Three new sesquiterpene glycosides from Dendrobium nobile with immunomodulatory activity

Article abstract of DOI:10.1021/np0102612

Dendroside A (1) and dendronobilosides A and B (2 and 3), three new sesquiterpene glycosides, have been isolated from the stems of Dendrobium nobile, a plant used in Chinese traditional medicine, Their structures and stereochemistry were determined as 10β,12,14-trihydroxyalloaromadendrane 14-O-β-D-glucopyranoside (1), 10,12-dihydroxypicrotoxane 10,12-di-O-β-D-glucopyranoside (2), and 6α, 10,12-trihydroxypicrotoxane 10-O-β-D-glucopyranoside (3), respectively, on the basis of spectroscopic and chemical methods. Quantum chemistry calculations were used in support of the structural determination of 1. Compounds 1 and 2 were found to stimulate the proliferation of murine T and B lymphocytes in vitro, while compound 3 showed inhibitory activity in this same assay.

Full text of DOI:10.1021/np0102612

1
196
J . Nat. Prod. 2001, 64, 1196-1200
Th r ee New Sesqu iter p en e Glycosid es fr om Den d r obiu m n obile w ith
Im m u n om od u la tor y Activity
Weimin Zhao,†,‡ Qinghua Ye, Xiaojian Tan, Hualiang J iang, Xiaoyu Li, Kaixian Chen, and
A. Douglas Kinghorn*
†
§
§
⊥
§
,
‡
Shanghai Institute of Materia Medica, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences,
Shanghai 200031, People’s Republic of China, and Program for Collaborative Research in the Pharmaceutical Sciences and
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago,
Chicago, Illinois 60612
Received May 30, 2001
Dendroside A (1) and dendronobilosides A and B (2 and 3), three new sesquiterpene glycosides, have
been isolated from the stems of Dendrobium nobile, a plant used in Chinese traditional medicine. Their
structures and stereochemistry were determined as 10â,12,14-trihydroxyalloaromadendrane 14-O-â-D-
glucopyranoside (1), 10,12-dihydroxypicrotoxane 10,12-di-O-â-D-glucopyranoside (2), and 6R,10,12-
trihydroxypicrotoxane 10-O-â-D-glucopyranoside (3), respectively, on the basis of spectroscopic and chemical
methods. Quantum chemistry calculations were used in support of the structural determination of 1.
Compounds 1 and 2 were found to stimulate the proliferation of murine T and B lymphocytes in vitro,
while compound 3 showed inhibitory activity in this same assay.
Ta ble 1. 13C NMR (100 MHz) Data of 1-3 in C5D5N (ppm)
The stems of Dendrobium nobile Lindl. (Chinese name
“
J in-Chai-Shi-Hu”) (Orchidaceae) are a renowned tradi-
tional Chinese medicine used as a tonic, and extracts of
the plant have been reported to possess immunostimulant
position
1
1 (DMSO-d6)
2
3
1
2
3
4
5
6
7
8
9
54.4, d
24.4, t
29.4, t
38.4, d
40.1, d
23.6, d
29.6, d
18.5, t
33.0, t
75.1, s
25.1, s
62.8, t
24.5, q
79.8, t
16.6, q
106.0, d
75.1, d
78.4, d
71.4, d
78.3, d
62.4, t
53.4, d
32.1, t
28.7, t
38.9, d
39.5, d
22.7, d
28.9, d
17.8, t
23.8, t
74.1, s
24.2, s
61.6, t
24.1, q
78.3, t
16.4, q
104.2, d
73.6, d
76.4, d
70.1, d
76.9, d
61.1, t
36.6, d
39.4, d
20.2, t
27.3, t
42.0, s
51.7, d
26.5, t
22.7, t
48.7, d
71.5, t
27.1, d
71.6, t
23.9, q
22.0, q
15.6, q
105.3, d
75.4, d
78.9, d
72.0, d
78.7, d
63.1, t
104.9, d
75.3, d
78.8, d
72.0, d
78.6, d
63.2, t
36.7, d
39.3, d
20.4, t
30.3, t
46.7, s
85.7, s
34.6, t
21.7, t
45.5, d
71.9, t
27.1, t
78.7, d
18.1, q
21.9, q
15.6, q
105.3, d
75.2, d
78.8, d
72.0, d
78.7, d
63.1, t
1
2-4
activity. Previously, several sesquiterpene alkaloids
phenanthrenoids5 have been identified from this species.
Two phenanthrenoids from the aerial parts of the plant
have been reported to exhibit antitumor activity. In the
present investigation to find additional biologically active
compounds from D. nobile, three new sesquiterpene gly-
cosides, named dendroside A (1) and dendronobilosides A
and
,6
6
1
0
(2) and B (3), have been isolated and characterized. Herein,
11
12
we report the isolation, structure determination, and in
vitro immunomodulatory activities of compounds 1-3.
