40.1
56.8
18.8
36.0
41.4
51.4
37.3
22.1
27.6
43.6
51.0
32.0
26.2
46.1
15.9
16.8
79.3
179.8
41.1
74.4
125.8
138.8
25.9
18.5
28.4
17.0
16.8
105.5
83.8
78.5
72.0
78.3
63.0
106.5
77.6
78.7
72.0
78.7
63.2
39.7
27.2
89.3
40.1
56.7
18.8
36.1
41.0
51.4
37.3
22.1
28.4
45.3
50.5
32.1
26.6
45.6
16.0
16.6
81.5
178.7
39.4
75.6
124.2
139.8
26.0
18.5
28.4
16.9
16.7
105.5
83.8
78.5
72.0
78.3
63.0
106.4
77.5
78.7
72.0
78.7
63.2
39.6
27.1
89.2
40.0
56.7
18.7
36.0
40.9
51.5
37.3
22.1
28.1
45.1
50.5
32.0
26.3
46.1
15.9
16.6
81.4
178.9
33.4
80.0
77.7
72.1
27.5
28.1
28.4
17.1
16.8
107.3
76.1
79.1
72.2
78.7
63.4
39.2
27.2
89.1
44.7
56.8
19.0
36.1
40.8
51.2
36.9
22.2
27.5
43.4
50.8
31.9
25.8
46.0
15.7
16.3
78.6
179.7
35.9
78.5
78.1
72.0
27.4
28.1
23.6
63.5
16.7
106.5
75.9
79.0
72.1
79.0
63.2
39.2
27.2
89.2
44.6
56.7
18.9
36.0
40.7
51.1
36.9
22.1
28.3
44.9
50.3
31.8
26.1
45.9
15.6
16.3
81.3
178.8
33.3
79.8
77.5
72.0
27.9
27.3
23.5
63.4
16.4
106.3
75.2
77.4
71.7
78.5
70.7
105.7
75.5
78.6
71.9
78.7
62.8
39.7
27.3
89.5
40.1
56.7
18.7
36.0
40.9
51.4
37.3
22.1
28.5
45.1
50.5
32.0
26.3
46.1
15.9
16.6
81.4
178.9
33.5
80.0
77.7
72.1
28.1
27.5
28.4
17.0
16.9
105.4
83.6
77.4
71.8
78.3
70.7
106.4
77.0
78.6
72.1
78.5
63.1
105.9
75.4
78.9
72.0
78.8
63.1
39.6
27.1
89.4
40.1
56.7
18.7
36.0
40.8
51.4
37.2
22.0
28.4
45.1
50.5
32.0
26.3
46.0
15.9
16.5
81.4
178.9
33.3
79.9
77.6
72.0
28.0
27.4
28.5
17.0
16.8
105.3
83.5
78.3
71.8
78.3
63.1
106.1
77.0
78.3
72.0
77.3
70.2
105.6
75.5
78.5
71.8
78.6
63.1
39.7
27.3
89.6
40.1
56.8
18.8
36.0
40.9
51.4
37.3
22.1
28.5
45.1
50.5
32.0
26.3
46.1
15.9
16.6
81.4
178.9
33.5
80.0
77.7
72.1
28.5
27.3
28.1
17.1
16.9
105.3
83.2
77.4
71.7
78.4
70.4
106.1
77.0
78.5
72.0
77.0
70.6
105.7
75.6
78.6
72.0
78.7
63.1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
′
′
′
′
′
′
″
″
″
″
″
″
‴
‴
‴
‴
‴
‴
a
b
Chemical shifts (ppm) referenced to pyridine-d (δ 135.9) at 125 MHz. Data for Glc-IV of 13: δ 105.8 (C-1⁗), 75.5 (C-2⁗), 78.7 (C-3⁗), 72.0
C-4⁗), 78.8 (C-5⁗), 63.2 (C-6⁗).
5
C
(
proton at δ 5.07 (d, J = 7.8 Hz) indicated a β-configuration for
The configuration of the dammarane skeleton was assumed
H
to be consistent with those of previously reported com-
the sugar unit. The HMBC data (Supporting Information)
allowed the connections of the aforementioned moieties to be
established, and the structure of 1 was determined to be
8a,9
pounds. The large coupling constant of H-3 (δ 3.61, dd, J =
H
1
1.6, 4.9 Hz) suggested an α-axial orientation. In the ROESY
spectrum, the correlation of H -29/H -19 showed that the
2
3
3
,20,23,29-tetrahydroxydammar-24-en-21-oic acid-21,23-lac-
hydroxy group was located at C-29. In addition, the correlation
8
tone. In addition, the glucose moiety was attached at C-3, as
supported by the HMBC correlation of H-1′/C-3. Therefore,
the planar structure of 1 was established.
of H-24/H -27 indicated that they were cofacial. Previously, the
3
configurations of C-20 and C-23 had been determined by
comparing either the NMR or the electronic circular dichroism
1
202
dx.doi.org/10.1021/np500077z | J. Nat. Prod. 2014, 77, 1201−1209

Journal of Natural Products
Article
1
a
Table 2. H NMR Data for Compounds 1−7 in Pyridine-d (δ in ppm, J in Hz)
5
H
no.
