Anti-inflammatory phenanthrene derivatives from stems of Dendrobium denneanum

Article abstract of DOI:10.1016/j.phytochem.2013.08.008

Cultivated Dendrobium denneanum has been substituted for other endangered Dendrobium species in recent years, but there have been few studies regarding either its chemical constituents or pharmacological effects. In this study, three phenanthrene glycosid

Full text of DOI:10.1016/j.phytochem.2013.08.008

Phytochemistry 95 (2013) 242–251
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Anti-inflammatory phenanthrene derivatives from stems of Dendrobium
denneanum
Yuan Lin a,b,c, Fei Wang a, Li-juan Yang a, Ze Chun a, Jin-ku Bao b, Guo-lin Zhang a,
⇑
a Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, PR China
b Key Laboratory of Bio-resources and Eco-environment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu 610064, PR China
c University of Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 May 2013
Received in revised form 3 August 2013
Available online 13 September 2013
Cultivated Dendrobium denneanum has been substituted for other endangered Dendrobium species in
recent years, but there have been few studies regarding either its chemical constituents or pharmacolog-
ical effects. In this study, three phenanthrene glycosides, three 9,10-dihydrophenanthrenes, two 9,10-
dihydrophenanthrenes glycosides, and four known phenanthrene derivatives, were isolated from the
stems of D. denneanum. Their structures were elucidated on the basis of MS and NMR spectroscopic data.
Ten compounds were found to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-activated
Keywords:
Dendrobium denneanum
Orchidaceae phenanthrene
Anti-inflammatory activity
iNOS
NF-jB
MAPKs
mouse macrophage RAW264.7 cells with IC50 values of 0.7–41.5
RAW264.7, HeLa, or HepG2 cells. Additionally, it was found that 2,5-dihydroxy-4-methoxy-phenanthrene
2-O-b- -glucopyranoside, and 5-methoxy-2,4,7,9S-tetrahydroxy-9,10-dihydrophenanthrene suppressed
LPS-induced expression of inducible NO synthase (iNOS) inhibited phosphorylation of p38, JNK as well
as mitogen-activated protein kinase (MAPK), and inhibitory kappa B- (I ). This indicated that both
lM, and exhibited no cytotoxicity in
D
a
jBa
Cytotoxic
RAW264.7 cell
compounds exert anti-inflammatory effects by inhibiting MAPKs and nuclear factor
pathways.
jB (NF-jB)
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
been reported as having antioxidant, anti-inflammatory (Zhang
et al., 2007), antifibrotic (Yang et al., 2007a), immunomodulatory
(Zhao et al., 2001) and anti-platelet aggregation activities (Fan
et al., 2001).
The genus Dendrobium Sw. (Orchidaceae) contains more than
1000 species that are wildly distributed in tropical and subtropical
regions in Asia and the Pacific islands. South of the Tsinling Moun-
tains of China, 74 species and two varieties of Dendrobium can be
found (Zhang et al., 2003). Wild Dendrobium plants are nearly ex-
tinct due to increasing clinical uses in China, and have been listed
as an endangered species in China and The United Nations. Stems
of Dendrobium nobile Lindl., Dendrobium chrysotoxum Lindl., Dendr-
obium fimbriatum Hook. var. oculatum Hook., and its additional
congeneric species, are recorded in the Chinese Pharmacopoeia
(2010) as ‘‘Shi-Hu’’. These are widely used in traditional Chinese
medicine for recovery of gastric motility and promoting gastric
acid secretion in the stomach, as well as promoting secretion of
saliva, improving eyesight, and reducing fever (Shu et al., 2004).
The plants of this genus contain bibenzyls (Li et al., 2009), phenan-
threnes (Lee et al., 2009; Wang et al., 2009), anthraquinones (Lin
et al., 2001), fluoro-derivatives (Zhang et al., 2007), coumarins
(Zhang et al., 2005), sesquiterpenes (Zhang et al., 2008) and alka-
loids (Wang and Zhao, 1985). Some of these compounds have also
Dendrobium denneanum, wildly distributed in the Sichuan Prov-
ince of China, is the major source of ‘‘Shi-Hu’’. Twelve compounds,
including bibenzyls, phenanthrenes, benzoquinones, coumarins,
cinnamic acid derivatives, b-sitosterol, and daucosterol, were pre-
viously isolated from the stems of D. denneanum (Yang et al.,
2006b, 2007b; Liu et al., 2009). Due to the successful cultivation
of D. denneanum in recent years, this plant has been a substitute
for the other endangered Dendrobium species in clinical use. How-
ever, the compounds responsible for its pharmacological effects are
still unknown.
Nitric oxide (NO) is an inflammatory mediator that plays an
important role in a variety of inflammatory diseases such as arthri-
tis, asthma, multiple sclerosis, colitis, inflammatory bowel dis-
eases, and atherosclerosis (Guzik et al., 2003). NO is synthesized
from L-arginine in a reaction catalyzed by a family of nitric oxide
synthase (NOS) enzymes. Unlike other constitutively expressed
NOS, inducible NOS (iNOS) is transcriptionally induced in response
to bacterial endotoxins such as lipopolysaccharide (LPS), and
pro-inflammatory cytokines in macrophages and various other
cell types (Korhonen et al., 2005). Transcription of iNOS in
⇑
Corresponding author. Tel./fax: +86 28 8522 9742.
E-mail address: zhanggl@cib.ac.cn (G.-l. Zhang).
0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2013.08.008

Y. Lin et al. / Phytochemistry 95 (2013) 242–251
243
macrophages is mediated by different signaling pathways, includ-
ing nuclear factor B (NF- B) and mitogen-activated protein
NMR resonances at d 102.7, 78.6, 78.2, 75.2, 71.8 and 62.8 could be
located at C-2 from the HMBC correlations of H-10 with C-2 (d
157.9) (Fig. 2). From the IR absorption at 3427 cmꢀ1 and the molec-
ular formula, a hydroxyl group was postulated, which could be lo-
cated at C-5 in view of the HMBC correlations of H-9 with C-8, and
H-8 with C-9. Structure 1 was thus elucidated as 2,5-dihydroxy-4-
j
j
kinase (MAPKs) (Thirunavukkarasu et al., 2006; Chen et al., 1998).
In this study, phytochemical experiments were carried out with
the 95% ethanol extract of dried D. denneanum stems. Twelve phen-
anthrene derivatives were isolated and their structures character-
ized using NMR but MS spectroscopic data. Six of the isolated
compounds inhibited nitric oxide (NO) production in lipopolysac-
charide (LPS)-activated mouse macrophage RAW264.7 cells with
methoxy-phenanthrene 2-O-b-D-glucopyranoside.
Compound 2, obtained as a yellowish colloidal solid, has the
molecular formula C26H30O12 as determined by the quasi molecu-
lar ion peak at 557.1629 in the HR-ESI-MS ([M+Na]+). The presence
of hydroxyl groups was suggested by the IR absorption at
3434 cmꢀ1. Its UV maxima absorptions at 214.5, 254.0, 283.5 and
313.5 nm also indicated a phenanthrene derivative (Ito et al.,
2010). Its NMR spectroscopic data (Table 1) of compound 2 were
very similar to those of compound 1, except that 2 contained one
more sugar residue. A b-apiofuranosyl was provided by the 1H
NMR signal at d 5.00 and the 13C NMR signals at d 111.1, 78.2,
IC50 values of 0.7–41.5
lM, but exhibited no cytotoxicity on
RAW264.7, HeLa and HepG2 cells. Two compounds exert an anti-
inflammatory effect by inhibiting MAPKs and NF-jB pathways.
2. Results and discussion
The air-dried stems of Dendrobium denneanum were extracted
with 95% ethanol, and the dried extract was partitioned with
petroleum ether, ethyl acetate and n-butanol, respectively. The
ethyl acetate fraction then was subjected to repeated silica gel col-
umn chromatography and HPLC to yield compounds 1–12. The
structures of these compounds are shown in Fig. 1.
