Resveratrol oligomers from Vatica albiramis

Article abstract of DOI:10.1021/np1002675

Five new stilbenoids, vatalbinosides A-E (1-5), and 13 known compounds (6-18) were isolated from the stem of Vatica albiramis. The effects of these new compounds on interleukin-1β-induced production of matrix metalloproteinase-1 (MMP-1) in human dermal fibroblasts were examined. Three resveratrol tetramers, (-)-hopeaphenol (6), vaticanol C (13), and stenophyllol C (14), were identified as strong inhibitors of MMP-1 production.

Full text of DOI:10.1021/np1002675

J. Nat. Prod. 2010, 73, 1499–1506
1499
Resveratrol Oligomers from Vatica albiramis
Naohito Abe,† Tetsuro Ito,† Kenji Ohguchi,‡ Minori Nasu,† Yuichi Masuda,† Masayoshi Oyama,† Yoshinori Nozawa,‡,§
Masafumi Ito,‡ and Munekazu Iinuma*,†
Laboratory of Pharmacognosy, Gifu Pharmaceutical UniVersity, 1-25-4 Daigaku-nishi, Gifu Gifu 501-1196, Japan, Gifu International Institute
of Biotechnology, 1 Naka-Fudogaoka, Kakamigahara, Gifu 504-0838, Japan, and Tokai Gakuin UniVersity, 5-68 Naka-Kirinocho,
Kakamigahara, Gifu 504-8511, Japan
ReceiVed April 23, 2010
Five new stilbenoids, vatalbinosides A-E (1-5), and 13 known compounds (6-18) were isolated from the stem of
Vatica albiramis. The effects of these new compounds on interleukin-1ꢀ-induced production of matrix metalloproteinase-1
(MMP-1) in human dermal fibroblasts were examined. Three resveratrol tetramers, (-)-hopeaphenol (6), vaticanol C
(13), and stenophyllol C (14), were identified as strong inhibitors of MMP-1 production.
The genus Vatica belongs to the family Dipterocarpaceae with
about 65 species distributed throughout Southeast Asia, especially
in Indonesia and Malaysia,1 south and east India, and Sri Lanka.
This genus is well documented to be a good source of biologically
active stilbenoids. These stilbenoids have antimicrobial,2 antitumor,3-5
and anti-HIV activities,2 as well as regulate endoplasmic reticulum
stress.6 Various types of stilbene oligomers have been reported by
us as constituents of the two species V. rassak and V. pauciflora.7-11
Previous articles have reported that homogeneous oligomerization
of resveratrols results in the production of stilbene oligomers, and
these oligomers sometimes occur in the O-glucoside form. These
oligmers are of special interest due to the numerous stereoisomers
resulting from many asymmetric carbons and various frameworks
(heterocyclic, bicyclic system). However, polar components of
stilbene oligomers and their absolute configuration have not been
discussed.
Our fundamental study of the phytochemical properties of the
stem of V. albiramis (Dipterocarpaceae) has been undertaken to
search for bioactive compounds. This paper reports the isolation
of five new stilbene glucosides (1-5) along with 13 known
compounds (6-17 and bergenin). Their structures were elucidated
by spectroscopic methods, including 1D (1H and 13C) and 2D NMR
(DQF-COSY, HMQC, HMBC, NOESY, and ROESY) experiments
and high-resolution (HR) FABMS analysis.
Matrix metalloproteinases (MMPs) are a superfamily of structur-
tography using silica gel, Chromatorex DMS, Sephadex LH-20,
ally related matrix-degrading enzymes that play important roles in
and Chromatorex ODS to give 13 known compounds and five new
tissue-destructive processes of various diseases such as cancer.12
stilbene glucosides, named vatalbinosides A-E (1-5). All five
compounds showed a positive reaction with the Gibbs reagent.
In skin dermal tissue, expression and activity of MMPs increase
with aging and, thus, cause degradation and remodeling of the
Structures of the known compounds were identified by comparing
structural extracellular matrix.13 MMP-1 (collagenase-1) and
obtained spectroscopic data with those in the literature. The
MMP-3 (stromelysin-1) play a major role in the multiple stages of
compounds were identified as (-)-hopeaphenol (6),15 vaticanol B
cutaneous photoaging.14 In the course of screening plant extracts
(7),11 vaticasides B (8) and C (9),10 (E)-(-)-ε-viniferin (10),16
that have an inhibitory effect on MMP-1 production in human
dermal fibroblasts, we recently found that an acetone extract of
balanocarpol (11),17 (-)-ampelopsin A (12),18 vaticanol C (13),11
stenophyllol C (14),19 piceid (15), (Z)-(-)-ε-viniferin (16),20
the stem of V. albiramis exhibited a strong inhibitory effect on
vateriaphenol A (17),21 and bergenin (18).3
MMP-1 production. In the present study, we examined the inhibitory
effect of stilbenoids isolated from V. albiramis on MMP-1
production in human dermal fibroblasts.
Results and Discussion
The dried and ground stem of V. albiramis was extracted with
acetone. The resulting extract was separated by column chroma-
* To whom correspondence should be addressed. Tel: +81-58-230-8100.
Fax: +81-58-230-8105. E-mail: iinuma@gifu-pu.ac.jp.
† Gifu Pharmaceutical University.
‡ Gifu International Institute of Biotechnology.
§ Tokai Gakuin University.
Vatalbinoside A (1) was obtained as a yellow, amorphous solid.
The molecular formula was deduced to be C62H52O17 using the
10.1021/np1002675  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/24/2010

1500 Journal of Natural Products, 2010, Vol. 73, No. 9
Abe et al.
pseudomolecular ion [M - H]- at m/z 1067.3134 from HRFABMS.
