Effects of stilbene constituents from rhubarb on nitric oxide production in lipopolysaccharide-activated macrophages

Article abstract of DOI:10.1016/S0960-894X(99)00702-7

Two new anthraquinone glucosides [chrysophanol 8-O-β-D-(6'-galloyl)-glucopyranoside, aloe-emodin 1-O-β-D-glucopyranoside] together with various known stilbenes and their glucosides, anthraquinone glucosides, and a naphthalene glucoside were isolated from the rhizome of Rheum undulatum L. Three stilbenes (rhapontigenin, piceatannol, resveratrol), a naphthalene glucoside (torachrysone 8-O-β-D-glucopyranoside), and two stilbene glucoside gallates (rhaponticin 2'-O-gallate, rhaponticin 6'-O-gallate) showed inhibitory activity of NO production in lipopolysaccharide-activated macrophages. (IC₅₀ = 11-69 μM). The oxygen functions (-OH,-OCH₃) at the benzene ring were found to be essential to show the activity. Whereas, the glucoside moiety reduced the activity, while the α,β-double bond did not affect the activity. Furthermore, the active stilbenes (rhapontigenin, piceatannol, resveratrol) inhibited iNOS induction. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(99)00702-7

Bioorganic & Medicinal Chemistry Letters 10 (2000) 323±327
E€ects of Stilbene Constituents from Rhubarb on Nitric Oxide
Production in Lipopolysaccharide-Activated Macrophages
Hisashi Matsuda, Tadashi Kageura, Toshio Morikawa, Iwao Toguchida,
Shoichi Harima and Masayuki Yoshikawa*
Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan
Received 28 September 1999; accepted 9 December 1999
0
AbstractÐTwo new anthraquinone glucosides [chrysophanol 8-O-b-d-(6 -galloyl)-glucopyranoside, aloe-emodin 1-O-b-d-gluco-
pyranoside] together with various known stilbenes and their glucosides, anthraquinone glucosides, and a naphthalene glucoside
were isolated from the rhizome of Rheum undulatum L. Three stilbenes (rhapontigenin, piceatannol, resveratrol), a naphthalene
0
0
00
glucoside (torachrysone 8-O-b-d-glucopyranoside), and two stilbene glucoside gallates (rhaponticin 2 -O-gallate, rhaponticin 6 -O-
gallate) showed inhibitory activity of NO production in lipopolysaccharide-activated macrophages. (IC50=11±69 mM). The oxygen
3
functions (-OH,-OCH ) at the benzene ring were found to be essential to show the activity. Whereas, the glucoside moiety reduced
the activity, while the a,b-double bond did not a€ect the activity. Furthermore, the active stilbenes (rhapontigenin, piceatannol,
resveratrol) inhibited iNOS induction. # 2000 Elsevier Science Ltd. All rights reserved.
A Korean rhubarb, the rhizome of Rheum undulatum L.,
is used as the remedy for the blood stagnation syndrome
In the course of our studies on the bioactive con-
stituents from the traditional medicine, the methanolic
extract from the dried rhizome of R. undulatum was
found to show inhibitory activity on NO production in
LPS-activated mouse peritoneal macrophages. Through
bioassay-guided separation, we isolated ten known stil-
bene constituents from the active fraction together with a
known naphthalene glucoside and two known and two
new anthraquinone glucosides. This communication
deals with the isolation and characterization of active
constituents from the rhizome of R. undulatum, and
structural requirements of active constituents for the
activity. In addition, we examined the inhibitory e€ects
of the principal active constituents on iNOS induction.
(
`Oketsu syndrome' in Japanese traditional medicine) as
well as a purgative agent. This rhubarb is considered to
have a less purgative e€ect but more potent e€ect on
the Oketsu syndrome than other rhubarbs such as R.
1
palmatum. Previously, anti-allergic and anti-in¯amma-
tory e€ects of the hot water extract were reported as its
1
anti-Oketsu e€ect. However, its pharmacological
property and bioactive constituents have not been stu-
died suciently.
Nitric oxide (NO), an inorganic free radical, has been
implicated in the physiological or pathological process,
such as vasodilation, non-speci®c host defense, ischemia
reperfusion injury, and chronic or acute in¯ammation.