1
2
3
4
5
6
7
1
2
3
5
6
7
9
1.48 m, 0.72 m
2.17 m, 2.00 m
1.52 m, 0.77 m
2.20 m, 2.01 m
1.44 m, 0.70 m
2.05 m, 1.95 m
1.34 m, 0.64 m
2.30 m, 1.87 m
1.40 m, 0.70 m
2.33 m, 1.88 m
1.43 m, 0.71 m
2.21 m, 1.82 m
1.49 m, 0.77 m
2.25 m, 1.86 m
3.61 dd (11.6, 4.9) 3.63 dd (11.8, 4.1) 3.62 dd (11.6, 4.7)
3.43 dd (11.7, 4.8) 3.44 dd (11.7, 4.8) 3.29 dd (11.8, 4.5) 3.32 dd (11.8, 4.5)
0.85 m
0.88 m
0.84 m
0.76 m
0.77 m
0.66 m
0.69 m
1.45 m, 1.21 m
1.47 m, 1.22 m
1.27 m
1.49 m, 1.20 m
1.51 m, 1.23 m
1.28 m
1.64 m, 1.46 m
1.46 m, 1.20 m
1.26 m
1.48 m, 1.25 m
1.60 m, 1.08 m
1.19 m
1.49 m, 1.26 m
1.54 m, 1.07 m
1.21 m
1.46 m, 1.39 m
1.42 m, 1.21 m
1.24 m
1.48 m, 1.21 m
1.47 m, 1.21 m
1.28 m
1
1
1
1
1
1
1
2
3
5
6
7
1.64 m, 1.46 m
2.42 m, 1.36 m
2.09 m
1.51 m, 1.28 m
2.53 m, 1.49 m
1.86 m
1.45 m, 1.20 m
2.44 m, 1.37 m
2.08 m
1.34 m, 1.16 m
2.38 m, 1.31 m
2.05 m
1.43 m, 1.21 m
2.49 m, 1.43 m
1.81 m
1.43 m, 1.24 m
2.41 m, 1.34 m
2.07 m
1.50 m, 1.28 m
2.53 m, 1.48 m
1.86 m
1.64 m, 1.12 m
2.02 m, 1.70 m
1.57 m, 1.11 m
2.10 m, 1.34 m
1.60 m, 1.08 m
2.02 m, 1.70 m
1.39 m, 1.14 m
1.99 m, 1.67 m
1.53 m, 1.07 m
2.05 m, 1.30 m
1.45 m, 1.19 m
1.64 m, 1.10 m
1.58 m, 1.09 m
2.08 m, 1.34 m
2.55 ddd (10.5, 5.3, 2.72 ddd (10.6, 5.6, 2.58 ddd (10.6, 5.1,
2.52 ddd (10.5, 5.4, 2.68 ddd (10.8, 5.8, 2.54 ddd (10.6, 5.2, 2.72 ddd (10.6, 5.5,
5.3)
5.6)
0.97 s
0.77 s
5.1)
0.92 s
0.71 s
5.4)
0.86 s
0.57 s
5.8)
0.92 s
0.63 s
5.2)
0.92 s
0.75 s
5.5)
1.00 s
0.81 s
1
1
2
8
9
2
0.92 s
0.71 s
2.72 dd (13.2, 7.2) 2.61 dd, (13.2, 5.6) 2.76 dd (13.2, 7.4)
.30 dd (13.2, 6.2) 2.14 dd (13.2, 9.8) 2.36 dd (13.2, 6.0)
2.71 dd (13.2, 7.2) 2.59 dd (13.3, 5.6) 3.29 dd (12.5, 4.5) 2.61 dd (13.3, 5.6)
2.28 dd (13.2, 6.3) 2.11 dd (13.3, 9.6) 2.72 dd (12.5, 5.2) 2.13 dd (13.3, 9.9)
2
23
5.50 ddd (8.9, 7.2, 5.72 ddd (9.8, 8.7, 5.66 ddd (9.0, 7.4, 6.0) 5.49 ddd (8.5, 7.2, 5.70 ddd (9.6, 8.8, 5.49 ddd (8.9, 5.2, 5.72 ddd (9.9, 8.9,
6.2)
5.6)
6.3)
5.6)
4.5)
5.6)
2
2
4
6
5.59 d (8.9)
1.70 s
5.42 d (8.7)
1.69 s
6.26 d (9.0)
5.58d (8.5)
5.40 d (8.8)
1.68 s
5.59 d (8.9)
1.70 s
5.42 d (8.9)
1.69 s
4.30 d (12.0), 4.28 d 1.62 s
12.0)
(
2
2
2
7
8
9
1.63 s
1.64 s
1.87 s
1.69 s
1.63 s
1.62 s
1.29 s
1.11 s
1.64 s
1.30 s
1.13 s
1.55 s
1.55 s
1.55 s
1.35 s
1.34 s
4.37 d (11.1)
4.39 d (11.2)
3.68 d (11.2)
0.92 s
4.40 d (11.1)
3.67 d (11.1)
0.88 s
4.28 d (11.4)
3.36 d (11.4)
0.85 s
4.28 d (9.7)
3.37 d (9.7)
0.86 s
3.66 d (11.1)
3
1
2
3
4
5
0
0.89 s
0.89 s
0.89 s
′
′
′
′
5.07 d (7.8)
5.07 d (7.7)
4.03 dd (8.8, 7.7)
4.27 dd (8.9, 8.8)
4.20 dd (9.2, 8.9)
5.08 d (8.0)
4.05 dd (8.4, 7.8)
4.09 dd (9.1, 8.4)
4.23 dd (9.1, 9.1)
4.88 d (7.7)
4.09 dd (8.4, 7.7)
4.20 dd (8.5, 8.4)
4.39 m
4.86 d (7.8)
4.07 dd (8.8, 7.8)
4.17 dd (9.2, 8.8)
4.36 m
4.94 d (7.5)
4.25 dd (8.5, 7.5)
4.26 dd (9.0, 8.5)
4.35 m
4.95 d (7.5)
4.27 dd (8.8, 7.5)
4.28 dd (8.8, 8.6)
4.37 m
4.03 dd (8.5, 7.8)
4.27 dd (9.1, 8.5)
4.21 dd (9.1, 9.1)
′
4.06 ddd (9.1, 5.6, 4.06 ddd (9.2, 5.6, 4.28 ddd (9.1, 5.9, 2.4) 3.73 m
.5) 2.5)
3.71 m
3.94 m
3.95 m
2
6′
4.64 dd (11.7, 2.4) 4.64 dd (11.6, 2.5) 4.67 dd (11.7, 2.4)
.43 dd (11.7, 5.6) 4.42 dd (11.6, 5.6) 4.45 dd (11.7, 5.9)
4.38 m
4.38 m
4.48 dd (11.6, 3.8) 4.50 dd (11.5, 3.8)
4.35 dd (11.6, 2.6) 4.36 dd (11.5, 2.3)
4
4.33 m
4.30 m
1″
2″
3″
4″
5″
5.57 d (7.3)
4.10 dd (8.5, 7.3)
4.29 dd (8.9, 8.5)
4.17 dd (8.9, 8.7)
5.54 d (7.8)
4.08 dd (8.8, 7.8)
4.27 dd (9.1, 8.8)
4.15 dd (9.2, 9.1)
5.39 d (7.6)
5.40 d (7.7)
4.13 dd (8.9, 7.9)
4.33 dd (8.9, 8.9)
4.15 dd (9.4, 9.1)
4.15 dd (8.7, 7.7)
4.34 dd (8.9, 8.7)
4.16 dd (9.4, 8.9)
3.94 ddd (8.7, 5.9, 3.91 m
.4)
3.94 ddd (9.1, 4.2, 3.95 ddd (9.4, 3.3,
2.4) 2.5)
2
6″
4.60 dd (11.8, 2.4) 4.58 (12.1, 2.4)
.58 dd (11.8, 5.9) 4.35 m
4.59 dd (11.8, 2.4) 4.59 dd (11.9, 2.5)
4.50 dd (11.8, 3.4) 4.50 dd (11.9, 3.3)
4
a
Chemical shifts (ppm) referenced to pyridine-d (δ 7.58) at 500 MHz.
5
H
8
(
ECD) data. X-ray diffraction analysis of crystals obtained by
NMR data with the data for (3β,20S,23S)-3,20,23-trihydrox-
ydammar-24-en-21-oic acid-21,23-lactone 3-O-[α-L-rhamnopyr-
anosyl-(1→2)]-[β-D-xylopyranosyl(1→3)]-α-L-arabinopyrano-
side, and these assignments were confirmed by the ECD
spectrum (Supporting Information). Thus, the structure of 2
was established as shown.
recrystallization from MeOH not only confirmed the gross
structure of 1 but also established its absolute configuration as
3β, 20S, 23R) (Figure 1), which agreed with the ECD
results. Thus, the structure of 1 was determined as
3β,20S,23R)-3,20,23,29-tetrahydroxydammar-24-en-21-oic
8a
(
8
a
(
acid-21,23-lactone 3-O-β-D-glucopyranoside.