NMR data of 1 [1H and 13C NMR spectra: Table 1 (in acetone-d6)
and Table S1 (in MeOH-d4); HMBC and NOESY spectra: Table
S2 (Supporting Information)] revealed the presence of four 4-hy-
droxyphenyl groups (designated as A1-D1), four 3,5-dioxygenated-
1,2-disubstituted benzene rings (A2-D2), and one O-ꢀ-glucopyr-
anose moiety. The presence of four sets of mutually coupled
aliphatic protons (H-7a/H-8a, H-7b/H-8b, H-7c/H-8c, H-7d/H-8d)
and a ꢀ-glucopyranose moiety [δC 105.1, 74.8, 77.7, 71.3, 76.7,
and 62.4; anomeric proton at δH 4.15 (J ) 7.6 Hz)] was supported
by NMR data, and this led to the composition C56H42O12 for the
1
aglycone of 1. The H NMR spectrum (in DMSO-d6) indicated
phenolic OH groups (δH 8.40-9.57), which disappeared upon the
addition of D2O. In the HMBC spectrum, the significant 3J
correlations H-2a(6a)/C-7a, H-14a/C-8a, H-2b(6b)/C-7b, H-14b/
C-8b, H-2c(6c)/C-7c, H-14c/C-8c, H-2d(6d)/C-7d, and H-14d/C-
8d indicated that rings A1, A2, B1, B2, C1, C2, D1, and D2 are attached
to C-7a, C-8a, C-7b, C-8b, C-7c, C-8c, C-7d, and C-8d, respectively.
Thus, 1 was composed of four resveratrol units, A-D (unit A: ring
A1-C-7a-C-8a-ring A2). Further correlations, H-8a/C-11b, H-7b/
C-11a, H-7c/C-11d, and H-8d/C-11c, supported respective single
bonds C-8a/C-10b, C-7b/C-10a, C-7c/C-10d, and C-8d/C-10c. The
C-C bond (C-8b/C-8c) was not proved by a COSY correlation
because the protons (H-8b and H-8c) were in close proximity. The
assignment of H-signals (H-8b and H-8c) by 1J HMQC cross-peaks
3
(H-8b/C-8b and H-8c/C-8c) and J HMBC cross-peaks (H-8b/C-
on the two dihydrobenzofuran rings (ring E and ring G) are trans-
oriented. The coupling constants, 11.8 Hz (H-7a/H-8a) and 12.4
Hz (H-7d/H-8d), are in agreement with diaxial orientations. In the
NOESY spectrum, H-2b(6b) displayed significant cross-peaks with
H-8a and H-8b, which indicated that ring B1, H-8a, and H-8b are
situated on the same side of the reference plane in ring F. These
results suggested that 1A has the same partial structure as
hopeaphenol {(1R*,6S*,7R*,11bR*)-1,6,7,11b-tetrahydro-4,8,10-
trihydroxy-1,7-bis(4-hydroxyphenyl)benzo[6,7]cyclohepta[1,2,3-cd]-
benzofuran-6-yl group}. Furthermore, identical relative stereostruc-
ture in 1B was shown by NOESY cross-peaks. These two units
(1A and 1B) may lead to two possible relative configurations of
the aglycone, hopeaphenol and neohopeaphenol. The former [(-)-
hopeaphenol (6) ([R]28D -402.9)]22 has no plane of symmetry, while
the latter (meso form) has a plane of symmetry [neohopeaphenol
([R]D 0)].23 The negative specific rotation of the aglycone can be
2
14b and H-8c/C-14c) enabled the distinction of J HMBC cross-
peaks (H-8b/C-8c and H-8c/C-8b), which made it possible to clarify
C-8b/C-8c connectivity (Figure 1). Although no long-range cor-
relations between H-7a/C-11b and H-7d/C-11d were observed, the
presence of two dihydrobenzofuran moieties (ring E: C-7a-C-
8a-C-10b-C-11b-O, ring G: C-7d-C-8d-C-10c-C-11c-O) was
deduced after considering the molecular formula of the aglycone
(C56H42O12). The long-range correlation between the anomeric
proton (δH 4.06) and C-13b (δC 158.18) in the HMBC spectrum
(in MeOH-d4) confirmed that the glucosyloxy group was at C-13b.
The relative configuration of 1 was determined by NOESY
experiments and analysis of coupling constants with the assistance
of computer-aided molecular modeling (Figure 2). The significant
NOEs observed among H-2a(6a)/H-8a, H-7a/H-14a, H-2d(6d)/H-
8d, and H-7d/H-14d suggested that the two sets of methine protons

ResVeratrol Oligomers from Vatica albiramis
Journal of Natural Products, 2010, Vol. 73, No. 9 1501
vatalbinoside B (2)
Table 1. 1H and 13C NMR Data for Vatalbinosides A (1) and B (2)a
vatalbinoside A (1)
position
1a
2a(6a)
3a(5a)
4a
δ(1H)
δ(13C)
δ(1H)
δ(13C)
130.5
130.2
116.0
158.3
88.3
130.7h
130.2
116.0i
158.6
90.4
7.16, d (8.4)
6.82, d (8.4)
7.23, d (8.8)
6.79, d (8.8)
7a
8a
5.83, d (12.0)
4.30, d (12.0)
5.77, d (12.4)
4.45, d (12.4)
49.5
48.7
9a
10a
11a
12a
13a
14a
1b
2b(6b)
3b(5b)
4b
141.8
121.0
158.6
101.1
157.2d
106.9
134.9e
129.1
115.2i
155.6
41.0
141.7
124.5
155.7j
101.6
156.4
105.7
133.5
130.7h
115.5
155.7j
37.0
6.55, d (2.0)
6.42 (br s)
6.28, d (2.0)
6.11, d (2.0)
6.91, d (8.8)
6.59, d (8.8)b
7.18, d (9.2)
6.71, d (9.2)
7b
5.79 (br s)c
5.23, d (3.6)
8b
9b
10b
11b
12b
13b
14b
1c
2c(6c)
3c(5c)
4c
7c
8c
9c
10c
11c
12c
13c
14c
1d
2d(6d)
3d(5d)
4d
7d
8d
4.02 (dd, J ) 10.2,4.2)
48.0
3.08 (br d, J ) 11.6)
53.6
140.5
122.9
158.4
100.8
157.9g
116.2
134.9e
129.2
115.2f
155.5
41.2
143.2
115.7
158.8
96.5
154.9
122.4
131.7
129.3
115.9
155.9
57.3
6.05, d (2.0)
5.43, d (2.0)
6.07 (s)
6.95, d (8.8)
6.59, d (8.8)b
6.41, d (8.8)
6.56, d (8.8)
5.79 (br s)c
4.02 (dd, J ) 10.2,4.2)
4.11 (dd, J ) 11.6, 10.8)
4.61, d (10.8)
48.1
48.6
140.4
118.7
159.2
95.5
141.2
122.5
155.9
106.0
157.8
108.3
134.5
128.3
116.0f
158.0
94.6
5.79, d (2.0)c
5.07, d (2.0)
157.2a
111.1
130.9
130.4
115.9
157.9g
88.0
6.43 (s)
7.23, d (8.4)
6.79, d (8.4)
7.21, d (8.8)
6.71, d (8.8)
6.18, d (12.4)
4.28, d (12.4)
5.43, d (5.8)
4.75, d (5.8)
49.4
57.9
9d
10d
11d
12d
13d
14d
glucose-1
glucose-2
glucose-3
glucose-4
glucose-5
glucose-6
141.9
120.9
158.7
101.2
157.2d
106.4
105.1
74.8
77.7
71.3
76.7
62.4
147.8
107.9
159.8
102.2
159.8
107.9
77.1
73.6
79.5
71.0
81.7
6.16 (br d)
6.58 (br s)
6.33 (t, J ) 2.0)
6.32 (br s)
4.15, d (7.6)
6.16 (br d)
4.75, d (9.6)
3.75 (dd, J ) 9.6, 9.2)
3.58 (dd, J ) 9.2, 8.8)
3.55 (dd, J ) 9.2, 8.4)
3.47 (m)
3.32-3.36 (m)
3.67 (dd, J ) 9.6, 9.2)
3.49 (dd, J ) 9.6)
3.32-3.36 (m)
3.80 (br d),
3.88 (dd, J ) 12.4, 2.8),
3.81 (dd, J ) 12.4, 4.8)
62.2
3.72 (dd, J ) 10.8, 4.0)
a In acetone-d6; at 400 (1H) and 100 (13C) MHz, δ in ppm, J in Hz. b-j Overlapping.