NO is produced by the oxidation of l-arginine by a NO
synthase (NOS). In the family of NOS, especially indu-
cible NOS (iNOS) is involved in the pathological aspect
with overproduction of NO, and can be expressed in
response to pro-in¯ammatory agents [interleukin-1b,
tumor necrosis factor-a, lipopolysaccharide (LPS)] in
various cells including macrophages, endothelial cells,
Materials and Methods
Isolation of chemical constituents from the rhizome of R.
undulatum
The dried rhizome of R. undulatum (5.8 kg, cultivated in
Korea) was extracted with methanol at room tempera-
ture (24 h, Â3). The methanolic extract (33.8% from the
natural medicines) was subjected to Diaion HP-20 col-
2
and smooth muscle cells. Inhibition of iNOS enzyme
activity or the induction may have provided therapeutic
3
bene®ts in various types of in¯ammation.
umn chromatography (H O!MeOH!acetone) to give
2
the H O-eluated fraction (13.3%), MeOH-eluated frac-
2
tion (18.7%), and acetone-eluated fraction (1.8%). The
active fraction (MeOH eluated fraction) was subjected to
silica-gel column chromatography [CHCl :MeOH (10:1,
*Corresponding author. Tel.: +81-75-595-4633; fax: +81-75-595-4768;
e-mail: shoyaku@mb.kyoto-phu.ac.jp
3
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(99)00702-7

3
24
H. Matsuda et al. / Bioorg. Med. Chem. Lett. 10 (2000) 323±327
0
v/v)!(4:1)!CHCl :MeOH:H O (10:3:1, lower layer)!
was observed between 1 -proton and 1-carbon. Conse-
3
2
MeOH] to give ®ve fractions (fr.1±fr.5). Each fraction
was subjected to ODS column chromatography (MeOH±
H O) and ®nally HPLC [YMC-pack R&D ODS-5-A,
quently, 25 was determined to be 1-O-b-d-glucopyr-
anosyl-aloe-emodin.
2
2
50Â20 mm i.d., MeOH±H O or CH CN±H O] to give
2
3
2
Reagents
4
0
rhaponticin (1, 3.5%), piceatannol 3 -O-b-d-glucopyr-
4
4
anoside (2, 2.0%), desoxyrhaponticin (3, 0.048%), iso-
rhapontin (4, 0.36%),5 rhapontigenin (5, 0.58%),4
piceatannol (6, 0.073%),4 desoxyrhapontigenin (7,
Lipopolysaccharide (LPS, from Salmonella enteritidis),
G
N -monomethyl-l-arginine (l-NMMA), and trans-
stilbene were purchased from Sigma; 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
from Dojin, Japan; ca€eic acid phenethylester (CAPE)
and guanidinoethyldisul®de (GED) from Calbiochem;
RPMI 1640 from Gibco; protease inhibitor cocktail
(Complete Mini) from Boehringer Mannheim; fetal calf
serum (FCS) from Bio Whittaker; nitrocellulose mem-
brane (0.25 mm) from Bio Rad; 96-well microplate and
culture dish (6 cm) from Nunc; anti-mouse iNOS anti-
body (monoclonal) from Transduction Laboratories;
anti-mouse IgG antibody conjugated to horse radish
peroxidase and the enhanced chemiluminescense (ECL)
kit from Amersham; thioglycolate (TGC) medium from
Nissui Seiyaku; and all other chemicals were from Wako.
6
7
00
0.015%), resveratrol (8, 0.048%), rhaponticin 2 -O-gal-
4
00
4
8
9
late (9, 0.12%), rhaponticin 6 -O-gallate (10, 0.087%),
torachrysone 8-O-b-d-glucopyranoside (19, 0.12%),
chrysophanol 1-O-b-d-glucopyranoside (21, 0.25%),
chrysophanol 8-O-b-d-glucopyranoside (22, 0.16%), and
two new anthraquinone glucosides, chrysophanol 8-O-b-
d-(6 -galloyl)-glucopyranoside (23, 0.092%) and aloe-
emodin 1-O-b-d-glucopyranoside (25, 0.0065%). Rela-
ted compounds 12, 20 and 24 were derived by enzymatic
hydrolysis of 4, 21 and 25; 13±16 by CH I-methylation
of 1, 2, 5, 8; 17 and 18 by methanolysis of 13 and 14;
dihydrostilbenes (1a, 2a, 5a, 6a, 8a, 11a) by hydrogena-
9
0
10
10
3
1
1
tion of 1, 2, 5, 6, 8 and 11.
