Euscaphinin, a new ellagitannin dimer from Euscaphis japonica (Thunb.) Kanitz

Article abstract of DOI:10.1248/cpb.57.421

A new ellagitannin dimer named euscaphinin was isolated from the leaves of Euscaphis japonica (Thunb.) Kanitz. The structure of this dimer was determined on the basis of spectroscopic and chemical evidence. The resulting structure indicated that it was pr

Full text of DOI:10.1248/cpb.57.421

April 2009
Notes
Chem. Pharm. Bull. 57(4) 421—423 (2009)
421
Euscaphinin, a New Ellagitannin Dimer from Euscaphis japonica (THUNB.)
KANITZ
a,b
a
,a
,a
Hajime MAEDA, Yosuke MATSUO, Takashi TANAKA,* and Isao KOUNO*
a
Graduate School of Biomedical Sciences, Nagasaki University; 1–14 Bunkyo-machi, Nagasaki 852–8521, Japan: and
Nagasaki Agriculture and Forestry Experiment Station; 3118 Kaidzu, Isahaya, Nagasaki 854–0063, Japan.
b
Received December 10, 2008; accepted January 10, 2009; published online January 14, 2009
A new ellagitannin dimer named euscaphinin was isolated from the leaves of Euscaphis japonica (THUNB.)
KANITZ. The structure of this dimer was determined on the basis of spectroscopic and chemical evidence. The re-
sulting structure indicated that it was produced by intermolecular C–O oxidative coupling between the galloyl
groups of molecules of 1(b)-O-galloyl pedunculagin, the major ellagitannin of the leaves. The ellagitannins were
not detected in the bark and wood part of the plant. This is the first example of the isolation of ellagitannins
from Staphyleaceaeous plants.
Key words Euscaphis japonica; Staphyleaceae; ellagitannin; pedunculagin; tannin
Recent studies on plant polyphenols have revealed various The sugar was confirmed to be D-glucose by the HPLC
1,2)
biological activities for ellagitannins. Distribution of ellagi- analysis of the thiocarbamoyl-thiazolidine derivative of the
20)
tannins in plants is relatively limited compared with that of acid hydrolysis products. The chemical shifts (d 3.7—6.2)
3)
condensed tannins (proanthocyanidins). In this study, we and the large coupling constants of the sugar proton signals
proposed to find new plant sources of functional polyphenols were similar to those of 1 and indicated that the anomeric po-
and examined the leaves, bark and wood of 39 plant samples sitions were b-configurations and that all the hydroxyl
belonging to 32 genera of 29 families, distributed in the Na- groups of the pyranose ring were acylated. In the aromatic
gasaki region of Japan, by thin-layer chromatography (TLC) region of the spectrum, nine singlets [d 7.16, 6.66, 6.65,
and high performance liquid chromatography (HPLC). Plants 6.51, 6.50, 6.47, 6.46, 6.33, 6.33] and two mutually coupled
4)
5)
belonging to the Elaeocarpaceae, Myricaceae, Anacar- doublets [d 7.30, 6.84 (each d, Jꢁ1.9 Hz)] were observed.
6)
7)
8)
9—11)
diaceae, Juglandaceae, Stachyuraceae, and Fagaceae
The hetero-nuclear multiple-bond connectivity (HMBC) cor-
families were shown to contain high concentrations of relations (Fig. 2) of these aromatic protons indicated that all
polyphenols, substances that have already been studied of the protons are attached to the 3,4,5-trihydroxybenzoyl
1
3
chemically. Unexpectedly, our study disclosed accumulation ester moieties. The C-NMR spectrum showed signals at-
of ellagitannins in the leaves of Euscaphis japonica (THUNB.) tributable to ten ester carbon signals, among which eight
KANITZ (Staphyleaceae). E. japonica is a deciduous tree dis- were observed in the range of 167 to 169 and remaining two
tributed in western Japan, and little is known about the phe- appeared upfield (d 162.4, 165.0). The ester signal at d 165.0
1
2—16)
nolic constituents of this plant.
