Structures of new cyclic diarylheptanoids and inhibitors of nitric oxide production from Japanese folk medicine Acer nikoense

Article abstract of DOI:10.1021/np020351m

Three new cyclic diarylheptanoids, acerosides B₁ and B₂ and aceroketoside, were isolated together with 20 known compounds from a Japanese folk medicine, the stem bark of Acer nikoense. The absolute stereostructures of the new compounds were determined on the basis of chemical and physicochemical evidence. In addition, the principal diarylheptanoid constituents were found to exhibit inhibitory activity on nitric oxide production in lipopolysaccharide-activated macrophages.

Full text of DOI:10.1021/np020351m

86
J . Nat. Prod. 2003, 66, 86-91
Str u ctu r es of New Cyclic Dia r ylh ep ta n oid s a n d In h ibitor s of Nitr ic Oxid e
P r od u ction fr om J a p a n ese F olk Med icin e Acer n ikoen se1
Toshio Morikawa, J ing Tao, Iwao Toguchida, Hisashi Matsuda, and Masayuki Yoshikawa*
Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, J apan
Received J uly 31, 2002
Three new cyclic diarylheptanoids, acerosides B1 and B2 and aceroketoside, were isolated together with
20 known compounds from a J apanese folk medicine, the stem bark of Acer nikoense. The absolute
stereostructures of the new compounds were determined on the basis of chemical and physicochemical
evidence. In addition, the principal diarylheptanoid constituents were found to exhibit inhibitory activity
on nitric oxide production in lipopolysaccharide-activated macrophages.
The stem bark of the Aceraceae plant Acer nikoense
Maxim., which is indigenous to J apan, has been used as a
folk medicine for hepatic disorders and eye diseases. As
the chemical constituents of this plant, diarylheptanoids
and phenolic compounds were characterized from the stem
bark,2 while tannin, coumarinolignans, flavonoids, sterols,
and triterpenes were identified from the leaves and wood.3
The methanolic extract of the stem bark was reported to
show protective effects on hepatic injury induced by carbon
tetrachloride in rats. Through bioassay-guided separation,
(+)-rhododendrol was isolated as the active principal
compound.4
As a part of our studies on bioactive constituents of
natural medicines, we previously reported that the biphe-
nyl-type diarylheptanoids from Myrica rubra Seib. et Zucc.
showed inhibitory activities on the release of â-hexosamini-
dase from RBL-2H3 cells5 and on nitric oxide (NO) produc-
tion in lipopolysaccharide (LPS)-activated macrophages,6
as well as the absolute stereostructures of six new biphenyl-
type diarylheptanoids. As a continuation of the character-
ization studies on bioactive diarylheptanoids, we have
isolated three new cyclic diarylheptanoids named aceros-
ides B1 (1) and B2 (2) and aceroketoside (3) from the stem
bark of A. nikoense. This paper deals with the isolation and
structure elucidation of three new cyclic diarylheptanoids
(1-3). Furthermore, we describe the inhibitory effects of
the principal diarylheptanoids on NO production in LPS-
activated mouse peritoneal macrophages.
acid18 (0.0003%), 3,4,5-trimethoxybenzyl alcohol18 (0.0011%),
2-hydroxy-5-methoxybenzaldehyde18 (0.0002%), 4-hydroxy-
2,6-dimethoxybenzoic acid18 (0.0002%), and 2,4′-dihydroxy-
5-carboxydiphenyl ether19 (0.0006%).
Aceroside B1 (1) was isolated as a white powder with
24
negative optical rotation ([R]D -94.0°). The positive-ion
FABMS of 1 showed a quasimolecular ion peak at m/z 483
[M + Na]+, while a quasimolecular ion peak was observed
at m/z 459 [M - H]- and a fragment ion peak at m/z 297
[M - C6H11O5]- in the negative-ion FABMS. The molecular
formula C25H32O8 of 1 was determined from the quasimo-
lecular ion peak [M + Na]+ and by HRFABMS measure-
ment. The IR spectrum of 1 showed absorption bands at
3400, 1590, 1508, 1458, and 1074 cm-1 assignable to
hydroxyl, aromatic ring, and ether functions, whereas in
the UV spectrum, it showed an absorption maximum at
272 nm (log ꢀ 3.24), which was suggestive of a diphenyl
ether-type diarylheptanoid structure.20 On acid hydrolysis
with 1 M hydrochloric acid (HCl), 1 liberated D-glucose,
which was identified by HPLC analysis using an optical
rotation detector.21 Enzymatic hydrolysis of 1 with â-
glucosidase in acetate buffer (pH 4.4) yielded (-)-(R)-
acerogenin B (1a ), which was obtained as a colorless fine
24
crystal with negative optical rotation ([R]D -95.0°). The
spectral data of 1a were in agreement with those of racemic
acerogenin B (5).9 The absolute configuration at the 9-
position in 1a was determined by an application of the
modified Mosher’s method.22 Namely, diazomethane me-
thylation of 1a furnished the 2-O-methyl derivative (1b),
which was derivated to the 9-(R)- and 9-(S)-2-methoxy-2-
trifluorophenylacetate (MTPA ester, 1c and 1d ). As shown
in Figure 1, the signals due to the protons attached to the
10, 11, 12, 13, 15, and 19-carbons in the 9-(S)-MTPA ester
(1d ) were observed at lower fields compared with those of
the 9-(R)-MTPA ester (1c) [∆δ: positive], while signals due
to the protons of the 4, 6, 7, and 8-carbons in 1d were
observed at higher fields compared with those of 1c [∆δ:
negative]. Consequently, the absolute stereostructure at
the 9-position of 1a has been determined to be the R
configuration.
Resu lts a n d Discu ssion
The methanolic extract from the stem bark of A. nikoense
collected in Miyagi Prefecture, J apan, was partitioned into
an ethyl acetate (EtOAc)-water mixture to furnish the
EtOAc-soluble portion and aqueous phase. The aqueous
phase was further extracted with n-butanol (n-BuOH) to
give the n-BuOH-soluble and H2O-soluble portions. The
EtOAc-soluble and n-BuOH-soluble portions were subjected
to normal-phase and reversed-phase silica gel column
chromatography and repeated HPLC to give 1 (0.0003%,
from the dried material), 2 (0.0003%), and 3 (0.0011%)
together with acerogenins A7,8 (4, 0.0078%), B9 (5, 0.0035%),
E10 (6, 0.0005%), and K11 (7, 0.0012%), (-)-centrolobol8 (8,
0.0025%), (+)-rhododendrol2 (0.019%), epirhododendrin12
(0.0052%), 4-(4-hydroxyphenyl)-2-butanone13 (0.0030%),
(+)-catechin14 (0.0032%), (-)-epicatechin14 (0.0008%), clem-
iscosin D15 (0.0009%), aquillochin15 (0.0050%), scopoletin16
(0.0070%), fraxetin17 (0.0002%), fraxin17 (0.0021%), vanillic
The 1H NMR (pyridine-d5) and 13C NMR (Table 1)
spectra23 of 1 showed signals assignable to a (-)-(R)-
acerogenin B moiety [δ 0.96, 1.48 (1H each, both m, H2-
10), 1.18, 1.48 (1H each, both m, H2-11), 1.48, 1.68 (1H each,
both m, H2-12), 1.66, 1.73 (1H each, both m, H2-8), 2.48,
2.64 (1H each, both m, H2-13), 2.69, 2.99 (1H each, both
m, H2-7), 3.35 (1H, m, H-9), 5.95 (1H, d, J ) 2.1 Hz, H-6),
6.74 (1H, dd, J ) 2.1, 8.3 Hz, H-4), 6.91, 7.20 (1H each,
both dd, J ) 2.1, 8.3 Hz, H-16, 18), 7.11, 7.26 (1H each,
* To whom correspondence should be addressed. Tel: +81-75-595-4633.