0
Chemical structures of chrysophanol 8-O-ꢀ-D-(6 -galloyl)-
glucopyranoside (23) and aloe-emodin 1-O-ꢀ-D-glucopyr-
anoside (25)
Bioassay methods
Screening for NO production
0
Chrysophanol 8-O-b-d-(6 -galloyl)-glucopyranoside (23),
ꢀ
25
ꢀ
yellow needles (MeOH), mp 207±210 C, [a] +95.0
Peritoneal exudate cells were collected from the peritoneal
cavities of male ddY mice by washing with 6±7 mL of ice-
d
(
c=0.1, MeOH), C H O , UV [MeOH, nm (log E)]:
28
24 13
5
2
20 (4.58), 260 (4.32), 284 (sh, 4.17), 409 (3.79), IR
�
cold PBS, and cells (5Â10 cells/well) were suspended in
1
(KBr, cm ): 3410, 2923, 1709, 1641, 1637, 1466, 1075,
+
showed quasimolecular ion peaks at m/z 591 (M+Na)
200 mL of RPMI 1640 supplemented 5% fetal calf serum
(FCS), penicillin (100 units/mL) and streptomycin (100
1
13
ꢀ
in its positive-ion FAB±MS. The H and C NMR
DMSO-d ) spectra of 23 showed signals due to a gal-
mg/mL), and pre-cultured in a 96-well microplate at 37 C
(
in 5% CO2 in air for 1 h. Nonadherent cells were
removed by washing the cells with PBS, and the adherent
cells (more than 95% macrophages using Giemza staining)
were cultured in the fresh medium containing 10 mg/mL
LPS and various concentrations of test compound for
6
0
0
00
cosylanthraquinone (22) moiety. Comparison of the H-
loyl group [d 6.99 (2H, s, galloyl-2 ,6 -H)] and the glu-
1
1
3
and C NMR data for 23 with those for 22 disclosed an
acylation shift at the 6 -position [d 4.27, 4.50 (1H each,
0
both m, Glc-6 -H )]. Furthermore, the position of gal-
0
loyl group in 23 was determined by the HMBC experi-
20 h. NO production in each well was assessed by mea-
�
13
2
suring the accumulation of nitrite (NO ) in the culture
2
0
proton and 7 -carbon. On the basis of this evidence, 23
ment, which showed long-range correlation between 6 -
0
medium using Griess reagent. Cytotoxicity was deter-
0
mined by MTT colorimetric assay. Namely, after 20-h
incubation with test compounds, MTT (10 mL, 5 mg/mL
in PBS) solution was added to the wells. After 4-h culture,
the medium was removed, and isopropanol containing
0.04 N HCl was then added to dissolve the formazan
produced in the cells. The optical density of the formazan
solution was measured by microplate reader at 570 nm
(reference: 655 nm). CAPE, l-NMMA, and GED were
used for reference compounds. Each test compound was
dissolved in DMSO, and the solution was added to the
medium (®nal DMSO concentration was 0.5%). Inhibi-
tion (%) was calculated by the following formula and
IC50 was determined graphically (N=4).
0
was characterized as 6 -galloyl-8-O-b-d-glucopyranosyl-
chrysophanol.