80.6, 75.1 and 65.7. The absolute configuration of the D-apiofurano-
syl moiety in 2 was confirmed by the molecular rotation difference
([M]D of 2 ꢀ [M]D of 1 = ꢀ501.2) (Takayanagi et al., 2003). The
HMBC correlations of H-1with C-6 (d 68.9) indicated that com-
pound 2 contains a b-
D-apiofuranosyl-(1–6)-b-D-glucopyranosyl
Compound 1 was obtained as a colorless colloidal solid. Its
molecular formula was determined to be C21H22O8 from the quasi
molecular ion peak at 425.1208 in the HR-ESI-MS ([M+Na]+). Its UV
maxima absorptions at 214.5, 254.0, 283.5, and 313.5 nm were
similar to phenanthrene derivatives (Ito et al., 2010). Its 1H NMR
(Table 1) signals at 7.44 (t, J = 7.6 Hz, H-7), 7.40 (d, J = 7.6 Hz, H-
8), 7.13 (dd, J = 7.6, 1.4 Hz, H-6) showed a 1,2,3-trisubstituted phe-
nyl ring. The 1H NMR resonances at d 7.54 and 7.62 (each d,
J = 8.8 Hz) could be respectively assigned to H-9 and H-10 of the
phenanthrene. The other two aromatic protons were observed at
d 7.33 and 7.20 (each d, J = 2.0 Hz, H-1 and H-3). A methoxy group
moiety (Fig. 2). Thus, compound 2 was determined to be 2,5-dihy-
droxy-4-methoxy-phenanthrene 2-O-b-D-apiofuranosyl-(1–6)-b-D-
glucopyranoside.
Compound 3, was obtained as a yellowish colloidal solid, and
molecular formula was determined to be C27H32O12, through the
quasi molecular ion peak at 571.1794 in the HR-ESI-MS
([M+Na]+). Its IR absorption at 3441 cmꢀ1 showed the presence
of hydroxyl groups. Its UV maxima absorptions at 214.5, 254.0,
283.5 and 313.5 nm again indicated a phenanthrene derivative
(Ito et al., 2010). The NMR spectroscopic data confirmed that com-
pound 3 possessed the same skeleton and substitution mode as
compound 1, except that it contained an extra sugar residue. The
was determined based on the 1H NMR signals at d 4.10 and the 13
C
a
-rhamnopyranosyl group was deduced from the 1H NMR signal
at d 4.71 and the 13C NMR signals at d 102.3, 72.3, 72.7, 74.3,
70.1 and 18.1, respectively. Its absolute configuration of -rhamno-
NMR resonance at d 59.1 (Table 1). The NOESY correlation between
H-1 and H-10, and between –OCH3 and H-3, suggested that the
methoxy group is at C-4. Acid hydrolysis of 1 afforded
which was determined by GC–MS and by the molecular rotation
([M]D ꢀ209.0) of 1 and methyl-b- -glucopyranoside ([M]D ꢀ66.3)
(Devkota et al., 2012; Khurelbat et al., 2010) according to Klyne’s
rule (Klyne, 1950). The -glucopyranosyloxyl moiety supported
by the H NMR signal at d 5.11 (d, J = 7.3 Hz, H-10) and the 13C
D-glucose,
L
pyranosyl moiety in 2 was confirmed by the molecular rotation dif-
ference ([M]D of 3 ꢀ [M]D of 1 = ꢀ580.1) (Okamura et al., 1981).
The HMBC correlation between H-100 (d 4.71) with C-60 (d 68.0)
D
D
indicated that compound 3 contains an
a-L-rhamnopyranosyl-(1–
1
6)-b- -glucopyranosyl moiety (Fig. 2). Thus, compound 3 was
D
Fig. 1. Compounds 1–12 isolated from Dendrobium denneanum.

244
Y. Lin et al. / Phytochemistry 95 (2013) 242–251
Table 1
1H and 13C NMR (600 and 150 MHz, MeOH-d4) spectroscopic data of compounds 1–3 (d in ppm, multiplicities, J in Hz).
Position
1
2
3
dC
dH
dC
dH
dC
dH
1
2
3
4
4a
4b
5
6
7
8
8a
9
10
10a
4-OCH3
109.7
157.9
104.0
156.7
116.5
119.9
155.4
117.4
128.3
121.8
136.0
130.3
127.7
137.4
59.1
7.33 d (2.0)
–
7.20 d (2.0)
–
–
–
–
7.13 dd (7.6, 1.4)
7.44 t (7.6)
7.40 d (7.6)
–
7.62 d (8.8)
7.54 d (8.8)
–
109.7
157.8
104.2
156.7
116.5
119.9
155.4
117.4
128.3
121.7
136.0
130.3
127.8
137.4
59.2
7.32 d (2.4)
–
7.17 d (2.4)
–
–
109.6
157.8
104.3
156.8
116.6
119.9
155.4
117.4
128.3
121.8
136.0
130.5
127.8
137.4
59.3
7.31 d (2.4)
–
7.17 d (2.4)
–
–
–
–
7.12 dd (7.6, 1.4)
7.45 t (7.6)
7.41 dd (7.6, 1.1)
–
7.67 d (8.8)
7.56 d (8.8)
–
–
–
7.13 dd (7.6, 1.4)
7.45 t (7.6)
7.40 d (7.6)
–
7.64 d (8.8)
7.56 d (8.8)
–
4.10 s
4.10 s
4.10 s
10
20
30
40
50
60
102.7
75.2
78.2
71.8
78.6
5.11 d (7.3)
3.57 m
3.57 m
3.42 m
3.57 m
3.97 dd (12.1, 2.2)
102.6
75.1
78.1
71.8
77.3
5.09 d (7.2)
3.54 m
3.54 m
3.20 m
3.72 m
102.5
75.1
78.2
71.7
77.2
5.10 d (7.2)
3.54 m
3.54 m
3.41 m
3.69 m
62.8
68.9
4.10 m
68.0
4.10 m
3.73 dd (12.1, 6.3)
3.65 dd (11.0, 6.7)
5.00 d (2.4)
3.94 d (2.4)
3.62 dd (11.0, 6.5)
4.71 d (1.2)
3.85 m
3.73 dd (9.5, 3.4)
3.35 m
100
200
300
400
–
–
–
–
–
–
–
–
111.1
78.2
80.6
75.1
102.3
72.3
72.7
74.3
4.00 d (9.6)
3.75 d (9.6)
3.58 s
500
600
–
–
–
–
65.7
–
70.1
18.1
3.66 dd (9.5, 6.1)
1.17 d (6.2)
–
Fig. 2. Key 1H–1H COSY, HMBC and NOESY correlations of compounds 1–3.
2,5-dihydroxy-4-methoxy-phenanthrene 2-O-
a
-
L
-rhamnopyrano-
3.9 Hz, H-10), 2.68 (dd, J = 13.8, 11.0 Hz, H-10), and one methine
proton at d 4.47 (dd, J = 11.0, 3.9 Hz). A methoxy group was pre-
sumed from the 1H NMR resonances at d 3.90 and the 13C NMR sig-
nal at d 58.0 (Table 2). The NOESY correlations between H-10 and
H-1, H-8 and H-9, OCH3 and H-6 confirmed the location of the 5-
OCH3 group (Fig. 3). Based on the IR absorption at 3420 cmꢀ1, its
molecular formula and oxygenated C-atoms (d 158.4, 154.2,
158.8, 70.1), four hydroxyl groups were assumed. The HMBC corre-
lations (H-1/C-2, C-3, C-4a; H-3/C-4, C-4a; H-6/C-4b, C-5, C-7; H-8/
C-4b, C-6, C-7; H-10/C-4a, C-8a, C-10a) suggested that the four hy-
droxyl groups were located at C-2, C-4, C-7 and C-9. The CD spectra
syl-(1–6)-b- -glucopyranoside.
D
Compound 4 was obtained as a yellowish powder. Its molecular
formula was determined to be C15H14O5 from the quasi molecular
ion peak at 297.0736 in the HR-ESI-MS ([M+Na]+). Its UV maxima
absorptions at 211.0, 274.5 and 307.0 nm were similar to those
of 9,10-dihydrophenanthrene derivatives (Yoshikawa et al.,
2012). Its 1H NMR spectrum (Table 2) exhibited signals for two
pairs of meta-coupled aromatic protons at d 6.35 (d, J = 2.5 Hz, H-
1), 6.30 (d, J = 2.5 Hz, H-3), 6.56 (d, J = 2.4 Hz, H-6) and 6.82 (d,
J = 2.4 Hz, H-8), two methylene protons at d 2.76 (dd, J = 13.8,

Y. Lin et al. / Phytochemistry 95 (2013) 242–251
245
Table 2
1H and 13C NMR (600 and 150 MHz, MeOH-d4) spectroscopic data of compounds 4–6 (d in ppm, multiplicities, J in Hz).