Fax: +81-75-595-4768. E-mail: shoyaku@mb.kyoto-phu.ac.jp.
10.1021/np020351m CCC: $25.00
© 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/17/2002

Cyclic Diarylheptanoids from Acer nikoense
J ournal of Natural Products, 2003, Vol. 66, No. 1 87
Ch a r t 1
F igu r e 1.
both dd, J ) 2.1, 8.3 Hz, H-15, 19), 7.54 (1H, d, J ) 8.3
Hz, H-3)] and a â-D-glucopyranosyl part [δ 5.82 (1H, d, J
) 7.3 Hz, H-1′)]. Comparison of the carbon signals in the
13C NMR spectrum for 1 with those for 1a disclosed a
glycosilation shift around the 2-position of 1. Furthermore,
the position of the â-D-glucopyranosyl part in 1 was
confirmed by the heteronuclear multiple bond connectivity
(HMBC) experiment of 1, which showed a long-range
correlation between the 1′-proton and 2-carbon. On the
basis of those findings, the structure of 1 including the
absolute configuration was characterized to be as shown.
Aceroside B2 (2) was also obtained as a white powder
with positive optical rotation ([R]D24 +24.5°). The molecular
formula C25H32O8, which was the same as that of 1, was
determined from the positive-ion and negative-ion FABMS
and by HRFABMS analysis. That is, a quasimolecular ion
peak was observed at m/z 483 [M + Na]+ in the positive-
ion FABMS, while the negative-ion FABMS showed a
quasimolecular ion peak and a fragment ion peak at m/z
459 [M - H]- and 297 [M - C6H11O5]-. The IR spectrum
of 2 showed absorption bands at 3400, 1590, 1508, 1458,
and 1074 cm-1 assignable to hydroxyl, aromatic ring, and
ether functions, whereas in the UV spectrum, it showed
an absorption maximum at 273 nm (log ꢀ 3.24), which was
suggestive of a diphenyl ether-type diarylheptanoid struc-
ture.20 The acid hydrolysis of 2 liberated D-glucose,21 and
the enzymatic hydrolysis of 2 furnished (+)-(S)-acerogenin
24
B (2a ) with positive optical rotation ([R]D +84.1°), which
was identified by comparison of the spectral data with those
of racemic acerogenin B (5).9 To confirm the absolute
configuration at the 9-position, the modified Mosher’s
method was also applied to both of the 9-MTPA esters (2c
and 2d ), which were prepared using the 2-methyl ether
(2b) by the same procedure as that of 1. As shown in Figure
1, the proton signals due to the 10, 11, 12, 13, 15, and 19-
positions in the 9-(S)-MTPA ester (2d ) appeared at a higher
field than those of the 9-(R)-MTPA ester (2c) [∆δ: nega-
tive], while the proton signals ascribable to the 4, 6, 7, and

88 J ournal of Natural Products, 2003, Vol. 66, No. 1
Morikawa et al.
Ta ble 1. 13C NMR Data for Acerosides B1 (1) and B2 (2),
(-)-R-Acerogenin B (1a ), (+)-S-Acerogenin B (2a ), and
Aceroketoside (3)
1a
2a
1a b
2a b
3a
C-1
152.4
145.2
117.0
122.6
136.5
116.8
28.9
152.3
145.2
117.0
122.5
136.7
116.7
29.0
149.1
142.8
115.1
122.5
133.7
114.7
30.4
149.2
142.8
115.1
122.5
134.1
114.7
30.4
150.7
145.4
117.3
122.7
132.6
116.9
32.3
C-2
C-3
C-4
C-5
C-6
C-7
C-8
37.2
37.3
36.3
36.4
28.3
C-9
70.7
70.7
71.8
71.8
25.1
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-1′
C-2′
C-3′
C-4′
C-5′
C-6′
C-1′′
C-2′′
C-3′′
C-4′′
C-5′′
39.7
39.7
38.8
38.8
36.9
23.2
23.1
22.4
22.4
79.3
30.9
30.9
28.3
28.4
39.5
35.3
35.3
35.2
35.3
32.3
F igu r e 2.
139.7
*132.2c
**123.8
156.4
**123.6
*131.1
102.6
74.9
139.7
*132.1
**123.8
156.4
**123.6
*131.1
102.7
75.0
139.7
*131.8
**123.5
155.3
**123.6
*130.7
139.7
*131.8
**123.2
155.3
**123.3
*130.7
140.0
*130.4
**123.5
156.8
**124.3
*132.5
105.0
78.4
Hz, H-2′), 4.76 (1H, d, J ) 4.9 Hz, H-4′), 4.85 (1H, d, J )
7.8 Hz, H-1′)], and an apiofuranosyl part [δ 5.77 (1H, d, J
) 2.4 Hz, H-1′′)]. In the HMBC experiment on 3, long-range
correlations were observed between the following protons
and carbons: H-11 and C-1′; H-1′ and C-11,3′; H-2′ and
C-3′; H-4′ and C-3′; H-5′ and C-3′; H2-6′ and C-1′′; H-1′′ and
78.6
78.6
207.1
74.0
76.6
68.5
71.4
71.3
1
C-6′ (Figure 2). Comparison of the H NMR and 13C NMR
78.9
78.9
data for 3 with those for aceroside III suggested that 3 was
the 3′-keto derivative of aceroside III. Finally, reduction
of 3 with sodium borohydride (NaBH4) in methanol yielded
a mixture of aceroside III and its 3′-epimer, which was
subjected to acid hydrolysis with 1 M HCl to give D-glucose,
D-allose, and D-apiose. On the basis of this evidence, the
structure of aceroketoside (3) was elucidated as shown.