Presence of ellagitan- [dehydrodigalloyl (DHDG) C-7] showed three HMBC corre-
nins in Staphyleaceae has not yet been reported. This paper
describes the isolation and structural determination of two el-
lagitannins, including a new dimeric ellagitannin.
Results and Discussion
An aqueous acetone extract of the fresh leaves of E. japon-
ica was subjected to a combination of Diaion HP20SS,
Sephadex LH-20, and Chromatorex ODS column chroma-
tography to furnish two major phenolic compounds. One of
the products was identified as 1(b)-O-galloyl pedunculagin
17,18)
1
(1)
by comparisons of H-NMR spectral data, HPLC re-
tention time and UV absorption with those of an authentic
sample.
Compound 2 was isolated as a white amorphous powder
and produced a dark-blue coloration upon reaction with
FeCl and a brown coloration upon reaction with NaNO –
3
2
19)
AcOH reagent, suggesting this compound is an ellagitan-
nin. The Rf value on silica gel TLC was much smaller than
that of 1 and the matrix-assisted laser desorption/ionization-
ꢀ
time-of-flight (MALDI-TOF) MS showed a [MꢀNa] peak
at m/z 1893, indicating that 2 is a dimeric ellagitannin. This
was supported by appearance of signals attributable to two
sugar anomeric protons [d 6.16 (1H, d, Jꢁ8.5 Hz, Glc-1),
1
6
.06 (1H, d, Jꢁ8.7 Hz, Glc-1ꢂ)] in the H-NMR spectrum. Fig. 1. Structures of Compounds 1, 2, and 3
∗
To whom correspondence should be addressed. e-mail: t-tanaka@nagasaki-u.ac.jp
© 2009 Pharmaceutical Society of Japan

4
22
Vol. 57, No. 4
lagitannin was assigned to 2, and named euscaphinin. This
tannin was deduced to be produced by intermolecular simple
oxidative coupling between the two galloyl groups of 1.
Some dimeric ellagitannins having m-dehydrodigalloyl
2
3,24)
groups were already isolated from Rosaseous,
Tamarica-
25)
26)
27)
30)
ceous, Coriariaceous, Euphorbiaceous and Nymphaea-
2
8,29)
ceous
plants. Among them, agrimoniin is the only iso-
mer of 2 composed of 1 and its isomer differing only in the
anomeric configuration.
Fig. 2. Selected HMBC Correlations for 2
The ellagitannins 1 and 2 were only found in the leaves of
E. japonica and were not detected in the bark and wood.
HPLC analysis of the aqueous acetone extract of the bark
showed a large peak corresponding to a major phenolic com-
pound, which was isolated and identified to be 3,3ꢂ-di-O-
31)
methyl ellagic acid (3). The ellagitannin 1 was reported to
be a selective inhibitor of the b-regulatory subunit of A-ki-
32)
nase and an effective autophosphorylation inhibitor of C-
33)
kinase. Since 1 was found to be accumulated in the leaves
in high concentration (1.28% of fresh leaves), E. japonica is
expected to be a potent plant source of this biologically ac-
tive ellagitannin.
Fig. 3. Structures of 2a and 2b
lation peaks with one of the glucose anomeric proton signal
[
d 6.16 (Glc-1)], and the meta-coupled aromatic doublets at Experimental
General UV spectra were obtained with a Jasco V-560 UV/Vis spec-
d 7.30 and 6.84. In turn, the ester carbon signal at d 162.4
trophotometer and optical rotations were measured with a Jasco DIP-370
digital polarimeter. H- and C-NMR spectra were measured in acetone-d6
(
[
DHDG C-7ꢂ) was correlated with the other anomeric proton
d 6.06 (Glc-1ꢂ)] and the aromatic singlet at d 7.16. The re-
1
13
at 27 °C with a Varian Unity plus 500 spectrometer operating at 500 MHz
1
13
maining eight ester carbons showed correlation peaks with for H and at 125 MHz for C. Coupling constants are expressed in Hz and
glucose H-2—H-6 protons and the aromatic singlets res- chemical shifts are given on a d (ppm) scale. MS were recorded on a Voy-
ager DE-PRO (Applied Biosystems) and a JEOL JMS-700N spectrometer.