1
1
1
3
4
5
Glc-1
Glc-2
Glc-3
Glc-4
Glc-5
Glc-6
Glc′-1
Glc′-2
Glc′-3
Glc′-4
Glc′-5
Glc′-6
ture. After evaporation of ethanol in vacuo, the aqueous
residue was extracted with petroleum ether, ethyl acetate,
and n-butanol, successively. The n-butanol extract was
subjected to a series of column chromatographic steps,
using reversed-phase C18 silica gel with a MeOH-water
gradient, and finally passage over Sephadex LH-20 with
EtOH, to afford pure compounds 1 (40 mg, 0.0016%), 2 (15
mg, 0.0006%), and 3 (70 mg, 0.0028%).
Resu lts a n d Discu ssion
The fresh stems of Dendrobium nobile (2.5 kg) were
percolated with 95% EtOH three times at room tempera-
Compound 1 was obtained as white amorphous powder,
with an elemental formula of C21
H
36 8
O determined by
*
To whom correspondence should be addressed. Tel: (312) 996-0914.
+
HRFABMS (m/z 439.2321, [M + Na] ). There was evidence
Fax: (312) 996-4107. E-mail: kinghorn@uic.edu.
-
1
†
of hydroxyl absorption in the IR spectrum at 3396 cm .
Department of Phytochemistry, Shanghai Institute of Materia Medica.
University of Illinois at Chicago.
Department of Medicinal Chemistry, Shanghai Institute of Materia
‡
13
In the C NMR spectrum (Table 1), 21 carbon signals were
observed, constituted by two methyls, seven methylenes,
ten methines, and two quaternary carbons. An anomeric
§
Medica.
⊥
Department of Pharmacology, Shanghai Institute of Materia Medica.
1
0.1021/np0102612 CCC: $20.00
© 2001 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/06/2001

Sesquiterpene Glycosides from Dendrobium
J ournal of Natural Products, 2001, Vol. 64, No. 9 1197
Ta ble 2. 1H NMR (400 MHz) Data of 1-3 in C5D5N (ppm, J in Hz)
position
1
1 (DMSO-d6)
2
3
1
2.38, m
1.81, m
2.10, m
1.28, m
1.09, m, 1.35, m
1.10, m, 1.15, m
2.12, m
1.32, m
1.18, m, 1.40, m
1.16, m, 1.36, m
2
3
4
5
6
7
8
9
1
1
1
1.65 (R), m, 1.81 (â), m
1.37 (R), m, 1.82 (â), m
2.00, m
2.40, m
0.40, dd (9.2, 9.2)
0.93, m
1.40 (R), m, 1.68 (â), m
1.19 (R), m, 1.70 (â), m
1.88, m
1.85, m
0.18, dd (8.9, 9.0)
0.65, m
2.05, m
1.53, m, 1.92, m
1.57, m, 1.86, m
2.15, m
3.90, m, 3.90, m
1.90, m
3.62, dd (8.9, 8.6)
4.32, m
1.07, s
2.02, m
1.65, m, 2.08, m
3.00, m
3.88, m, 4.02, m
1.88, m
3.88, m, 4.02, m
1.95 (R), m, 2.25 (â), m
1.70 (R), m, 2.08 (â), m
0.94 (R), m, 1.55 (â), m
1.40 (R), m, 1.52 (â), m
0
1
2
4.07, d (11.2)
3.44, m,
3.48, d (4.3)
1.02, s
3.14, d (10.0)
3.51, d (10.4)
0.92, m
4.10, d (7.8)
2.95, m
3.14, m
3.04, m
3.06, m
3.64, m, 3.42, m
4
.16, d (11.2)
1
1
3
4
1.45, s
3.78, d (10.4)
1.30, s
0.88, d (6.7)
0.92, d (6.8)
4
.06, d (10.2)
1
5
1.13, d (6.7)
5.00, d (7.8)
4.12, m
4.30, m
4.29, m
0.82, d (6.8)
4.90, d (7.7)
4.09, m
4.31, m
4.27, m
0.78, d (6.7)
4.88, d (7.6)
4.00, m
4.25, m
4.25, m
4.00, m
4.40, dd (5.3, 5.3)
4.58, dd (2.5, 2.3)
Glc-1
Glc-2
Glc-3
Glc-4
Glc-5
Glc-6
4.05, m
4.45, dd (11.7, 5.4)
4.04, m
4.46, m, 4.64, m
4
.63, dd (11.7, 1.9)
Glc′-1
Glc′-2
4.90, d (7.7)
4.09, m
Glc′-3
4.31, m
Glc′-4
4.27, m
Glc′-5
4.04, m
Glc′-6
4.46, m, 4.64, m
OH-10
OH-12
Glc-2OH
Glc-3OH
Glc-4OH
Glc-6OH
3.97, s
4.23, m
5.06, d (3.9)
4.92, m
4.88, m
4.50, t (11.5)
carbon signal at δ
C
106.0 (d) indicated the existence of one
signals between OH-10 and H-1 and between OH-10 and
H-5 in the NOESY spectrum suggested that OH-10 is in
the â configuration.
sugar moiety in the structure of 1. Enzymatic hydrolysis
of 1 gave the aglycon 1a , and glucose was determined as
the sugar moiety by co-TLC with an authentic sample. In
To confirm the C-10 stereochemistry of 1, quantum
chemistry calculations were carried out on the two possible
isomers, with the C-10 hydroxyl in either an R or a â
configuration. The density functional theory method
1
the H NMR spectrum of 1 (Table 2), an anomeric proton
signal was found at δ 5.00 (1H, d, J ) 7.8 Hz), which
H
indicated the glucose unit to be in the â-glycosidic form.