Position
4
5
6
dC
dH
dC
dH
dC
dH
1
2
3
4
4a
4b
5
6
7
109.8
158.4
105.2
154.2
113.6
114.3
156.0
101.4
158.8
107.6
145.9
70.1
6.35 d (2.5)
–
6.30 d (2.5)
–
–
–
–
6.56 d (2.4)
–
6.82 d (2.4)
–
4.47 dd (11.0, 3.9)
2.76 dd (13.8, 3.9)
111.7
158.7
101.3
156.1
113.6
112.7
155.8
105.5
158.7
106.1
143.3
70.2
6.50 d (2.3)
–
6.53 d (2.3)
–
–
–
–
6.34 d (2.6)
–
6.64 brs
–
4.47 dd (11.0, 3.9)
2.76 dd (13.8, 3.9)
121.8
128.8
118.8
154.5
121.7
113.7
156.8
101.1
160.0
108.4
147.8
70.1
6.83 m
7.08 t (7.7)
6.83 m
–
–
–
–
6.59 d (2.3)
–
6.83 d (2.3)
–
4.49 dd (11.0, 3.9)
2.85 dd (13.8, 3.9)
8
8a
9
10
41.3
41.0
57.9
2.68 dd (13.8, 11.0)
2.68 dd (13.8, 11.0)
2.68 dd (13.8, 11.0)
10a
4-OCH3
5-OCH3
139.0
–
58.0
–
–
139.6
57.9
–
–
137.9
–
57.9
–
–
3.91 s
–
3.90 s
3.93 s
Fig. 3. Key HMBC and NOESY correlations of compounds 4–8.
of both 4 and (9S)-hydroxy-9,10-dihydrophenanthrene (Resnick
and Gibson, 1996) showed a negative Cotton effect at 234 nm
and a positive Cotton effect at 268 nm, indicating a 9S configura-
tion of 4. Therefore, compound 4 was 5-methoxy-2,4,7,9S-tetra-
hydroxy-9,10-dihydrophenanthrene.
ion peak at 281.0784 in the HR-ESI-MS ([M+Na]+). Its maxima IR
absorption at 3434 cmꢀ1 indicated the presence of hydroxyl
groups, whereas UV maxima absorptions at 212.0, 272.5 and
303.0 nm were similar to other 9,10-dihydrophenanthrene deriva-
tives (Yoshikawa et al., 2012). Its NMR (Table 2), NOESY and HMBC
spectra (Fig. 3) supported the structure of 6. The CD spectra of 6
and (+)-(9S)-hydroxy-9,10-dihydrophenanthrene (Resnick and
Gibson, 1996) showed a negative Cotton effect at 234 nm and a po-
sitive Cotton effect at 268 nm. Therefore, structure 6 was 5-meth-
oxy-4,7,9S-trihydroxy-9,10-dihydrophenanthrene.
Compound 7 was obtained as a yellowish colloidal solid. Its
molecular formula was determined to be C21H24O9 from the quasi
molecular ion peak at 443.1318 in the HR-ESI-MS ([M+Na]+). The IR
spectrum indicated hydroxyl groups (3398 cmꢀ1). The UV maxima
absorptions at 211.0, 270.0 and 302.0 nm were similar to 9,10-
dihydrophenanthrene derivatives (Yoshikawa et al., 2012). Two
methylene protons at 2.85 (dd, J = 14.3, 3.4 Hz, H-10), 2.79 (dd,
J = 14.3, 9.6 Hz, H-10), one methoxy proton at 3.96 (s), and one
methine proton at 4.57 (brs) were deduced from the 1H NMR spec-
trum. Its NOESY and HMBC correlations (Fig. 3) supported the
Compound 5, obtained as a yellowish powder, has the molecu-
lar formula C15H14O5, as established by the quasi molecular ion
peak at 297.0736 in the HR-ESI-MS ([M+Na]+). Its IR spectrum
showed the presence of hydroxyl groups (3439 cmꢀ1). Its UV max-
ima absorptions at 211.5, 274.5 and 306.0 nm are similar to 9,10-
dihydrophenanthrene derivatives (Yoshikawa et al., 2012). The
mass and NMR spectroscopic data were similar to those of com-
pound 4. The NMR (Table 2), NOESY and HMBC spectra (Fig. 3) sup-
ported the structure of 5. The CD spectra of 5 and (+)-(9S)-hydroxy-
9,10-dihydrophenanthrene (Resnick and Gibson, 1996) are similar,
with a negative Cotton effect at 234 nm and a positive Cotton effect
at 268 nm. Thus, compound 5 was elucidated to be 4-methoxy-
2,5,7,9S-tetrahydroxy-9,10-dihydrophenanthrene.
Compound 6 was obtained as a yellowish powder. Its molecular
formula was established to be C15H14O4 from the quasi molecular

246
Y. Lin et al. / Phytochemistry 95 (2013) 242–251
Table 3
Table 5
1H and 13C NMR (600 and 150 MHz, MeOH-d4) spectroscopic data of compounds 7, 8
(d in ppm, multiplicities, J in Hz).
Effects of compounds 1–12 on LPS-Induced NO production in RAW264.7 cells.
Compounds
% Inhibition on LPS-induced NO production
IC50 (lM)
Position
7
8
50
10
1
dC
dH
dC
dH
1
2
3
4
5
6
7
8
92
76
62
90
86
58
68
92
95
82
95
74
2
4
1
7
2
8
2
5
6
5
3
6
78
44
30
80
78
50
60
77
75
39
69
43
4
7
3
2
3
9
1
2
4
1
2
3
38
24
24
27
42
44
40
62
30
21
38
27
3
5
1
8
4
5
8
2
1
3
4
5
4.6
16.9
41.5
3.1
4.2
–
1
2
3
4
4a
4b
5
6
7
112.5
159.3
102.2
156.5
117.4
120.5
154.8
119.6
129.4
118.3
143.8
69.8
6.79 brs
–
6.87 brs
–
–
–
–
6,91 dd (7.7, 1.2)
7,21 t (7.7)
7.11 d (7.7)
–
109.9
159.3
105.4
156.4
113.4
123.2
152.8
116.3
128.6
120.2
143.5
69.9
6.38 d (2.2)
–
6.34 d (2.4)
–
–
–
–
7.29 m
7.29 m
7.29 m
–
4.57 dd (10.2, 4.0)
2.78 dd (13.8, 4.0)
2.71 dd (13.8, 10.2)
–
–
0.7
6.3
27.4
7.6
32.7
9
10
11
12
8
8a
9
4.57 brs
Curcumina
84
3
72
2
30
4
6.2
10
40.7
2.85 dd (14.3, 3.4)
2.79 dd (14.3,9.6)
–
3.96 s
5.07 d (7.2)
3.49 m
3.45 m
3.37 m
3.45 m
3.93 dd (12.1, 2.0)
3.68 dd (12.1, 6.5)
41.2
The values are mean SEM (n = 3).
a
10a
140.2
57.8
102.5
75.1
78.1
71.8
78.5
62.9
139.7
–
Curcumin was used as a positive control.
4-OCH3
Glc-1
Glc-2
Glc-3
Glc-4
Glc-5
Glc-6
–
102.1
74.9
78.2
71.3
78.5
62.7
5.10 d (7.6)
3.47 m
3.47 m
3.40 m
3.47 m
Acid hydrolysis of 8 afforded
by GC–MS and by its optical rotation {[
The b-
-glucopyranosyloxyl provided by the 1H NMR signal at d
D-glucose, which was determined
25
a]
+51.4 (c 0.4, H2O)}.