Aloe-emodin 1-O-b-d-glucopyranoside (25), yellow
ꢀ
24
ꢀ
needles (MeOH), mp 174±177 C, [a]d � 56.4 (c=0.1,
MeOH), C H O , UV [MeOH, nm (log E)]: 222
2
1
20 10
(
cm ): 3410, 2924, 1645, 1638, 1603, 1458, 1072, showed
4.55), 257 (4.37), 282 (sh, 4.06), 407 (3.84), IR (KBr,
1
�
+
quasimolecular ion peaks at m/z 455 (M+Na) in the
1
positive-ion FAB±MS. The H NMR (DMSO-d ) spec-
6
trum of 25 indicated the presence of aromatic protons [d
7.36 (1H, dd, J=1.2, 8.2 Hz, 7-H), 7.62 (1H, d, J=1.2
Hz, 2-H), 7.68 (1H, dd, J=1.2, 7.6 Hz, 5-H), 7.75 (1H,
dd, J=7.6, 8.2 Hz, 6-H), 7.91 (1H, d, J=1.2 Hz, 4-H)],
A � B
hydroxymethyl protons [d 4.65 (2H, s, 3-CH OH)], and
2
Inhibition ꢀ%† ˆ
 100
0
an anomeric proton [d 5.14 (1H, d, J=7.6 Hz, Glc-1 -
A � C
H)]. The carbon signals of the aglycone part in the 13
NMR spectrum of 25 were superimposable on those of
C
�
A ꢁ C : NO concentration ꢀꢀM†
2
‰
A : LPSꢀ‡†; Sampleꢀ� †; B : LPSꢀ‡†;
1
2
the aloe-emodin derivative, except for the 1-position. In
the HMBC experiment of 25, long-range correlation
Sampleꢀ‡†; C : LPSꢀ� †; Sampleꢀ� †ꢂ

H. Matsuda et al. / Bioorg. Med. Chem. Lett. 10 (2000) 323±327
325
Detection of iNOS
As shown in Table 2, three stilbenes [rhapontigenin (5),
piceatannol (6), resveratrol (8)], two stilbene glucoside
0
0
00
In this experiment, peritoneal exudate cells were obtained
from the peritoneal cavities of male ddY mice that
had been peritoneally injected with 4% TGC medium 4
days previously to ensure a large number of the cells.
gallates [rhaponticin 2 -O-gallate (9), rhaponticin 6 -O-
gallate (10)], and a naphthalene glucoside [torachrysone
8-O-b-D-glucopyranoside (19)] inhibited the LPS-
induced NO production (IC =11±69 mM). Whereas,
50
6
Cells (7.5Â10 cells/3 mL/dish) were pre-cultured in a
other stilbene constituents (1±4) and anthraquinone
constituents (20±23, 25) showed little activity (IC50
>100 mM). Related compounds (5a, 6a, 8a, 12±18) also
showed inhibitory activity (IC =5.8±76 mM), though
culture dish (6 cm i.d.) for 1 h, and the adherent cells
(
more than 95% macrophages) were collected as descri-
bed above. After washing, culture medium was exchan-
ged for fresh medium containing 5% FCS, 20 mg/ml
LPS and test compound for 12 h. Cells were collected in
the lysis bu€er [100 mM NaCl, 10 mM Tris, Complete
Mini (1 tab/10 mL), 0.1% Triton X-100, 2 mM EGTA]
and sonicated. After determination of protein con-
50
trans-stilbene (11) and dihydrostilbene (11a) lacking the
oxygen functions (-OCH and-OH) showed little activ-
3
ity. Dihydrostilbene derivatives (5a, 6a, 8a) also showed
equipotent activities as compared with their corre-
sponding stilbenes, while the glucosides (1a, 2a) did not.
Permethylated stilbene glucosides (13, 14) also showed
the activity (IC =5.8, 27 mM). Furthermore, two gal-
centration of each suspension by the BCA method
TM
(
BCA
Protein Assay Kit, Pierce), the suspension was
50
14
boiled in Laemmli bu€er. For SDS±PAGE, aliquots of
0 mg protein of each sample were subjected to electro-
lates (9, 10) showed more potent activity than 1 and 26.
Test compounds (100 mM) did not show the cytotoxicity
using MTT asssay, except for desoxyrhapontigenin (7,
100 mM), 17 (100 mM), 18 (100 mM), and aloe-emodin
(24, 30 mM).
5
phoresis in 10% polyacrylamide gel. Following electro-
phoresis, the proteins were electrically transferred onto
a nitrocellulose membrane. The membrane were incu-
bated with 5% nonfat dried milk in Tris bu€ered saline
(
7
TBS, 100 mM NaCl, 10 mM Tris, 0.1% Tween 20, pH
.4) and probed with a mouse monoclonal IgG (dilution
E€ects of stilbene constituents on iNOS induction
of 1:1000) against iNOS. The blots were washed in TBS
and probed with secondary antibody, anti-mouse IgG
antibody conjugated to horseradish peroxidase (dilution
of 1 : 5000). Detection was performed using ECL kit
and X-ray ®lm (Hyper Film, Amersham).
iNOS was detected at 130 kDa after 12 h incubation
with LPS using SDS±PAGE-Western blotting method.
Three principal stilbenes [rhapontigenin (5), piceatannol
(6), resveratrol (8)] at 100 mM apparently inhibited
iNOS induction, in consistent with above results, as
shown in Figure 1.