onated in the range of d 6.66—6.33. These aromatic proton
signals correlated with the aromatic carbon signals, the
chemical shifts of which indicated the presence of four hexa-
2,5-Dihydroxybenzoic acid and glycerol were used as the matrix for
MALDI-TOF-MS and FAB-MS measurements, respectively. Column chro-
matography was performed using Diaion HP20SS (Mitsubishi Chemical),
hydroxydiphenoyl (HHDP) ester groups similar to those of 1, Sephadex LH-20 (25—100 mm; Sigma), and Chromatorex ODS (100—200
though complete assignments were difficult due to overlap- mesh; Fuji Silysia Chemical) columns. TLC was performed on precoated
Kieselgel 60 F254 plates (0.2 mm thick; Merck) with toluene–ethyl formate–
formic acid (1 : 7 : 1, v/v) as a solvent, and spots were detected by UV illu-
ping signals.
The acyl groups were confirmed by chemical degradation.
First, 2 was methylated with CH N and the product was hy-
mination (254 nm) and by spraying with 2% ethanolic FeCl and 10% sulfu-
3
2
2
ric acid reagent, followed by heating.
drolyzed under alkaline conditions. The resulting carboxylic
Plant Material The leaves of E. japonica were collected in May 2007,
acids were converted to methyl esters with CH N to yield di- and a voucher specimen was deposited at the Nagasaki Agriculture and
2
2
Forestry Experiment Station, Japan.
methyl pentamethyl dehydrodigallate (2a) and dimethyl (S)-
,4ꢂ,5,5ꢂ,6,6ꢂ-hexamethoxydiphenoate (2b) (Fig. 3). The lat-
ter showed a negative optical rotation value ([a] ꢃ24.0°);
Extraction and Separation The fresh leaves of E. japonica (500 g)
4
were crushed with acetone–H O (4 : 1, v/v, 1 l) in a Warring blender and fil-
2
D
tered. The plant debris remaining on the filter paper was further extracted
thus the atropisomerism of the biphenyl bond of 2b was with the same solvent at room temperature two times. The three extracts
21)
were combined and acetone was removed by evaporation under reduced
pressure (ca. 40 °C). The resulting dark green precipitates, consisting mainly
of chlorophylls and waxes, were removed by filtration. The filtrate was sub-
jected to Diaion HP20SS column chromatography (4.0 cm i.d.ꢅ30 cm) with
determined to be S-configuration.
The aforementioned
HMBC correlations indicated that the two ester carbonyls of
the dehydrodigalloyl groups were attached to the both the
anomeric hydroxyl groups. The HHDP ester groups are be- water containing increasing proportions of MeOH (10%, 20%, 30%, 40%,
lieved to bridge over the 2,3-hydroxy and 4,6-hydroxy groups 50%, 70%, 100%) to give three fractions containing polyphenols; fr. 1
(
3.2 g), fr. 2 (12.8 g), and fr. 3 (5.45 g). The fr. 2, which showed dark blue
based on the large coupling constants of the sugar proton sig-
coloration with 2% ethanolic FeCl3 reagent, was further separated by
Sephadex LH-20 column chromatography (4.0 cm i.d.ꢅ25 cm) with aqueous
MeOH–H O–acetone (3 : 2 : 0, 4 : 1 : 0, 1 : 0 : 0, 18 : 1 : 1, 8 : 1 : 1, 3 : 1 : 1, and
nals (J ꢁJ ꢁJ ꢁ9—10 Hz) indicating that the glucose
2
,3
3,4
4,5
4
adapted a C conformation. In addition, the large chemical
1
2
shift differences (Dd ꢄ1.4) for the glucose methylene pro- then 0 : 1 : 1, v/v/v, each 300 ml) to yield 1(b)-O-galloyl pedunculagin (1)
tons [d 5.31, 5.29 (each 1H, dd, Jꢁ6.9, 13.5 Hz, Glc-6a, (6.41 g, eluted with MeOH–H O–acetone, 4 : 1 : 0 and 1 : 0 : 0) and fraction
-2 (1.05 g, eluted with MeOH–H O–acetone, 8 : 1 : 1 and 3 : 1 : 1) which
