Inhibitors from rhubarb on lipopolysaccharide-induced nitric oxide production in macrophages: Structural requirements of stilbenes for the activity

Article abstract of DOI:10.1016/S0968-0896(01)00093-1

By bioassay-guided separation, three stilbenes (rhapontigenin, piceatannol, and resveratrol), two stilbene glucoside gallates (rhaponticin 2″-O-gallate and rhaponticin 6″-O-gallate), and a naphthalene glucoside (torachrysone 8-O-β-D-glucopyranoside) with inhibitory activity against nitric oxide (NO) production in lipopolysaccharide-activated macrophages were isolated (IC₅₀=11-69μM). The oxygen functions (-OH, -OCH₃) of stilbenes at the benzene ring were essential for the activity. The glucoside moiety reduced the activity, while the α,β-double bond had no effect. Furthermore, the active stilbenes (rhapontigenin, piceatannol, and resveratrol) did not inhibit inducible NO synthase activity, but they inhibited nuclear factor-κB activation following expression of iNOS. Copyright

Full text of DOI:10.1016/S0968-0896(01)00093-1

Bioorganic & Medicinal Chemistry 9 (2001) 1887–1893
Inhibitors from Rhubarb on Lipopolysaccharide-Induced Nitric
Oxide Production in Macrophages: Structural Requirements of
Stilbenes for the Activity
Tadashi Kageura, Hisashi Matsuda, Toshio Morikawa, Iwao Toguchida,
Shoichi Harima, Mamiko Oda and Masayuki Yoshikawa*
Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
Received 11 January 2001; accepted 24 March 2001
Abstract—By bioassay-guided separation, three stilbenes (rhapontigenin, piceatannol, and resveratrol), two stilbene glucoside gal-
lates (rhaponticin 200-O-gallate and rhaponticin 600-O-gallate), and a naphthalene glucoside (torachrysone 8-O-b-d-glucopyranoside)
with inhibitory activity against nitric oxide (NO) production in lipopolysaccharide-activated macrophages were isolated (IC50=11–
69 mM). The oxygen functions (–OH, –OCH3) of stilbenes at the benzene ring were essential for the activity. The glucoside moiety
reduced the activity, while the a,b-double bond had no effect. Furthermore, the active stilbenes (rhapontigenin, piceatannol, and
resveratrol) did not inhibit inducible NO synthase activity, but they inhibited nuclear factor-kB activation following expression of
inducible NO synthase. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
(iNOS) has been shown to be involved in pathological
processes via overproduction of NO, and is expressed in
response to pro-inflammatory agents [interleukin-1b
(IL-1b), tumor necrosis factor-a (TNF-a), and lipo-
polysaccharide (LPS), etc.] in various cell types includ-
ing macrophages, endothelial cells and smooth muscle
cells.3 Nuclear factor (NF)-kB is a major transcription
factor involved in iNOS, TNF-a, IL-1b, and IL-8 gene
expression. NF-kB is present as an inactive form due to
combination with an inhibitory subunit, IkB, which
keeps NF-kB in the cytoplasm, thereby preventing acti-
vation of the target gene in the nucleus. Cellular signals
lead to phosphorylation of IkB following elimination of
IkB from NF-kB by proteolytic degradation. Then, the
activated-NF-kB is released and translocated into the
nucleus to activate transcription of its target genes.4
Inhibition of iNOS enzyme activity or iNOS induction
and inhibition of NF-kB activation may be of
therapeutic benefit in various types of inflammation.3À5
Korean rhubarb, the rhizome of Rheum undulatum L., is
used as a remedy for the blood stagnation syndrome
(‘Oketsu syndrome’ in Japanese traditional medicine) as
well as a purgative agent. This rhubarb is considered to
have a lesser purgative effect but more potent effect on
Oketsu syndrome than other rhubarbs such as R. pal-
matum.1 Previously, anti-allergic and anti-inflammatory
effects of the hot water extract were reported to be
responsible for its anti-Oketsu effect.1 However, its
pharmacological properties and bioactive constituents
have not been studied in sufficient detail. Recently, we
reported the isolation and structure elucidation of anti-
oxidant constituents from the rhizome of R. undulatum
and elucidated the structural requirements for anti-oxidant
activity (Chart 1).2
Nitric oxide (NO) has been implicated in a number of
diverse physiological or pathological processes, such as
vasodilation, nonspecific host defense, ischemia reper-
fusion injury, and chronic or acute inflammation.