explained by the specific rotation of 1 ([R]25 -320), even if the
to detect NOESY cross-peaks between C2 units.15 A further problem
is the difficulty in distinguishing cross-peaks such as H-2a(6a)/H-
8a and H-2a(6a)/H-8d because H-8a and H-8d are equivalent. These
problems not only prevent the determination of the stereostructure
of the C1 unit but also make it impossible to comprehend the
relationship between the two C1 units. In the case of 1, the
equivalent C2 system of the aglycone is destroyed by the glycosyl
group. The original number of signals observed, which enabled
analysis of NOEs between units 1A and 1B, were as follows: H-14b/
H-7c, H-14b/H-7d, H-14b/H-14d, H-14c/H-7a, H-14c/H-14a, and
H-14c/H-7b (Table S2). To ascertain these results, a 3D structure
of 1 (Figure 2) was generated by employing the PCMODEL 9.1
molecular modeling program software using the MMFF94 force
field (MM2 type) for energy minimization.25 The model reasonably
D
influence of the glucosyloxy group is considered. The absolute
configuration of 6 was determined on the basis of an X-ray
crystallographic analysis.24 Enzymatic hydrolysis of 1 with ꢀ-glu-
cosidase led to the generation of 6. Therefore, vatalbinoside A (1)
was elucidated as (-)-hopeaphenol 13b-O-ꢀ-glucopyranoside
{(1R,6S,7R,11bR)-1,6,7,11b-tetrahydro-1,7-bis(4-hydroxyphenyl)-
4-(ꢀ-D-glucopyranosyloxy)-6-[(1R,6S,7R,11bR)-1,6,7,11b-tetrahy-
dro-4,8,10-trihydroxy-1,7-bis(4-hydroxyphenyl)benzo[6,7]cyclohepta-
[1,2,3-cd]benzofuran-6-yl]benzo[6,7]cyclohepta[1,2,3-cd]benzofu-
ran-8,10-diol}. A molecule with C2 symmetry shows equivalence
of the NMR resonances of the two units. This results in half the
number of signals with double the intensity. It also complicates
definition of the connectivity of the C2 units and makes it impossible

1502 Journal of Natural Products, 2010, Vol. 73, No. 9
Abe et al.
The NMR data of 2 (Table 1) analyzed by 2D experiments exhibited
signals of a C-ꢀ-glucopyranose [δC 77.1, 73.6, 79.5, 71.0, 81.7,
and 62.2; anomeric proton at δH 4.75 (J ) 9.6 Hz)] and four
resveratrol units. Data analysis confirmed that the aglycone was
vaticanol B (7). The position of the C-glucopyranosyl moiety was
defined as C-12c from results of the HMBC spectrum, which
displayed cross-peaks between the anomeric proton and three
aromatic carbons (C-11c, C-12c, and C-13c: δC 155.9, 106.0, and
157.8) (Table S2). Therefore, vatalbinoside B (2) was elucidated
as vaticanol B 12c-C-ꢀ-D-glucopyranoside {(3S*,4S*,4aR*,5R*,
9bR*,10R*)-3-[(2R*,3R*)-3-(3,5-dihydroxyphenyl)-2,3-dihydro-6-hy-
droxy-7-(ꢀ-glucopyranosyl)-2-(4-hydroxyphenyl)benzofuran-4-yl]-
3,4,4a,5,9b,10-hexahydro-4,5,10-tris(4-hydroxyphenyl)benz[5,6]azuleno-
[7,8,1-cde]benzofuran-2,6,8-triol}. The absolute configuration of
vaticanol B (7) has not been elucidated, and that of its glucosides
[vatalbinoside B (2) and vaticasides B (8) and C (9)] is difficult to
determine. Because they possessed the same 1,2-diaryldihydroben-
zofuran chromophore as (2R,3R)-3-(3,5-dihydroxyphenyl)-6-hy-
droxy-2-(4-hydroxyphenyl)-2,3-dihydrobenzofuran-4-carbalde-
hyde (19), whose absolute configuration is known,26 the CD spectra
of 19 and 2 were compared (Figure 3). The CD spectrum of 2 and
19 showed a negative Cotton effect around 236 nm, which indicates
a 7dR, 8dR configuration. Because there is no chiroptical data of
3,4,4a,5,9b,10-hexahydro-3,4,5,10-tetrakis(aryl)benz[5,6]azuleno[7,8,1-
cde]benzofuran, the absolute configurations of the other asymmetric
carbons could not be defined. The understanding of the chiroptical
properties of 2 requires a spectroscopic database of diastereomeric
and enantiomeric core skeletons of bicyclo[5.3.0]decadienes. Since
it is difficult to obtain a single crystal of 2, X-ray analysis was not
performed. The absolute structures of the aglycones of 2, 8, and
9swhich are glucosides of vaticanol B (7)sare identical to the
absolute structure of the agylcone of 7, which was evidenced by
the negative Cotton effect at 236 nm.