The absolute configuration of glucose in 1 was found to
belong to the D series by GC analysis of a peracetylated
1
0
(B3LYP / 6-31G*) was employed to optimize the structures
using the Gaussian98 program,11 then NMR calculations
were performed on the optimized geomeries at the same
7
thiazolidine derivative. Since there were 15 carbon signals
belonging to the aglycon, this was consistent with 1 being
1
2
level using the gauge-independent atomic orbital (GIAO)
a sesquiterpene glycoside. Preliminary inspection of the
H- H COSY, TOCSY, and HMQC NMR spectra of 1
method. Meanwhile, the same procedure was applied on
TMS to calculate the relative chemical shifts of these two
isomers. The plots were made using experimental data
versus theoretical values, and it was found that the
1
1
enabled the deduction of the following structural fragment
in its aglycon: -C-9, C-8, C-7, C-6, C-5, C-4(C-15), C-3,
C-2, C-1, C-5-. Further analysis of the 1H- H COSY,
TOCSY, HMQC, and HMBC spectra of 1 led to the
assignment of an alloaromadendrane skeleton for the
aglycon 1a 8 and assignment of all proton and carbon
1
2
13
1
correlation coefficients (R ) for the C and H NMR spectra
with the OH-10â configuration were 0.9908 and 0.9622,
respectively. These values were higher than those for a OH-
10R configuration (0.9900 and 0.9477). Some important
computational results are summarized in Table 3. It can
be seen from Table 3 that for the OH-10â configuration
the chemical shifts deduced by the B3LYP/6-31G* program
are closer to the experimental data actually obtained for
1, while the corresponding values for the OH-10R config-
uration were more distant from the experimental values.
In the OH-10â configuration, since the hydrogens having
the same orientation as the hydroxyl are proximal to the
oxygen atom, this would result in more downfield chemical
shifts due to the deshielding effect of the oxygen atom.
Comparing the experimental and theoretical data, it was
apparent that the sesquiterpene glycoside 1 has a greater
probability of adopting the OH-10â configuration. A ses-
quiterpene with a structure identical to that of 1a has been
,9
5 5
signals in the parent molecule in C D N (Tables 1 and 2).
In the NOESY spectrum of 1, correlation signals were
found between H-6 and H-7; H-6 and H-15; H-1 and H-5;
H-4 and H-5; H-14 and Hglc-1; H-13 and H-6; and H-13 and
H-7. Therefore, the relative configuration of all chiral
carbons in the molecule could be determined except for that
of the quaternary chiral center at C-10.
To propose the C-10 stereochemistry of 1, NMR spectra
6
were run with DMSO-d as solvent. On the basis of the
analysis of various 2D NMR spectra, all proton and carbon
signals of 1 could be assigned (Tables 1 and 2). Due to the
formation of a hydrogen bond between the hydroxyl group
and DMSO-d
6
, the OH-10 proton signal was observed at
δ
H
3.97 (1H, s) in 1H NMR spectrum of 1. Correlation

1
198 J ournal of Natural Products, 2001, Vol. 64, No. 9
Zhao et al.
Ta ble 3. Experimentally Observed NMR Data for 1 and
Values Obtained by Quantum Chemistry Calculations (ppm)
Ta ble 4. Effect of Compounds 1-3 on Murine Lymphocyte
Proliferation Induced by Concanavalin A (ConA) or
Lipopolysaccharide (LPS) (5 µg/mL)a
1H
13C
OD at 570 nm
calcd values
calcd values
exptl
exptl
group
with Con A
with LPS
no. value OH-10R OH-10â value OH-10R OH-10â
concentration (M) mean ( SD
mean ( SD
1
2
2.38
1.65
2.66
2.29
1.65
1.59
1.55
2.04
1.96
1.16
1.00
1.77
1.61
1.09
2.21
2.34
2.14
2.21
1.49
1.77
2.07
1.83
0.79
0.65
1.86
2.21
1.51
1.88
54.4
24.4
46.4
26.8
59.4
27.3
control
0.57 ( 0.03
0.58 ( 0.02
0.65 ( 0.05
10-
10
10
7
0.59 ( 0.04
1
.81
1.37
.82
astragaloside I
-
6
5
3
29.4
32.4
31.6
0.63 ( 0.02v 0.65 ( 0.01v
0.63 ( 0.04v 0.63 ( 0.02v
-
1
4
5
6
7
8
2.00
2.40
0.40
0.93
1.95
38.4
40.1
23.6
29.6
18.5
41.0
43.2
25.2
27.1
22.9
39.6
43.1
23.3
28.0
19.9
control
1
0.47 ( 0.01
0.45 ( 0.01
-
-
-
-
-
-
7
6
5
7
6
5
10
10
10
10
10
10
0.64 ( 0.03vv 0.45 ( 0.02
0.62 ( 0.03v 0.52 ( 0.01vv
0.50 ( 0.03
0.46 ( 0.02
0.53 ( 0.03
0.45 ( 0.04
0.49 ( 0.00v
0.50 ( 0.03
2
2
.25
1.70
.08
9
33.0
40.7
37.5
0.60 ( 0.03v 0.56 ( 0.03vv
0.80 ( 0.02 0.56 ( 0.03
2
control
3
-
-
7
6
5
1
1
0
1
75.1
25.1
74.3
28.6
73.6
25.4
10
10
1
0.68 ( 0.04V 0.54 ( 0.04
0.68 ( 0.05V 0.51 ( 0.06
0.71 ( 0.02V 0.57 ( 0.04
0-
a
For protocols used, see Experimental Section (n ) 3; v, V p <
.05; vv, VV p < 0.01 compared with control group).