The inorganic free radical NO has been implicated in
physiological and pathological processes such as vasodila-
tion, nonspecific host defense, ischemia reperfusion injury,
and chronic or acute inflammation.24 NO is produced by
the oxidation of L-arginine catalyzed by NO synthase
(NOS). In the NOS family, inducible NOS in particular is
involved in pathological overproduction of NO and can be
expressed in response to pro-inflammatory agents such as
interleukin-1â, tumor necrosis factor-R, and LPS in various
cell types including macrophages, endothelial cells, and
smooth muscle cells.
As part of our studies to characterize the bioactive
components of natural medicines, we have reported several
NO production inhibitors, i.e., higher unsaturated fatty
acids,25 polyacetylenes,26,27 coumarins,26 flavonoids,27 stil-
benes,28 lignans,29 sesquiterpenes,30-32 diterpenes,33 trit-
erpenes,6,34 and biphenyl-type diarylheptanoids.6 As a
continuatiion of these studies, we examined the effects of
the five constituents from the stem bark of A. nikoense on
NO production from LPS-activated macrophages, and the
results are summarized in Table 2.
Five diarylheptanoid constituents were examined and
found to show inhibitory activity (IC50 ) 24-88 µM; Table
2) without cytotoxic effects in the MTT assay (data not
shown). Among them, the inhibitory activities of biphenyl-
type diarylheptanoids, acerogenins E (6, IC50 ) 24 µM) and
K (7, IC50 ) 25 µM), were equivalent to that of NG-
monomethyl-L-arginine (L-NMMA), a nonselective NOS
inhibitor (IC50 ) 28 µM). Comparison of the inhibitory
activity for acerogenins A (4, IC50 ) 74 µM) and B (5, IC50
) 88 µM) and (-)-centrolobol (8, IC50 ) 73 µM) with those
of 6 and 7 suggested that biphenyl-type diarylheptanoids
had stronger NO production inhibitory activity than diphe-
nyl ether-type and straight chain-type diarylheptanoids.
62.5
62.5
111.0
77.6
80.3
75.1
65.3
a
Measured in pyridine-d5 at 125 MHz. bMeasured in CDCl3
at 125 MHz. c *,** May be interchangeable within the same
column.
8-positions of 2d were observed at lower fields compared
with those of 2c [∆δ: positive]. This evidence suggests the
absolute stereostructure at the 9-position in 2a to be the
S configuration. The H NMR (pyridine-d5) and 13C NMR
(Table 1) spectra of 2 showed the presence of a (+)-(S)-
acerogenin B moiety and a â-D-glucopyranosyl part [δ 5.85
(1H, d, J ) 7.1 Hz, H-1′)]. Comparison of the 13C NMR data
for 2 with those for 2a suggested a glycosidation shift at
the 2-position, and the HMBC experiment on 2 showed a
long-range correlation between the 1′-proton and 2-carbon.
Consequently, the absolute stereostructure of aceroside B2
(2) was determined to be the 9-isomer of 1.
1
Aceroketoside (3), which was obtained as a white powder
with negative optical rotation ([R]D -82.2°), showed
24
absorption bands at 3403, 1736, 1590, 1502, 1460, and 1041
cm-1 ascribable to hydroxyl, carbonyl, aromatic ring, and
ether functions. The UV spectrum of 3 exhibited an
absorption maximum at 278 nm (log ꢀ 3.41). The positive-
ion FABMS of 3 showed a quasimolecular ion peak at m/z
613 [M + Na]+, while a quasimolecular ion peak was
observed at m/z 589 [M - H]- together with a fragment
ion peak at m/z 297 [M - C11H17O9]- in the negative-ion
FABMS, and HRFABMS analysis revealed the molecular
formula of 3 to be C30H38O12. Enzymatic hydrolysis of 3
with naringinase provided acerogenin A (4).7,8 The 1H NMR
(pyridine-d5) and 13C NMR (Table 1) spectra23 of 3 showed
signals assignable to an acerogenin A moiety [δ 0.90, 1.08
(1H each, both m, H2-9), 1.13, 1.50 (1H each, both m, H2-
10), 1.20, 1.38 (1H each, both m, H2-8), 1.55, 2.34 (1H each,
both m, H2-12), 2.41, 2.49 (1H each, both m, H2-7), 2.90,
3.08 (1H each, both m, H2-13), 3.57 (1H, m, H-9), 5.94 (1H,
d, J ) 2.0 Hz, H-6), 6.71 (1H, dd, J ) 2.0, 8.1 Hz, H-4),
7.22 (1H, d, J ) 8.1 Hz, H-3), 7.23 (2H, dd, J ) 2.0, 8.1 Hz,
H-16, 18), 7.25, 7.69 (1H each, both dd, J ) 2.0, 8.1 Hz,
H-15, 19)], a 3-ketohexopyranosyl part [δ 4.06 (1H, m, H-5′),
4.27, 4.73 (1H each, both m, H2-6′), 4.70 (1H, d, J ) 7.5
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. The following in-
struments were used to obtain physical data: specific rota-

Cyclic Diarylheptanoids from Acer nikoense
J ournal of Natural Products, 2003, Vol. 66, No. 1 89
Ta ble 2. Inhibitory Effects of Constituents from A. nikoense on NO Production in LPS-Activated Mouse Peritoneal Macrophagesa
inhibition (%)
0 µM
1 µM
3 µM
10 µM
30 µM
100 µM
IC50 (µM)
acerogenin A (4)
acerogenin B (5)
acerogenin E (6)
acerogenin K (7)
(-)-centrolobol (8)
L-NMMA
0.0 ( 0.9
0.0 ( 1.4
0.0 ( 2.1
0.0 ( 3.8
0.0 ( 1.9
0.0 ( 1.1
6.7 ( 0.7
1.9 ( 1.1
0.1 ( 3.8
6.6 ( 3.6
-1.3 ( 2.6
4.4 ( 2.0
5.8 ( 2.6
-0.5 ( 3.3
3.3 ( 2.5
2.1 ( 4.4
1.3 ( 3.0
2.0 ( 1.6
11.6 ( 2.1**,b
22.2 ( 1.9**
17.6 ( 1.3**
59.4 ( 1.2**
56.6 ( 2.9**
10.7 ( 1.4**
52.3 ( 1.5**
61.4 ( 0.9**
54.1 ( 2.1**
81.2 ( 1.8**
91.3 ( 0.9**
67.2 ( 0.7**
79.2 ( 0.9**
74
88
24
25
73
28
-0.2 ( 2.1
13.6 ( 3.1**
24.3 ( 3.7**
-1.1 ( 2.3
17.7 ( 2.8**
a
b
Each value represents the mean ( SEM (N ) 4). Significantly different from the control, **p < 0.01.
tions, Horiba SEPA-300 digital polarimeter (l ) 5 cm); UV
spectra, Shimadzu UV-1600 spectrometer; IR spectra, Shi-
madzu FTIR-8100 spectrometer; EIMS and HREIMS, J EOL
J MS-GCMATE mass spectrometer; FABMS and HRFABMS,
J EOL J MS-SX 102A mass spectrometer; 1H NMR spectra,
J EOL LNM-500 (500 MHz) spectrometer; 13C NMR spectra,
J EOL LNM-500 (125 MHz) spectrometer with tetramethylsi-
lane as an internal standard; HPLC, Shimadzu RID-6A
refractive index detector.