Figure 1. Selected correlations in the 2D NMR of 1.
Figure 2. Stereostructure and shielding of protons (acetone-d6) by
anisotropy in 1.
explains the anisotropic effects of rings A2 and D2, which cause
shielding of aromatic protons H-14c (δH 5.07) and H-14b (δH 5.43),
respectively. The anisotropy of ring C2 also causes shielding of
the anomeric proton (H-Glc-1, δH 4.06).
Vatalbinoside B (2) was obtained as a pale yellow, amorphous
solid. The molecular formula was deduced to be C62H52O17 from
the [M - H]- ion at m/z 1067.3134 in the negative-ion HRFABMS,
which corresponds to a monoglucoside of a resveratrol tetramer.
Vatalbinoside C (3) was obtained as a pale yellow, amorphous
solid. The molecular formula was deduced to be C40H42O16 from
the [M - H]- ion at m/z 777.2400 from the negative-ion
HRFABMS, which corresponded to a diglucoside of a resveratrol
dimer.27,28 The NMR data supported the presence of two ꢀ-glu-
copyranosyloxy groups (Table 2). The 1H and 13C NMR data of 3,
except for the ꢀ-glucopyranosyloxy group, showed close similarity
to those of ε-viniferin (10). The HMBC, NOESY (Table S3), and
Figure 3. CD spectra of vatalbinosides A-E (1-5) and (2R,3R)-3-(3,5-dihydroxyphenyl)-6-hydroxy-2-(4-hydroxyphenyl)-2,3-dihydroben-
zofuran-4-carbaldehyde (19) in MeOH.

ResVeratrol Oligomers from Vatica albiramis
Journal of Natural Products, 2010, Vol. 73, No. 9 1503
Table 2. 1H and 13C NMR Data of Vatalbinosides C-E (3-5)a
vatalbinoside C (3)
vatalbinoside D (4)
δ(1H)
vatalbinoside E (5)
δ(1H)
position
1a
2a(6a)
3a(5a)
4a
7a
8a
9a
10a
11a
12a
13a
14a
δ(1H)
δ(13C)
δ(13C)
δ(13C)
133.7
128.1
116.2
158.5
94.7
133.3f
130.6
116.4
158.5d
93.6
134.1
130.0
117.7
159.2
88.7
7.10 (d, J ) 7.4)
6.73 (d, J ) 7.4)
7.52 (d, J ) 8.6)
6.96 (d, J ) 8.6)
7.00 (d, J ) 8.8)
6.88 (d, J ) 8.8)
5.37 (d, J ) 5.4)
4.44 (d, J ) 5.4)
5.73 (d, J ) 9.6)
5.24 (d, J ) 9.6)
5.63 (d, J ) 11.2)
3.92 (d. J ) 11.2)
57.8
52.2
50.3
147.3
108.3
159.9
103.6
160.6e
109.9
136.9
128.9
116.4
158.4d
131.1
123.2
130.2
122.7
162.4
98.4
142.7
120.2
157.3
101.9
156.8
106.7
133.3f
131.4
114.3
155.6
50.0
143.1
122.8
160.1
104.1
157.9
108.4
132.6
129.1
115.7
156.3
44.3
6.38 (br s)
6.44 (d, J ) 1.6)
6.31 (br s)
6.20 (d, J ) 2.2)
6.28 (d, J ) 2.2)
6.61 (d, J ) 2.0)
6.20 (br s)
1b
2b(6b)
3b(5b)
4b
7b
8b
9b
10b
11b
12b
7.03 (d, J ) 7.6)
6.64 (d, J ) 7.6)
6.75 (d, J ) 8.8)
6.45 (d, J ) 8.8)
6.68 (d, J ) 8.8)
6.42 (d, J ) 8.8)
6.87 (d, J ) 16.2)
6.54 (d, J ) 16.2)
4.95 (br s)
5.45 (br s)
5.40 (d, J ) 4.9)
5.29 (d, J ) 4.9)
73.0
71.6
140.3
116.9
159.3
97.3
159.7
106.7
102.5
74.4
77.6
71.1
77.2
62.5
139.4
119.4
159.4
97.7
160.5
110.9
104.8
74.7
6.56 (br s)
6.24 (d, J ) 2.0)
6.01 (d, J ) 2.2)
13b
14b
160.6e
106.1
102.0
74.9c
78.2c
71.4c
77.9c
62.5c
6.95 (br s)
4.80 (br d)b
3.52 (m)c
6.30 (d, J ) 2.0)
4.67 (d, J ) 7.6)
3.34 (m)
3.44 (dd, J ) 8.4, 8.0)
3.42 (dd, J ) 8.8, 8.0)
3.38 (m)
6.40 (d, J ) 2.2)
4.76 (d, J ) 7.3)
3.34 (m)
3.29 (m)
3.28 (m)
glucose1-1
glucose1-2
glucose1-3
glucose1-4
glucose1-5
glucose1-6
3.52 (m)c
78.1
71.2
3.43 (m)c
3.44 (m)c
3.29 (m)
3.75, 3.56 (m)
77.9
3.75, 3.94 (m)c
3.67 (dd, J ) 12.0 5.0)
3.81 (dd, J ) 12.0 2.8)
62.5g
glucose2-1
glucose2-2
glucose2-3
glucose2-4
glucose2-5
glucose2-6
4.98 (d, J ) 7.6)
3.44 (m)c
102.6
74.7c
77.8c
70.9c
77.7c
62.1c
4.76 (d, J ) 7.3)
3.30 (m)
3.29 (m)
3.28 (m)
3.29 (m)
102.0
74.9
78.0
71.3
78.3
62.5g
3.44 (m)c
3.46 (m)c
3.31 (m)c
3.69, 3.78 (m)c
3.75, 3.56 (m)
a In methanol-d4; at 400 (1H) and 100 (13C) MHz, δ in ppm, J in Hz. b Masked by large H2O signal. Assigned by HMQC spectrum. c Assigned
glucose units (H atoms and C atoms) are interchangeable between Glc1 and Glc2. Protons were assigned by HMQC spectrum. d Interchangeable.
e-g Overlapping.