Among the NO synthase (NOS) family, inducible NOS
In the course of our characterization studies on anti-
Oketsu effect in traditional medicine, we reported vari-
ous constituents of natural medicines that act as inhibi-
tors of overproduction of NO.6 In our continuing study,
the methanolic extract from the dried rhizome of R.
undulatum was found to show inhibitory activity against
NO production in LPS-activated mouse peritoneal
*Corresponding author. Tel.:+81-75-595-4633; fax: +81-75-595-
4768; e-mail: shoyaku@mb.kyoto-phu.ac.jp
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00093-1

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T. Kageura et al. / Bioorg. Med. Chem. 9 (2001) 1887–1893
macrophages. By bioassay-guided separation, we iso-
lated 10 known stilbene constituents from the active
fraction together with a naphthalene glucoside and four
anthraquinone glucosides. This report describes the
inhibitory effects of constituents from the rhizome of R.
undulatum, and structural requirements of active con-
stituents for the activity.7 In addition, we examined the
inhibitory effects of the principal active constituents on
iNOS enzyme activity, iNOS induction and NF-kB
activation to clarify their mechanisms of action.
Results and Discussion
Isolation of NO production inhibitors from the rhizome
of R. undulatum
First, the effects of the methanolic extract on nitrite
accumulation from LPS-activated macrophages were
examined. Nitrite, an oxidative product of NO, was
accumulated in the medium after 20 h of incubation
with LPS. Nitrite concentration in the medium without
inhibitors (control group) was 28.3Æ5.7 mM, and that in
Chart 1. Chemical constituents from the rhizome of R. undulatum.

T. Kageura et al. / Bioorg. Med. Chem. 9 (2001) 1887–1893
1889
the medium without LPS (unstimulated group) was
2.9Æ2.1 mM (meanÆSD of 38 experiments). Reference
compounds, CAPE (an inhibitor of NF-kB activa-
tion),5b l-NMMA (a non-selective inhibitor of NOS),8
and GED (an inhibitor of iNOS)9 potently inhibited
nitrite accumulation in the medium. The methanolic
extract (33.8% from the natural medicine) of the dried
rhizome of R. undulatum significantly inhibited the
nitrite accumulation (Table 1).
stronger inhibitory effect on nitrite accumulation than
the H2O-eluted and acetone-eluted fractions (Table 1),
the MeOH-eluated fraction was subjected to silica gel
column chromatography (CHCl3–MeOH!CHCl3–
MeOH–H2O!MeOH) to give five fractions (fr. 1–fr. 5).
Each fraction was subjected to ODS column chromato-
graphy (MeOH–H2O) and finally HPLC (ODS,
250Â20 mm i.d., MeOH–H2O or CH3CN–H2O) to give
rhaponticin (1,10 3.5%), piceatannol 30-O-b-d-glucopyr-
anoside (2,10 2.0%), desoxyrhaponticin (3,10 0.048%), iso-
rhapontin (4,11 0.36%), rhapontigenin (5,10 0.58%),
piceatannol (6,10 0.073%), desoxyrhapontigenin (7,12
0.015%), resveratrol (8,13 0.048%), rhaponticin 200-O-
gallate (9,10 0.12%), rhaponticin 600-O-gallate (10,10
0.087%), aloe-emodin 1-O-b-d-glucopyranoside (11,2,7
The methanolic extract was subjected to Diaion HP-20
column chromatography (H2O!MeOH!acetone) to
give the H2O-eluted fraction (13.3%), MeOH-eluted
fraction (18.7%), and acetone-eluted fraction (1.8%).