The following experimental conditions were used for chro-
matography: normal-phase silica gel column chromatography,
silica gel BW-200 (Fuji Silysia Chemical, Ltd., 150-350 mesh);
reversed-phase silica gel column chromatography, Chroma-
torex ODS DM1020T (Fuji Silysia Chemical, Ltd., 100-200
mesh); TLC, precoated TLC plates with silica gel 60F254
(Merck, 0.25 mm) (normal-phase) and silica gel RP-18 F254S
(Merck, 0.25 mm) (reversed-phase); reversed-phase HPTLC,
precoated TLC plates with silica gel RP-18 WF254S (Merck, 0.25
mm); detection was achieved by spraying with 1% Ce(SO4)2-
10% aqueous H2SO4 followed by heating.
g), 3-4 (1.06 g), 3-5 (3.06 g), 3-6 (1.95 g), 3-7 (0.87 g), 3-8 (5.28
g)]. Fraction 3-3 (0.72 g) was further purified by HPLC
[MeOH-H2O (30:70, v/v)] to give epirhododendrin (265 mg,
0.0052%), (+)-catechin (163 mg, 0.0032%), (-)-epicatechin (42
mg, 0.0008%), and fraxin (109 mg, 0.0021%). Fraction 3-6 (1.95
g) was purified by HPLC [(1) MeOH-H2O (60:40, v/v), (2) CH3-
CN-H2O (25:75, v/v)] to give acerosides B1 (1, 11 mg, 0.0003%)
and B2 (2, 12 mg, 0.0003%) and aceroketoside (3, 61 mg,
0.0011%).
The EtOAc-soluble fraction (40 g) was subjected to normal-
phase silica gel column chromatography [1.2 kg, n-hexane-
EtOAc (1:1)-MeOH] to give three fractions [Fr. 1 (2.7 g), Fr.
2 (3.1 g), Fr. 3 (34.2 g)]. Fraction 2 (3.1 g) was separated by
reversed-phase silica gel column chromatography [90 g, MeOH-
H2O (30:70-70:30, v/v)-MeOH] to give five fractions [Fr. 2-1
(0.47 g), 2-2 (0.28 g), 2-3 (0.76 g), 2-4 (0.67 g), 2-5 (0.92 g)].
Fraction 2-2 (0.28 g) was further purified by HPLC [MeOH-
H2O (40:60, v/v)] to give fraxetin (9 mg, 0.0002%), 3,4,5-tri-
methoxybenzyl alcohol (55 mg, 0.0011%), 2-hydroxy-5-methoxy-
benzaldehyde (9 mg, 0.0002%), and 4-hydroxy-2,6-dimethoxy-
benzoic acid (11 mg, 0.0002%). Fraction 2-3 (0.76 g) was sub-
jected to HPLC [MeOH-H2O (40:60 or 45:55, v/v)] to give (+)-
rhododendrol (424 mg, 0.0087%), 4-(4-hydroxyphenyl)-2-butan-
one (40 mg, 0.0008%), scopoletin (167 mg, 0.0035%), and 2,4′-
dihydroxy-5-carboxydiphenyl ether (28 mg, 0.0006%). Fraction
2-4 (0.67 g) was purified by HPLC [MeOH-H2O (65:35, v/v)]
to furnish 4 (248 mg, 0.0056%), 5 (113 mg, 0.0025%), 6 (13
mg, 0.0003%), 7 (35 mg, 0.0008%), and 8 (84 mg, 0.0019%).
P la n t Ma ter ia l. The stem bark of Acer nikoense was
collected in Miyagi Prefecture, J apan, and identified by one
of the authors (M.Y.). A voucher of the plant is on file in our
laboratory.
E xt r a ct ion a n d Isola t ion . The dried stem bark of A.
nikoense (8.5 kg) was finely cut and extracted three times with
methanol (MeOH) under reflux for 3 h. Evaporation of the
solvent under reduced pressure provided the methanolic
extract (232 g, 2.7%). The methanolic extract (196 g) was
partitioned in an EtOAc-H2O (1:1, v/v) mixture. The aqueous
layer was extracted with n-BuOH, and removal of the solvent
in vacuo from the EtOAc-, n-BuOH-, and H2O-soluble portions
yielded 60 g (0.8%), 103 g (1.4%), and 34 g (0.5%) of the residue,
respectively.
The known compounds were identified by comparison of
their physical data ([R]D, IR, 1H NMR, 13C NMR, MS) with
reported values,2,7-13,15-17,19 authentic samples,14 or commercial
samples.18
24
Acer osid e B1 (1): a white powder; [R]D -94.0° (c 0.40,
EtOH); UV (EtOH) λmax (log ꢀ) 272 (3.24); IR (KBr) νmax 3400,
2928, 1590, 1508, 1458, 1074 cm-1; 1H NMR (pyridine-d5, 500
MHz) δ 0.96, 1.48 (1H each, both m, H2-10), 1.18, 1.48 (1H
each, both m, H2-11), 1.48, 1.68 (1H each, both m, H2-12), 1.66,
1.73 (1H each, both m, H2-8), 2.48, 2.64 (1H each, both m, H2-
13), 2.69, 2.99 (1H each, both m, H2-7), 3.35 (1H, m, H-9), 5.82
(1H, d, J ) 7.3 Hz, H-1′), 5.95 (1H, d, J ) 2.1 Hz, H-6), 6.74
(1H, dd, J ) 2.1, 8.3 Hz, H-4), 6.91, 7.20 (1H each, both dd, J
) 2.1, 8.3 Hz, H-16, 18), 7.11, 7.26 (1H each, both dd, J ) 2.1,
8.3 Hz, H-15, 19), 7.54 (1H, d, J ) 8.3 Hz, H-3); 13C NMR data,
see Table 1; positive-ion FABMS m/z 483 [M + Na]+; negative-
ion FABMS m/z 459 [M - H]-, 297 [M - C6H11O5]-; HR-
FABMS m/z 483.1986 (calcd for C25H32O8Na [M + Na]+,
483.1995).
Normal-phase silica gel column chromatography [BW-200
(Fuji Silysia Co., Ltd., 1.6 kg), CHCl3-MeOH (20:1-10:1, v/v)-
CHCl3-MeOH-H2O (7:3:1, lower layer-6:4:1, v/v/v)-MeOH]
of the n-BuOH-soluble portion (80 g) gave six fractions [Fr. 1
(2.6 g), 2 (13.3 g), 3 (17.0 g), 4 (30.4 g), 5 (10.2 g), 6 (6.5 g)].