CD spectra (Figure 3) confirmed that the aglycone of 3 was (-)-
ε-viniferin (10), which was further supported by the generation of
10 by enzymatic hydrolysis of 3 with ꢀ-glucosidase. The positions
of the glucosyloxy groups were determined to be C-11a and C-13b
by HMBC and NOESY correlations of aromatic atoms in rings A2
and B2 with anomeric protons (H-Glc1-1 and H-Glc2-1). Vatalbi-
noside C (3) was elucidated as 1-(ꢀ-D-glucopyranosyloxy)-5-
{(2R,3R)-6-(ꢀ-glucopyranosyloxy)-2-(4-hydroxyphenyl)-4-[(E)-4-
hydroxystyryl]-2,3-dihydrobenzofuran-3-yl}benzene-3-ol.Theaglycone
of 3 is an enantiomer of tingitanol isolated from Iris tingitana
(Iridaceae).28
not be elucidated. Takaya et al. determined the stereostructure of
(+)-ampelopsin A, which has the absolute configurations 7aS and
8aS in a 1,2-diaryl-dihydrobenzofuran skeleton.29 The CD curve
of 5 was similar to that of (-)-ampelopsin A. Thus, vatalbinoside
E (5) was elucidated as (-)-ampelopsin A 4a,11a-O-ꢀ-D-diglu-
copyranoside ((1R,6S,7R,11bR)-1,6,7,11b-tetrahydro-8-(ꢀ-D-glu-
copyranosyloxy)-4,6,10-trihydroxy-7-(4-(ꢀ-D-glucopyranosyloxy)-
phenyl)-1-(4-hydroxyphenyl)benzo[6,7]cyclohepta[1,2,3-
cd]benzofuran).
The absolute structures of most stilbenoids from Dipterocar-
paceae plants have not been determined, except for (-)-hopeaphenol
(6),24 (-)-ε-viniferin (10),30 shorealactone,31 and uliginosides
A-C.26 In the present study, we have determined the absolute
configuration of three new glucosides of resveratrol oligomers 1,
3, and 5.
Glucosides of stilbenoids and their oligomers as well as their aglycones
have been isolated from Dipterocarpaceae,2,3,5-11,16,17,21-24,26,27,31-35
Vitaceae,18,36 Gnetaceae,37,38 Welwitschiaceae,39 Iridaceae,28 and
Umbelliferae.40 Each family has a regiospecific biosynthetic
pathway for glycosylation to the stilbene unit(s), represented by
blocking units, as listed in Table 3. Among these units, piceid (1-
(ꢀ-D-glucopyranosyloxy)-5-[(1E)-2-(4-hydroxyphenyl)ethenyl]ben-
zene-3-ol; 15), 2-(ꢀ-D-glucopyransoyl)-5-[(1E)-2-(4-hydroxyphe-
nyl)ethenyl]benzene-1,3-diol, and 4-(ꢀ-D-glucopyransoyl)-5-[(1E)-
2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol are found in Dipter-
ocarpaceae. In dipterocarpaceaeous plants, C-glucosides have been
found only in the genera Shorea26,27 and Hopea,33 and O-glucosides
have been found to have only one glucopyranosyl moiety.8-10,21,34,35
Vatalbinosides D (4) and E (5) were obtained as pale yellow,
amorphous solids. Their molecular formulas were deduced to be
C34H32O12 ([M - H]-, m/z 631.1809) and C40H42O17 ([M - H]-,
m/z 793.2336), respectively, from negative-ion HRFABMS. The
presence of one and two ꢀ-glucopyranosyloxy group(s) in
the respective compounds was supported by their NMR spectra.
1
The H and 13C NMR data of 4 and 5 (Table 2), except for the
ꢀ-glucopyranosyloxy group(s), showed close similarity to those of
balanocarpol (11)17 and ampelopsin A (12),18 respectively. The
HMBC and NOESY spectra (Table S3) and the respective genera-
tion of aglycones (11 and 12) by enzymatic hydrolysis of 4 and 5
with ꢀ-glucosidase confirmed the aglycones (balanocarpol for 4;
ampelopsin A for 5). The positions of the O-ꢀ-D-glucopyranosyl
moieties were defined by NOEs (C-13b for 4; C-4a and C-11a for
5). Using CD spectroscopy, and for the same reasons as described
for 2, the configuration of two methine stereogenic centers in the
1,2-diaryl-dihydrobenzofuran skeleton of 4 and balanocarpol could

1504 Journal of Natural Products, 2010, Vol. 73, No. 9
Abe et al.
Table 3. Blocking Units as Glucosylated Resveratrol in Oligostilbenoid and Distribution
The novel aspects found in these new compounds (1-5) are as
follows: (1) vatalbinoside B (2) is the first example of a C-
glucopyranoside from the genus Vatica and the second occurrence
of a C-glucopyranoside of a resveratrol tetramer;41 (2) vatalbino-
sides C (3) and E (5) have two O-D-glucopyranosyl moieties, which
is the first case in the Dipterocarpaceae; and (3) the blocking unit
(1-(ꢀ-D-glucopyranosyloxy)-5-[(1E)-2-(4-(ꢀ-D-glucopyranosyloxy)-
phenyl)ethenyl]benzene-3-ol) found in 5 is the first instance in this
family. Our present structural characterization of 1-5 not only adds
new features to the biogenesis of stilbene oligomers but also
demonstrates the additional diversity of resveratrol oligomers in
dipterocarpaceaeous plants.
including inflammatory cytokines, such as interleukin (IL)-1ꢀ. Thus,
MMP-1 release from human dermal fibroblasts was initiated by
the addition of IL-1ꢀ and was examined by western blot analysis
of the conditioned medium. As shown in Figure 4, treatment with
several resveratrol oligomers (1, 2, 6, 7, 13, 14, and 17) at 1 µM
blocked IL-1ꢀ-induced MMP-1 production, but did not induce
inhibitory activities of cell growth (data not shown). Three
resveratrol oligomers (6, 13, and 14) displayed significant inhibitory
effects on MMP-1 production. These results suggest the following
important points of SAR: (1) the active compounds have four or
more resveratrol units; (2) the structure of each active resveratrol
tetramer (1, 2, 6, 7, 13, and 14) has two trans-oriented dihydroben-
zofuran rings and a sequence of four -CH- groups; (3) resveratrol
dimers (3-5, 10-12, and 16) have no or less effect than active
tetramers on MMP-1 production; and (4) compounds 2 and 17 have
a partial structure of 6, which has a strong potency. This indicates
In the present study, each constituent isolated from the stem of
V. albiramis was examined for its ability to inhibit MMP-1
production. MMP expression levels are relatively low in unstimu-
lated cells, but some are induced by various extracellular stimuli
Figure 4. Inhibition of IL-1ꢀ-induced MMP-1 production in human dermal fibroblasts.