Results
Discussion
E€ects on NO production
The results in the present study demonstrated that the
extract of the rhizome of R. undulatum and three stil-
benes [rhapontigenin (5), piceatannol (6), resveratrol
Nitrite, a NO product, was accumulated in the medium
after 20-h incubation with LPS. Nitrite concentration in
the medium without inhibitors (control group) was
0
0
(8)], two stilbene glucoside gallates [rhaponticin 2 -O-
0
0
28.3‹5.7 mM and that in without LPS (un-stimulated
gallate (9), rhaponticin 6 -O-gallate (10)], and a naph-
thalene glucoside [torachrysone 8-O-b-d-glucopyrano-
side (19)] inhibited the LPS-induced NO production
(IC =11±69 mM). Whereas, other stilbene and anthra-
group) was 2.9‹2.1 mM (mean‹SD of 38 experiments).
Reference compounds, CAPE (an inhibitor of nuclear
1
5
factor kB activation), l-NMMA (a non-selective inhi-
50
bitor of NOS), and GED (an inhibitor of iNOS)17
potently inhibited the nitrite accumulation in the medium.
The methanolic extract and methanol-eluated fraction
showed the inhibition of nitrite accumulation more than
1
6
quinone constituents showed little activity (IC50 > 100
mM). Related compounds (5a, 6a, 8a, 12±18) also
showed inhibitory activity, except for trans-stilbene (11)
and 11a lacking oxygen functions (-OH and-OCH ) and
3
the H O-eluated fraction and acetone-eluated fraction
2
two dihydrostilbene glucosides (1a, 2a). Dihydrostilbene
derivatives (5a, 6a, 8a) having the oxygen functions
showed equipotent activities as compared with their
corresponding stilbenes. Permethylated stilbene gluco-
sides (13, 14) also show the activity. These results indi-
cate the following structural requirements of stilbenes
(
Table 1).
2
Table 1. Inhibitory e€ects of MeOH extract, H O-eluated, MeOH-
eluated, and acetone-eluated fractions from the rhizome of R. undula-
tum on NO production in LPS-activated mouse peritoneal macro-
phages
for the activity: (1) the oxygen functions (-OH,-OCH3)
Compounds
mg/mL Inhibition Compounds IC50
(%)
(mM)
MeOH extract
H O-eluated fraction
2
MeOH-eluated fraction
Acetone-eluated fraction
100
100
100
100
23
27
51
� 4
CAPE
l-NMMA
GED
4.0
28
1.4
Figure 1. E€ects of stilbenes (5, 6, 8) on iNOS induction in LPS-
activated macrophages. Un: unstimulation.

3
26
H. Matsuda et al. / Bioorg. Med. Chem. Lett. 10 (2000) 323±327
Table 2. E€ects of stilbene, naphthalene, and anthraquinone constituents from the rhizome of R. undulatum and related compounds on NO pro-
duction in LPS-activated mouse peritoneal macrophages
a±b
R1
R2
R3
R4
IC50 (mM)
Rhaponticin (1)
C=C
C� C
C=C
C� C
C=C
C=C
C=C
C� C
C=C
C=C
C=C
C=C
C� C
C=C
C=C
C=C
C� C
C=C
C=C
C=C
C=C
C=C
C=C
C=C
O-Glc
O-Glc
OH
OH
O-Glc
O-Glc
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
OH
OH
O-Glc
O-Glc
H
OCH
OH
OH
OH
OH
H
H
H
OH
OH
H
OCH
OCH
OH
3
>100 (25)
>100 (8)
>100 (10)
>100 (6)
>100 (13)
>100 (5)
48
49
23
32
>30 (13)
1
Piceatannol 3 -O-Glc (2)
a
3
0
2
Desoxythaponticin (3)
Isorhapontin (4)
a
OH
OCH
OH
3
3
Rhapontigenin (5)
OCH
OCH
OH
3
5
Piceatannol (6)
a
3
6