Fraction 2 (13.3 g) was separated by reversed-phase silica gel
column chromatography [Chromatorex ODS DM1020T (Fuji
Silysia Co., Ltd., 300 g), MeOH-H2O (50:50-60:40-70:30,
v/v)-MeOH] to furnish seven fractions [Fr. 2-1 (0.20 g), 2-2
(0.57 g), 2-3 (2.80 g), 2-4 (1.00 g), 2-5 (0.70 g), 2-6 (0.50 g), 2-7
(7.53 g)]. Fraction 2-1 (0.20 g) was further separated by HPLC
[YMC-Pack ODS-A (YMC Co., Ltd., Kyoto, J apan, 250 × 20
mm i.d.), MeOH-H2O (30:70, v/v)] to give vanillic acid (4 mg,
0.0003%). Fraction 2-2 (0.57 g) was separated by HPLC
[MeOH-H2O (45:55, v/v)] to give (+)-rhododendrol (57 mg,
0.0099%), 4-(4-hydroxyphenyl)-2-butanone (12 mg, 0.0022%),
and scopoletin (20 mg, 0.0035%). Fraction 2-3 (2.80 g) was
separated by HPLC [MeOH-H2O (35:65, v/v)] to give cleo-
moiscosin D (28 mg, 0.0009%) and aquillochin (292 mg,
0.0050%). Fraction 2-5 (0.61 g) was purified by HPLC [MeOH-
H2O (35:65, v/v)] to give acerogenins A (4, 122 mg, 0.0022%)
and B (5, 58 mg, 0.0010%) and (-)-centrolobol (8, 34 mg,
0.0006%). Fraction 2-6 (0.40 g) was subjected to HPLC
[MeOH-H2O (35:65, v/v)] to furnish acerogenins E (6, 10 mg,
0.0002%) and K (7, 26 mg, 0.0004%). Fraction 3 (15.0 g) was
subjected to reversed-phase silica gel column chromatography
[450 g, MeOH-H2O (30:70-50:50-70:30, v/v)-MeOH] to
furnish eight fractions [Fr. 3-1 (1.68 g), 3-2 (0.38 g), 3-3 (0.72
24
Acer osid e B2 (2): white powder; [R]D +24.5° (c 0.50,
EtOH); UV (EtOH) λmax (log ꢀ) 273 (3.24); IR (KBr) νmax 3400,
2926, 1590, 1508, 1458, 1074 cm-1; 1H NMR (pyridine-d5, 500
MHz) δ 0.98, 1.47 (1H each, both m, H2-10), 1.18, 1.45 (1H
each, both m, H2-11), 1.51, 1.65 (1H each, both m, H2-12), 1.67,
1.75 (1H each, both m, H2-8), 2.50, 2.63 (1H each, both m, H2-
13), 2.68, 3.00 (1H each, both m, H2-7), 3.37 (1H, m, H-9), 5.85
(1H, d, J ) 7.1 Hz, H-1′), 5.95 (1H, d, J ) 2.1 Hz, H-6), 6.73
(1H, dd, J ) 2.1, 8.3 Hz, H-4), 6.91, 7.21 (1H each, both dd, J
) 2.1, 8.3 Hz, H-16, 18), 7.11, 7.26 (1H each, both dd, J ) 2.1,
8.3 Hz, H-15, 19), 7.54 (1H, d, J ) 8.3 Hz, H-3); 13C NMR data,
see Table 1; positive-ion FABMS m/z 483 [M + Na]+; negative-
ion FABMS m/z 459 [M - H]-, 297 [M - C6H11O5]-; HR-
FABMS m/z 483.2002 (calcd for C25H32O8Na [M + Na]+,
483.1995).

90 J ournal of Natural Products, 2003, Vol. 66, No. 1
Morikawa et al.
24
Acer ok etosid e (3): white powder; [R]D -82.2° (c 0.20,
chromatography [0.5 g, n-hexane-EtOAc (5:1, v/v)] to give 1b
(3.0 mg, 96%) or 2b (3.7 mg, quant.).
EtOH); UV (EtOH) λmax (log ꢀ) 278 (3.41); IR (KBr) νmax 3403,
2932, 1736, 1590, 1502, 1460, 1041 cm-1; 1H NMR (pyridine-
d5, 500 MHz) δ 0.90, 1.08 (1H each, both m, H2-9), 1.13, 1.50
(1H each, both m, H2-10), 1.20, 1.38 (1H each, both m, H2-8),
1.55, 2.34 (1H each, both m, H2-12), 2.41, 2.49 (1H each, both
m, H2-7), 2.90, 3.08 (1H each, both m, H2-13), 3.57 (1H, m,
H-9), 4.06 (1H, m, H-5′), 4.27, 4.73 (1H each, both m, H2-6′),
4.70 (1H, d, J ) 7.5 Hz, H-2′), 4.76 (1H, d, J ) 4.9 Hz, H-4′),
4.85 (1H, d, J ) 7.8 Hz, H-1′), 5.77 (1H, d, J ) 2.4 Hz, H-1′′),
5.94 (1H, d, J ) 2.0 Hz, H-6), 6.71 (1H, dd, J ) 2.0, 8.1 Hz,
H-4), 7.22 (1H, d, J ) 8.1 Hz, H-3), 7.23 (2H, dd, J ) 2.0, 8.1
Hz, H-16, 18), 7.25, 7.69 (1H each, both dd, J ) 2.0, 8.1 Hz,
H-15, 19); 13C NMR data, see Table 1; positive-ion FABMS m/z
613 [M + Na]+; negative-ion FABMS m/z 589 [M - H]-, 297
[M - C11H17O9]-; HRFABMS m/z 613.2269 (calcd for C30H38O12-
Na [M + Na]+, 613.2261).
Acid Hyd r olysis of Acer osid es B1 (1) a n d B2 (2). A
solution of 1 or 2 (5 mg each) in 1 M HCl (0.5 mL) was heated
under reflux for 2 h. After cooling, the reaction mixture was
poured into ice-water and neutralized with Amberlite IRA-
400 (OH- form), and the resin was removed by filtration. Then,
the filtrate was extracted with EtOAc. The aqueous layer was
subjected to HPLC analysis under the following conditions:
HPLC column, Shodex Asahipak NH-2P-50-4E, 4.6 mm i.d. ×
250 mm (Showa Denko Co., Ltd., Tokyo, J apan); detection,
optical rotation [Shodex OR-2 (Showa Denko Co., Ltd., Tokyo,
J apan)]; mobile phase, CH3CN-H2O (75:25, v/v); flow rate 0.8
mL/min; column temperature, room temperature. Identifica-
tion of D-glucose present in the aqueous layer was carried out
by comparison of its retention time and optical rotation with
that of an authentic sample. tR: 11.1 min (D-glucose, positive
optical rotation).