ResVeratrol Oligomers from Vatica albiramis
Journal of Natural Products, 2010, Vol. 73, No. 9 1505
NOESY correlations, Table S2; FABMS, m/z 1067 [M - H]-;
HRFABMS, m/z 1067.3132 (calcd for C62H51O17, 1067.3126) [M -
H]-.
Vatalbinoside C (3): pale yellow solid; UV (MeOH) 227 (4.65),
284 (4.14), 325 (4.29); CD (c 25.7 µM, MeOH) nm (∆ε) 236 (-33.5),
288 (-9.2); [R]25D -72 (c 0.1, MeOH); 1H and 13C NMR spectroscopic
data, Table 2; HMBC and NOESY correlations, Table S3; FABMS,
m/z 777 [M - H]-; HRFABMS, m/z 777.2400 (calcd for C40H41O16,
777.2394) [M - H]-.
that a substitution on 6 would weaken its effects, such as
O-glucosylation and further oligomerized resveratrols. The stem
of V. albiramis contains resveratrol tetramers 6, 7, and 13 in large
quantities, which explains the effect of the acetone extract. In
previous decades, resveratrol tetramers, including 6, 7, and 13, have
mostly been isolated from several dipterocarpaceaeous plants. These
stilbenoids and extracts containing resveratrol tetramers are con-
sidered to be useful resources for cosmetic agents. A limited number
of inhibitors of MMP-1 production have been identified from nature,
and the current discovery is the first report of oligostilbenoids as
candidates for preventing cutaneous photoaging. Further studies at
the molecular level are required to explain the detailed mechanisms
underlying the effect of stilbenoids on MMP-1 production and are
currently underway in our laboratory.
Vatalbinoside D (4): pale yellow solid; UV (MeOH) 227 (4.71),
283 (4.13); CD (c 31.6 µM, MeOH) nm (∆ε) 237 (-27.4); [R]25D -44
(c 0.1, MeOH); 1H and 13C NMR spectroscopic data, Table 2; HMBC
and NOESY correlations, Table S3; FABMS, m/z 631 [M - H]-;
HRFABMS, m/z 631.1809 (calcd for C34H31O12, 631.1816) [M - H]-.
Vatalbinoside E (5): pale yellow solid; UV (MeOH) 226 (4.57),
285 (3.77); CD (c 25.2 µM, MeOH) nm (∆ε) 236 (-19.6); [R]25D -142
(c 0.1, MeOH); 1H and 13C NMR spectroscopic data, Table 2; HMBC
and NOESY correlations, Table S3; FABMS, m/z 793 [M - H]-;
HRFABMS, m/z 793.2360 (calcd for C40H41O17, 793.2343) [M - H]-.
Enzymatic Hydrolysis of 1 and 3-5. Separate solutions of 1 (1
mg), 3 (1 mg), 4 (1 mg), and 5 (1 mg) in phosphate buffer (pH 6, 1.0
mL) were treated with ꢀ-glucosidase (1 mg/mL) for 24 h at 37 °C.
Each reaction solution was evaporated to dryness, and the resultant
residue was purified by PTLC (EtOAc/CHCl3/MeOH/H2O, 80:40:11:
2) to obtain the respective aglycones [6 (0.3 mg), 10 (0.3 mg), 11 (0.2
Experimental Section
General Experimental Procedures. The following instruments were
used: optical rotations: JASCO P-1020 polarimeter; UV spectra:
Shimadzu UV-3100 spectrophotometer (in MeOH solution); CD spectra:
JASCO J-820 spectrometer (in MeOH solution); 1H and 13C NMR
spectra: JEOL JNM AL-400 (chemical shift values in 1H NMR spectra
are presented as δ values with TMS as the internal standard); FABMS:
JEOL JMS-DX-300 instrument.
The following adsorbents were used for purification: analytical TLC:
Merck Kieselgel 60 F254 (0.25 mm); preparative TLC: Merck Kieselgel
60 F254 (0.5 mm); column chromatography: Merck Kieselgel 60,
Pharmacia Fine Chemicals AB Sephadex LH-20, and Fuji Silysia
Chemical Chromatorex, Waters Sep-Pak C18 cartridges; vacuum liquid
chromatography (VLC): Merck Kieselgel 60; medium-pressure column
chromatography (MPLC): Fuji Silysia Chemical Chromatorex ODS
(100-200 mesh).
1
mg), and 12 (0.3 mg)]. The structures were confirmed by H NMR
spectra.
Materials for MMP-1 Assay. The antibody against MMP-1 was
obtained from Daiichi Fine Chemical (Toyama, Japan). Anti-mouse
antibody conjugated with horseradish peroxidase and the ECL western
blotting detection system were obtained from GE Healthcare (Piscat-
away, NJ). IL-1ꢀ was obtained from Chemicon (Temecula, CA). Other
reagents used were of the highest quality available.
Cell Culture. Human dermal foreskin fibroblasts were purchased
from Kurabo (Osaka, Japan). Cells were cultured in Dulbecco’s
modified Eagle’s medium supplemented with 10% (v/v) fetal bovine
serum at 37 °C in a humidified, CO2-controlled (5%) incubator.
Measurement of MMP-1 Secretion. MMP-1 secretion was analyzed
by western blot analysis. In brief, cells (2 × 105 cells/mL) were seeded
and cultured until confluent, treated for 24 h with stilbenoids isolated
from V. albiramis, and stimulated with IL-1ꢀ (10 ng/mL) for 24 h.
Supernatants were collected and concentrated by acetone precipitation.
Aliquots of concentrated supernatants were subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. Proteins were then trans-
ferred electrophoretically to a polyvinylidene fluoride membrane
(Hybond-P; GE healthcare). After blocking in Tris-buffered saline
containing 5% skim milk and 0.05% Tween-20 for 1 h at room
temperature, the blots were incubated with the appropriate primary
antibody overnight at 4 °C and further incubated with horseradish
peroxidase-conjugated secondary antibody for 1 h at room temperature.
The proteins were visualized using an ECL western blot detection
system according to the manufacture’s instructions, and gel images were
obtained with a LAS 4000 imaging system (Fuji Film, Tokyo, Japan).