reported recently in two independent studies on the
microbial biotransformation of alloaromadendrane-type
0
sesquiterpene substrates.8,9 Comparison of the H and
1
13
C
Compound 3 was obtained as white amorphous powder,
3
NMR data of 1a (in CD OD) with these reported data
with an elemental formula of C21
H
38
+
O
8
determined by
further confirmed the structure of the aglycon of 1. A
similar alloaromadendrane sesquiterpene with a hydroxyl
group substituted at C-13 instead of C-12 has been identi-
13
HRFABMS (m/z 441.2451, [M + Na] ). In the C NMR
spectrum, 21 carbon signals including three methyls, seven
methylenes, nine methines, and two quaternary carbons
were observed (Table 1). On enzymatic hydrolysis of 3,
1
3
fied from Wyethia arizonica by Bohlmann et al. Accord-
ingly, compound was determined structurally as
0â,12,14-trihydroxyalloaromadendrane 14-O-â-D-glucopy-
1
1
glucose was obtained as the only sugar component. The H
1
NMR spectrum of 3 revealed only one anomeric proton
ranoside. Since this is a new sesquiterpene glycoside, it has
been assigned the trival name dendroside A.
signal at δ 4.88 (1H, d, J ) 7.6 Hz). Therefore, the glucose
H
1
3
unit was assigned in the â configuration. The C NMR data
of 3 were similar to those of 2, except for the loss of a
methine group and a set of signals belonging to a glucose
moiety, along with the appearance of a hydroxyl-substi-
Compound 2 was obtained as white amorphous powder,
with an elemental formula of C27
48
H O12 determined by
+
HRFABMS (m/z 587.3017, [M + Na] ). An IR hydroxyl
-1
13
peak was observed at 3386 cm . In the C NMR spectrum
of 2, 27 carbon signals including three methyls, eight
methylenes, 15 methines, and one quaternary carbon were
observed (Table 1). Enzymatic hydrolysis of 2 yielded
C
tuted quaternary carbon signal at δ 85.7 (s). Analysis of
the 1H- H COSY and HMQC spectra of 3 led to the
1
deduction of the fragments -C-14, C-11, C-15- and -C-4,
C-3, C-2, C-1(C-10), C-9, C-8, C-7-. Further analysis of the
1
glucose and its aglycon 2a . In the H NMR spectrum of 2
HMBC spectrum of 3 permitted the aglycon to also be
(
Table 2), two anomeric proton signals were found to
overlap at δ 4.90, with both appearing as a doublet with
an identical coupling constant (J ) 7.7 Hz). In turn, in the
14,15
assigned with a picrotoxane-type skeleton.
The differ-
H
ence between the aglycons of 2 and 3 lay in the hydroxyl
substitution at C-6 in the structure of 3. The relative
configuration of the aglycon of 3 was determined according
to the NOESY spectrum, in which correlation signals were
found between H-10 and H-2; H-9 and H-1; H-13 and H-1,
H-9, and H-12; and H-1 and H-14 and H-15. The aglycon
of 3 obtained by enzymatic hydrolysis also is a new
1
3
C NMR spectrum (Table 1), two anomeric carbon signals
were observed at δ 105.3 (d) and 104.9 (d), respectively.