The molecular formula of 3 was determined to be C H O
3
6
58 11
Compound 2 was defined as an isomer of 1, as indicated by
HRESI(+)MS analysis and the spectroscopic data. Analysis of
the NMR data (Tables 1 and 2) supported this conclusion, as
most of the resonances of 2 were nearly superimposable on
those of 1, with the exception of the resonances of ring E. The
HMBC spectrum (Supporting Information) established that 2
had the same planar structure as 1. The (20S, 23S)
from the 13C NMR and HRESI(+)MS data, which showed a
+
molecular ion at m/z 689.3883 [M + Na] (calcd 689.3871), 16
amu greater than the molecular weight of 1. The NMR data for
3 (Tables 1 and 2) were similar to those for 1, except for an
additional hydroxy group at C-27, thus accounting for the
difference in molecular weight. These observations were further
supported by the HMBC correlations between H -27/C-24 and
2
1
13
configuration was proposed by comparing the H and
C
C-25. The ROESY spectrum showed correlations between H-
1
203
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Journal of Natural Products
Article
the data for 2, the resonances of the olefinic bond in 2 were
replaced by resonances for one methine and one quaternary
carbon (both oxygenated), suggesting that the double bond had
been dihydroxylated. In addition, C-29 was deoxygenated.
These observations were supported by the HMBC correlations
of H -26/C-24, C-25, and C-27; H -27/C-24, C-25, and C-26;
3
3
H -28/C-3, C-4, and C-5; and H -29/C-3, C-4, and C-5. In
3
3
addition, these observations were consistent with the chemical
shifts of these protons and carbons and with the molecular
formula. Single-crystal X-ray diffraction analysis permitted
definition of the absolute configuration (Figure 2) of 8 as
(
(
20S, 23S, 24R). Thus, the structure of 8 was established as
3β,20S,23S,24R)-3,20,23,24,25-pentahydroxydammaran-21-oic
acid-21,23-lactone 3-O-β-D-glucopyranoside.
Compound 9 displayed a molecular formula of C H O , as
3
6
60 12
13
deduced from the C NMR spectroscopic data and the
HRESI(+)MS ion at m/z 707.3991 [M + Na] (calcd
Figure 1. X-ray ORTEP drawing of compound 1.
+
7
07.3977), which suggested that 9 was a hydroxy analogue of
8
. The hydroxy group was located at C-29 based on the HMBC
2
4/H -27 and H -19/H -29, which established the relative
2 3 2
correlations of H -29/C-4 and C-28. The chemical shifts for C-
configuration. The configurations of C-20 and C-23 were
established by comparing the NMR and ECD data with the
data for 1. Thus, the structure of 3 was defined as
2
2
0 (δ 78.6), C-22 (δ 35.9), C-23 (δ 78.5), and C-24 (δ
C C C C
7
8.1) showed minor differences from the data for 8, which
suggested that 9 had a different C-23 configuration than 8.
Fortunately, a single crystal of 9 was obtained via slow
evaporation from a ternary solvent system of H O/pyridine/
MeOH (2:1:7). The X-ray diffraction of a single crystal of 9
permitted definition of the absolute configuration of 9 as (20S,
(
3β,20S,23R)-3,20,23,26,29-pentahydroxydammar-24-en-21-oic
acid-21,23-lactone 3-O-β-D-glucopyranoside.
Compounds 4 and 5 shared the same molecular formula of
2
1
3
C H O , as determined by the C NMR data and
HRESI(+)MS ions at m/z 835.4452 [M + Na] (calcd
4
2
68 15
+
+
1
2
3R, 24R) (Figure 3). Therefore, the structure of compound 9
835.4450) and 835.4447 [M + Na] , respectively. The H
13
was defined as (3β,20S,23R,24R)-3,20,23,24,25,29-hexahydrox-
ydammaran-21-oic acid-21,23-lactone 3-O-β-D-glucopyranoside.
and C NMR data (Tables 1 and 2) of 4 and 5 were similar to
the data for 1 and 2, respectively, except for additional
Compound 10 had a molecular formula of C H O , as
resonances resulting from a hexosyl unit (H-1″ of 4, δ 5.57, d,
42 70 17
H
determined by the 13C NMR and HRESI(+)MS data, which
showed an ion at m/z 869.4508 [M + Na] (calcd 869.4505).
The NMR resonances for 10 were similar to those for 8, except
for the presence of an additional hexosyl moiety (δ 105.7,
78.7, 78.6, 75.5, 71.9, and 62.8) and a C-29 hydroxy substituent.
Acid hydrolysis of 10 yielded only D-glucose and the aglycone,
and the coupling constants of the anomeric protons at δ 5.00
J = 7.3 Hz; H-1″ of 5, δ 5.54, d, J = 7.8 Hz). Acid hydrolysis of
H
+
4
4
yielded only D-glucose and the aglycone. The sugar moiety of
was determined to be β-D-glucopyranosyl-(1→2)-β-D-
glucopyranosyl based on the HMBC correlations of H-1″ (δ
C
H
5
.57, d, J = 7.3 Hz)/C-2′ (δ 82.8) and H-1′ (δ 4.88, d, J = 7.7
C
H
Hz)/C-3 (δC 91.0). Therefore, the structure of 4 was
established as (3β,20S,23R)-3,20,23,29-tetrahydroxydammar-
H
2
4-en-21-oic acid-21,23-lactone 3-O-β-D-glucopyranosyl-(1→
(d, J = 7.8 Hz) and 5.10 (d, J = 7.7 Hz) suggested β-configured
glucosyl moieties. The sugar chain was determined to be β-D-
glucopyranosyl-(1→6)-β-D-glucopyranosyl based on the
HMBC correlation of H-1″/C-6′. Thus, the structure of 10
was established as (3β,20S,23S,24R)-3,20,23,24,25,29-hexahy-
droxydammaran-21-oic acid-21,23-lactone 3-O-β-D-glucopyra-
nosyl-(1→6)-β-D-glucopyranoside.
2)-β-D-glucopyranoside. The structure of 5 was elucidated in
a similar manner and named (3β,20S,23S)-3,20,23,29-tetrahy-
droxydammar-24-en-21-oic acid-21,23-lactone 3-O-β-D-gluco-
pyranosyl-(1→2)-β-D-glucopyranoside.
Compounds 6 and 7 gave sodiated molecular ions at m/z [M
+
+
Na] 819.4513 and 819.4518, respectively, by HRESI(+) MS,
1
3
which together with the C NMR spectroscopic data suggested
the same molecular formula of C H O (calcd 819.4501).
This formula suggests that compounds 6 and 7 are deoxy
derivatives of 4 and 5, respectively. The NMR data of 6 and 7
Compound 11 had a molecular formula of C48H O21 based
80
on the 13C NMR and HRESI(+)MS data ([M + Na] m/z
1015.5064, calcd 1015.5084). Comparisons between the NMR
data of 11 and 8 showed similar resonances but with two
additional β-D-glucopyranosyl groups, which was confirmed by
2D NMR spectroscopic data and acid hydrolysis of 11. In
addition, HMBC correlations of H-1″/C-2′ and H-1‴/C-6′
established the linkage of the sugar moieties. Thus, the
structure of compound 11 was defined as (3β,20S,23S,24R)-
3,20,23,24,25-pentahydroxydammaran-21-oic acid-21,23-lac-
tone 3-O-[β-D-glucopyranosyl-(1→2)]-[β-D-glucopyranosyl-
(1→6)]-β-D-glucopyranoside.