D
D
3.87 d (11.4)
3.68 dd (12.2, 5.5)
5.10 (d, J = 7.6 Hz, H-1¢) and the 13C NMR signals at d 102.1, 78.5,
78.2, 74.9, 71.3 and 62.7 and which could be located at C-2 from
the HMBC correlations of H-1¢ with C-5. Based on the molecular
formula C20H22O9, three hydroxyl groups could be presumed, and
are likely located at C-2, C-4 and C-9 on the basis of the NOESY
and HMBC experiments. The CD spectrum of 8 (negative/positive
Cotton effects: 234 nm/268 nm) is opposite to that, (+)-(9S)-hydro-
xy-9,10-dihydrophenanthrene (Resnick and Gibson, 1996). Thus,
structure 8 was 1,2,5,9R-tetrahydroxy-9,10-dihydrophenanthrene
Table 4
Effects of phenanthrene derivatives (1–12) on cell viability in RAW 264.7 cells, HeLa
cells and HepG2 cells.
Compounds
RAW 264.7(%)
HeLa(%)
50
98
HepG2(%)
50
72
5-O-b-D-glucopyranoside.
100
l
M
50
l
M
1
l
M
lM
l
M
Compounds 1 and 9 might be aromatized from compounds 7
and 10 during the silica gel column chromatography. Thus, a test
was carried out to study the impact of silica gel and solvent on
the stability of compounds 1 and 9. Thus, a mixture of compound
7 (or 10, 0.5 mg), silica gel (25.0 mg) and methanol (0.3 mL) in a
test tube was heated at 45 °C for 24 h. Each suspension was filtered
and the filtrate so obtained was analyzed by HPLC (Fig. S44 and
S45). These results indicated that dehydration had not occurr in
presence of silica gel in our test and provided preliminary evidence
toward the natural occurrence of compounds 1 and 9.
1
2
3
4
5
6
7
8
9
61
58
92
51
53
67
55
56
47
54
64
88
2
124
93
90
105
93
124 15
93
102
86
82
83
9
101
93
89
97
95
97
3
3
9
2
3
3
3
3
1
3
2
5
2
6
4
3
1
2
8
2
2
8
4
95
96
105
94
96
89
95
106 11
96
95
96
3
5
7
8
8
8
2
87
99
4
2
5
2
2
5
1
3
5
3
3
100
95
92
9
6
2
7
1
2
106 14
100
109
89
105
79
80
97
94
4
8
9
3
6
10
11
12
4
3
1
109
92
96
93
Cytotoxic effects of compounds 1–12 were examined in mouse
macrophage RAW264.7 cells, human cervical cancer HeLa cells and
human hepatoma HepG2 cells. As shown in Table 4, compounds 1–
The values are mean SEM (n = 3).
12 had no cytotoxic activity in these cells at 50
2, and 4–11 showed weak cytotoxic activity in RAW264.7 macro-
phages at 100 M.
lM. Compounds 1,
assignment of the 4-OCH3, 5-OH and 9-OH. Acid hydrolysis of 7
afforded -glucose, which was determined by GC–MS and by its
optical rotation {[
D
l
25
a
]
+51.1 (c 0.3, H2O)}. The b-
D
-glucopyrano-
Phenanthrenes, the major components of D. denneanum, were
also obtained from species of the Orchidaceae family, including
Bulbophyllum, Eria, Maxillaria, Bletilla, Coelogyna, Cymbidium,
Ephemerantha and Epidendrum (Adriána et al., 2008). Phenan-
threnes have been found to have various biological activities such
as anti-inflammatory (Yang et al., 2006a), cytotoxic (Lee et al.,
2009), antifibrotic (Yang et al., 2007a), anticancer (Lee et al.,
1995), and antimicrobial (Adriána et al., 2008) effects. To deter-
mine if the isolated compounds were responsible for the anti-
inflammatory effects of this plant in clinical use, compounds 1–
12 (Fig. 1) were tested for their effects on LPS-induced NO produc-
tion in RAW264.7 cells. As shown in Table 5, compounds 1, 4, 5, 8, 9
and 11 potently inhibited NO production with IC50 values of 0.7–
D
syloxyl moiety was presumed based on the 1H NMR signal at d
5.07 (d, J = 7.2 Hz, H-1) and 13C NMR resonances at d 102.5, 78.5,
78.1, 75.1, 71.8 and 62.9, which could be located at C-2 from the
HMBC correlation of H-10 and C-2. The CD spectrum of 7 (nega-
tive/positive Cotton effects: 234 nm/268 nm) is opposite to that
of (+)-(9S)-hydroxy-9,10-dihydrophenanthrene (Resnick and Gib-
son, 1996). Thus, structure 7 was 4-methoxy-2,5,9R-trihydroxy-
9,10-dihydrophenanthrene 2-O-b-D-glucopyranoside.
Compound 8, obtained as a colorless colloidal solid, has the
molecular formula C20H22O9, as established by the molecular ion
peak at 428.1156 in the HR-ESI-MS ([M+Na]+). Its IR spectrum
showed the presence of hydroxyl groups (3420 cmꢀ1). Its UV max-
ima absorptions at 211.0, 275.0 and 302.0 nm, were similar to
9,10-dihydrophenanthrene derivatives (Yoshikawa et al., 2012).
The 1H NMR and 13C NMR spectroscopic data (Table 3) were similar
to those of compound 7, but with no methoxyl group detected.
7.6
ate inhibitory activity with IC50 values of 16.9–41.5
itory effects of compounds 9 and 10 on NO production (IC50 = 6.3,
27.4 M) were similar as those reported (IC50 = 6.4, 29.1 M) (Ito
l
M, whereas compounds 2, 3, 10, and 12 showed only moder-
lM. The inhib-
l
l

Y. Lin et al. / Phytochemistry 95 (2013) 242–251
247
Fig. 4. Effect of compounds 1 and 4 on the expression of iNOS. RAW264.7 cells were pretreated with compounds 1 and 4 (1, 10, 50
lM) and a positive control (BAY) for 2 h,
and then treated with LPS for 18 h. Cell lysates were immunoblotted with an anti-iNOS antibody. GAPDH staining is shown as a loading control. The quantitative results are
depicted. Cont. (control), DMSO; LPS, 1 g/mL lipopolysaccharide; BAY, 10
l
l
M BAY 11-7082. ⁄⁄⁄p < 0.001 compared with the LPS group.
Fig. 5. Effect of compounds 1 and 4 on the phosphorylation of p38 and JNK. (A) RAW264.7 cells were pretreated with compounds 1 and 4 (1, 10, 50
for 2 h and treated with LPS for 30 min. Cell lysates were immunoblotted with an anti-phospho-p38 antibody (Thr180/Tyr182). The total p38 staining was used as an internal
control. The quantitative results are depicted. Cont., DMSO; LPS, 1 g/mL lipopolysaccharide; SB, 10 M SB203580. (B) RAW264.7 cells were pretreated with compounds 1
and 4 (1, 10, 50 M) and a positive control for 2 h and treated with LPS for 30 min. Cell lysates were immunoblotted with an anti-phospho-JNK antibody (Thr183/Tyr185). The
lM) and a positive control
l
l
l
total JNK staining was used as an internal control. The quantitative results are depicted. Cont., DMSO; LPS, 1
compared with the LPS group.
lg/mL lipopolysaccharide; SP, 20 l
M SP600125. ⁄⁄⁄p < 0.001
et al., 2010) in LPS-activated RAW264.7 cells, thus indicating that
our assay is suitable for the evaluation of NO production.
NO is a well-known pro-inflammatory cytokine involved in
many inflammatory diseases (Guzik et al., 2003). Compounds 1–
12 may act as the anti-inflammatory chemical compounds in D.
denneanum, and therefore, may be suitable substitutes for other
endangered species for the prevention and treatment of inflamma-
tory diseases in a clinical setting (Chinese Pharmacopoeia, 2010).
The anti-inflammatory activities of these phenanthrenes (1–3, 9)
suggest that attached disaccharide moieties could reduce their
activity: compounds 2 and 3, with a disaccharide, were much less
active (IC50 = 16.9, 41.5 lM) than compounds 1 and 9, with or

248
Y. Lin et al. / Phytochemistry 95 (2013) 242–251
Fig. 6. Effect of compounds 1 and 4 on the phosphorylation of I
j
B
a
. RAW264.7 cells were pretreated with compounds 1 and 4 (1, 10, 50
antibody (Ser32/36). GAPDH staining is shown as a loading control.