C
Therefore, it was concluded that the two glucose units both
exhibit a â-glycosidic linkage to the aglycon 2a . Analysis
1
1
of H- H COSY, TOCSY, and HMQC spectra of 2 led to
1
13
the deduction of the following fragments in its aglycon:
compound, and assignments of H and C NMR signals of
3a have been made on the basis of its 1H, C, H- H
13
1
1
-C-14, C-11, C-15-; -C-4, C-3, C-2, C-1(C-10), C-8, C-9-,
1
3
1
and -C-12, C-6, C-7-. Further analysis of the NMR data
of 2 revealed a picrotoxane-type sesquiterpene skeleton.14,15
The relative configuration of 2 was established according
to its NOESY spectrum, in which correlation signals were
found between H-10 and H-2; H-1 and H-11, H-13, H-14,
and H-15; and H-13 and H-9 and H-12. To the best of our
knowledge, the aglycon of 2 obtained by enzymatic hy-
COSY, HMQC, and HMBC NMR spectra. The C- H long-
range correlation signal between C-10 and Hglc-1 in the
HMBC spectrum and the correlation signal between H10
and Hglc-1 in the NOESY spectrum of 3 indicated the
linkage of the glucose unit to C-10 of the aglycon. Accord-
ingly, the structure of 3 was established as 6R,10,12-
trihydroxypicrotoxane 10-O-â-D-glucopyranoside. Com-
pound 3 is a new sesquiterpene glycoside and has been
given the trival name dendronobiloside B.
1
drolysis is a new compound, and assignments of the H and
1
3
C NMR signals of 2a have been made on the basis of its
1H, C, H- H COSY, HMQC, and HMBC NMR spectra.
The positions of attachment of the two glucose units with
the aglycon were determined to be at C-10 and C-12
according to correlation signals in the HMBC spectrum of
13
1
1
In a preliminary in vitro pharmacological evaluation
(Table 4), it was found that dendroside A (1) stimulated
significantly (p < 0.01) the proliferation of T lymphocytes
-
7
of mice at concentrations of 1 × 10 M. Dendroside A (1)
and dendronobiloside A (2) stimulated significantly (p <
0.01) the proliferation of B lymphocytes of mice at concen-
2
. Thus, the structure of 2 was assigned as 10,12-dihy-
droxypicrotoxane 10,12-di-O-â-D-glucopyranoside. Com-
pound 2 is a new sesquiterpene diglycoside and has been
given the trival name dendronobiloside A.
-
5
-6
trations of 1 × 10 and 1 × 10 M, respectively. In
contrast, dendronobiloside B (3) was found to inhibit the

Sesquiterpene Glycosides from Dendrobium
J ournal of Natural Products, 2001, Vol. 64, No. 9 1199
Com p ou n d 1a : colorless gum; [R] 24
IR νmax 3417 (br, OH), 2953, 2868, 1647, 1454, 1383, 1111,
-15.5° (c 0.2, MeOH);
proliferation of T lymphocytes of mice at concentrations of
D
-
7
-6
-5
16
1
× 10 , 1 × 10 , and 1 × 10
M (p < 0.05).
-
1
1
13
1
080, 1028 cm ; H and C NMR data of 1a in CD
consistent with those of 10â,12,14-trihydroxyalloaromaden-
drane reported in the literature:8 1H NMR (C
N, 400 MHz)
3
OD were
Astragaloside I was used as a positive control and exhibited
stimulant activity toward the proliferation of T and B
D
5
-
5
-6
5
lymphocytes at concentrations of 1 × 10 and 1 × 10
M
δ 4.15 (1H, d, J ) 11.1 Hz, H-12a), 4.05 (1H, d, J ) 11.1 Hz,
H-12b), 3.78 (1H, d, J ) 10.3 Hz, H-14a), 3.68 (1H, d, J ) 10.3
Hz, H-14b), 2.41 (1H, m, H-5), 2.40 (1H, m, H-1), 2.25 (1H, m,
H-8a), 2.08 (1H, m, H-9a), 2.00 (1H, m, H-4), 1.95 (1H, m,
H-8b), 1.82 (1H, m, H-2a), 1.81 (1H, m, H-3a), 1.79 (1H, m,
H-9a), 1.65 (1H, m, H-2b), 1.40 (3H, s, H-13), 1.37 (1H, m,
H-3b), 1.07 (3H, d, J ) 6.8 Hz, H-15), 0.90 (1H, m, H-7), 0.38
(
p < 0.05).17 Previously, polysaccharides from Dendrobium
species have been reported to stimulate the proliferation
of T and B lymphocytes of mice.18 This is the first report
of small molecules with immunomodulatory activities from
a Dendrobium species.
(
7
1H, dd, J ) 9.1, 9.1 Hz, H-6); 13C NMR (C
5
D
5
N, 100 MHz) δ
Exp er im en ta l Section
6.1 (s, C-10), 71.3 (t, C-14), 63.2 (t, C-12), 54.5 (d, C-1), 40.4
Gen er a l Exp er im en ta l P r oced u r es. The melting point
uncorrected) was determined on a Kofler apparatus. The
optical rotations were measured with a Horiba Sepa-300
polarimeter. The IR spectra were recorded using a Perkin-
(d, C-5), 38.9 (d, C-4), 33.1 (t, C-9), 29.9 (d, C-7), 29.8 (t, C-3),
25.4 (s, C-11), 24.9 (t, C-2), 24.8 (q, C-13), 24.0 (d, C-6), 19.1
(t, C-8), 17.0 (q, C-15); ESIMS (negative-ion mode) m/z 253
(
-
+
[
M - H] ; APCIMS (positive-ion mode) m/z 255 [M + H] .