+
4
2
68 14
(
Tables 1 and 2) were similar to the data for 4 and 5, except
that the C-4 hydroxymethyl resonances in 4 and 5 were
replaced by methyl resonances in 6 and 7. The configurations
of 4 and 5 were identical to those of 6 and 7, respectively.
Therefore, the structures of compounds 6 and 7 were
established as (3β,20S,23R)-3,20,23-trihydroxydammar-24-en-
2
1-oic acid-21,23-lactone 3-O-β-D-glucopyranosyl-(1→2)-β-D-
glucopyranoside and (3β,20S,23S)-3,20,23-trihydroxydammar-
2
2
4-en-21-oic acid-21,23-lactone 3-O-β-D-glucopyranosyl-(1→
)-β-D-glucopyranoside, respectively.
Compound 12 showed the same molecular formula as 11, as
determined by the 13C NMR spectroscopic data and the
+
Compound 8 exhibited a sodiated molecular ion at m/z
91.4020 (calcd 691.4028) by HRESI(+)MS analysis, which
HRESI(+)MS ion at m/z 1015.5064 ([M + Na] , calcd
6
1015.5084), indicating that they are isomers. D-Glucose and the
aglycone were obtained from hydrolysis. Detailed analysis of
the NMR data indicated that 12 had the same aglycone as 11,
1
3
together with the C NMR data suggested a molecular formula
of C H O . Comparing the NMR data (Tables 1 and 3) with
36
60 11
1
204
dx.doi.org/10.1021/np500077z | J. Nat. Prod. 2014, 77, 1201−1209

Journal of Natural Products
Article
1
a
Table 3. H NMR Data for Compounds 8−13 in Pyridine-d (δ in ppm, J in Hz)
5
H
b
no.
8
9
10
11
12
13
1
2
3
5
6
7
9
1.47 m, 0.77 m
2.27 m, 1.85 m
3.40 dd (11.8, 4.4)
0.72 m
1.41 m, 0.66 m
2.15 m, 1.92 m
3.58 dd (11.7, 4.8)
0.82 m
1.58 m, 0.89 m
2.37 m, 2.04 m
1.60 m, 0.87 m
2.50 m, 1.41 m
3.26 dd (11.7, 4.5)
0.61 m
1.45 m, 0.74 m
2.22 m, 1.87 m
3.28 dd (11.7, 4.4)
0.64 m
1.60 m, 0.84 m
2.34 m, 1.94 m
3.25 dd (11.6, 4.6)
0.62 m
3.59 dd (11.8, 4.8)
0.79 m
1.43 m, 1.12 m
1.43 m, 1.12 m
1.26 m
1.60 m, 1.41 m
1.44 m, 1.17 m
1.22 m
1.59 m, 1.41 m
1.40 m, 1.12 m
1.24 m
1.38 m, 1.28 m
1.38 m, 1.10 m
1.21 m
1.44 m, 1.31 m
1.41 m, 1.12 m
1.23 m
1.44 m, 1.34 m
1.39 m, 1.09 m
1.24 m
1
1
1
1
1
1
1
1
2
1
2
3
5
6
7
8
9
2
1.46 m, 1.20 m
2.56 m, 1.48 m
1.95 m
1.38 m, 1.10 m
2.38 m, 1.34 m
2.06 m
1.24 m, 1.16 m
2.50 m, 1.42 m
1.93 m
1.49 m, 1.22 m
2.38 m, 1.92 m
1.93 m
1.46 m, 1.24 m
2.55 m, 1.46 m
1.95 m
1.49 m, 1.25 m
2.49 m, 1.44 m
1.94 m
1.48 m, 0.96 m
2.02 m, 1.66 m
2.76 m
1.57 m, 1.06 m
1.97 m, 1.72 m
2.54 m
1.46 m, 0.96 m
2.00 m, 1.66 m
2.72 m
1.48 m, 0.95 m
2.01 m, 1.65 m
2.70 m
1.53 m, 0.99 m
2.02 m, 1.65 m
2.74 m
1.51 m, 0.99 m
2.02 m, 1.65 m
2.73 m
0.84 s
0.90 s
0.84 s
0.85 s
0.86 s
0.84 s
0.73 s
0.66 s
0.73 s
0.79 s
0.78 s
0.81 s
3.17 dd (13.4, 9.5),
3.21 dd (13.5, 5.9),
2.78 dd (13.5, 7.6)
3.13 dd (13.4, 9.5),
2.75 dd (13.4, 6.0)
3.14 dd (13.4, 9.5),
2.77 dd (13.4, 5.9)
3.12 dd (13.4, 9.4),
2.75 dd (13.4, 6.0)
3.13 dd (13.4, 9.4),
2.75 dd (13.4, 5.8)
2.78 dd (13.4, 5.9)
2
2
2
2
2
2
3
4
6
7
8
9
5.67 ddd (9.5, 5.9, 2.6) 5.37 ddd (7.6, 5.9, 3.0) 5.64 ddd (9.5, 6.0, 2.5) 5.65 ddd (9.5, 5.9, 2.5) 5.65 ddd (9.4, 6.0, 2.4) 5.64 ddd (9.4, 5.8, 2.1)
4.34 d (2.6)
1.60 s
4.19 d (3.0)
1.56 s
4.32 d (2.5)
1.59 s
4.33 d (2.5)
1.60 s
4.33 d (2.4)
1.59 s
4.30 d (2.1)
1.60 s
1.56 s
1.56 s
1.55 s
1.55 s
1.55 s
1.55 s
1.30 s
1.52 s
1.48 s
1.23 s
1.28 s
1.25 s
0.99 s
4.33 d (11.2)
4.37 d (10.1)
3.64 d (10.1)
0.82 s
1.11 s
1.13 s
1.15 s
3
.63 d (11.2)
30
1′
2′
3′
4′
5′
6′
0.84 s
0.83 s
0.82 s
0.83 s
0.83 s
4.96 d (7.7)
5.04 d (7.8)
5.00 d (7.8)
4.05 m
4.88 d (7.5)
4.16 m
4.92 d (7.3)
4.25 m
4.87 d (7.5)
4.19 m
4.06 dd (8.3, 7.7)
4.03 dd (8.9, 8.3)
4.23 dd (9.0, 8.9)
3.99 dd (8.4, 7.8)
4.25 dd (9.1, 8.4)
4.17 dd (9.1, 9.0)
4.32 m
4.33 m
4.30 m
4.23 m
4.23 m
4.06 m
4.20 m
4.10 m
4.28 ddd (9.0, 5.5, 2.5) 4.21 ddd (9.0, 5.6, 2.4) 4.22 m
4.23 m
3.91 m
4.03 m
4.63 dd (11.7, 2.5)
.42 dd (11.7, 5.5)
4.62 dd (11.8, 2.4)
4.39 dd (11.8, 5.6)
4.91 dd (11.3, 1.9)
4.30 dd (11.3, 5.2)
5.10 d (7.7)
3.95 m
4.85 m
4.53 m
4.78 m
4
4.29 m
4.35 m
4.47 m
1″
2″
3″
4″
5″
6″
5.33 d (7.7)
4.05 m
5.33 d (7.6)
4.07 m
5.28 d (7.6)
4.22 m
4.18 m
4.23 m
4.23 m
4.24 m
4.08 m
4.23 m
4.16 m
4.32 m
3.93 m
3.93 m
4.03 m
4.03 m
4.51 dd (11.8, 2.7),
4.49 m,
4.37 m
4.78 dd (11.3, 2.7),
4.47 dd (11.3, 4.3)
5.12 d (7.7)
4.06 m
4.81 m,
4.30 m
4
.38 dd (11.8, 5.3)
1‴
2‴
3‴
4‴
5‴
6‴
5.08 d (7.7)
4.06 m
5.11 d (7.7)
4.05 m
4.23 m
4.23 m
4.23 m
4.34 m
4.32 m
4.21 m
3.93 m
3.91 m
3.93 m
4.49 m,
4.53 m,
4.51 m,
4.37 m
4
.37 m
4.35 m
a
b
Chemical shifts (ppm) referenced to pyridine-d (δ 7.58) at 500 MHz. Data for Glc-IV of 13: δ 5.07 d (7.7 Hz, H-1⁗), 4.05 m (H-2⁗), 4.22 m
5
H
(
H-3⁗), 4.21 m (H-4⁗), 3.90 m (H-5⁗), 4.51 m (H-6a⁗), 4.36 m (H-6b⁗).