1b: a white powder; 1H NMR (CDCl3, 500 MHz) δ 0.85, 1.22
(1H each, both m, H2-10), 1.00, 1.28 (1H each, both m, H2-11),
1.49 (2H, m, H2-8), 1.55, 1.74 (1H each, both m, H2-12), 2.58,
2.64 (1H each, both m, H2-7), 2.64, 2.78 (1H each, both m, H2-
13), 3.13 (1H, m, H-9), 3.95 (3H, s, OCH3), 5.57 (1H, d, J ) 2.2
Hz, H-6), 6.67 (1H, dd, J ) 2.2, 8.4 Hz, H-4), 6.82 (1H, d, J )
8.4 Hz, H-3), 6.97, 7.13 (1H each, both dd, J ) 2.4, 8.2 Hz,
H-16, 18), 7.22, 7.28 (1H each, both dd, J ) 2.1, 8.2 Hz, H-15,
19); EIMS m/z 312 [M+] (68), 294 [M+ - H2O] (100); HREIMS
m/z 312.1708 (calcd for C20H24O3, 312.1725).
2b: a white powder; 1H NMR (CDCl3, 500 MHz) δ 0.85, 1.22
(1H each, both m, H2-10), 1.05, 1.28 (1H each, both m, H2-11),
1.49 (2H, m, H2-8), 1.56, 1.74 (1H each, both m, H2-12), 2.58,
2.64 (1H each, both m, H2-7), 2.64, 2.80 (1H each, both m, H2-
13), 3.13 (1H, m, H-9), 3.95 (3H, s, OCH3), 5.57 (1H, d, J ) 2.2
Hz, H-6), 6.67 (1H, dd, J ) 2.2, 8.1 Hz, H-4), 6.82 (1H, d, J )
8.1 Hz, H-3), 6.97, 7.13 (1H each, both dd, J ) 2.5, 8.1 Hz,
H-16, 18), 7.23, 7.28 (1H each, both dd, J ) 2.2, 8.1 Hz, H-15,
19); EIMS m/z 312 [M+] (100), 294 [M+ - H2O] (39); HREIMS
m/z 312.1717 (calcd for C20H24O3, 312.1725).
P r ep a r a tion of th e (R)-MTP A Ester s (1c, 2c) a n d (S)-
MTP A Ester s (1d , 2d ) fr om 1b a n d 2b. A solution of 1b
(1.0 mg) or 2b (1.0 mg) in CH2Cl2 (1.0 mL) was treated with
(R)-2-methoxy-2-trifluoromethylphenylacetic acid [(R)-MTPA,
7.5 mg] in the presence of 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC‚HCl, 6.2 mg) and 4-(dimethylamino)-
pyridine (4-DMAP, 2.0 mg), and the mixture was stirred under
reflux for 6 h. After cooling, the reaction mixture was poured
into ice-water, and the whole was extracted with AcOEt. The
AcOEt extract was successively washed with 5% aqueous HCl,
saturated aqueous NaHCO3, and brine, then dried over MgSO4
powder and filtrated. Removal of the solvent from the filtrate
under reduced pressure furnished a residue, which was
purified by silica gel column chromatography [0.5 g, n-hexane-
EtOAc (5:1, v/v)] to give 1c (1.4 mg, 83%) or 2c (1.3 mg, 77%),
respectively. Using a similar procedure, (S)-MTPA esters [1d
(1.5 mg, 89%) or 2d (1.0 mg, 59%)] were obtained from 1b (1.0
mg) and 2b (1.0 mg) using (S)-MTPA (7.5 mg), EDC‚HCl (6.2
mg), and 4-DMAP (2.0 mg).
En zym a tic Hyd r olysis of Acer osid es B1 (1) a n d B2 (2).
A solution of 1 (6.0 mg) or 2 (7.0 mg) in 0.2 M acetate buffer
(pH 4.4, 2.0 mL) was treated with â-glucosidase (10 mg from
almond, Oriental Yeast Co., Ltd., Tokyo, J apan), and the
mixture was stirred at 38 °C for 4 h. After the addition of EtOH
to the reaction mixture, the solvent was removed under
reduced pressure. The crude product was purified by silica gel
column chromatography [0.5 g, n-hexane-EtOAc (1:1, v/v)] to
give (-)-(R)-acerogenin B (1a , 3.5 mg, 90%) or (+)-(S)-acero-
genin B (2a , 3.9 mg, 86%).
1
1c: H NMR (CDCl3, 500 MHz) δ 0.93, 1.40 (1H each, both
m, H2-10), 1.03, 1.39 (1H each, both m, H2-11), 1.63, 1.73 (1H
each, both m, H2-8), 1.45, 1.72 (1H each, both m, H2-12), 2.48,
2.51 (1H each, both m, H2-7), 2.63, 2.73 (1H each, both m, H2-
13), 3.50, 3.96 (3H each, both s, OCH3), 4.64 (1H, m, H-9), 5.52
(1H, d, J ) 2.1 Hz, H-6), 6.62 (1H, dd, J ) 2.1, 8.3 Hz, H-4),
6.80 (1H, d, J ) 8.3 Hz, H-3), 7.05, 7.13 (1H each, both dd, J
) 2.5, 8.3 Hz, H-16, 18), 7.23, 7.29 (1H each, both dd, J ) 2.2,
8.3 Hz, H-15, 19), 7.39-7.49 (5H, m, Ph-H).
(-)-(R)-Acer ogen in B (1a ): colorless fine crystals; mp
182.0-184.5 °C (from MeOH); [R]D -95.0° (c 0.10, EtOH);
22
UV (EtOH) λmax (log ꢀ) 276 (3.32); IR (KBr) νmax 3460, 2951,
1592, 1507, 1456 cm-1; 1H NMR (CDCl3, 500 MHz) δ 0.79, 1.23
(1H each, both m, H2-10), 1.00, 1.30 (1H each, both m, H2-11),
1.50 (2H, m, H2-8), 1.53, 1.76 (1H each, both m, H2-12), 2.57,
2.63 (1H each, both m, H2-7), 2.63, 2.80 (1H each, both m, H2-
13), 3.07 (1H, m, H-9), 5.57 (1H, d, J ) 1.8 Hz, H-6), 6.63 (1H,
dd, J ) 1.8, 8.2 Hz, H-4), 6.84 (1H, d, J ) 8.2 Hz, H-3), 6.93,
7.13 (1H each, both dd, J ) 2.5, 8.2 Hz, H-16, 18), 7.23, 7.30
(1H each, both dd, J ) 2.1, 8.2 Hz, H-15, 19); 13C NMR data,
see Table 1; EIMS m/z 298 [M+] (100), 280 [M+ - H2O] (75);
HREIMS: m/z 298.1561 (calcd for C19H22O3, 298.1569).