2
2
6
aꢂ), 3.82, 3.77 (each 1H, dd, Jꢁ1.4, 13.5 Hz, Glc-6b, 6bꢂ)]
2
mainly contained euscaphinin. Fraction 2-2 was successively applied to a
Sephadex LH-20 column (3.0 cm i.d.ꢅ20 cm) with EtOH–H O–acetone
were observed. This spectroscopic phenomenon was com-
monly observed in the spectrum of ellagitannins having (S)-
2
(
1 : 0 : 0, 9 : 1 : 0, 4 : 1 : 0, 3 : 4 : 0, 27 : 18 : 5, 12 : 8 : 5 and then 6 : 4 : 5, v/v/v,
4
HHDP esters at the C-4 and C-6 hydroxyl groups of the C - each 200 ml, 2 was eluted with 27 : 18 : 5 and 12 : 8 : 5) and a Chromatorex
1
22)
glucopyranose. The location of the esters was also sup- ODS column (3.0 cm i.d.ꢅ20 cm) with water containing increasing propor-
tions of MeOH (5—40%, 5% stepwise elution) to give euscaphinin (2)
ported by the partial hydrolysis of 2 in hot water, which
yielded pedunculagin [a mixture of a and b form of 2,3,4,6-
bis-(S)-HHDP-D-glucose]. On the basis of these spectro-
(0.30 g, eluted with 30% MeOH).
Separation of 3,3ꢀ-Di-O-methyl Ellagic Acid from Bark Fresh bark of
E. japonica (500 g) was extracted 3 times with 80% acetone at room temper-
scopic and chemical results, the structure of this dimeric el- ature, and the resulting extract was concentrated under reduced pressure.

April 2009
423
After removal of insoluble precipitates by filtration, the filtrate was separated No. 18510189 from the Japan Society for the Promotion of Science.
by Diaion HP20SS column chromatography (0—100% MeOH, 10% step-
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0
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1
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1
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3
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2
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(
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1
1
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2
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2
2
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D
3
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R
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anomers of pedunculagin.
254—264 (1994).
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mixture was neutralized by addition of Amberlite IRA400 (OH form) and
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containing L-cysteine methyl ester (10 mg/ml) and reacted at 60 °C for 1 h.
To this mixture a solution (0.2 ml) of o-torylisothiocyanate in pyridine
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ꢃ
(10 mg/ml) was added and the solution was heated at 60 °C for 1 h. The final
mixture was directly analyzed by HPLC [Cosmosil 5C18 AR II (250ꢅ4.6
mm i.d., Nacalai Tesque Inc.); 25% CH CN in 50 mM H PO ; flow rate,
3
3
4
0.8 ml/min; detection, 250 nm]. The tR of the peak at 17.48 min coincided
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Acknowledgments The authors are grateful to Mr. Hiroyuki Soeyama 32) Kosuge S., Maekawa T., Saito C., Tanaka T., Kouno I., Ohtsuki K., J.
for collection of plant samples. They are also indebted to Mr. K. Inada and Biochem. (Tokyo), 129, 403—409 (2001).
Mr. N. Yamaguchi (Nagasaki University) for the NMR and MS measure- 33) Ueno T., Miyanaga T., Kawakami F., Okano M., Tanaka T., Ohtsuki
ments. This work was supported by a Grant-in-aid for Scientific Research K., Biol. Pharm. Bull., 25, 1401—1404 (2002).