suggesting that the difference between compounds 11 and 12 is
hydrolysis of 13 yielded only D-glucose and the aglycone. The
the linkage of the sugar units. The HMBC correlations of H-
coupling constants of the anomeric protons at δ 4.87 (H-1′, d,
H
1
‴/C-6″ and H-1″/C-2″ specified the linkages of the sugar
units. Therefore, compound 12 was defined as
3β,20S,23S,24R)-3,20,23,24,25-pentahydroxydammaran-21-oic
J = 7.5 Hz), 5.28 (H-1″, d, J = 7.6 Hz), 5.07 (H-1‴, d, J = 7.7
Hz), and 5.11 (H-1⁗, d, J = 7.7 Hz) suggested they are all β-
configured. The NMR resonance assignments for 13 were
confirmed by 2D NMR data analysis. In particular, the HMBC
correlations of H-1⁗/C′-6, H-1‴/C″-6, and H-1″/C′-2
specified the linkages of the sugar units. Thus, compound 13
was determined as (3β,20S,23S,24R)-3,20,23,24,25-pentahy-
droxydammaran-21-oic acid-21,23-lactone 3-O-{[β-D-glucopyr-
(
acid-21,23-lactone 3-O-{[β-D-glucopyranosyl-(1→6)]-β-D-glu-
copyranosyl-(1→2)}-β-D-glucopyranoside.
The molecular formula of 13 was identified as C H O by
5
4
90 26
13
the C NMR and HRESI(+)MS data. The NMR spectra of 13
were similar to those of 12, except for the presence of
resonances resulting from an additional hexosyl moiety. Acid
1
205
dx.doi.org/10.1021/np500077z | J. Nat. Prod. 2014, 77, 1201−1209

Journal of Natural Products
Article
MeOH at room temperature. The X-ray diffraction data
showed that compounds 14, 15, and 8 had identical E-rings.
Thus, the structures of gentirigenic acid and gentirigeosides A−
E should be revised as (3β,20S,23S,24R)-3,20,23,24,25,29-
hexahydroxydammaran-21-oic acid-21,23-lactone,
(
3β,20S,23S,24R)-3,20,23,24,25,29-hexahydroxydammaran-21-
oic acid-21,23-lactone 3-O-β-D -glucopyranoside,
3β,20S,23S,24R)-3,20,23,24,25,29-hexahydroxydammaran-21-
(
oic acid-21,23-lactone 3-O-β-D-glucopyranosyl-(1→2)-β-D-glu-
copyranoside, (3β,20S,23S,24R)-3,20,23,24,25,29-hexahydroxy-
dammaran-21-oic acid-21,23-lactone 3-O-β-D-glucopyranosyl-
Figure 2. X-ray ORTEP drawing of compound 8.
2
3
9 -O -β -D - g l u c o p yr a n o s i d e , ( 3 β , 20 S, 2 3 S, 2 4R )-
,20,23,24,25,26,29-heptahydroxydammaran-21-oic acid-21,23-
lactone 3-O-β-D-glucopyranoside, and (3β,20S,23S,24R)-
3
,20,23,24,25-pentahydroxydammaran-21-oic acid-21,23-lac-
tone 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside, re-
spectively.
Compounds 1−17 were examined for anti-inflammatory
activity by evaluating the inhibition of LPS-induced NO
production in RAW 264.7 macrophages. Only gentirigeoside
A (15) inhibited NO production in RAW 264.7 macrophages,
with an IC50 value of 6.6 ± 2.1 μM, while indomethacin, the
positive control, showed an IC50 value of 1.25 ± 0.52 μM.
EXPERIMENTAL SECTION
■
Figure 3. X-ray ORTEP drawing of compound 9.
General Experimental Procedures. Optical rotations were
measured on a PerkinElmer 341 polarimeter. UV data were acquired
on a Shimadzu UV-2550 spectrophotometer. IR spectra were recorded
on a PerkinElmer 577 spectrometer using KBr disks. NMR
experiments were recorded in pyridine-d5 on a Bruker AM-500
spectrometer referenced to solvent peaks (δH 7.58; δC 135.9).
HRESIMS analyses were carried out on an Agilent 6224 TOF mass
spectrometer with an ESI interface. Semipreparative HPLC was
performed on an Agilent 1100 series HPLC system using a YMC-Pack
ODS-A column (250 × 10 mm, S-5 μm). Silica gel (200−300 mesh,
Qingdao Haiyang Chemical Co. Ltd.), C18 reversed-phase (RP-18)
anosyl-(1→6)]-β-D-glucopyranosyl-(1→2)}-β-D-glucopyrano-
syl-(1→6)-β-D-glucopyranoside.
The IR, NMR, and HRESIMS spectrometric data and the
specific rotations for compounds 14−17 (Supporting Informa-
tion) were similar to the data for the known compounds
gentirigenic acid and gentirigeosides A, B, and E, respectively.
1
13
However, the characteristic resonances in the H and C NMR
spectra for ring E were also similar to the resonances of 8,
which was surprising. Thus, we reexamined the chemical
silica gel (20−45 μm; Fuji Silysia Chemical Ltd.), C reversed-phase
6
8
structures of these reported compounds and also obtained
silica gel (20−45 μm; Fuji Silysia Chemical Ltd.), CHP20P MCI gel
crystals of compounds 14 (Figure 4) and 15 (Figure 5) from
(
75−150 μm, Mitsubishi Chemical Industries Co., Ltd.), Toyopearl
HW-40F (32−63 μm; Tosoh), and Sephadex LH-20 gel (Amersham
Biosciences) were used for column chromatography (CC). Precoated
silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd.) were
used for TLC. All solvents used for CC were of analytical grade
(
Shanghai Chemical Reagents Co., Ltd.), and the solvents used for
HPLC were of HPLC grade (TEDIA Ltd.).
Plant Material. The aerial parts of G. azurea were collected in May
012 from Yunnan Province, People’s Republic of China, and
2
authenticated by Prof. He-Ming Yang. A voucher specimen (No.