Since the methanol (MeOH)-eluted fraction showed a
Table 1. Inhibitory effects of MeOH extract, H2O-eluted, MeOH-eluted, and acetone-eluted fractions from the rhizome of R. undulatum on NO
production in LPS-activated mouse peritoneal macrophagesa
Concentration
(mg/mL)
Inhibitionb
(%)
IC50
(mM)
Control
MeOH extract
H2O-eluted fraction
MeOH-eluted fraction
Acetone-eluted fraction
—
0.0Æ5.6
CAPE
l-NMMA
GED
4.0
28
1.4
100
100
100
100
22.8Æ4.7**
26.7Æ8.3**
50.6Æ4.3**
À3.5Æ4.4
aAsterisks denote significant differences from control, **p<0.01.
bValues indicate the meansÆSEM (N=4).
Table 2. Effects of stilbene constituents (1–10) from the rhizome of R. undulatum and related compounds (16–23, 1a, 2a, 5a, 6a, 8a, 16a) on NO
production in LPS-activated mouse peritoneal macrophagesa,b
a—b
R1
R2
R3
R4
IC50 (mM) (%)
Rhaponticin (1)
1a
C¼C
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
OCH3
OH
OH
OH
OH
H
H
H
OH
OH
H
OCH3
OCH3
OH
OH
OCH3
OH
OCH3
OCH3
OH
>100 (25)
>100 (8)
>100 (10)
>100 (6)
Piceatannol 30-O-Glc (2)
2a
C¼C
C–C
Desoxyrhaponticin (3)
Isorhapontin (4)
Rhapontigenin (5)
5a
Piceatannol (6)
6a
Desoxyrhapontigenin (7)
Resveratrol (8)
8a
Rhaponticin 200-O-gallate (9)
Rhaponticin 600-O-gallate (10)
trans-Stilbene (16)
16a
17
18
19
20
21
22
23
C¼C
C¼C
C¼C
C–C
>100 (13)
>100 (5)
48
49
C¼C
2
32
3
C–C
OH
C¼C
C¼C
C–C
C¼C
C¼C
C¼C
C–C
C¼C
C¼C
C¼C
C¼C
C¼C
C¼C
C¼C
OCH3
OH
OH
OCH3
OCH3
H
>30 (13)c
68
76
13
11
OH
O-Glc(2-gallate)
O-Glc(6-gallate)
H
H
OH
O-Glc(CH3)4
OCH3
OCH3
>100 (21)
>100 (26)
H
OH
H
H
OH
OCH3
OCH3
O-Glc(CH3)4
OCH3
H
63
20
27
28
22
19c
23c
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OCH3
OCH3
OH
aValues in parentheses represent the inhibition (%) at 30 or 100 mM.
bGlc: b-d-glucopyranosyl.
cCytotoxic effects were observed at 100 mM.

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T. Kageura et al. / Bioorg. Med. Chem. 9 (2001) 1887–1893
0.0065%),
chrysophanol
1-O-b-d-glucopyranoside
reference compound, l-NMMA, inhibited iNOS
(12,14 0.25%), chrysophanol 8-O-b-d-glucopyranoside
(13,14 0.16%), chrysophanol 8-O-b-d-(60-galloyl)-gluco-
pyranoside (14,2,7 0.092%), and torachrysone 8-O-b-d-
glucopyranoside (15,15 0.12%).
enzyme activity with an IC50 of 13 mM, but compounds
5, 6, and 8 showed weak inhibitory effects on iNOS
enzyme activity; their inhibitory effects at 100 mM were
9.3, 22, and 1.2%, respectively. iNOS protein was
detected at 130 kDa after a 12-h incubation with LPS on
SDS-PAGE-Western blotting analysis (Fig. 1). Three
stilbenes (5, 6, 8) concentration-dependently inhibited
iNOS induction, consistent with the above results.