20
Den d r on obilosid e A (2): white amorphous powder; [R]
D
Elmer 577 spectrometer. NMR spectra were run in C
5 5
D N,
-
1
1
62.9° (c 0.6, MeOH); IR νmax 3386 (br, OH), 2956, 2873, 1645,
DMSO-d , or CDCl on a Bruker AM-400 spectrometer with
6
3
-
1 1 13
466, 1385, 1078, 1031 cm ; H and C NMR data, see Tables
TMS as internal standard. LRFABMS measurements were
made with a Varian MAT 212 instrument, and the HRFABMS
data were obtained on a VG 7070-HF spectrometer. ESIMS
+
and 2; FABMS m/z 587 [M + Na] ; HRFABMS m/z 587.3017
+
[M + Na] (calcd for C27
H
48
O
12Na, 587.3042).
2
0
Den d r on obilosid e B (3): white amorphous powder; [R]
D
(
negative-ion mode) and APCIMS (positive-ion mode) were
-
1
63.6° (c 0.5, MeOH); IR νmax 3396 (br, OH), 2929, 2874, 1645,
measured using a Finnigan LCQ-DECA instrument. GC
analysis was performed with a Shimadzu model GC-9A instru-
ment equipped with an OV-17 column (3.1 mm i.d. × 2 m)
and a He flame ionization detector and temperature program-
ming from 170 to 210 °C at a rate of 10 °C/min; injection
temperature, 270 °C; carrier gas, He (50 mL/min). Column
chromatographic separations were carried out using silica gel
H60 (Qingdao Haiyang Chemical Group Corporation, Qingdao,
People’s Republic of China) and RP-18 (100-200 mesh, Tianjin
No. 2 Chemical Reagent Factory, Tianjin, People’s Republic
of China) as packing materials. HSGF254 silica gel TLC plates
-
1
1
13
456, 1383, 1163, 1078, 1028 cm ; H and C NMR data, see
+
Tables 1 and 2; FABMS m/z 419 [M + H] and m/z 441 [M +
+
+
Na] ; HRFABMS m/z 441.2451 [M + Na] (calcd for C21
38 8
H O -
Na, 441.2463).
En zym a tic Hyd r olysis of Den d r on obilosid es A (2) a n d
B (3). Compounds 2 (10 mg) and 3 (15 mg) were dissolved in
5 mL of H O, and â-cellulase (10 mg) was added to each
2
solution and kept at 37 °C for 7 days. The aqueous solutions
were then extracted with EtOAc, and each EtOAc extract was
chromatographed over silica gel, with CHCl
used to afford 2a (3 mg) and CHCl -MeOH (9:1) used to afford
3a (8 mg). Each residual aqueous solution was concentrated
to dryness and chromatographed over Si gel, with CHCl
O (7:3:0.5) as eluent, to give glucose (3 mg) from
3
-acetone (5:1)
3
(
Yantai Chemical Industrial Institute, Yantai, People’s Re-
public of China) were used for analytical TLC.
3
-
MeOH-H
2
P la n t Ma ter ia l. The plant material was collected in the
suburbs of Chongqing in September 1998 and identified by
Prof. Ming Zhang of Chongqing Institute of Traditional
Chinese Medicine, Chongqing, Sichuan Province, People’s
Republic of China. A voucher specimen (No. SIMMW9809) is
deposited at the herbarium of Shanghai Institute of Materia
Medica, Chinese Academy of Sciences.
both 2 and 3, which were identified by co-TLC with an
authentic standard.
Com p ou n d 2a : colorless gum; [R] 20
-22.0° (c 0.25, MeOH);
D
IR νmax 3331 (br, OH), 2955, 2872, 1674, 1464, 1385, 1082, 1032
-
1
1
cm ; H NMR (CDCl , 400 MHz) δ 3.82 (1H, m, H-12), 3.77
3
(
1H, m, H-10), 3.51 (1H, dd, J ) 8.8, 8.8 Hz, H-10), 3.40 (1H,
dd, J ) 9.5, 9.6 Hz, H-12), 1.91 (1H, m, H-8), 1.88 (1H, m,
Extr a ction a n d Isola tion . The fresh stems of Dendrobium
nobile (2.5 kg) were percolated with 95% EtOH (4000 mL ×
H-9), 1.84 (1H, m, H-7), 1.82 (1H, m, H-6), 1.80 (1H, m, H-11),
1
.78 (1H, m, H-1), 1.64 (1H, m, H-7), 1.45 (1H, m, H-3), 1.42
3
) at room temperature. The filtrate was concentrated in vacuo.