SIMMPZ323) was deposited at the Herbarium of Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, People’s Republic of
China.
Extraction and Isolation. Air-dried and pulverized G. azurea plant
material (2.6 kg) was soaked in 95% ethanol (3 × 30 L; 24 h each
time) at room temperature. After removing the solvent under reduced
pressure at 40 °C, the combined extracts were concentrated to afford
Figure 4. X-ray ORTEP drawing of compound 14.
the crude extract (321.1 g), which was partitioned between H O and
2
CHCl . The CHCl (131.7 g) fraction was loaded onto a silica gel
3
3
column and eluted with a gradient of petroleum ether/acetone (20/1,
1
0/1, 5/1, 3/1, 1/1, and 0/1) to give fractions 1−7. Fraction 7 (4.3 g)
was subjected to a column of reversed-phase silica gel eluting with a
MeOH/H O (50/50 to 100/0) gradient to yield subfractions 7a−7c.
2
Subfraction 7c (67 mg) was purified by semipreparative HPLC with
8
2% MeOH in H O as the mobile phase to yield compounds 1 (16
2
mg) and 2 (17 mg).
The water-soluble fraction (171.3 g) was subjected to MCI gel
column chromatography (MeOH/H O, 0/100 to 100/0) to afford
2
Figure 5. X-ray ORTEP drawing of compound 15.
fractions A−G. Fraction B was separated using a C column (MeOH/
8
1
206
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Journal of Natural Products
Article
1
H O, 10/90 to 50/50) to afford subfractions B1−B5. Subfraction B2
NMR, Table 1; H NMR, Table 3; HRESIMS m/z 691.4020 [M +
2
+
was further separated on a Toyopearl HW-40F column (MeOH/H O,
Na] (calcd for C H O , 691.4028).
2
36 60 11
0
1
/100 to 30/70) to yield 17 (89 mg). Compounds 15 (132 mg) and
6 (44 mg) were obtained from subfractions B3 and B4, respectively,
(3β,20S,23R,24R)-3,20,23,24,25,29-Hexahydroxydammaran-21-
oic acid-21,23-lactone 3-O-β-D-glucopyranoside (9): colorless
2
0
using the same method as that used for B1. Fraction C (17.8 g) was
further purified through repeated C and Sephadex LH-20 columns to
afford compound 14 (12 mg). Fraction D (7.0 g) was purified by
chromatography using a C18 column eluting with a 30/70 to 80/20
MeOH/H O gradient. The major fraction (MeOH/H O = 40/60, 3.4
g) was purified by semipreparative HPLC eluting with 58% MeOH in
H O to yield compounds 10 (12 mg), 11 (57 mg), and 12 (132 mg),
and the minor fraction (MeOH/H O = 50/50, 300 mg) was purified
through repeated LH-20 columns with MeOH to yield compound 13
crystals (H
2
O/pyridine/MeOH, 2:1:7); mp 256−258 °C; [α]
+7
D
(c 0.1, MeOH); IR (KBr) νmax 3419, 2939, 1761, 1595, 1454, 1377,
1076, 1030 cm ; C NMR, Table 1; H NMR, Table 3; HRESIMS
8
−1
13
1
+
m/z 707.3991 [M + Na] (calcd for C36H O12, 707.3977).
60
(3β,20S,23S,24R)-3,20,23,24,25,29-Hexahydroxydammaran-21-
oic acid-21,23-lactone 3-O-β-D-glucopyranosyl-(1→6)-β-D-gluco-
2
2
pyranoside (10): amorphous powder; [α]2 −1 (c 0.1, MeOH); IR
0
D
2
−1 13
(
KBr) νmax 3396, 2944, 1761, 1454, 1379, 1205, 1076, 1030 cm ; C
NMR, Table 1; H NMR, Table 3; HRESIMS m/z 869.4508 [M +
2
1
+
Na] (calcd for C H O , 869.4505).
(
22 mg). Fraction F was separated using a C18 column eluting with
42 70 17
(
3β,20S,23S,24R)-3,20,23,24,25-Pentahydroxydammaran-21-oic
MeOH/H O (50/50 to 100/0) to yield three subfractions (F1−F8).
Subfraction F1 (5.0 g) was then subjected to a Toyopearl HW-40F
column eluting with MeOH/H O (0/100 to 50/50) to give
subfractions F1A−F1E. Subfraction F1A was purified by semi-
preparative HPLC with 65% MeCN in H O as the mobile phase to
yield compounds 8 (15 mg), 9 (9 mg), and 3 (4 mg). Subfraction F1B
was purified by semipreparative HPLC with 76% MeOH in H O as the
2
acid-21,23-lactone 3-O-[β-D-glucopyranosyl-(1 → 2)]-[β-D-gluco-
pyranosyl-(1→6)]-β-D-glucopyranoside (11): amorphous powder;
α] +2 (c 0.1, MeOH); IR (KBr) ν
620, 1354, 1076 cm ; C NMR, Table 1; H NMR, Table 3;
HRESIMS m/z 1015.5064 [M + Na] (calcd for C H O ,
2
20
D
[
1
3396, 2943, 2779, 1761,
max
−1 13
1
2
+
4
8
80 21
1
015.5084).
2
(
3β,20S,23S,24R)-3,20,23,24,25-Pentahydroxydammaran-21-oic
mobile phase to yield compounds 4 (21 mg), 5 (48 mg), 6 (14 mg),
and 7 (8 mg).
acid-21,23-lactone 3-O-{[β-D-glucopyranosyl-(1→6)]-β-D-glucopyr-
20
anosyl-(1→2)}-β-D-glucopyranoside (12): amorphous powder; [α]D
(
3β,20S,23R)-3,20,23,29-Tetrahydroxydammar-24-en-21-oic
+
1
7 (c 0.1, MeOH); IR (KBr) ν 3406, 2943, 1761, 1641, 1454, 1377,
max
acid-21,23-lactone 3-O-β-D-glucopyranoside (1): colorless crystals
−1
1
13
207, 1167, 1076, 1028 cm ; H NMR, Table 3; C NMR, Table 1;
HRESIMS m/z 1015.5064 [M + Na] (calcd for C H O ,
2
0
(
2
1
MeOH); mp 189−191 °C; [α] −8 (c 0.1, MeOH); ECD (MeOH)
+
D
4
8
80 21
27 (Δε +1.05), 204 (Δε −3.89); IR (KBr) ν 3423, 2940, 1764,
max
1
015.5084).
3β,20S,23S,24R)-3,20,23,24,25-Pentahydroxydammaran-21-oic
−1 13 1
452, 1378, 1200, 1076, 1031 cm ; C NMR, Table 1; H NMR,
(
+
Table 2; HRESIMS m/z 673.3917 [M + Na] (calcd for C H O ,
6
36
58 10
acid-21,23-lactone 3-O-{[β-D-glucopyranosyl-(1→6)]-β-D-glucopyr-
anosyl-(1→2)}-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside
(13): amorphous powder; [α]2 −25 (c 0.1, MeOH); IR (KBr) ν
73.3922).