(+)-(S)-Acer ogen in B (2a ): colorless fine crystals; mp
1d : 1H NMR (CDCl3, 500 MHz) δ 1.03, 1.46 (1H each, both
m, H2-10), 1.12, 1.45 (1H each, both m, H2-11), 1.60, 1.62 (1H
each, both m, H2-8), 1.48, 1.74 (1H each, both m, H2-12), 2.34,
2.40 (1H each, both m, H2-7), 2.66, 2.76 (1H each, both m, H2-
13), 3.50, 3.96 (3H each, both s, OCH3), 4.64 (1H, m, H-9), 5.49
(1H, d, J ) 2.1 Hz, H-6), 6.58 (1H, dd, J ) 2.1, 8.3 Hz, H-4),
6.80 (1H, d, J ) 8.3 Hz, H-3), 7.06, 7.13 (1H each, both dd, J
) 2.5, 8.3 Hz, H-16, 18), 7.26, 7.30 (1H each, both dd, J ) 2.2,
8.3 Hz, H-15, 19), 7.39-7.49 (5H, m, Ph-H).
22
181.3-183.4 °C (from MeOH); [R]D +84.1° (c 0.20, EtOH);
UV (EtOH) λmax (log ꢀ) 276 (3.34); IR (KBr) νmax 3400, 2951,
1590, 1508, 1456 cm-1; 1H NMR (CDCl3, 500 MHz) δ 0.80, 1.22
(1H each, both m, H2-10), 1.00, 1.30 (1H each, both m, H2-11),
1.48 (2H, m, H2-8), 1.50, 1.76 (1H each, both m, H2-12), 2.57,
2.62 (1H each, both m, H2-7), 2.63, 2.80 (1H each, both m, H2-
13), 3.07 (1H, m, H-9), 5.57 (1H, d, J ) 2.0 Hz, H-6), 6.63 (1H,
dd, J ) 2.0, 8.2 Hz, H-4), 6.84 (1H, d, J ) 8.2 Hz, H-3), 6.93,
7.14 (1H each, both dd, J ) 2.5, 8.3 Hz, H-16, 18), 7.25, 7.30
(1H each, both dd, J ) 2.2, 8.3 Hz, H-15, 19); 13C NMR data,
see Table 1; EIMS m/z 298 [M+] (100), 280 [M+ - H2O] (70);
HREIMS m/z 298.1564 (calcd for C19H22O3, 298.1569).
1
2c: H NMR (CDCl3, 500 MHz) δ 1.02, 1.45 (1H each, both
m, H2-10), 1.08, 1.43 (1H each, both m, H2-11), 1.60, 1.63(1H
each, both m, H2-8), 1.52, 1.75 (1H each, both m, H2-12), 2.33,
2.42 (1H each, both m, H2-7), 2.66, 2.76 (1H each, both m, H2-
13), 3.50, 3.96 (3H each, both s, OCH3), 4.64 (1H, m, H-9), 5.49
(1H, d, J ) 2.1 Hz, H-6), 6.58 (1H, dd, J ) 2.1, 8.3 Hz, H-4),
6.81 (1H, d, J ) 8.3 Hz, H-3), 7.06, 7.13 (1H each, both dd, J
) 2.3, 8.3 Hz, H-16, 18), 7.24, 7.30 (1H each, both dd, J ) 2.3,
8.3 Hz, H-15, 19), 7.39-7.49 (5H, m, Ph-H).
2d : 1H NMR (CDCl3, 500 MHz) δ 0.93, 1.38 (1H each, both
m, H2-10), 1.03, 1.37 (1H each, both m, H2-11), 1.45, 1.72 (1H
each, both m, H2-12), 1.62, 1.72 (1H each, both m, H2-8), 2.48,
2.51 (1H each, both m, H2-7), 2.63, 2.72 (1H each, both m, H2-
13), 3.50, 3.96 (3H each, both s, OCH3), 4.64 (1H, m, H-9), 5.51
(1H, d, J ) 2.1 Hz, H-6), 6.62 (1H, dd, J ) 2.1, 8.3 Hz, H-4),
6.81 (1H, d, J ) 8.3 Hz, H-3), 7.06, 7.13 (1H each, both dd, J
Dia zom eth a n e Meth yla tion of 1a a n d 2a . A solution of
1a (3.0 mg) or 2a (3.5 mg) in MeOH (2.0 mL) was treated with
ethereal diazomethane (CH2N2‚Et2O) until the yellow color
persisted. The reaction solution was stirred at room temper-
ature for 2 h. Removal of the solvent under reduced pressure
furnished a residue, which was purified by silica gel column

Cyclic Diarylheptanoids from Acer nikoense
J ournal of Natural Products, 2003, Vol. 66, No. 1 91
Refer en ces a n d Notes
) 2.3, 8.3 Hz, H-16, 18), 7.23, 7.29 (1H each, both dd, J ) 2.1,
8.3 Hz, H-15, 19), 7.39-7.49 (5H, m, Ph-H).
(1) This paper is number 30 in our series “Medicinal Foodstuffs”. For
paper number 29, see: Morikawa, T.; Matsuda, H.; Ninomiya, K.;
Yoshikawa, M. Biol. Pharm. Bull. 2002, 25, 627-631.
(2) Inoue, T.; Ishidate, Y.; Fujita M.; Kubo, M.; Fukushima, M.; Nagai,
M. Yakugaku Zasshi 1978, 98, 41-46.
Na BH4 Red u ction a n d Acid Hyd r olysis of Acer ok eto-
sid e (3). A solution of 3 (5.0 mg) in MeOH (1.0 mL) was treated
with NaBH4 (1.0 mg), and the mixture was stirred at room
temperature for 1 h. The reaction mixture was quenched with
acetone, and then removal of the solvent under reduced
pressure yielded a reduction mixture. Aceroside III35 was
detected in the reduction mixture by TLC analysis [solvent:
CHCl3-MeOH-H2O (10:3:1, lower layer, v/v/v)]. Then, a solu-
tion of the reduction mixture in 1 M HCl (0.5 mL) was heated
under reflux for 2 h. After cooling, the reaction mixture was
poured into ice-water and neutralized with Amberlite IRA-
400 (OH- form), and the residue was removed by filtration.
Then the reaction mixture was extracted with EtOAc. The
aqueous layer of the above mixture was subjected to HPLC
analysis under the following conditions: HPLC column, Sho-
dex Asahipak NH-2P-50-4E, 4.6 mm i.d. × 250 mm; detection,
optical rotation; mobile phase, CH3CN-H2O (75:25, v/v); flow
rate 0.8 mL/min; column temperature, room temperature.