Desoxyrhapontigenin (7)
Resveratrol (8)
a
OH
#
OCH
OH
3
68
76
13
11
8
Rhaponticin 2 -O-gallate (9)
a
OH
O-Glc(2-gallate)
O-Glc(6-gallate)
OH
0
0
0
0
OCH
OCH
H
3
Rhaponticin 6 -O-gallate (10)
trans-Stilbene (11)
3
H
H
>100 (21)
>100 (26)
1
1
1
1
1
1
1
1
1a
2
3
4
5
6
7
8
H
H
OCH
OCH
H
OH
OH
O-Glc(CH
OH
OCH
OCH
OCH
OCH
OCH
OCH
3
3
63
5.8
27
28
22
3
)
4
3
3
3
3
3
3
OCH
OCH
OCH
OCH
OCH
OCH
3
3
3
3
3
3
OCH
OCH
OCH
OH
3
3
3
3 4
O-Glc(CH )
OCH
H
OCH
OH
3
3
#
19
23
#
OCH
3
R1
R2
R3
IC50 (mM)
Chrysophanol (20):
Chrysophanol 1-O-Glc (21):
H
Glc
H
H
H
Glc
H
H
H
H
OH
OH
H
H
>100 (2)
>100 (20)
>100 (31)
>100 (31)
>10 (38)
>100 (46)
Chrysophanol 8-O-Glc (22):
0
Glc
Glc(6-gallate)
Chrysophanol 8-O-(6 -galloyl)Glc (23):
Aloe-emodin (24):
Aloe-emodin 1-O-Glc (25):
#
#
H
H
Glc
Glc
:
b-d-Glucopyranosyl.
: b-d-Glucopyranosyl.
(
)
( )
:
, ##
Values in parentheses represent the inhi b: t Vi o an lu( e%s )i na tp a1 r0 e no tr h 1e 0s 0e smr Me p.resent the inhibtion (%) at 10 or 100 mM.
#
#, ##
:
7, 17, 18 (100 mM) and 24 (30 mM) show:e 7d , c1 y7 t, o 1t o8 x( i1c 0i t0y .m M) and 24 (30 mM) showed cytotoxicity.
at the benzene rings are essential for the activity; (2) the
glucoside moiety reduced the activity; (3) the a,b-double
bond did not a€ect the activity; (4) the galloyl ester and
permethylated glucoside tended to potentiate the activity.
stilbenes (5, 6, 8) inhibited iNOS induction. These
results suggest that active stilbenes may, at least in part,
inhibit the pathway from stimulation by LPS to iNOS
induction, thereby preventing NO production.
As to possible mechanisms of active stilbene con-
stituents, we examined the e€ects of stilbenes on iNOS
induction using three active stilbenes [rhapontigenin (5),
piceatannol (6), and resveratrol (8)], since iNOS is
responsible for the high-output production of NO by
various cells after exposure to LPS. As a result, the
References and Notes
1. Kubo, M.; Ko, S.; Hiraba, K.; Harima, S.; Matsuda, H.;
Kim, I. J. Traditional Medicines 1997, 14, 237.
2. Xia, Q.; Nathan, C. J. Leukocyte Biol. 1994, 56, 576.

H. Matsuda et al. / Bioorg. Med. Chem. Lett. 10 (2000) 323±327
327
�
3
4
. Nussler, A. K.; Billiar, T. R. J. Leukocyte Biol. 1993, 54, 171.
. Kashiwada, Y.; Nonaka, G.; Nishioka, I. Chem. Pharm.
Bull. 1984, 32, 3501.
Negative-ion FAB±MS: m/z 567 (M-H) . 25: High-resolution
+
positive-ion FAB±MS: calcd for C H O Na (M+Na) :
21
20 10
1
455.0954, found: 455.0952, H NMR (500 MHz, DMSO-d
0
6
, d)
OH), 5.14 (1H, d, J=7.6 Hz, Glc-1 -H),
5
6
2
7
. Pan, H.; Lundgren, L. N. Phytochemistry 1995, 39, 1423.
. Banks, H. J.; Cameron, D. W. Aust. J. Chem. 1971, 24,
427.
: 4.65 (2H, s, 3-CH
2
7.36 (1H, dd, J=1.2, 8.2 Hz, 7-H), 7.62 (1H, d, J=1.2 Hz, 2-
H), 7.68 (1H, dd, J=1.2, 7.6 Hz, 5-H), 7.75 (1H, dd, J=7.6,
8.2 Hz, 6-H), 7.91 (1H, d, J=1.2 Hz, 4-H), 12.90 (1H, br s, 8-
, dc) : 158.3 (C-1), 119.2
(C-2), 151.8 (C-3), 118.0 (C-4), 118.3 (C-5), 136.1 (C-6), 124.2
(C-7), 161.3 (C-8), 187.6 (C-9), 182.1 (C-10), 134.5 (C-4a),
. Nakajima, K.; Taguchi, H.; Endo, T.; Yoshioka, I. Chem.