Band intensity was quantified by a densitometer (ATTO densitograph).
Plant Material. V. albiramis was collected from Borneo Island,
Malaysia, in April 2002 and identified by J. Josue, head of the forest
product branch at the Forest Research Center, Sandakan Sabah,
Malaysia. A voucher specimen, number DP-026, has been deposited
at the herbarium of Gifu Pharmaceutical University.
Extraction and Isolation. The dried and ground stem of V. albiramis
(30 kg) was extracted with acetone (120 L, 24 h × 2, at room
temperature). Evaporation produced a dry solid (2.3 kg). A part (2.0
kg) of the acetone extract was divided into five fractions (400 g each),
and each one was subjected to column chromatography (120 × 12 cm)
on silica gel (5 kg), eluted with a mixture of CHCl3/MeOH, increasing
in polarity. Nine fractions (Frs.1-9) were obtained from the initial five
fractions, and these five fractions were combinations of appropriate
fractions according to TLC (Gibbs test) (fraction: solvent system (total
solvent eluted), amount; Fr.1: CHCl3 (30 L), 222 g, Fr.2: CHCl3/MeOH,
10:1 (30 L), 30 g, Fr.3: CHCl3/MeOH, 10:1 (30 L), 155 g, Fr.4: CHCl3/
MeOH, 8:1 (20 L), 82 g, Fr.5: CHCl3/MeOH, 7:1 (50 L), 412 g, Fr.6:
CHCl3/MeOH, 6:1 (50 L), 450 g, Fr.7: CHCl3/MeOH, 5:1 (25 L), 84 g,
Fr.8: CHCl3/MeOH, 5:1 (40 L), 234 g, Fr.9: MeOH (30 L), 430 g).
Compounds 10 (10 g), 11 (2 g), 12 (2 g), and 16 (20 mg) were obtained
from Fr.3 by further purification by column chromatography over
Sephadex LH-20 (MeOH) and Sep-Pak C18 (MeOH/H2O system). After
purification using column chromatography over Sephadex LH-20
(MeOH) and ODS (MeOH/H2O system), compounds 7 (250 g), 13 (21
g), 15 (1.2 g), and bergenin (25 g) were obtained from Fr.5 and
compounds 6 (120 g) and 14 (4 g) were obtained from Fr.6. Purification
of the ninth fraction by silica gel column chromatography (CHCl3/
MeOH gradient system), Sephadex LH-20 (MeOH), Sep-Pak C18
(MeOH/H2O system), VLC (EtOAc/CHCl3/MeOH/H2O system), reversed-
phase MPLC (MeOH/H2O system), and PTLC (EtOAc/CHCl3/MeOH/
H2O system) achieved the isolation of compounds 1 (78.8 mg), 2 (24.4
mg), 3 (59.6 mg), 4 (19.1 mg), 5 (21.8 mg), 8 (2 g), 9 (2 g), and 17 (3
g).
Acknowledgment. The authors are grateful to Mrs. M. Hosokawa
and Mrs. M. Hayashi of Gifu Pharmaceutical University for their
measurement of FABMS data.
Note Added after ASAP Publication: This paper was published
on the Web on August 24, 2010, with Table S1 missing from the
Supporting Information file. The revised SI was reposted on September
3, 2010.
Supporting Information Available: 1D NMR spectra (1H and 13
C
NMR spectra); tables of HMBC and NOESY data for compounds 1-5.
This information is available free of charge via the Internet at http://
pubs.acs.org.
Vatalbinoside A (1): pale yellow solid; UV (MeOH) 226 (4.97),
282 (4.28); CD (c 18.7 µM, MeOH) nm (∆ε) 236 (-11.8); [R]25D -320
(c 0.1, MeOH); 1H and 13C NMR spectroscopic data, Table 1; HMBC
and NOESY correlations, Table S2; FABMS, m/z 1067 [M - H]-;
HRFABMS, m/z 1067.3134 (calcd for C62H51O17, 1067.3126) [M -
H]-.
References and Notes
(1) Ashton, P. S. Flora Malesiana, Ser. I, Spermatophyta; Steenis,
C. G. G. J., Ed.; Martivus Nijhoff Publishers: Leiden, The Netherlands,
1982; Vol. 9, pp 237-552.
(2) Zgoda-Pols, J. R.; Freyer, A. J.; Killmer, L. B.; Porter, J. R. J. Nat.
Prod. 2002, 65, 1554–1559.
Vatalbinoside B (2): pale yellow solid; UV (MeOH) 284 (4.50);
CD (c 18.7 µM, MeOH) nm (∆ε) 236 (-24.5); [R]25 -16 (c 0.1,
D
1
MeOH); H and 13C NMR spectroscopic data, Table 1; HMBC and

1506 Journal of Natural Products, 2010, Vol. 73, No. 9
Abe et al.
(3) Seo, E.-K.; Chai, H.; Constant, H. L.; Santisuk, T.; Reutrakul, V.;
Beecher, C. W. W.; Farnsworth, N. R.; Cordell, G. A.; Pezzuto, J. M.;
Kinghorn, A. D. J. Org. Chem. 1999, 64, 6976–6983.
(4) Ito, T.; Akao, Y.; Yi, H.; Ohguchi, K.; Matsumoto, K.; Tanaka, T.;
Iinuma, M.; Nozawa, Y. Carcinogenesis 2003, 24, 1489–1497.
(5) Shibata, M. A.; Akao, Y.; Shibata, E.; Nozawa, Y.; Ito, T.; Mishima,
S.; Morimoto, J.; Otsuki, Y. Cancer Chemother. Pharmacol. 2007,
60, 681–691.
(6) Tabata, Y.; Takano, K.; Ito, T.; Iinuma, M.; Yoshimoto, T.; Miura,
H.; Kitao, Y.; Ogawa, S.; Hori, O. Am. J. Physiol. 2007, 293, C411–
418.
(7) Ito, T.; Tanaka, T.; Iinuma, M.; Nakaya, K.; Takahashi, Y.; Sawa,
R.; Murata, J.; Darnaedi, D. J. Nat. Prod. 2004, 67, 932–937.
(8) Ito, T.; Tanaka, T.; Iinuma, M.; Iliya, I.; Nakaya, K. i.; Ali, Z.;
Takahashi, Y.; Sawa, R.; Shirataki, Y.; Murata, J. Tetrahedron 2003,
59, 5347–5363.