0
(
3β,20S,23S)-3,20,23,29-Tetrahydroxydammar-24-en-21-oic
D
max
−1
13
acid-21,23-lactone 3-O-β-D-glucopyranoside (2): amorphous pow-
3
406, 2939, 1761, 1645, 1456, 1377, 1203, 1074 cm ; C NMR,
Table 1; H NMR, Table 3; HRESIMS m/z 1177.5614 [M + Na]
der; [α]2 +26 (c 0.1, MeOH); ECD (MeOH) 232 (Δε −2.09), 202
0
D
1
+
(
1
6
Δε +4.12); IR (KBr) ν 3423, 2938, 1754, 1452, 1379, 1201, 1076,
max
(calcd for C H O , 1177.5613).
54
90 26
−1 13 1
032 cm ; C NMR, Table 1; H NMR, Table 2; HRESIMS m/z
Gentirigenic acid (14): colorless crystals (MeOH); mp 166−168
+
20
73.3917 [M + Na] (calcd for C H O , 673.3922).
°C; [α]D +18 (c 0.1, MeOH); IR (KBr) ν 3425, 2929, 1761, 1620,
36
58 10
max
−
1
1
13
(
3β,20S,23R)-3,20,23,26,29-Pentahydroxydammar-24-en-21-oic
1466, 1097 cm
Information; HRESIMS m/z 1045.7182 [2 M + H] (calcd for
;
H NMR and C NMR see Supporting
acid-21,23-lactone 3-O-β-D-glucopyranoside (3): amorphous pow-
+
der; [α]2 −6 (c 0.1, MeOH); ECD (MeOH) 226 (Δε +2.08), 200
0
D
C H O , 1045.7186).
6
0
100 14
(
1
6
Δε −9.90); IR (KBr) ν 3423, 2945, 1765, 1452, 1379, 1211, 1078,
max
Gentirigeoside A (15): colorless crystals (MeOH); mp 220−222
−1 13 1
030 cm ; C NMR, Table 1; H NMR, Table 2; HRESIMS m/z
20
°
C; [α] +8 (c 0.1, MeOH); IR (KBr) ν 3421, 2933, 1761, 1456,
D
max
13
+
89.3883 [M + Na] (calcd for C H O , 689.3871).
−1 1
36
58 11
1379, 1205, 1076, 1028 cm ; H NMR and C NMR see Supporting
(
3β,20S,23R)-3,20,23,29-Tetrahydroxydammar-24-en-21-oic
Information; HRESIMS m/z 707.4002 [M + Na]+ (calcd for
acid-21,23-lactone 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyra-
C H O , 707.3977).
Gentirigeoside B (16): amorphous powder; [α]D +16 (c 0.1,
noside (4): amorphous powder; [α]2 −4 (c 0.1, MeOH); IR (KBr)
0
36 60 12
D
20
−
1 13
νmax 3419, 2942, 1765, 1452, 1379, 1200, 1076, 1028 cm ; C NMR,
MeOH); IR (KBr) ν 3406, 2943, 1763, 1454, 1379, 1203, 1076,
1
+
max
Table 1; H NMR, Table 2; HRESIMS m/z 835.4452 [M + Na]
−1
1
13
1
028 cm ; H NMR and C NMR see Supporting Information;
HRESIMS m/z 869.4511 [M + Na] (calcd for C H O , 869.4505).
Gentirigeoside E (17): amorphous powder; [α]D +10 (c 0.1,
MeOH); IR (KBr) νmax 3419, 2939, 1761, 1595, 1454, 1377, 1076,
1030 cm ; H NMR and C NMR see Supporting Information;
(calcd for C H O , 835.4450).
+
42
68 15
42
70 17
20
(
3β,20S,23S)-3,20,23,29-Tetrahydroxydammar-24-en-21-oic
acid-21,23-lactone 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyra-
noside (5): amorphous powder; [α]20 +22 (c 0.1, MeOH); IR
D
−1
1
13
−1
13
(
KBr) νmax 3396, 2943, 1761, 1452, 1379, 1200, 1076, 1026 cm ; C
+
1
HRESIMS m/z 853.4574 [M + Na] (calcd for C42H O16, 853.4556).
70
NMR, Table 1; H NMR, Table 2; HRESIMS m/z 835.4447 [M +
Na] (calcd for C H O , 835.4450).
+
X-ray Diffraction Analysis. The X-ray crystallographic data were
obtained on a Bruker APEX-II CCD detector employing graphite-
monochromated Cu Kα radiation operated in the ϕ−ω scan mode.
The structures were solved by the direct method using SHELXS-97
(Sheldrick 2008) and refined with full-matrix least-squares calculations
42
68 15
(
3β,20S,23R)-3,20,23-Trihydroxydammar-24-en-21-oic acid-
2
1,23-lactone 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside
20
(
6): amorphous powder; [α]D −2 (c 0.1, MeOH); IR (KBr) ν 3423,
max
−1
13
1
2
939, 1763, 1452, 1379, 1200, 1076 cm ; C NMR, Table 1; H
2
+
on F using SHELXL-97 (Sheldrick 2008). All non-hydrogen atoms
NMR, Table 2; HRESIMS m/z 819.4513 [M + Na] (calcd for
C H O , 819.4501).
were refined anisotropically. The hydrogen atom positions were
geometrically idealized and allowed to ride on their parent atoms.
42
68 14
(
3β,20S,23S)-3,20,23-Trihydroxydammar-24-en-21-oic acid-
2
1,23-lactone 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside
Crystallographic data for 1: C36H O10, M = 650.82, size 0.25 mm
58
20
(
7): amorphous powder; [α]D +26 (c 0.1, MeOH); IR (KBr) ν
× 0.08 mm × 0.06 mm, monoclinic, space group P2 , a = 15.2138(3)
max
1
−1 13
3
421, 2943, 1761, 1452, 1379, 1201, 1076 cm ; C NMR, Table 1;
Å, b = 8.0132(2) Å, c = 16.3249(3) Å, α = 90°, β = 116.9640(10)°, γ =
1
+
3
3
H NMR, Table 2; HRESIMS m/z 819.4518 [M + Na] (calcd for
90°, V = 1773.84(7) Å , T = 140(2) K, Z = 2, d = 1.219 Mg/m , λ(Cu
Kα) = 1.541 78 Å, F(000) = 708, reflections collected/unique 13 834/
5381 [R(int) = 0.0245], h (−18/18), k (−7/9), l (−19/19), theta
range 3.037° to 69.676°, completeness 98.2%, data/restrains/
C H O , 819.4501).
42
68 14
(
3β,20S,23S,24R)-3,20,23,24,25-Pentahydroxydammaran-21-oic
acid-21,23-lactone 3-O-β-D-glucopyranoside (8): colorless crystals
2
0
(
3
MeOH); mp 261−263 °C; [α] +28 (c 0.1, MeOH); IR (KBr) ν
parameters 5381/1/427, final R indices R1 = 0.0364 and wR2
=
D
max
13
−1
423, 2945, 1778, 1749, 1639, 1377, 1217, 1076, 1032 cm ;
C
0.1031 (I > 2σ(I)), R = 0.0370 and wR = 0.1038 (all data), GOF =
1
2
1
207
dx.doi.org/10.1021/np500077z | J. Nat. Prod. 2014, 77, 1201−1209

Journal of Natural Products
Article
−
3
1
.028, largest diff peak and hole, 0.382 and −0.245 e Å , absolute
structure parameter 0.05(6).