As shown in Table 2, three stilbenes [rhapontigenin (5),
piceatannol (6), and resveratrol (8)], two stilbene gluco-
side gallates [rhaponticin 200-O-gallate (9) and rhaponti-
cin 600-O-gallate (10)], and a naphthalene glucoside
[torachrysone 8-O-b-d-glucopyranoside (15)] inhibited
LPS-induced NO production (IC50=11–69 mM). Other
stilbene constituents (1–4) and anthraquinones (11–14)
showed weak inhibitory effects (IC50>100 mM) (Table 3).
Finally, the effects of 5, 6, and 8 on activation of NF-kB
were analyzed by electrophoretic mobility shift assay.
The cells were incubated with or without LPS and the
test sample for 4 h, and proteins of the cell lysate were
added to reaction mixtures containing NF-kB consensus
oligonucleotide labeled with 32P-ATP. The oligonucleo-
tide–protein complex was separated electrophoretically
and visualized by autoradiography. Recent studies
demonstrated that compound 8, a bioactive polyphenol
found in grapes and wine, inhibited LPS-induced NO
generation and LPS- and TNF-a-induced activation of
NF-kB.16 Similarly, compounds 5 and 6 also inhibited
LPS-induced NF-kB activation (Fig. 2). These findings
indicated that the active constituents (5, 6, 8), at least in
part, inhibited the upstream signaling pathway of NF-
kB activation following iNOS expression, thereby
preventing NO production.
Strucutural requirements of stilbenes and anthraquinones
for NO production inhibitory activity
To clarify structure–activity relationships of stilbenes
and anthraquinones, related compounds 17, 26, and 27
were derived by enzymatic hydrolysis of 4, 11, and 12;
18–21 by CH3I-methylation of 1, 2, 5, 8; 22 and 23 by
methanolysis of 18 and 19; dihydrostilbenes (1a, 2a, 5a,
6a, 8a, 16a) by hydrogenation of 1, 2, 5, 6, 8, and 16 as
reported previously.2
Related stilbene compounds (5a, 6a, 8a, 17–23) showed
inhibitory activity (IC50=19–76 mM), although trans-
stilbene (16) and dihydrostilbene (16a) lacking the oxy-
gen functions (ÀOCH3 and ÀOH) showed little activity.
Dihydrostilbene derivatives (5a, 6a, 8a) also showed
equipotent activities as compared with their corre-
sponding stilbenes (5, 6, 8), while the glucosides (1a, 2a)
did not. Permethylated stilbene glucosides (18, 19) also
showed activity (IC50=20, 27 mM). Furthermore, two
gallates (9, 10) showed more potent activity than 1 and
gallic acid (31). With exception of desoxyrhapontigenin
(7), 22, and 23, test compounds at 100 mM did not show
cytotoxicity in MTT assay.
In conclusion, three stilbenes [rhapontigenin (5), picea-
tannol (6), and resveratrol (8)], two stilbene glucoside
gallates [rhaponticin 200-O-gallate (9) and rhaponticin
600-O-gallate (10)], and a naphthalene glucoside [tor-
achrysone 8-O-b-d-glucopyranoside (15)] with inhibi-
tory activity against NO production in LPS-activated
macrophages were isolated from the rhizome of
R. undulatum (IC50=11–68 mM). The oxygen functions
These results indicated the following structural require-
ments of stilbenes for the activity: (1) the oxygen func-
tions (–OH, –OCH3) at the benzene rings are essential
for the activity; (2) the glucoside moiety reduced the
activity but permethylation of the glucose moiety
restored the activity; (3) the a,b-double bond did not
affect the activity; and (4) the galloyl ester tended to
potentiate the activity.
Sennosides A (24) and B (25) and anthraquinones (26–
29) showed weak inhibitory effects or cytotoxic effects,
although rhein (30) showed inhibitory effect
(IC50=70 mM). Compounds 14 with a galloyl ester and
13 showed equipotent effect. This result indicated that
the galloyl ester of anthraquinone glucoside did not
potentiate the activity.