Pharm. Bull. 1978, 26, 3050.
. Tuboi, M.; Minami, M.; Nonaka, G.; Nishioka, I. Chem.
Pharm. Bull. 1977, 25, 2708.
OH), 13C NMR (125 MHz, DMSO-d
6
8
9
1
1
. Okabe, H.; Matsuo, K.; Nishioka, I. Chem. Pharm. Bull.
973, 21, 1254.
0. The structures of new compounds, 23 and 25, were eluci-
2
116.7 (C-8a), 119.0 (C-9a), 132.4 (C-10a), 62.1 (3-CH OH),
0
0
0
0
100.5 (Glc-1 ), 73.2 (Glc-2 ), 76.5 (Glc-3 ), 69.3 (Glc-4 ), 77.2
0
0
(Glc-5 ), 60.4 (Glc-6 ), Nagative-ion FAB±MS : m/z 431 (M-
�
dated on the basis of chemical and physiochemical evidence.
The full characteristics will be presented in our full paper.
23: High-resolution positive-ion FAB±MS: calcd for
+
C H O Na (M+Na) : 591.1114, found: 591.1168,
NMR (500 MHz, DMSO-d
4
H) . Known compounds were identi®ed by comparison of
their physical data with those of authentic samples (1, 6, 8, 20,
24) or with reported value.6
±9
1
H
), 4.27,
11. The details will be presented in our full paper.
12. Ko, S. K.; Whang, W. K.; Kim, I. H. Arch. Pharm. Res.
1995, 18, 282.
13. Ding, A. H.; Nathan, D. F.; Stuehr, D. J. J. Immunol.
1988, 141, 2407.
14. Khalkhai-Ellis, Z. Prep. Biochem. 1995, 25, 1.
15. Natarajan, K.; Singh, S.; Burke, Jr., T. R.; Grunberger, D.;
Aggarwal, B. B. Proc. Natl Acad. Sci. USA 1996, 20, 9090.
16. Rees, D. D.; Palmer, R. M.; Schulz, R.; Hodson, H. F.;
Moncada, S. Br. J. Pharmacol. 1990, 101, 746.
17. Szabo, C.; Bryk, R.; Zingarelli, B.; Southern, G. J.; Gah-
man, T. C.; Bhat, V.; Salzman, A. L.; Wol€, D. J. Br. J.
Pharmacol. 1996, 118, 1659.
2
8
24 13
6
, d): 2.40 (3H, s, 3-CH
3
0
0
.50 (1H each, both m, Glc-6 -H
2
), 5.24 (1H, d, J=7.7, Glc-1 -
0
0
00
H), 6.99 (2H, s, galloyl-2 ,6 -H), 7.16 (1H, br s, 4-H), 7.46
1H, br s, 2-H), 7.67 (1H, d, J=8.6 Hz, 5-H), 7.72 (1H, dd,
J=7.6, 8.6 Hz, 6-H), 7.82 (1H, d, J=7.6 Hz, 7-H), 12.82 (1H,
(
1
3
br s, 1-OH), C NMR (125 MHz, DMSO-d
6
, dc): 161.5 (C-1),
19.3 (C-2), 147.5 (C-3), 123.9 (C-4), 122.2 (C-5), 138.5 (C-6),
20.5 (C-7), 157.8 (C-8), 187.4 (C-9), 181.9 (C-10), 132.0 (C-
a), 119.3 (C-8a), 114.6 (C-9a), 134.7 (C-10a), 21.4 (3-CH ),
00.2 (Glc-1 ), 73.2 (Glc-2 ), 76.3 (Glc-3 ), 69.7 (Glc-4 ), 74.0
1
1
4
1
3
0
0
0
0
0 0 00 00 00
(
Glc-5 ), 63.3 (Glc-6 ), 119.2 (galloyl-1 ), 108.6 (galloyl-2 , 6 ),
00 00 00 00
45.5 (galloyl-3 , 5 ), 135.6 (galloyl-4 ), 165.1 (galloyl-7 ),
1