Identification of D-apiose,36 D-allose, and D-glucose present in
the aqueous layer was carried out by comparison of their
retention time and optical rotation with those of authentic
samples. tR: 6.4 min (D-apiose, positive optical rotation), 9.7
min (D-allose, positive optical rotation), and 11.1 min (D-
glucose, positive optical rotation).
(3) Furukawa, N.; Nagumo, S.; Inoue, T.; Nagai, M. Shoyakugaku Zasshi
1988, 42, 163-165.
(4) Shinoda, M.; Ohta, S.; Kumasaka, M.; Fujita, M.; Nagai, M.; Inoue,
T. Shoyakugaku Zasshi 1986, 40, 177-181.
(5) Matsuda, H.; Morikawa, T.; Tao, J .; Ueda, K.; Yoshikawa, M. Chem.
Pharm. Bull. 2002, 50, 208-215.
(6) Tao, J .; Morikawa, M.; Toguchida, I.; Ando, S.; Matsuda, H.;
Yoshikawa, M. Bioorg. Med. Chem., in press.
(7) Nagai, M.; Kubo, M.; Fujita, M.; Inoue, T.; Matsuo, M. Chem. Pharm.
Bull. 1978, 26, 2805-2810.
(8) Nagai, M.; Kenmochi, N.; Fujita, M.; Furukawa, N.; Inoue, T. Chem.
Pharm. Bull. 1986, 34, 1056-1060.
(9) Kubo, M.; Inoue, T.; Nagai, M. Chem. Pharm. Bull. 1980, 28, 1300-
1303.
(10) Nagumo, S.; Kaji, N.; Inoue, T.; Nagai, M. Chem. Pharm. Bull. 1993,
41, 1255-1257.
(11) Nagumo, S.; Ishizawa, S.; Nagai, M.; Inoue, T. Chem. Pharm. Bull.
1996, 44, 1086-1089.
(12) Kubo, M.; Nagai, M.; Inoue, T. Chem. Pharm. Bull. 1983, 31, 1917-
1922.
(13) Marco, J . A.; Barbera, O.; Rodriguez, S.; Domingo, C.; Adell, J .
Phytochemistry 1988, 27, 3155-3159.
(14) (a) (+)-Catechin and (-)-epicatechin were identified by comparison
of their physical data ([R]D, IR, 1H NMR, 13C NMR, MS) with
authentic samples. (b) Yoshikawa, H.; Morikawa, T.; Fujiwara, E.;
Ohgushi, T.; Asao, Y.; Matsuda, H. Heterocycles 2001, 55, 1653-1657.
(15) Tanaka, H.; Ishihara, M.; Ichino, K.; Ito, K. Chem. Pharm. Bull. 1988,
36, 3833-3837.
En zym a tic Hyd r olysis of Acer ok etosid e (3). A solution
of 3 (8.0 mg) in 0.2 M acetate buffer (pH 4.8, 2.0 mL) was
treated with naringinase (10 mg from Penicillium decumbens,
Sigma Co., Ltd., St. Louis, MO), and the mixture was stirred
at 38 °C for 4 day. After the addition of EtOH to the reaction
mixture, the solvent was removed under reduced pressure. The
crude product was purified by silica gel column chromatogra-
phy [0.5 g, CHCl3-MeOH-H2O (20:3:1, lower layer, v/v/v)] to
give acerogenin A (4, 1.7 mg, 42%).10
(16) Barbera, O.; Marco, J . A.; Sanz, J . F.; Sanchez-Parareda, J . Phy-
tochemistry 1986, 25, 2357-2360.
(17) Tsukamoto, H.; Hisada, S.; Nishibe, S. Chem. Pharm. Bull. 1985, 33,
4069-4073.
(18) The known compounds were identified by comparison of their physical
data ([R]D, IR, 1H NMR, 13C NMR, MS) with commercial samples.
(19) Coutts, I. G. C.; Hamblin, M. R.; Welsby, S. E. J . Chem. Soc., Perkin
Trans. 1 1981, 493-497.
(20) Inoue, T. Yakugaku Zasshi 1993, 113, 181-197.
(21) (a) Matsuda, H.; Nishida, N.; Yoshikawa, M. Chem. Pharm. Bull.
2002, 50, 429-431. (b) Yokosuka, A.; Mimoki, Y.; Sashida, Y. J . Nat.
Prod. 2000, 63, 1239-1243.
NO P r od u ct ion fr om Ma cr op h a ges St im u la t ed b y
LP S. Peritoneal exudate cells were collected from the perito-
neal cavities of male ddY mice by washing with 6-7 mL of
ice-cold phosphate-buffered saline (PBS), and cells (5 × 105
cells/well) were suspended in 200 µL of RPMI 1640 supple-
mented with 5% fetal calf serum, penicillin (100 units/mL),
and streptomycin (100 µg/mL) and pre-cultured in 96-well
microplates 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 as determined
by Giemsa staining) were cultured in fresh medium containing
10 µg/mL LPS and test compound (10, 30, and 100 µM) for 20
h. NO production in each well was assessed by measuring the
accumulation of nitrite in the culture medium using Griess
reagent.
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(23) The 1H and 13C NMR spectra of 1-3 were assigned with the aid of
homo- and hetero-correlation spectroscopy (1H-1H, 13C-1H COSY),
distortionless enhancement by polarization transfer (DEPT), and
heteronuclear multiple bond connectivity (HMBC) experiments.
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Cytotoxicity was determined by 3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric as-
say. Briefly, after 20 h incubation with test compounds, MTT
(10 µL, 5 mg/mL in PBS) solution was added to the wells. After
4 h culture, the medium was removed, and 2-propanol con-
taining 0.04 M HCl was then added to dissolve the formazan
produced in the cells. The optical density of the formazan
solution was measured with a microplate reader at 570 nm
(reference, 655 nm). NG-Monomethyl-L-arginine (L-NMMA)
was used as a reference compound. Each test compound was
dissolved in dimethyl sulfoxide (DMSO), and the solution was
added to the medium (final DMSO concentration was 0.5%).
Inhibition (%) was calculated by the following formula, and
the IC50 was determined graphically (N ) 4):
A - B
A - C
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Yoshikawa, M. Bioorg. Med. Chem. Lett. 1999, 9, 3081-3086.
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Pharmaceutical Society of J apan, Chiba, J apan, March 2002, Vol. 2,
p 125.
inhibition (%) )
× 100
-
A - C: NO2 concentration (µM) [A: LPS (+), sample (-); B:
LPS (+), sample (+); C: LPS (-), sample (-)].
(36) Authentic D-apiose was obtained by acid hydrolysis of 1,2,3,5-di-O-
isopropylidene-R-D-apiose (Funakoshi Co., Ltd., Tokyo, J apan).
Sta tistics. Values are expressed as means ( SEM. One-
way analysis of variance followed by Dunnett’s test was used
for statistical analysis.
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