Effects of stilbenes (5, 6, 8) on iNOS enzyme activity,
iNOS induction, and NF-ꢀB activation
Figure 1. Effects of stilbenes (5, 6, 8) on LPS-induced induction of
iNOS protein in macrophages. iNOS was detected at 130 kDa after
12-h incubation with 20 mg/mL LPS by SDS-PAGE-Western blotting
analysis.
Next, the effects of three principal stilbenes [rhaponti-
genin (5), piceatannol (6), and resveratrol (8)] on iNOS
enzyme activity and iNOS induction were examined. A

T. Kageura et al. / Bioorg. Med. Chem. 9 (2001) 1887–1893
1891
(–OH, –OCH3) at the benzene ring of stilbenes were
essential for the activity, and the glucoside moiety
reduced the activity but permethylation of the glucose
moiety restored the activity, while the a,b-double bond
had no effect. Furthermore, the active stilbenes [rha-
pontigenin (5), piceatannol (6), and resveratrol (8)]
inhibited iNOS induction and activation of NF-kB, but
not iNOS enzyme activity.
Figure 2. Effects of stibenes (5, 6, 8) on LPS-induced activation of NF-kB in macrophages. The cells were incubated with or without LPS and test
sample for 4 h, and proteins of the cell lysate were added to reaction mixtures containing NF-kB consensus oligonuclotide labeled with 32P-ATP.
The oligonucleotide–protein complex was separated electrophoretically and visualized by autoradiography.
Table 3. Effects of anthraquinones (11–14), a naphthalene (15) and related compounds (24–31) on NO production in LPS-activated mouse peri-
toneal macrophagesa,b
10–100
IC50 (mM) (%)
Sennoside A (24)
Sennoside B (25)
threo
erythro
>100 (31)
>100 (37)
R1
R2
R3
R4
IC50 (mM) (%)
Aloe-emodin 1-O-Glc (11)
Chrysophanol 1-O-Glc (12)
Chrysophanol 8-O-Glc (13)
Chrysophanol 8-O-(60-galloyl)-Glc (14)
Aloe-emodin (26)
Chrysophanol (27)
Emodin (28)
Physcion (29)
Rhein (30)
Glc
Glc
H
H
H
H
H
H
H
CH2OH
CH3
CH3
H
H
H
H
H
H
H
>100 (46)
>100 (20)
>100 (31)
>100 (31)
>10 (38)c
>100 (2)
>30 (3)d
>100 (À9)
70
Glc
Glc(6-gallate)
CH3
CH2OH
CH3
CH3
CH3
COOH
H
H
H
H
H
H
OH
OCH3
H
torachrysone 8-O-Glc (15): IC50=69 mM
gallic acid (31): IC50=63 mM
aGlc: b-d-glucopyranosyl.
bValues in parentheses represent the inhibition (%) at 10, 30 or 100 mM.
cCytotoxic effect was observed at 30 mM.
dCytotoxic effect was observed at 100 mM.

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T. Kageura et al. / Bioorg. Med. Chem. 9 (2001) 1887–1893
Materials and Methods
Assay Kit) were from Promega (Madison, WI, USA);
Aquasol-2was from Packard (Meriden, CT, USA);
emodine (28), physcion (29), and rhein (30) were from
Funakoshi (Tokyo, Japan); sennosides A (24) and B
(25) and other chemicals were from Wako (Osaka,
Japan). Nitrocellulose membranes (0.25 mm) were pur-
chased from Bio Rad (Tokyo, Japan); 96-well micro-
plates and culture dishes (6 cm) were from Nunc
(Naperville, IL, USA); and spin columns (UFC30SV00)
were from Millipore (Bedford, MA, USA).