(1H, m, H-8), 1.28 (1H, m, H-2), 1.20 (1H, m, H-4), 1.12 (1H,
The residue was partitioned with water and petroleum ether,
EtOAc, and n-BuOH (1000 mL × 3), successively. The n-BuOH
extract (15.0 g) was subjected to column chromatography over
RP-18 eluted with water, and 20%, 40%, 60%, and 100%
MeOH-water in sequence to afford five fractions (F1-F5). F4
was chromatographed further over an RP-18 column with
m, H-3), 1.06 (3H, s, H-13), 0.90 (3H, d, J ) 7.0 Hz, H-14),
13
0
6
.80 (3H, d, J ) 6.6 Hz, H-15); C NMR (CDCl
3
, 100 MHz) δ
4.6 (t, C-10), 64.5 (t, C-12), 53.9 (d, C-6), 48.3 (d, C-9), 41.9
(s, C-5), 38.9 (d, C-2), 38.4 (d, C-1), 27.3 (t, C-4), 26.9 (d, C-11),
2
5.6 (t, C-8), 23.9 (q, C-13), 22.4 (t, C-7), 21.7 (q, C-14), 19.8
+
(
t, C-3), 15.4 (q, C-15); EIMS m/z 240 [M] and m/z 222 [M -
4
0%-60% MeOH in water as eluent and finally purified
+
H
2
O] .
Com p ou n d 3a : colorless gum; [R]
IR νmax 3415 (br, OH), 2955, 1655, 1456, 1381, 1159, 1068, 1047
through a Sephadex LH-20 column eluted with EtOH to afford
dendroside A (1, 40 mg, 0.0016% w/w) and dendronobilosides
A (2, 15 mg, 0.0006%) and B (3, 70 mg, 0.0028%).
20
D
-68.5° (c 0.21, MeOH);
-1
1
cm ; H NMR (C
5 5
D N, 400 MHz) δ 4.09 (1H, dd, J ) 4.7, 4.8
Den d r osid e A (1): white amorphous powder; mp 145-147
Hz, H-10), 4.02 (1H, d, J ) 10.9 Hz, H-12), 3.90 (1H, d, J )
1
m, H-9), 2.10 (1H, m, H-8), 2.08 (2H, m, H-7), 2.04 (1H, m,
H-11), 2.00 (1H, m, H-1), 1.76 (1H, m, H-8), 1.44 (1H, m, H-3),
1
4
°
C; [R]
D
-48.6° (c 0.1, MeOH); IR νmax 3396 (br, OH), 2949,
0.7 Hz, H-12), 3.65 (1H, t, J ) 9.2, 9.9 Hz, H-10), 3.11 (1H,
1
1
641, 1454, 1383, 1265, 1163, 1078, 1028, 631 cm-1; H and
1
3
C NMR data, see Table 1; LRFABMS (positive ion mode) m/z
+
+
4
17 [M + H] and 439 [M + Na] ; HRFABMS (positive-ion
1
.42 (1H, m, H-4), 1.40 (1H, m, H-2), 1.38 (3H, s, H-13), 1.22
+
mode) m/z 439.2321 [M + Na] (calcd for C21
36 8
H O Na, 439.2307).
(
1H, m, H-3), 1.20 (1H, m, H-4), 0.91 (3H, d, J ) 6.9 Hz, H-14),
1
3
En zym a tic Hyd r olysis of Den d r osid e A (1). Compound
(10 mg) and â-cellulase (10 mg; Lizhu Dongfeng Bio-Tech
5 5
0.82 (3H, d, J ) 7.0 Hz, H-15); C NMR (C D N, 100 MHz) δ
1
85.3 (s, C-6), 65.9 (t, C-12), 63.2 (t, C-10), 46.4 (s, C-5), 45.2
(d, C-9), 39.2 (d, C-2), 38.8 (d, C-1), 34.4 (t, C-7), 30.0 (t, C-4),
26.8 (d, C-11), 21.5 (q, C-14), 21.3 (t, C-8), 20.2 (t, C-3), 17.8
Co. Ltd., Shanghai, People’s Republic of China) were dissolved
in 5 mL of H O and kept at 37 °C for 3 days. The aqueous
solution was then concentrated to dryness and chromato-
graphed over silica gel, with CHCl -acetone (1:1) as eluent,
to give 1a (5 mg), and then with CHCl -MeOH-water (7:3:
.5) as eluent, to give glucose (2 mg), which was compared with
an authentic standard by co-TLC (EtOAc-MeOH-H O-
HOAc, 13:3:3:4, R 0.46).
2
+
(q, C-13), 15.3 (q, C-15); ESIMS m/z 279 [M + Na] .
3
Con figu r a tion of D-Glu cose Obta in ed on th e Hyd r oly-
sis of 1-3. A solution in pyridine (100 µL) of each hydrolyzed
sugar from 1-3 (0.04 mol/L) and L-cysteine methyl ester
hydrochloride (0.06 mol/L) was mixed and warmed at 60 °C
for 1 h. Acetic anhydride (150 µL) was then added, and each
3
0
2
f

1
200 J ournal of Natural Products, 2001, Vol. 64, No. 9
Zhao et al.
mixture warmed at 90 °C for another 1 h. After evaporation
of pyridine and acetic anhydride in vacuo, each residue was
dissolved in acetone (350 µL) and the solution (1 µL) was
and identification of the plant material. We acknowledge Dr.