M BAY 11-7082. ⁄⁄⁄p < 0.001 compared with the LPS group.
lM) and a positive control (BAY) for
2 h, and then treated with LPS for 20 min. Cell lysates were immunoblotted with an anti-phospho-I
The quantitative results are depicted. Cont., DMSO; LPS, 1 g/mL lipopolysaccharide; BAY, 10
jBa
l
l
without a sugar moiety (IC50 = 4.6, 6.3
l
M). For the class of 9-hy-
tory cytokines (Sung et al., 2009; Khan et al., 2011). Thus, the ef-
fects of compounds 1 and 4 on I phosphorylation were
investigated. As shown in Fig. 6, LPS significantly activated the
phosphorylation of I . BAY 11-7082, an I B inhibitor, inhibited
the phosphorylation of I induced by LPS. Compounds 1 and 4
(1–50 M) significantly inhibited the LPS-stimulated I phos-
phorylation in a concentration-dependent manner. This result
indicated that these compounds may decrease NF- B-mediated
pro-inflammatory cytokine production by inhibiting I phos-
phorylation. NF- B transcription factors play a key role in regulat-
ing the expression of numerous genes involved in inflammation.
Activation of NF- B depends on phosphorylation of I and sub-
sequent ubiquitination and degradation of I proteins. After
degradation of I , NF- B translocates to the nucleus and exerts
its transcription function (Quivy and Lint, 2004). In this study, it
was found that I phosphorylation was significantly inhibited
by compounds 1 and 4, which prevents the degradation of I
and subsequent NF- B activation. Therefore, compounds 1 and 4
may decrease the expression of pro-inflammatory cytokines by
inhibiting the activation of NF- B mediated by phosphorylation
of I . Previously 2,5-dimethoxy-1,7-dihydroxy-9,10-dihydroph-
enanthrene was found to inhibit the MAPK and NF- B pathways,
droxy-9,10-dihydrophenanthrenes (4–8, 10), a hydroxyl group at
C-2 was necessary for increasing activity: the activity of com-
pounds 4, 5, 8 and 10 were more potent than compounds 6 and 7.
To further study the mechanism of these compound-mediated
inhibition of NO production, the protein expression of iNOS, the
major enzyme catalyzing the formation of NO, was examined. As
shown in Fig. 4, LPS treatment significantly increased iNOS expres-
jBa
j
B
a
jBa
j
l
jBa
j
sion. BAY 11-7082, a NF-
(Mendes et al., 2009), inhibited LPS-induced iNOS expression. Pre-
treatment with compounds 1 and 4 (1–50 M), significantly
jB inhibitor, used as a positive control
jBa
j
l
blocked LPS-Induced iNOS expression in a concentration-depen-
dent manner, indicating that these compounds could decrease
NO production in LPS-activated RAW264.7 cells by inhibiting iNOS
expression.
Phosphorylation of p38 and JNK MAPK has been shown to reg-
ulate expression of iNOS and other pro-inflammatory cytokines
(Sung et al., 2009; Thirunavukkarasu et al., 2006); thus, the effects
of compounds 1 and 4 on p38 and JNK MAPK phosphorylation
were examined. As shown in Fig. 5A, LPS significantly increased
phosphorylation of p38. SB203580, a specific p38 MAPK inhibitor
(Lee et al., 1994; Badger et al., 1998), inhibited the p38 phosphor-
ylation stimulated by LPS. Pretreatment with compounds 1 and 4
j
jBa
jBa
jB
a
j
jBa
jBa
j
j
jBa
j
mimicking a TLR4 (toll-like receptor 4 antagonist) (Datla et al.,
2010); hence, further study is needed to examine if compounds 1
and 4 exert their anti-inflammatory effects by inhibiting LPS-in-
duced toll-like receptor activation.
(1–50 lM) also significantly inhibited LPS-stimulated p38 MAPK
phosphorylation in a concentration-dependent manner. LPS can
also stimulate JNK phosphorylation in RAW264.7 cells, as previ-
ously reported. As shown in Fig. 5B, SP600125, a specific JNK
MAPK inhibitor, significantly blocked LPS-activated JNK phosphor-
ylation (Bennett et al., 2001). Pretreatment with compounds 1
3. Conclusion
and 4 (1–50 lM) also significantly inhibited LPS-stimulated JNK
phosphorylation in a concentration-dependent manner. These re-
sults suggest that these compounds could decrease iNOS expres-
sion by inhibiting phosphorylation of p38 and JNK MAPK. AP-1
(activating proteins 1), sequencing specific transcription factors,
have also been shown to be key regulatory molecules in the con-
trol of inflammatory responses. MAPKs, including ERK, p38, and
JNK have been reported to facilitate binding of AP-1 transcription
factors with promoters of pro-inflammatory cytokines (Ono and
Han, 2000; Karin, 1995). Thus, it is possible that compounds 1
and 4 decrease pro-inflammatory cytokine expression via inhibi-
tion of AP-1, by inhibiting phosphorylation of p38 and JNK
MAPKs.
Eight new compounds were isolated from the stems of D. den-
neanum: three new phenanthrene glycosides (1–3), three new
9,10-dihydrophenanthrenes (4–6) and two new 9,10-dihydro-
phenanthrenes glycosides (7 and 8). The new compounds (1, 4, 5
and 8) showed potent anti-inflammatory activity in LPS-induced
NO production in RAW264.7 cells with IC50 values of 0.7–4.6
Compounds 1 and 4 inhibited NO production by blocking iNOS
expression, p38 MAPK phosphorylation and I phosphorylation.
lM.
jBa
These results indicated that the chemical composition of D. den-
neanum was similar to other species of Dendrobium, and validated
its clinical use as a substitute for other endangered Dendrobium
species. Compounds 1 and 4 warrant further investigation as
new pharmaceutical tools for prevention and treatment of inflam-
matory diseases.
Phosphorylation of inhibitory kappa B
a (IjBa) plays a key role
in regulation of NF- B function in transcription of pro-inflamma-
j

Y. Lin et al. / Phytochemistry 95 (2013) 242–251
249
4.3.1. 2, 5-Dihydroxy-4-methoxy-phenanthrene 2-O-b-D-
4. Experimental
glucopyranoside (1)
4.1. General experimental procedures
25
Colorless colloidal solid; mp 166–169 °C; [
a]
ꢀ52 (c 0.8,
D
MeOH); UV (MeOH) kmax (loge) 214.5, 254, 283.5 and 313.5 nm;
UV Spectra were recorded on a UV1902 spectrophotometer,
IR (KBr) mmax 3427, 1614, 1384, 1261, 1073, 819, and 595 cmꢀ1
;
whereas optical rotation [
a
]
D
values were determined with a
for 1H NMR and 13C spectroscopic data NMR, see Table 1; HR-
ESI-MS m/z 425.1208 (calcd for C21H22O8Na, 425.1208).
Perkin–Elmer 341 automatic polarimeter, and IR spectra were
measured on a Perkin–Elmer FT-IR spectrometer (KBr disc). CD
spectra were obtained on a JASCO J-810 spectrometer. NMR
spectra were recorded in MeOH-d4 on a Bruker Avance 600
NMR spectrometer. MS data was obtained on a Bruker Daltonics
Bio-TOF-Q mass spectrometer. Column chromatography (CC)
was carried out on silica gel (200–300 mesh, Qingdao Haiyang
Chemical CO., Ltd.). Silica gel GF 254 pre-coated plates (Qingdao
Haiyang Chemical Inc., Qingdao, P. R. China) were used for pre-
parative TLC. Sephadex LH-20 was purchased from Pharmacia
4.3.2. 2,5-Dihydroxy-4-methoxy-phenanthrene 2-O-b-
apiofuranosyl-(1–6)-b- -glucopyranoside (2)
Yellowish colloidal solid; mp 206–210 °C; [
MeOH); UV (MeOH) (log ) 215.0, 254, 283.5 and 315.0 nm;
D-
D
25
a
]
ꢀ133 (c 0.2,
D
e
max
IR (KBr) mmax 3434, 1615, 1430, 1261, 1068, 821, and 619 cmꢀ1
;
for 1H NMR and 13C NMR spectroscopic data, see Table 1; HR-
ESI-MS m/z 557.1629 (calcd for C26H30O12Na, 557.1629).