Extraction and isolation
The dried rhizome of R. undulatum (5.8 kg, cultivated in
Korea and purchased from MAE CHU Co. Ltd., Nara,
Japan), which was botanically identified by comparison
of the taxonomical features with authentic rhubarb
samples, was crushed and then extracted three times
with methanol at room temperature for 24 h. The
methanolic extract (33.8% from the natural medicine)
of the dried rhizome of R. undulatum (5.8 kg, cultivated
in Korea) was subjected to Diaion HP-20 column chro-
matography (H2O!MeOH!acetone) to give the H2O-
eluted fraction (13.3%), MeOH-eluted fraction
(18.7%), and acetone-eluted fraction (1.8%). The
active fraction (MeOH–eluted fraction) was subjected
to silica gel column chromatography [CHCl3–MeOH
(10:1, v/v)!(4:1)!CHCl3–MeOH–H2O (10:3:1, lower
layer)!MeOH] to give five fractions (fr. 1–fr. 5). Each
fraction was subjected to ODS column chromatography
(MeOH–H2O) and finally HPLC [YMC-pack R&D
ODS-5-A, 250Â20 mm i.d., MeOH–H2O or CH3CN–
H2O] to give rhaponticin (1, 3.5%), piceatannol 30-O-b-
d-glucopyranoside (2, 2.0%), desoxyrhaponticin (3,
0.048%), isorhapontin (4, 0.36%), rhapontigenin (5,
0.58%), piceatannol (6, 0.073%), desoxyrhapontigenin
(7, 0.015%), resveratrol (8, 0.048%), rhaponticin 200-O-
Screening for NO production
Peritoneal exudate cells were collected from the perito-
neal cavities of male ddY mice by washing with 6–7 mL
of ice-cold PBS, and cells (5Â105 cells/well) were sus-
pended in 200 mL of RPMI 1640 supplemented with 5%
fetal calf serum (FCS), penicillin (100 units/mL) and
streptomycin (100 mg/mL), and pre-cultured in 96-well
microplates at 37 ꢀC in 5% CO2 in air for 1 h. Non-
adherent cells were removed by washing with PBS, and
the adherent cells (more than 95% macrophages as
determined by Giemsa staining) were cultured in fresh
medium containing 10 mg/mL LPS and various con-
centrations of test compound for 20 h. NO production
in each well was assessed by measuring the accumula-
tion of nitrite (NOÀ2) in the culture medium using Griess
reagent.17 Cytotoxicity was determined by MTT colori-
metric assay. Briefly, after 20-h incubation with test
compounds, MTT (10 mL, 5 mg/mL in PBS) solution
was added to the wells. After a further 4 h in culture, the
medium was removed, and isopropanol containing
0.04 M HCl was then added to dissolve the formazan
produced in the cells. The optical density of the for-
mazan solution was measured with a microplate reader
at 570 nm (reference: 655 nm). CAPE, l-NMMA and
GED were used as reference compounds. Each test
compound was dissolved in DMSO, and the solution
was added to the medium (final DMSO concentration
was 0.5%). Inhibition (%) was calculated by the fol-
lowing formula and IC50 was determined graphically
(N=4).
gallate
(9,
0.12%),
rhaponticin
600-O-gallate
(10, 0.087%), aloe-emodin 1-O-b-d-glucopyranoside
(11, 0.0065%), chrysophanol 1-O-b-d-glucopyranoside
(12, 0.25%), chrysophanol 8-O-b-d-glucopyranoside
(13, 0.16%), chrysophanol 8-O-b-d-(60-galloyl)-gluco-
pyranoside (14, 0.092%) and torachrysone 8-O-b-d-
glucopyranoside (15, 0.12%). Related compounds 17,
26, and 27 were derived by enzymatic hydrolysis of 4, 11
and 12; 18–21 by CH3I-methylation of 1, 2, 5, 8; 22 and
23 by methanolysis of 18 and 19; dihydrostilbenes (1a,
2a, 5a, 6a, 8a, 16a) by hydrogenation of 1, 2, 5, 6, 8, and
16 as reported previously.2
Reagents
Lipopolysaccharide (LPS, from Salmonella enteritidis)
and NG-monomethyl-l-arginine (l-NMMA) were pur-