K. Fagerquist, Mass Spectrometry Facility, Department of
Chemistry, University of Minnesota, Minneapolis, MN, for the
high-resolution mass spectral data. The authors are grateful
to the Network Information Center, Chinese Academy of
Sciences, for providing computational facilities.
7
subjected to GLC. A peak for a peracetylated thiazolidine
derivative with retention time at 9.39 min was observed for
all three samples, which was identical to the derivative of
authentic D-glucose prepared in the same manner.
Lym p h ocyte P r olifer a tion Test. The prepared spleen
Refer en ces a n d Notes
5
cells of mice (4 ×10 ) were seeded into each well of a 96-well
(
1) J iangsu New Medical College. Dictionary of Chinese Medicines;
Shanghai Scientific and Technologic Press: Shanghai, 1986; pp 586-
590.
microplate, and 5 µg/mL of concanavalin A (Con A, from
Canavalia ensiformis Type III, Sigma) or lipopolysaccharide
(
2) Chen, K. K.; Chen, A. L. J . Biol. Chem. 1935, 111, 653-665.
(
LPS, from Escherichia coli, Sigma) was added alone or in
combination with various concentrations of compounds 1-3.
The plates were cultured at 37 °C with 5% CO in a humidified
atmosphere for 48 h. Cell proliferation was assayed by the
(3) Suzuki, I.; Nakashima, T.; Nagasawa, N. Chem. Pharm. Bull. 1965,
1
3, 713-715.
2
(
4) Okamota, T.; Natsume, M.; Onaka, T.; Uchimaru, F.; Shimizu, M.
Chem. Pharm. Bull. 1972, 20, 418-421.
(5) Talapatra, B.; Mukhopadhyay, P.; Chaudhury, P.; Talapatra, S. K.
1
6
MTT method. Briefly, 4 h before the termination of each
culture, 20 µL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphen-
yl tetrazolium bromide) was added to each well (the final MTT
concentration was 0.5 µg/mL). At the end of the culture stage,
Indian J . Chem. Soc. 1982, 21B, 386-387.
6) Lee, Y. H.; Park, J . D.; Baek, N. I.; Kim, S. I.; Ahn, B. Z. Planta Med.
(
1995, 61, 178-180.
(7) Hara, S.; Okabe, H.; Mihashi, K. Chem. Pharm. Bull. 1987, 35, 501-
5
06.
1
00 µL of dissolving solution (10% SDS-50% N,N-dimethyl-
(
8) Miyazawa, M.; Uemura, T.; Kameoka, H. Phytochemistry 1995, 40,
formamide) was added, then the plates were incubated for a
further 6-7 h in the incubator to let the purple formazan dye
dissolve. Absorbance at a wavelength of 570 nm in each well
was determined in a multiwell spectrophotometer. Results
were the means of triplicate samples ( SD. Independent two-
tailed Student t-tests were performed. Astragaloside I was
7
93-796.
(9) Lima, D. P.; Carnell, A. J .; Roberts, S. M. J . Chem. Res., Synop. 1999,
396-397.
(
(
(
10) Becke, A. D. J . Chem. Phys. 1993, 98, 5648-5652.
11) Gaussian 98, Revision A.7; Guassian, Inc.: Pittsburgh, PA, 1998.
12) Wolinski, K.; Hilton, J . F.; Pulay, P. J . Am. Chem. Soc. 1990, 112,
8251-8260.
1
7
(13) Bohlmann, F.; Zdero, C.; King, R. M.; Robinson, H. Planta Med. 1984,
used as a positive control.
5
0, 195-196.
(
14) Fischer, N. H.; Olivier, E. J .; Fischer, H. D. Prog. Chem. Org. Nat.
Ack n ow led gm en t. W.-M.Z. thanks the Chinese Academy
of Sciences for financial support. We gratefully acknowledge
additional financial support from the National Natural Science
Foundation of the People’s Republic of China (grant 29725203)
and the “863” High Technology Program of the People’s
Republic of China (grant 863-103-04-01). We thank Prof. Ming
Zhang of Chongqing Insitute of Traditional Chinese Medicine,
Sichuan Province, People’s Republic of China, for the collection
Prod. 1979, 38, 268-273.
(15) Inubushi, Y.; Tsuda, Y.; Katarao, E. Chem. Pharm. Bull. 1966, 14,
6
68-671.
(
(
16) Niks, M.; Otto, M. J . Immunol. Methods 1990, 130, 149-151.
17) Bedir, E.; Pugh, N.; Calis, I.; Pasco, D. S.; Khan, I. A. Biol. Pharm.
Bull. 2000, 23, 834-837.
(
18) Zhao, Y. L.; Wang, S. L.; Li, X. Y. Acta Bot. Yunnan. 1994, 16,
3
92-396.
NP0102612