Biotech (Sweden). HPLC analysis was performed using
a
250 mm ꢁ 20 mm, 5
l
m, Kromasil 100-10-C18 column on an
4.3.3. 2,5-Dihydroxy-4-methoxy-phenanthrene 2-O-
rhamnopyranosyl-(1–6)-b- -glucopyranoside (3)
Yellowish colloidal solid; mp 194–200 °C; [
MeOH); UV (MeOH) (log ) 214.5, 254, 283.5, and 314.5 nm;
a-L-
Agilent 1260 HPLC system.
D
25
a
]
ꢀ144 (c 0.5,
D
4.2. Plant material
e
max
IR (KBr) mmax 3441, 1615, 1454, 1268, 1063, 828, and 599 cmꢀ1
;
for 1H NMR and 13C NMR spectroscopic data, see Table 1; HR-
Fresh stems of Dendrobium denneanum were collected in April
2010 from Jiajiang, Sichuan Province, P. R. China and identified
by Prof. Ze Chun at the Chengdu Institute of Biology, Chinese Acad-
emy of Science (CAS). A voucher specimen (LD-0) is deposited in
the Herbarium of Chengdu Institute of Biology, CAS.
ESI-MS m/z 571.1794 (calcd for C27H32O12Na, 571.1794).
4.3.4. 5-Methoxy-2,4,7,9S-tetrahydroxy-9,10-dihydrophenanthrene
(4)
25
Yellowish powder; mp 157–161 °C; [
a]
+69 (c 0.1, MeOH);
D
4.3. Extraction and isolation
UV (MeOH)
(loge) 211.0, 274.5, and 307.0 nm; IR (KBr) mmax
max
3420, 1735, 1622,1455, 1156,1037, 873, and 672 cmꢀ1; for 1H
NMR and 13C NMR spectroscopic data, see Table 2; HR-ESI-MS m/
z 297.0736 (calcd for C15H14O5Na, 297.0736).
Air-dried stems of Dendrobium denneanum (15 kg) were ex-
tracted with EtO4–H2O (3 ꢁ 70 L, 95:5, v/v, each 6 days duration)
at room temperature. The filtrates were combined and concen-
trated under reduced pressure at 45 °C to obtain an EtO4 (1.1 kg).
The latter was suspended in H2O (4 L) and partitioned successively
with petroleum ether (6 ꢁ 4 L), EtoA2 (6 ꢁ 4 L) and n-butanol
(6 ꢁ 4 L). The EtoA2 fraction (150.0 g) was divided into 7 subfrac-
tions (Fr. 1–7) over a silica gel column eluted with CHCl3-MeOH
(30:1, 10:1, 0:1).
4.3.5. 4-Methoxy-2,5,7,9S-tetrahydroxy-9,10-dihydrophenanthrene
(5)
An isomer of compound 4: yellowish powder; mp 167–172 °C;
25
[a
]
D +62 (c 0.15, MeOH); UV (MeOH) max (loge) 211.5, 274.5 and
306.0 nm; IR (KBr) mmax 3439, 1636, 1615, 1438, 1161, 871, and
617 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data, see Table 2;
HR-ESI-MS m/z 297.0736 (calcd for C15H14O5Na, 297.0736).
Fr.3 (9.4 g) was separated by a Sephadex (LH-20) CC eluted
with CHCl3-MeOH (1:1) to yield Fr.3.1–Fr.3.2. Subsequently,
Fr.3.2. (2.1 g) was separated by a silica gel CC and eluted with
CHCl3-MeOH (25:1) to give
Separation of Fr.3.2.2 by HPLC (MeOH/H2O, 1:1) afforded
(1.5 mg).
Fr.6 (40.7 g) was separated using a Sephadex (LH-20) CC and
eluted with CHCl3-MeOH (1:1) to yield Fr.6.1–Fr.6.2. Fr.6.2
(21.9 g) was divided into 8 subfractions (Fr.6.2.1–Fr.6.2.8) over
silica gel using CHCl3-MeOH (7:1) as a solvent. Fr.6.2.4 was sep-
arated by HPLC (CH3CN/H2O, 17:83) to give 4 (21 mg) and 5
(17 mg), respectively. Separation of Fr.6.2.6 over a C18 column
6 subfractions (Fr.3.2.1–Fr.3.2.6).
4.3.6. 5-Methoxy-4,7,9S-trihydroxy-9,10-dihydrophenanthrene (6)
25
6
Yellowish powder; mp 166–172 °C; [
a]
+80 (c 1.5, MeOH);
D
UV (MeOH)
(loge) 212.0, 272.5 and 303.0 nm; IR (KBr) mmax
max
3434, 1626, 1458, 1384, 1161, and 617 cmꢀ1; for 1H NMR and
13C NMR spectroscopic data, see Table 1; HR-ESI-MS m/z
281.0784 (calcd for C15H14O4Na, 281.0784).
4.3.7. 4-Methoxy-2,5,9R-trihydroxy-9,10-dihydrophenanthrene 2-O-
b-
D
-glucopyranoside (7)
(MeOH/H2O, 45:55) yielded
6
subfractions (Fr.6.2.6.1–
25
Yellowish colloidal solid; mp 179–185 °C; [
a
]
ꢀ60 (c 0.9,
D
Fr.6.2.6.6). Fr.6.2.6.2 was separated by HPLC (CH3CN/H2O, 1:3)
to afford 1 (18 mg). Fr.6.2.7 was divided into 3 subfractions
(Fr.6.2.7.1–Fr.6.2.7.3) over a C18 column (MeOH/H2O, 35:65).
Fr.6.2.7.2 was separated by HPLC (CH3CN/H2O, 1:3) to give 7
(42 mg).
MeOH); UV (MeOH)
(loge) 211.0, 270.0 and 302.0 nm; IR
max
(KBr) mmax 3398, 1612, 1450, 1302, 1261, 1166, 905, 799, and
625 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data, see Table 1;
HR-ESI-MS m/z 443.1318 (calcd for C21H24O9Na, 443.1318).
Fr.7 (7.8 g) was separated using a Sephadex (LH-20) CC eluted
with CHCl3-MeOH (1:1) to yield Fr.7.1–Fr.7.4. Fr.7.2 (1.2 g) was
separated over a C18 column (MeOH/H2O, 35:65) to yield 3 sub-
fractions (Fr.7.2.1–Fr.7.2.3). Fr.7.2.3 was further purified by HPLC
(CH3CN/H2O, 1:2) to give 2 (17 mg) and 3 (3.5 mg), respectively.
Fr.7.3 was separated by silica gel CC using CHCl3-MeOH (12:1) to
afford 3 subfractions (Fr.7.3.1–Fr.7.3.3). Fr.7.3.1 was separated by
HPLC (MeOH/H2O, 1:4) to give 8 (35 mg).
4.3.8. 1,2,5,9R-Tetrahydroxy-9,10-dihydrophenanthrene 5-O-b-D-
glucopyranoside (8)
25
Yellowish colloidal solid; mp 164–168 °C; [
a
]
ꢀ46 (c 0.3,
D
MeOH); UV (MeOH) kmax (log
e
) 211.0, 275.0 and 302.0 nm; IR
(KBr) mmax 3420, 1622, 1459, 1384, 1232, 1156, 1073, and
618 cmꢀ1; for 1H NMR and 13C NMR spectroscopic data, see Table 1;
HR-ESI-MS m/z 443.1156 (calcd for C21H24O9Na, 443.1156).