chased from Sigma (St. Louis, MO, USA); 3-(4,5-dime-
thyl-2-thiazolyl) 2,5-diphenyl tetrazolium bromide
(MTT) was from Dojin (Kumamoto, Japan); caffeic
acid phenethyl ester (CAPE) and guanidino-
ethyldisulfide (GED) were from Calbiochem (San
Diego, CA, USA); RPMI 1640 was from Life Technol-
ogies (Rockville, MD, USA); protease inhibitor cocktail
(Complete Mini) was from Boehringer Mannheim
(Germany); fetal calf serum (FCS) was from Bio
Whittaker (Walkersville, MD, USA); anti-mouse iNOS
antibody (monoclonal) was from Transduction Labora-
tories (San Diego, CA, USA); anti-mouse IgG antibody
conjugated to horseradish peroxidase and the enhanced
chemiluminescense (ECL) kit, l-[U-14C]- arginine, g-[32P]-
ATP were from Amersham Pharmacia Biotech (Bucks,
UK); thioglycolate (TGC) medium was from Nissui
Seiyaku (Tokyo, Japan); iNOS was from OXIS Inter-
national (Portland, OR, USA); NF-kB consensus oli-
gonucleotide and T4 polynucleotide kinase (Gel Shift
A À B
Inhibition ð%Þ ¼
 100 AÀC : NOÀ2 concentration ðmMÞ
A À C
½A : LPS ðþÞ; sample ðÀÞ;
B : LPS ðþÞ; sample ðþÞ;
C : LPS ðÀÞ; sample ðÀÞꢀ
Detection of iNOS
In this experiment, peritoneal exudate cells were
obtained from the peritoneal cavities of male ddY mice
that had been intraperitoneally injected with 4% TGC
medium 4 days previously to ensure a large number of
the cells. Cells (7.5Â106 cells/3 mL/dish) were pre-cul-
tured in culture dishes (6 cm i.d.) for 1 h, and the
adherent cells (more than 95% macrophages) were col-
lected as described above. After washing, the culture
medium was exchanged for fresh medium containing
5% FCS, 20 mg/mL LPS and test compound for 12h.
Cells were collected in lysis buffer [100 mM NaCl,

T. Kageura et al. / Bioorg. Med. Chem. 9 (2001) 1887–1893
1893
10 mM Tris, Complete Mini (1 tab/10 mL), 0.1% Triton
X-100, 2mM EGTA] and sonicated. After determina-
tion of protein concentration of each suspension by the
BCA method (BCATM Protein Assay Kit, Pierce), the
suspension was boiled in Laemmli buffer.18 For SDS-
PAGE, aliquots of 50 mg of protein from each sample
were subjected to electrophoresis in 10% poly-
acrylamide gels. Following electrophoresis, the proteins
were electrophoretically transferred onto nitrocellulose
membrane. The membrane were incubated with 5%
nonfat dried milk in Tris-buffered saline (TBS, 100 mM
NaCl, 10 mM Tris, 0.1% Tween 20, pH 7.4) and probed
with mouse monoclonal IgG (dilution of 1:1000) against
iNOS. The blots were washed in TBS and probed with
secondary antibody, anti-mouse IgG antibody con-
jugated to horseradish peroxidase (dilution of 1:5000).
Detection was performed using an ECL kit and X-ray
film (Hyper Film, Amersham).
counter (LS 6500, Beckman). Test compound was dis-
solved in DMSO and diluted with Tris–HCl buffer (pH
7.4) (final concentration of DMSO: 2%).
Statistical analysis
Values were expressed as meansÆSD or SEM One-way
analysis of variance following Dunnett’s test for multi-
ple comparison analysis were used for statistical analy-
sis. Probability (p) values less than 0.05 were considered
significant.
References and Notes
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6. (a) Matsuda, H.; Ninomiya, K.; Morikawa, T.; Yoshikawa,
M. Bioorg. Med. Chem. Lett. 1998, 8, 339. (b) Yoshikawa, M.;
Murakami, T.; Shimada, H.; Yoshizumi, S.; Saka, M.;
Yamahara, J.; Matsuda, H. Chem. Pharm. Bull. 1998, 46,
1008. (c) Matsuda, H.; Murakami, T.; Kageura, T.; Ninomiya,
K.; Toguchida, I.; Nishida, N.; Yoshikawa, M. Bioorg. Med.