Crystallographic data for 8: C H O , M = 668.82, size 0.35 mm
cells/well) and allowed to adhere for 24 h. The stock solution for each
test sample was dissolved in DMSO and diluted with RPMI medium
1640 to a final concentration of less than 0.1%. To determine the
inhibitory activity of each compound, the cells were co-incubated with
fresh medium (200 μL/well) containing 200 ng/mL of LPS and the
tested compounds at various concentrations (0.1−50.0 μM) for 24 h.
For the positive control group, the cells were co-incubated with
36
60 11
×
7
=
0.04 mm × 0.02 mm, monoclinic, space group P2 2 2 , a =
1 1 1
.3172(10) Å, b = 23.8986(3) Å, c = 40.6627(5) Å, α = 90°, β = 90°, γ
3
3
90°, V = 7110.72(16) Å , T = 140(2) K, Z = 8, d = 1.296 Mg/m ,
λ(Cu Kα) = 1.541 78 Å, F(000) = 3024, reflections collected/unique
4
8 526/12 683 [R(int) = 0.0534], h (−8/6), k (−28/28), l (−48/49),
indomethacin. The Griess reagent was used to determine NO
−
theta range 2.144° to 69.650°, completeness 97.5%, data/restrains/
parameters 12 683/0/912, final R indices R = 0.0344 and wR =
production by measuring the accumulation of NO
2
in the culture
supernatant. Briefly, 100 μL of the supernatant from the incubate was
1
2
0
1
.0860 (I > 2σ(I)), R = 0.0382 and wR = 0.0890 (all data), GOF =
.052, largest diff peak and hole, 0.189 and −0.173 e Å , absolute
mixed with an equal volume of Griess reagent (0.2% naphthylenedia-
1
2
−3
mide dihydrochloride and 2% sulfanilamide in 5% H PO ) and
3 4
structure parameter −0.05(6).
agitated at room temperature. The absorbance was measured with a
microplate reader at 540 nm. Inhibition activity (%) was calculated
using the following equation, and the IC50 values were calculated using
Crystallographic data for 9: C H O , M = 684.84, size 0.29 mm
36
60 12
×
0.16 mm × 0.10 mm, monoclinic, space group P2 , a = 15.2751(3)
1
Å, b = 8.0457(2) Å, c = 15.9079(3) Å, α = 90°, β = 116.3950(10)°, γ =
the GraphPad Prism program: Inhibition activity (%) = (A − B)/(A −
3
3
−
9
0°, V = 1751.25(7) Å , T = 140(2) K, Z = 2, d = 1.299 Mg/m , λ(Cu
Kα) = 1.541 78 Å, F(000) = 744, reflections collected/unique 12 092/
840 [R(int) = 0.0333], h (−18/18), k (−7/9), l (−18/19), theta
range 3.230° to 69.571°, completeness 99.2%, data/restrains/
parameters 4840/1/447, final R indices R1 = 0.0468 and wR2
C) × 100, where A−C is the NO
2
concentration (μM) [A: LPS (+),
sample (−); B: LPS (+), sample (+); C: LPS (−), sample (−)].
4
ASSOCIATED CONTENT
Supporting Information
■
=
*
S
0
.1362 (I > 2σ(I)), R = 0.0472 and wR = 0.1368 (all data), GOF =
1
2
−3
NMR data for compounds 14−17. Copies of IR and 1D and
1
.027, largest diff peak and hole, 0.553 and −0.286 e Å , absolute
2
D NMR spectra for compounds 1−17. Copies of the ECD
structure parameter 0.11(9).
Crystallographic data for 14: C H O , M = 522.80, size 0.26 mm
spectra for compounds 1−3. This material is available free of
30
50
7
×
1
=
0.13 mm × 0.08 mm, orthorhombic, space group P2 2 2, a =
charge via the Internet at http://pubs.acs.org.
1
1
1.2093(3) Å, b = 38.8302(9) Å, c = 7.2838(2) Å, α = 90°, β = 90°, γ
3
3
90°, V = 3170.34(14) Å , T = 140(2) K, Z = 4, d = 1.267 Mg/m ,
AUTHOR INFORMATION
Corresponding Author
Tel and Fax: +86-21-20231968. E-mail: ljxuan@mail.shcnc.ac.
■
λ(Cu Kα) = 1.541 78 Å, F(000) = 1328, reflections collected/unique
1
7 922/5668 [R(int) = 0.0468], h (−11/13), k (−46/47), l (−8/6),
*
theta range 2.276° to 69.623°, completeness 96.5%, data/restrains/
parameters 5668/12/390, final R indices R = 0.0822 and wR =
cn.
1
2
0
1
.2311 (I > 2σ(I)), R = 0.0844 and wR = 0.2343 (all data), GOF =
.153, largest diff peak and hole, 0.823 and −0.927 e Å , absolute
1
2
Notes
−3
The authors declare no competing financial interest.
structure parameter 0.24(7).
Crystallographic data for 15: C H O , M = 684.81, size 0.23
mm × 0.12 mm × 0.08 mm, triclinic, space group P1, a = 7.1558(3) Å,
36
60 12
ACKNOWLEDGMENTS
■
b = 12.8203(4) Å, c = 21.0330(7) Å, α = 85.672(2)°, β = 88.636(2)°, γ
The authors gratefully acknowledge grants from the National
Science & Technology Major Project “Key New Drug Creation
and Manufacturing Program”, China (No. 2009ZX09301-001),
and the National Natural Sciences Foundation of China (No.
3
=
77.863 (2)°, V = 1880.95(12) Å , T = 140(2) K, Z = 1, d = 1.333
3
Mg/m , λ(Cu Kα) = 1.541 78 Å, F(000) = 822, reflections collected/
unique 23 575/10 540 [R(int) = 0.0442], h (−8/8), k (−15/15), l
(
−25/25), theta range 3.535° to 69.252°, completeness 95.6%, data/
3
0901851).
restrains/parameters 10 540/3/966, final R indices R = 0.0477 and
1
wR = 0.1226 (I > 2σ(I)), R = 0.0479 and wR = 0.1230 (all data),
GOF = 1.045, largest diff peak and hole, 0.540 and −0.289 e Å ,
absolute structure parameter 0.05(6).
2
1
2
−3
REFERENCES
■
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2
3
2
2
(
3 × 5 mL). The aqueous layer was desalted (Sephadex LH-20, H O)
2
to afford a sugar residue (0.5 mg). The sugar was confirmed to be D-
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pyridine/H O (6:1:5:4) as the development solution] with an
2
authentic sample of glucose (R 0.42) and by its specific rotation
f
0
(
1
[α]2 +43.6 c 0.04, H O). The constituent sugars of compounds 2−
D
2
3 were identified using the same method.
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10
humidified 5% CO /95% air atmosphere at 37 °C. The cells were
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harvested with trypsin-EDTA and diluted and suspended in fresh4
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208
dx.doi.org/10.1021/np500077z | J. Nat. Prod. 2014, 77, 1201−1209

Journal of Natural Products
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dx.doi.org/10.1021/np500077z | J. Nat. Prod. 2014, 77, 1201−1209