250
Y. Lin et al. / Phytochemistry 95 (2013) 242–251
4.4. Acid hydrolysis of compounds 1–3, 7 and 8
the cells were lysed in RIPA buffer (Beyotime, Haimen, China) sup-
plemented with a cocktail of protease and phosphatase inhibitors
(Pierce, Rockford, IL, USA). The protein concentrations were deter-
mined using a BCA protein assay kit (Pierce). Aliquots of total cell
Compounds 1–3 (1 mg) in dioxane (1 mL) and 10% H2SO4 (1 mL)
were heated at 95 °C for 3 h. After neutralization with 0.2 M
Ba(OH)2, the aglycone was extracted with CH2Cl2, with the aque-
ous residue filtered through absorbent gauze, and the resulting fil-
trate concentrated under reduced pressure. Each sugar mixture
was treated with Ac2O (0.5 mL) and pyridine (0.25 mL) at 95 °C
for 12 h. After cooling, each mixture was poured into ice-water,
stirred and stored for several hours. Each residue was then parti-
tioned between H2O and CH2Cl2. The CH2Cl2 layer was washed
with H2O (3 ꢁ 2.5 mL), with each CH2Cl2 soluble was analyzed by
GC–MS; compounds were identifier by comparison with deriva-
tives of authentic sugars (Antonov et al., 2009). The absolute con-
figuration of the sugar was determined by the Klyne’s rule
(Klyne, 1950).
lysates (50 lg) were mixed in loading buffer, boiled for 5 min,
and subjected to SDS–PAGE (10%). Following electrophoresis, the
proteins were transferred to nitrocellulose membranes, blocked
with 5% bovine serum albumin and then incubated at 4 °C over-
night with anti-phospho-p38 MAPK, anti-p38 MAPK, anti-GAPDH
(Epitomics, Burlingame, CA, USA), anti-iNOS, anti-phospho-JNK,
anti-JNK, or anti-phospho-IjBa (Cell Signaling Technology, Dan-
vers, MA, USA) antibodies. Membranes were then incubated with
a horseradish peroxidase-conjugated secondary antibody (Santa
Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h at room tempera-
ture and developed using an enhanced chemiluminescence detec-
tion system (Amersham Bioscience, Piscataway, NJ, USA). The
intensity of each signal was determined by a computer image anal-
ysis system (Quantity One, Bio-Rad, Hercules, CA, USA).
Compounds 7–8 (3 mg) were treated as described for com-
pounds 1–3 to give a sugar. The sugar was confirmed by compari-
son of its derivative with that of authentic sample on GC–MS and
by its optical rotation.
4.9. Statistical analysis
4.5. Cell culture
All statistical calculations were carried out using GraphPad
Prism 5.01. The results were expressed as the mean standard er-
ror of mean of 3 independent experiments with individual values.
The data were subjected to a one-way analysis of variance (ANO-
VA); p < 0.05 was considered to be statistically significant.
RAW264.7 cells and HeLa cells were grown in Dulbecco’s mod-
ified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA) sup-
plemented with 1% penicillin/streptomycin and 10% fetal calf
serum (Gibco-Invitrogen). HepG2 cells were maintained in RPMI
1640 medium (Invitrogen) supplemented with 1% penicillin/strep-
tomycin and 10% fetal calf serum at 37 °C in a humidified atmo-
sphere containing 5% CO2. For experiments of nitric oxide
content, RAW264.7 cells were maintained in medium devoid of
Phenol Red (Invitrogen).
Acknowledgments
This work was supported by National New Drug Innovation
Major Project of China (2011ZX09307-002-02), Key Projects in
the
National
Science
&
Technology
Pillar
Program
(2011BAC09B04), Knowledge Innovation Program of the Chinese
Academy of Sciences (No. XBCD-2011-007) and ‘‘Twelfth Five-
Year’’ Chinese Herbal Medicines Breeding Research of Sichuan
Province.
4.6. Cell proliferation assay
Cell proliferation was assayed as described previously (Yang
et al., 2011). In brief, 1 ꢁ 104 RAW264.7 cells, HeLa cells, and
HepG2 cells were seeded in 96-well plates, and treated with the
compounds for 24 h. After AlamarBlue reagent was added to each
well, and fluorescence intensity was measured with excitation at
544 nm and excitation at 590 nm using Thermo Scientific Varios-
kan Flash Multimode Reader. Cytotoxicity was defined as the ratio
of the fluorescence intensity in test wells compared to solvent con-
trol wells (0.1% DMSO). The assay was conducted 3 times in
triplicate.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.
2013.08.008.
References
Adriána, K., Andrea, V., Judit, H., 2008. Natural phenanthrenes and their biological
activity. Phytochemistry 69, 1084–1110.
4.7. Measurement of nitric oxide content
Antonov, A.S., Avilov, S.A., Kalinovsky, A., Anastyuk, S.D., Dmitrenok, P.S., Kalinin,
V.I., Taboada, S., Bosh, A., Avila, C., Stonik, V., 2009. Triterpene glycosides from
antarctic sea cucumbers. 2. Structure of achlioniceosides A1, A2 and A3 from the
sea cucumber Achlionice wiolaecuspidata (=Rhipidothuria racowitzai). J. Nat. Prod.
72, 33–38.
Badger, A.M., Cook, M.N., Lark, M.W., Newman-Tarr, T.M., Swift, B.A., Nelson, A.H.,
Barone, F.C., Kumar, S., 1998. SB 203580 inhibits p38 mitogen-activated protein
kinase, nitric oxide production, and inducible nitric oxide synthase in bovine
cartilage-derived chondrocytes. J. Immunol. 161, 467–473.
Bennett, B.L., Sasaki, D.T., Murray, B.W., Sakata, S.T., Xu, W., Leisten, J.C., Motiwala,
A., Pierce, S., Satoh, Y., Bhagwat, S.S., Manning, A.M., Anderson, D.W., 2001.
SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc. Natl.
Acad. Sci. USA 98, 13681–13686.
The RAW264.7 macrophages were seeded in 96-well plates at
1 ꢁ 104 cells/well, and pretreated with various concentrations of
compounds 1–12 for 2 h, followed by 1 lg/mL LPS for an additional
24 h. After that the cell culture medium was used for NO measure-
ments using a commercial available kit (Beyotime, Haimen, China).
Nitrite production was measured at OD550. The assay was con-
ducted 3 times in triplicate. Percentage inhibition was calculated
using the following equation:
%Inhibition ¼ ðA ꢀ BÞ=ðA ꢀ CÞ ꢁ 100
A: LPS (+), sample (ꢀ); B: LPS (+), sample (+); C: LPS (ꢀ), sample (ꢀ).
Chen, C.C., Wang, J.K., Lin, S.B., 1998. Antisense oligonucleotides targeting protein
kinase C-
synthase expression in RAW 264.7 macrophages: involvement of a nuclear
factor B-dependent mechanism. J. Immunol. 161, 6206–6214.
a, -bl, or -d but not -g inhibit lipopolysaccharide-induced nitric oxide
j
4.8. Western blotting
Datla, P., Kalluri, M.D., Basha, K., Bellary, A., Kshirsagar, R., Kanekar, Y., Upadhyay, S.,
Singh, S., Rajagopal, V., 2010. 9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-
diol, from Eulophia ochreata, inhibits inflammatory signalling mediated by Toll-
like receptors. Br. J. Pharmacol. 160, 1158–1170.
Devkota, H.P., Watanabe, M., Watanabe, T., Yahara, S., 2012. Diplomorphanins A and
B: new C-methyl flavonoids from Diplomorpha canescens. Chem. Pharm. Bull. 60,
554–556.
The RAW264.7 macrophages were pretreated with various con-
centrations (1, 10, 50
stimulated with LPS (1
pho-JNK), 20 min (phospho-I
l
l
M) of compounds 1 and 4 for 2 h and then
g/mL) for 30 min (phospho-p38 and phos-
), or 18 h (iNOS). Subsequently
jBa

Y. Lin et al. / Phytochemistry 95 (2013) 242–251
251
Fan, C., Wang, W., Wang, Y., Qin, G., Zhao, W., 2001. Chemical constituents from
Dendrobium densiflorum. Phytochemistry 57, 1255–1258.
Shu, Y., Guo, S.X., Chen, X.M., Wang, C.L., Yang, J.S., 2004. On the chemical
constituents of Dendrobium nobil. Chin. Pharm. J. 6, 421–422.
Guzik, T.J., Korbut, R., Adamek-Guzik, T., 2003. Nitric oxide and superoxide in
inflammation and immune regulation. J. Physiol. Pharmacol. 54, 469–487.
Ito, M., Matsuzaki, K., Wang, J., Daikonya, A., Wang, N., Yao, X., Kitanaka, S., 2010.
New phenanthrenes and stilbenes from Dendrobium loddigesii. Chem. Pharm.
Bull. 58, 628–633.
Sung, M.J., Davaatseren, M., Kim, W., Park, S.K., Kim, S., Hur, H.J., Kim, M.S., Kim, Y.,
Kwon, D.Y., 2009. Vitisin A suppresses LPS-induced NO production by inhibiting
ERK, p38, and NF-kappaB activation in RAW 264.7 cells. Int. Immunopharmacol.