Chem. Lett. 1998, 8, 2191. (d) Matsuda, H.; Kageura, T.;
Toguchida, I.; Murakami, T.; Kishi, A.; Yoshikawa, M.
Bioorg. Med. Chem. Lett. 1999, 9, 3081. (e) Yoshikawa, M.;
Morikawa, T.; Toguchida, I.; Harima, S.; Matsuda, H. Chem.
Pharm. Bull. 2000, 48, 651. (f) Matsuda, H.; Kageura, T.;
Toguchida, I.; Ueda, H.; Morikawa, T.; Yoshikawa, M. Life
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Electrophoretic mobility shift assay
TGC-induced peritoneal macrophages (7.5Â106 cells/
3 mL/dish) was prepared as described above. Cells were
cultured in RPMI 1640 supplemented with 5% FCS,
penicillin (100 units/mL) and streptomycin (100 mg/mL),
20 mg/mL LPS and test compound for 4 h. Cells were
collected in ice-cold PBS and resuspended in four cell
volumes of lysis buffer (420 mM NaCl, 1.5 mM MgCl2,
0.2 mM EDTA, 25% glycerol, 1% Nonidet P40, 20 mM
HEPES, pH 7.9). The cell lysate was incubated on ice
for 1 h, then centrifuged at 13,000 rpm at 4 ꢀC for 5 min.
The protein content of each supernatant was deter-
mined, and equal amounts of protein (20 mg) were added
to reaction mixtures containing 20 mg BSA and 32P-
labeled NF-kB consensus oligonucleotide. The oligonu-
cleotide–protein complex was separated by non-dena-
turing polyacrylamide gel electrophoresis (Gel Shift
Assay Kit, Promega), and autoradiography was per-
formed using an imaging analyzer (BAS 5000, Fuji
Film). 32P-Labeled NF-kB consensus oligonucleotide
was labeled using g-[32P]-ATP (3000 Ci/mmol) and T4
polynucleotide kinase.
7. This study was partly reported in our preliminary commu-
nication: Matsuda, H.; Kageura, T.; Morikawa, T.; Togu-
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iNOS Enzyme activity
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NOS activity was measured by monitoring the conversion
of l-[U-14C]-arginine to l-[U-14C]-citrulline. Briefly, test
sample solution (5 mL) and 40 mL of substrate and
coenzyme solution [100 mM arginine (containing 50 nCi
l-[U-14C]-arginine), 1 mM NADPH, 3 mM tetra-
hydrobiopterin (BH4), 1 mM flavin adenine dinucleotide
(FAD), 1 mM flavin mononucleotide (FMN) in 25 mM
Tris–HCl buffer (pH 7.4)] were pre-incubated at 37 ꢀC
for 10 min. iNOS (20 mU/5 mL) was then added to the
reaction mixture. After incubation at 37 ꢀC for 30 min,
the reaction was terminated by addition of 400 mL of
cold buffer containing 5 mM EDTA and 50 mM HEPES
(pH 5.5). The substrate was adsorbed on AG 50W X-8
ion-exchange resin (Na+ form, 60–70 mg) packed in
spin columns. The l-citrulline, which is ionically neutral
at pH 5.5, flowed through the column completely,19 and
was mixed with a scintillation cocktail (Aquasol-2) and
radioactivity was determined using a liquid scintillation
15. Tuboi, M.; Minami, M.; Nonaka, G.; Nishioka, I. Chem.
Pharm. Bull. 1977, 25, 2708.
16. (a) Tsai, S-H.; Lin-Shiau, S-Y.; Lin, J-K. Br. J. Pharma-
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Aggarwal, B. B. J. Immunol. 2000, 164, 6509. (c) Holmes-
McNary, M.; Baldwin, A. S., Jr. Cancer Res. 2000, 60, 3477.
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