(9) Ito, T.; Tanaka, T.; Nakaya, K. i.; Iinuma, M.; Takahashi, Y.;
Naganawa, H.; Ohyama, M.; Nakanishi, Y.; Bastow, K. F.; Lee, K. H.
Tetrahedron 2001, 57, 7309–7321.
(10) Ito, T.; Tanaka, T.; Ido, Y.; Nakaya, K.; Iinuma, M.; Takahashi, Y.;
Naganawa, H.; Riswan, S. Heterocycles 2001, 55, 557–567.
(11) Tanaka, T.; Ito, T.; Nakaya, K.; Iinuma, M.; Riswan, S. Phytochemistry
2000, 54, 63–69.
(12) Vincenti, M. P.; Brinckerhoff, C. E. J. Cell. Physiol. 2007, 213, 355–
364.
(13) Khorramizadeh, M. R.; Tredget, E. E.; Telasky, C.; Shen, Q.; Ghahary,
(21) Ito, T.; Tanaka, T.; Iinuma, M.; Nakaya, K. i.; Takahashi, Y.; Sawa,
R.; Naganawa, H.; Chelladurai, V. Tetrahedron 2003, 59, 1255–1264.
(22) Madhav, R.; Seshadri, T. R.; Subramanian, G. B. V. Phytochemistry
1967, 6, 1155–1156.
(23) Liu, J. Y.; Ye, Y. H.; Wang, L.; Shi, D. H.; Tan, R. X. HelV. Chim.
Acta 2005, 88, 2910–2917.
(24) Coggon, P.; King, T. J.; Wallwork, S. C. Chem. Commun. 1966, 439–
440.
(25) PCMODEL v 9.0; Serena Software: Bloomington, IN.
(26) Ito, T.; Abe, N.; Oyama, M.; Iinuma, M. Tetrahedron Lett. 2009, 50,
2516–2520.
(27) Ito, T.; Tanaka, T.; Ido, Y.; Nakaya, K.; Iinuma, M.; Riswan, S. Chem.
Pharm. Bull. 2000, 48, 1959–1963.
(28) Farag, S. F.; Takaya, Y.; Niwa, M. Phytochem. Lett. 2009, 2, 148–
151.
(29) Takaya, Y.; Yan, K. X.; Terashima, K.; Ito, J.; Niwa, M. Tetrahedron
2002, 58, 7259–7265.
(30) Kurihara, H.; Kawabata, J.; Ichikawa, S.; Mizutani, J. Agric. Biol.
Chem. 1990, 54, 1097–1099.
(31) Ito, T.; Tanaka, T.; Iinuma, M.; Nakaya, K.; Takahashi, Y.; Nakamura,
H.; Naganawa, H.; Riswan, S. HelV. Chim. Acta 2003, 86, 3394–3401.
(32) Sotheeswaran, S.; Pasupathy, V. Phytochemistry 1993, 32, 1083–1092.
(33) Tanaka, T.; Ito, T.; Ido, Y.; Son, T. K.; Nakaya, K.; Iinuma, M.;
Ohyama, M.; Chelladurai, V. Phytochemistry 2000, 53, 1015–1019.
(34) Ito, T.; Ali, Z.; Iliya, I.; Furusawa, M.; Tanaka, T.; Nakaya, K.;
Takahashi, Y.; Sawa, R.; Murata, J.; Darnaedi, D.; Iinuma, M. HelV.
Chim. Acta 2005, 88, 23–34.
A. Mol. Cell. Biochem. 1999, 194, 99–108.
(14) Brenneisen, P.; Sies, H.; Scharffetter-Kochanek, K. Ann. N.Y. Acad.
Sci. 2002, 973, 31–43.
(15) Kawabata, J.; Fukushi, E.; Hara, M.; Mizutani, J. Magn. Reson. Chem.
1992, 30, 6–10.
(16) Sultanbawa, M. U. S.; Surendrakumar, S.; Wazeer, M. I. M.; Bladon,
P. J. Chem. Soc., Chem. Commun. 1981, 1204–1206.
(17) Diyasena, M. N. C.; Sotheeswaran, S.; Surendrakumar, S.; Balasubra-
manian, S.; Bokel, M.; Kraus, W. J. Chem. Soc., Perkin Trans. 1 1985,
1807–1809.
(35) Ito, T.; Ali, Z.; Furusawa, M.; Iliya, I.; Tanaka, T.; Nakaya, K.; Murata,
J.; Darnaedi, D.; Iinuma, M. Chem. Pharm. Bull. 2006, 54, 363–367.
(36) Park, W. H.; Lee, S. J.; Moon, H. I. Antimicrob. Agents Chemother.
2008, 52, 3451–3453.
(37) Wang, L.-Q.; Zhao, Y.-X.; Hu, J. M.; Jia, A.-Q.; Zhou, J. HelV. Chim.
Acta 2008, 91, 159–164.
(38) Huang, K. S.; Wang, Y. H.; Li, R. L.; Lin, M. J. Nat. Prod. 2000, 63,
86–89.
(39) Murata, H.; Iliya, I.; Tanaka, T.; Furasawa, M.; Ito, T.; Nakaya, K.;
Oyama, M.; Iinuma, M. Chem. BiodiVersity 2005, 2, 773–779.
(40) De Marino, S.; Gala, F.; Borbone, N.; Zollo, F.; Vitalini, S.; Visioli,
F.; Iorizzi, M. Phytochemistry 2007, 68, 1805–1812.
(41) Saroyobudiono, H.; Juliawaty, L. D.; Syah, Y. M.; Achmad, S. A.;
Hakim, E. H.; Latip, J.; Said, I. M. J. Nat. Med. 2008, 62, 195–198.
(18) Oshima, Y.; Ueno, Y.; Hikino, H.; Ling-Ling, Y.; Kun-Ying, Y.
Tetrahedron 1990, 46, 5121–5126.
(19) Ohyama, M.; Tanaka, T.; Iinuma, M.; Burandt, C. L. Chem. Pharm.
Bull. 1998, 46, 663–668.
(20) Kim, H. J.; Chang, E. J.; Bae, S. J.; Shim, S. M.; Park, H. D.; Rhee,
C. H.; Park, J. H.; Choi, S. W. Arch. Pharm. Res. 2002, 25, 293–299.
NP1002675