9, 319–323.
Takayanagi, T., Ishikawa, T., Kitajima, J., 2003. Sesquiterpene lactone glucosides and
alkyl glycosides from the fruit of cumin. Phytochemistry 63, 479–484.
Thirunavukkarasu, C., Watkins, S.C., Gandhi, C.R., 2006. Mechanisms of endotoxin
Karin, M., 1995. The regulation of AP-1 activity by mitogen-activated protein
kinases. J. Biol. Chem. 270, 16483–16486.
Khan, S., Shin, E.M., Choi, R.J., Jung, Y.H., Kim, J., Tosun, A., Kim, Y.S., 2011.
induced NO, IL-6, and TNF-a production in activated rat hepatic stellate cells:
Suppression of LPS-induced inflammatory and NF-
jB responses by anomalin in
role of p38 MAPK. Hepatology 44, 389–398.
RAW 264.7 macrophages. J. Cell. Biochem. 112, 2179–2188.
Khurelbat, D., Densmaa, D., Sanjjav, T., Gotov, C., Kitamura, C., Shibuya, H., Ohashi,
K., 2010. Artemisioside, a new monoterpene glucoside from the aerial parts of
Artemisia ordosica (Asteraceae). J. Nat. Med. 64, 203–205.
Klyne, W., 1950. The configuration of the anomeric carbon atomsinsome cardiac
glycosides. Biochem. J. 47, Xii–XIii.
Korhonen, R., Lahti, A., Kankaanranta, H., Moilanen, E., 2005. Nitric oxide production
and signaling in inflammation. Curr. Drug Targets 4, 471–479.
Lee, J.C., Laydon, J.T., McDonnell, P.C., Gallagher, T.F., Kumar, S., Green, D., McNulty,
D., Blumenthal, M.J., Heys, R.J., Landvatter, S.W., Strickler, J.E., McLaughlin, M.M.,
Siemens, I.R., Fisher, S.M., Livi, G.P., White, J.R., Adams, J.L., Young, P.R., 1994. A
protein kinase involved in the regulation of inflammatory cytokine
biosynthesis. Nature 372, 739–746.
Wang, H.K., Zhao, T.F., 1985. Dendrobine and 3-hydroxy-2-oxodendrobine from
Dendrobium nobile. J. Nat. Prod. 48, 796–801.
Wang, X.Y., Ke, C.Q., Tang, C.P., Yuan, D., Ye, Y., 2009. 9,10-Dihydrophenanthrenes
and -henanthrenes from Juncus setchuensis. J. Nat. Prod. 72, 1209–1212.
Yang, L., Qin, L.H., Annie Bligh, S.W., Bashall, A., Zhang, C.F., Zhang, M., Wang, Z.T.,
Xu, L.S., 2006a. A new phenanthrene with a spirolactone from Dendrobium
chrysanthum and its anti-inflammatory activities. Bioorg. Med. Chem. 14, 3496–
3510.
Yang, L., Wang, Z., Xu, L., 2006b. Phenols and
a triterpene from Dendrobium
aurantiacum var. denneanum (Orchidaceae). Biochem. Syst. Ecol. 34, 658–
660.
Yang, H., Sung, S.H., Kim, Y.C., 2007a. Antifibrotic phenanthrenes of Dendrobium
nobile stems. J. Nat. Prod. 70, 1925–1929.
Lee, Y.H., Park, J.D., Baek, N.I., Kim, S.I., Ahn, B.Z., 1995. In vitro and in vivo
antitumoral phenanthrenes from the aerial parts of Dendrobium nobile. Planta
Med. 61, 178–180.
Lee, C.L., Chang, F.R., Yen, M.H., Yu, D., Liu, Y.N., Bastow, K.F., Susan, L., Morris, N.,
Wu, Y.C., Lee, K.H., 2009. Cytotoxic phenanthrenequinones and 9,10-
dihydrophenanthrenes from Calanthe arisanensis. J. Nat. Prod. 72, 210–213.
Li, Y., Wang, C.L., Wang, Y.J., Guo, S.X., Yang, J.S., Chen, X.M., Xiao, P.G., 2009. Three
new bibenzyl derivatives from Dendrobium candidum. Chem. Pharm. Bull. 57,
218–219.
Yang, L., Wang, Y., Zhang, G., Zhang, F., Zhang, Z., Wang, Z., Xu, L., 2007b.
Simultaneous quantitative and qualitative analysis of bioactive phenols in
Dendrobium aurantiacum var. denneanum by high-performance liquid
chromatography coupled with mass spectrometry and diode array detection.
Biomed. Chromatogr. 21, 687–694.
Yang, L.J., Chen, Q.F., Wang, F., Zhang, G.L., 2011. Antiosteoporotic compounds from
seeds of Cuscuta chinensis. J. Ethnopharmacol. 135, 553–560.
Yoshikawa, K., Ito, T., Iseki, K., Baba, C., Imagawa, H., Yagi, Y., Morita, H., Asakawa, Y.,
Kawano, S., Hashimoto, T., 2012. Phenanthrene derivatives from Cymbidium
great flower marie laurencin and their biological activities. J. Nat. Prod. 75, 605–
609.
Lin, T.H., Chang, S.J., Chen, C.C., Wang, J.P., Tsao, L.T., 2001. Two phenanthraquinones
from Dendrobium moniliforme. J. Nat. Prod. 64, 1084–1086.
Liu, Y., Jiang, J.H., Zhang, Y., Chen, Y.G., 2009. Chemical constituents of Dendrobium
aurantiacum var. denneanum. Chem. Nat. Compd. 45, 525–527.
Mendes, S.D.S., Candi, A., Vansteenbrugge, M., Pignon, M.R., Bult, H., Boudjeltia, K.Z.,
Munaut, C., Raes, M., 2009. Microarray analyses of the effects of NF-kappaB or
PI3K pathway inhibitors on the LPS-induced gene expression profile in
RAW264.7 cells: synergistic effects of rapamycin on LPS-induced MMP9-
overexpression. Cell. Signalling 21, 1109–1122.
Okamura, N., Nohara, T., Yagi, A., Nishioka, I., 1981. Studies on the constituents of
Zizyphi fructus. III. Structures of dammarane-type saponins. Chem. Pharm. Bull.
29, 676–683.
Zhang, G.N., Bi, Z.M., Wang, Z.T., Xu, L.S., Xu, G.J., 2003. Advances in studies on
chemical constitutents from plants of Dendrobium Sw. Chin. Tradit. Herbal
Drugs 34, S5–S8.
Zhang, G.N., Zhong, L.Y., Bligh, S.W.A., Guo, Y.L., Zhang, C.F., Zhang, M., Wang, Z.T.,
Xu, L.S., 2005. Bi-bicyclic and bi-tricyclic compounds from Dendrobium
thyrsiflorum. Phytochemistry 66, 1113–1120.
Zhang, X., Xu, J.K., Wang, J., Wang, N.L., Hiroshi, K., Sumumu, K., Yao, X.S., 2007.
Bioactive bibenzyl derivatives and fluorenones from Dendrobium nobile. J. Nat.
Prod. 70, 24–28.
Zhang, X., Tu, F.J., Yu, H.Y., Wang, N.L., Wang, Z., Yao, X.S., 2008. Copacamphane,
picrotoxane and cyclocopacamphane sesquiterpenes from Dendrobium nobile.
Chem. Pharm. Bull. 56, 854–857.
Ono, K., Han, J., 2000. The p38 signal transduction pathway activation and function.
Cell. Signalling 12, 1–13.
Quivy, V., Lint, C.V., 2004. Regulation at multiple levels of NF-
j
B-mediated
Zhao, W., Ye, Q., Tan, X., Jiang, H., Li, X., Chen, K., Kinghorn, A.D., 2001. Three new
sesquiterpene glycosides from Dendrobium nobile with immunomodulatory
activity. J. Nat. Prod. 64, 1196–1200.
transactivation by protein acetylation. Biochem. Pharmacol. 68, 1221–1229.
Resnick, S.M., Gibson, D.T., 1996. Regio- and stereospecific oxidation of fluorene,
dibenzofuran, and dibenzothiophene by naphthalene dioxygenase from
Pseudomonas sp. strain NCIB 9816-4. Appl. Environ. Microbiol. 62, 4073–4080.